Multisite receptors and signaling complexes

Abstract

The present invention provides adoptive T cell therapy with improved structures for targeting antigens and recruiting multimeric immune signaling complexes to treat, prevent, or ameliorate at least one symptom of cancer, infectious disease, autoimmune disease, inflammatory disease, and immune deficiency, or conditions associated therewith. The present disclosure relates generally to engineered immune receptor complexes capable of recognizing a target antigen and recruiting and activating natural or transgenic immune receptor signaling complexes, e.g., T cell receptor (TCR) signaling complexes, polynucleotides and polypeptides encoding same, compositions thereof, and methods of making and using same to treat a disorder or disease (e.g., cancer or autoimmunity).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 329,003, filed April 8, 2022, which is incorporated herein by reference in its entirety.

[0002] The present disclosure relates to adoptive cell therapy. More specifically, the present disclosure relates to improved immune receptor signaling molecules, cells, and methods of using the same to modulate the spatial and / or temporal control of immune receptor signal initiation and downstream responses during adoptive immunotherapy. [Background technology]

[0003] Description of related fields Cancer incidence doubled worldwide between 1975 and 2000. It is the second leading cause of morbidity and mortality worldwide, with approximately 14.1 million new cases and 8.2 million cancer-related deaths in 2012. The number of new cancer cases is expected to rise to 22 million within the next 20 years.

[0004] While chemotherapy and subsequent biologics have been the standard of care for decades, adoptive cell therapy (ACT) has emerged as a powerful paradigm for delivering complex biological signals to treat cancer. In contrast to small molecule and biologic drugs, ACT has the potential to exert unique therapeutic tasks through diverse sensory and response programs and further defined gene regulatory mechanisms. To realize such therapeutic value, cells must be equipped with mechanisms to understand and integrate chemical and / or biological information associated with their local physiological environment.

[0005] In recent years, adapting cells with chimeric antigen receptors (CARs) or transgenic T cell receptors (TCRs) has proven to be a powerful method for targeting immune cells to specific antigens (e.g., tumor antigens), stimulating T cell activation signaling, and ultimately attacking and killing antigen-engaged cells (e.g., cancer cells). Despite these successes, fundamental differences and limitations between the two structures remain, including i) sensitivity, ii) antigen recognition, iii) antigen-independent signaling activity, and iv) lack of readability. Thus, there remains a need for improved targeting and signaling mechanisms that more potently sense and respond to target antigens and associated cells. Summary of the Invention [Means for solving the problem]

[0006] The present disclosure relates generally to engineered immune receptor complexes that can recognize target antigens and recruit and activate natural or transgenic immune receptor signaling complexes, e.g., T cell receptor (TCR) signaling complexes, polynucleotides and polypeptides encoding same, compositions thereof, and methods of making and using same to treat disorders or diseases (e.g., cancer or autoimmunity).

[0007] In one aspect, disclosed herein is a non-naturally occurring cell comprising a signaling moiety comprising a first multimerization domain and an actuator domain; and a targeting moiety comprising an extracellular domain, a second multimerization domain, and a transmembrane domain.

[0008] In one embodiment, the second multimerization domain and the transmembrane domain are separated by a hinge domain. In some embodiments, the hinge domain is selected from the group consisting of a CD4 hinge, a CD8 hinge, a CD28 hinge, an IgG4 hinge, and any fragment, variant, or combination thereof. In some embodiments, the hinge domain is a CD4 hinge. In some embodiments, the CD4 hinge comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 41. In one embodiment, the CD4 hinge comprises the amino acid sequence set forth in SEQ ID NO: 41. In some embodiments, the hinge domain is a CD28 hinge. In some embodiments, the CD28 hinge comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 42. In one embodiment, the CD28 hinge comprises the amino acid sequence set forth in SEQ ID NO: 42. In some embodiments, the hinge domain is a CD8 hinge. In some embodiments, the CD8 hinge comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 44. In one embodiment, the CD8 hinge comprises the amino acid sequence set forth in SEQ ID NO: 44. In some embodiments, the hinge domain is an IgG4 hinge. In some embodiments, the IgG4 hinge comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 43. In one embodiment, the IgG4 hinge comprises the amino acid sequence set forth in SEQ ID NO: 43.

[0009] In some embodiments, the actuator domain is a CD3 polypeptide, an FcεR1γ polypeptide, an Igα / CD79a polypeptide, an Igβ / CD79b polypeptide, a DAP10 polypeptide, or a DAP12 polypeptide. In some embodiments, the CD3 polypeptide is a CD3 epsilon (CD3ε) polypeptide or a variant thereof, a CD3 gamma (CD3γ) or a variant thereof, or a CD3 delta (CD3δ) or a variant thereof. In some embodiments, the actuator domain is a CD3ε polypeptide or a variant thereof. In some embodiments, the actuator domain is a CD3ε polypeptide comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 32. In one embodiment, the actuator domain is a CD3ε polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 32. In some embodiments, the actuator domain is a CD3γ polypeptide or a variant thereof. In some embodiments, the actuator domain is a CD3γ polypeptide comprising an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 33. In one embodiment, the actuator domain is a CD3γ polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 33. In some embodiments, the actuator domain is a CD3δ polypeptide or a variant thereof. In some embodiments, the actuator domain is a CD3δ polypeptide comprising an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 34. In one embodiment, the actuator domain is a CD3δ polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 34. In some embodiments, the actuator domain is an FcεR1γ polypeptide or a variant thereof. In some embodiments, the actuator domain is an FcεR1γ polypeptide comprising an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 35. In one embodiment, the actuator domain is an FcεR1γ polypeptide comprising the amino acid sequence set forth in SEQ ID NO:35.In some embodiments, the actuator domain is an FcεR1γ polypeptide comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 36. In one embodiment, the actuator domain is an FcεR1γ polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 36. In some embodiments, the actuator domain is an Igα / CD79a polypeptide or a variant thereof. In some embodiments, the actuator domain is an Igα / CD79a polypeptide comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 37. In one embodiment, the actuator domain is an Igα / CD79a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 37. In some embodiments, the actuator domain is an Igβ / CD79b polypeptide or a variant thereof. In some embodiments, the actuator domain is an Igβ / CD79b polypeptide comprising an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 38. In one embodiment, the actuator domain is an Igβ / CD79b polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 38. In some embodiments, the actuator domain is a DAP10 polypeptide or a variant thereof. In some embodiments, the actuator domain is a DAP10 polypeptide comprising an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 39. In one embodiment, the actuator domain is a DAP10 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 39. In some embodiments, the actuator domain is a DAP12 polypeptide or a variant thereof. In some embodiments, the actuator domain is a DAP12 polypeptide comprising an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 40. In one embodiment, the actuator domain is a DAP12 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 40. In one embodiment, the actuator domain comprises both an extracellular portion and an intracellular portion.

[0010] In one embodiment, the first and second multimerization domains are localized extracellularly when the signaling component and the targeting component are expressed. In one embodiment, the first and second multimerization domains are different. In some embodiments, the multimerization domains of the signaling component and the targeting component are associated with a cross-linking agent selected from the group consisting of rapamycin or a rapalog thereof, gibberellin or a derivative thereof, abscisic acid (ABA) or a derivative thereof, methotrexate or a derivative thereof, cyclosporin A or a derivative thereof, FK506 / cyclosporin A or a derivative thereof, trimethoprim (Tmp) synthetic ligand for FK506 binding protein (FKBP) (SLF) or a derivative thereof, wherein the cross-linking agent promotes the formation of a polypeptide complex, and the cross-linking agent is associated with and disposed between the multimerization domains of the signaling component and the targeting component. In some embodiments, the first multimerization domain and the second multimerization domain are a pair selected from the group consisting of FK506-binding protein 1A (FKBP12) and FKBP12-rapamycin-binding (FRB), FKBP12 and calcineurin, FKBP and cyclophilin A or any other member of the peptidyl-prolyl cis-trans isomerase (PPIase) family, FKBP and dihydrofolate reductase (DHFR), calcineurin and cyclophilin A or any other member of the peptidyl-prolyl cis-trans isomerase (PPIase) family, and PYR1-like 1 (PYL1) and abscisic acid-insensitive 1 (ABI1). In one embodiment, the first multimerization domain comprises a first FRB polypeptide or a variant thereof, and the second multimerization domain comprises a first FKBP12 polypeptide or a variant thereof. In one embodiment, the first multimerization domain comprises a first FKBP12 polypeptide or a variant thereof, and the second multimerization domain comprises a first FRB polypeptide or a variant thereof. In one embodiment, the FRB polypeptide is the FRB T2098L variant. In some embodiments, the FRB polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the FKBP12 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4.

[0011] In some embodiments, the cross-linking agent is AP1903, AP20187, AP21967 (also known as C16-(S)-7-methylindolerapamycin), everolimus, novolimus, pimecrolimus, ridaforolimus, sirolimus, tacrolimus, temsirolimus, umirolimus, zotarolimus, or BPC015. In some embodiments, the first multimerization domain and the second multimerization domain are heterodimerization domains derived from a pair of antibodies.

[0012] In some embodiments, the first multimerization domain comprises a polypeptide or comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 5; and the second multimerization domain comprises a polypeptide or comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the first multimerization domain comprises a polypeptide or comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 6; and the second multimerization domain comprises a polypeptide or comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the first multimerization domain comprises a polypeptide or comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 7; and the second multimerization domain comprises a polypeptide or comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the first multimerization domain comprises a polypeptide or comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 8; and the second multimerization domain comprises a polypeptide or comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the first multimerization domain comprises a polypeptide having at least 90%, 95%, 96%, 97%, 98%, 99% identity to, or comprises an amino acid sequence set forth in SEQ ID NO: 9; and the second multimerization domain comprises a polypeptide having at least 90%, 95%, 96%, 97%, 98%, 99% identity to, or comprises an amino acid sequence set forth in SEQ ID NO: 10.In some embodiments, the first multimerization domain comprises a polypeptide having at least 90%, 95%, 96%, 97%, 98%, 99% identity to, or comprises an amino acid sequence set forth in SEQ ID NO: 10; and the second multimerization domain comprises a polypeptide having at least 90%, 95%, 96%, 97%, 98%, 99% identity to, or comprises an amino acid sequence set forth in SEQ ID NO: 9.

[0013] In some embodiments, the first multimerization domain and the actuator domain are separated by a first polypeptide linker between 2 and 40 amino acids in length. In some embodiments, the first polypeptide linker is selected from the group consisting of GG, GS, SG, SS, GSS, SSG, GSG, SGS, SGG, G4S, 2xG4S, 3xG4S, 4xG4S, 5xG4S, and any combination thereof. In one embodiment, the first polypeptide linker is a 3xG4S linker. In some embodiments, the first polypeptide linker comprises a polypeptide comprising an amino acid sequence set forth as any one of SEQ ID NOs: 16-31. In some embodiments, the extracellular domain and the second multimerization domain are separated by a second polypeptide linker between 2 and 40 amino acids in length. In some embodiments, the second polypeptide linker is selected from the group consisting of GG, GS, SG, SS, GSS, SSG, GSG, SGS, SGG, G4S, 2xG4S, 3xG4S, 4xG4S, and any combination thereof. In one embodiment, the second polypeptide linker is a G4S linker. In some embodiments, the second polypeptide linker comprises a polypeptide comprising an amino acid sequence set forth as any one of SEQ ID NOs: 16-31.

[0014] In some embodiments, the transmembrane domain is a CD4 transmembrane domain. In some embodiments, the CD4 transmembrane domain comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 45. In one embodiment, the CD4 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 45. In some embodiments, the transmembrane domain is a CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 46. In one embodiment, the CD28 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 46. In some embodiments, the transmembrane domain is a CD8 transmembrane domain. In some embodiments, the CD8 transmembrane domain comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 47. In one embodiment, the CD8 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 47.

[0015] In some embodiments, the targeting moiety further comprises an intracellular signaling or costimulatory domain derived from a protein selected from the group consisting of an antigen receptor, a costimulatory receptor, a growth receptor, a cytokine receptor, an adaptor signaling protein, an intracellular signaling protein, or any fragment or variant thereof. In some embodiments, the intracellular signaling or costimulatory domain on the targeting moiety is selected from the group consisting of Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain family member 11 (CARD11), CD2, CD3ε, CD3γ, CD3δ, CD4, CD7, CD8, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICAM), CD83 (ICAM), CD94 (ICAM), ...83 (ICAM), CD94 (ICAM), CD134 (OX40), CD137 (4-1BB), CD278 (ICAM), CD83 (ICAM), CD83 (ICAM), CD83 (ICAM), CD83 (ICAM), CD83 (ICAM), CD83 (ICAM), CD83 (ICAM), CD COS), common gamma chain cytokine, DNAX-activation protein 10 (DAP10), linker for activation of T-cell family member 1 (LAT), interleukin 2 receptor (IL-2R), IL-4R, IL-7R, IL-9R, IL-12R, IL-13R, IL-15R, IL-21R, SH2 domain-containing leukocyte protein 76 kD (SLP76), T cell receptor-associated transmembrane adaptor 1 (TRAT1), TNFR2, TNFRS14, TNFRS18, TNRFS25, and zeta chain of T cell receptor-associated protein kinase 70 (ZAP70). In some embodiments, the intracellular signaling or costimulatory domain on the targeting moiety is selected from the group consisting of 4-1BB, CD28, TNFR2, OX40, ICOS, and a DAP10 costimulatory domain. In one embodiment, the costimulatory domain on the targeting moiety is a 4-1BB costimulatory domain, optionally wherein the 4-1BB costimulatory domain comprises the amino acid sequence set forth in SEQ ID NO: 98. In one embodiment, the costimulatory domain on the targeting moiety is a CD28 costimulatory domain, optionally wherein the CD28 costimulatory domain comprises the amino acid sequence set forth in SEQ ID NO: 99. In some embodiments, the intracellular signaling or costimulatory domain is one or more cytokine receptor intracellular signaling domains.In some embodiments, the one or more cytokine receptor intracellular signaling domains are selected from the group consisting of an IL7Rα intracellular signaling domain, an IL2Rβ intracellular signaling domain, a common gamma chain intracellular signaling domain, and both an IL2Rβ and a common gamma chain intracellular signaling domain. In some embodiments, the IL7Rα intracellular signaling domain comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 103. In one embodiment, the IL7Rα intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 103. In some embodiments, the IL2Rβ intracellular signaling domain comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 104 or SEQ ID NO: 105. In one embodiment, the IL2Rβ intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 104 or SEQ ID NO: 105. In some embodiments, the common gamma chain intracellular signaling domain comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 106. In one embodiment, the common gamma chain intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 106. In some embodiments, the intracellular signaling or costimulatory domain is a LAT domain. In some embodiments, the LAT domain comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 102. In one embodiment, the LAT domain comprises the amino acid sequence set forth in SEQ ID NO: 102. In some embodiments, the intracellular signaling or costimulatory domain is a CD4 co-receptor domain. In some embodiments, the CD4 co-receptor domain comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 100. In one embodiment, the CD4 co-receptor domain comprises the amino acid sequence set forth in SEQ ID NO: 100. In some embodiments, the intracellular signaling or costimulatory domain is a CD8 co-receptor domain.In some embodiments, the CD8 co-receptor domain comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 101. In one embodiment, the CD8 co-receptor domain comprises the amino acid sequence set forth in SEQ ID NO: 101.

[0016] In some embodiments, the targeting moiety does not comprise a functional intracellular signaling or costimulatory domain. In some embodiments, the targeting moiety further comprises a truncated intracellular CD4 polypeptide. In some embodiments, the truncated intracellular CD4 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 48 or SEQ ID NO: 49. In some embodiments, the truncated intracellular CD4 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 48 or SEQ ID NO: 49. In some embodiments, the extracellular domain comprises a first targeting domain. In some embodiments, the first targeting domain comprises a single-chain variable fragment (scFv) or a single-domain antibody (sdAb). In some embodiments, the sdAb is a camelid VHH, a nanobody, or a heavy-chain-only antibody (HcAb). In one embodiment, the sdAb is a camelid VHH. In some embodiments, the scFv or sdAb is human or humanized. In some embodiments, the first targeting domain comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 50-83. In some embodiments, the first targeting domain comprises a sequence set forth in any one of SEQ ID NOs: 50-83. In some embodiments, the first targeting domain comprises a PD1 ectodomain, human A-proliferation-inducing ligand (APRIL), trimerized human APRIL, or NKG2D membrane protein. In some embodiments, the PD1 ectodomain comprises a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 84 or SEQ ID NO: 85. In one embodiment, the PD1 ectodomain comprises a sequence set forth in SEQ ID NO: 85. In some embodiments, APRIL comprises a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 86, or trimerized human APRIL comprises a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 87. In one embodiment, APRIL comprises the sequence set forth in SEQ ID NO: 86, or the trimerized human APRIL comprises the sequence set forth in SEQ ID NO: 87. In some embodiments, the NKG2D membrane protein comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 88.In one embodiment, the NKG2D membrane protein comprises the sequence shown as SEQ ID NO:88.

[0017] In some embodiments, the extracellular domain further comprises a second targeting domain. In some embodiments, the second targeting domain comprises a second single-chain variable fragment (scFv) or a second single-domain antibody (sdAb). In some embodiments, the second sdAb is a camelid VHH, a nanobody, or a heavy-chain-only antibody (HcAb). In one embodiment, the second sdAb is a camelid VHH. In some embodiments, the second scFv or second sdAb is human or humanized. In some embodiments, the second targeting domain comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 50-83. In some embodiments, the second targeting domain comprises a sequence set forth in any one of SEQ ID NOs: 50-83. In some embodiments, the second targeting domain comprises a PD1 ectodomain, human A-growth-inducing ligand (APRIL), trimerized human APRIL, or NKG2D membrane protein. In some embodiments, the PD1 ectodomain comprises a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 84 or SEQ ID NO: 85. In one embodiment, the PD1 ectodomain comprises the sequence set forth in SEQ ID NO: 85. In some embodiments, APRIL comprises a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 86, or trimerized human APRIL comprises a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 87. In one embodiment, APRIL comprises the sequence set forth in SEQ ID NO: 86, or trimerized human APRIL comprises the sequence set forth in SEQ ID NO: 87. In some embodiments, the NKG2D membrane protein comprises a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 88. In one embodiment, the NKG2D membrane protein comprises the sequence shown as SEQ ID NO:88.

[0018] In some embodiments, the targeting domain and the second targeting domain bind to the same antigen or different antigens. In some embodiments, the targeting domain and the second targeting domain are separated by a third polypeptide linker between 2 and 40 amino acids in length. In some embodiments, the third polypeptide linker is selected from the group consisting of GG, GS, SG, SS, GSS, SSG, GSG, SGS, SGG, G4S, 2xG4S, 3xG4S, 4xG4S, 5xG4S, any one of SEQ ID NOs: 16-31, and any combination thereof. In some embodiments, the signaling component comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, or SEQ ID NO: 115. In some embodiments, the signaling component comprises the sequence set forth as SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, or SEQ ID NO:115.

[0019] In some embodiments, the targeting moiety comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to the sequence set forth as SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, or SEQ ID NO:136. In some embodiments, the targeting moiety comprises the sequence set forth as SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, or SEQ ID NO:136.

[0020] In some embodiments, the signaling moiety and the targeting moiety further comprise a signal sequence, optionally the signal sequence is a CD8, IgK, or PD1 signal sequence.

[0021] In some embodiments, the signaling component further comprises a signal sequence, optionally wherein the signal sequence has or comprises at least 90%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:95, SEQ ID NO:96, or SEQ ID NO:97.

[0022] In some embodiments, the targeting moiety further comprises a signal sequence, optionally wherein the signal sequence has or comprises at least 90%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:95, SEQ ID NO:96, or SEQ ID NO:97.

[0023] In one embodiment, the signaling component further comprises a signal sequence. In one embodiment, the signal sequence is a CD8 signal sequence. In one embodiment, the CD8 signal sequence comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 96. In another embodiment, the CD8 signal sequence comprises the amino acid sequence set forth as SEQ ID NO: 96.

[0024] In another embodiment, the targeting moiety further comprises a signal sequence. In one embodiment, the signal sequence is an IgK signal sequence. In one embodiment, the IgK signal sequence comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 95. In one embodiment, the IgK signal sequence comprises the amino acid sequence set forth as SEQ ID NO: 95.

[0025] In one embodiment, the non-native cell comprises a fusion polypeptide comprising a targeting moiety and a signaling moiety. In some embodiments, the fusion polypeptide comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, or SEQ ID NO:170. In some embodiments, the fusion polypeptide comprises the sequence set forth as SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, or SEQ ID NO:170.

[0026] In some embodiments, the cell comprises a first nucleic acid molecule encoding a signaling component. In some embodiments, the cell comprises a second nucleic acid molecule encoding a targeting component. In some embodiments, the cell comprises nucleic acid molecules encoding both a signaling component and a targeting component. In some embodiments, the cell further expresses an exogenous costimulatory factor, an immunomodulatory factor, an agonist for a costimulatory factor, an antagonist for an immunosuppressive factor, an immune cell engager, a flip receptor, or any combination thereof.

[0027] In some embodiments, the cells further express an exogenous lymphocyte receptor or co-receptor. In some embodiments, the exogenous lymphocyte receptor or co-receptor is selected from the group consisting of TCR alpha (TCRα), TCR beta (TCRβ), TCR gamma (TCRγ), TCR delta (TCRδ), CD4, CD8, pre-T cell receptor alpha (pTα), Fc receptor alpha (FcRα), Fc receptor beta (FcRβ), Fc receptor gamma (FcRγ), natural killer group 2 member D (NKG2D), CD79A, CD79B, and any combination thereof. In one embodiment, the cells further express an exogenous TCR.In some embodiments, the exogenous TCR is selected from the group consisting of alpha-fetoprotein (AFP), B melanoma antigen (BAGE) family members, siblings of regulator of imprinted sites (BORIS), cancer testis antigen, cancer testis antigen 83 (CT-83), carbonic anhydrase IX (CA1X), carcinoembryonic antigen (CEA), cytomegalovirus (CMV) antigen, cytotoxic T cell (CTL)-recognized antigen against melanoma (CAMEL), Epstein-Barr virus (EBV) antigen, G antigen 1 ( GAGE-1), GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, GAGE-8, glycoprotein 100 (GP100), hepatitis B virus (HBV) antigen, hepatitis C virus (HCV) nonstructural protein 3 (NS3), human epidermal growth factor receptor 2 (HER-2), human papillomavirus (HPV)-E6, HPV-E7, human telomerase reverse transcriptase (hTERT), IGF2BP3 / A3, K-Ras, K-Ras G12C, K-Ras G12D, K-Ras G12V, latent membrane protein 2 (LMP2), melanoma antigen family A, 1 (MAGE-A1), MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, melanoma antigen recognized by T cells (MART-1), mesothelin (MSLN), mucin 1 (MUC1), mucin 16 (MUC16), New York esophageal squamous cell carcinoma-1 (NYESO-1), p53, P antigen (PAGE) family member, placenta-specific 1 (PLAC1), melanoma The exogenous TCR binds to a target antigen selected from the group consisting of preferentially expressed antigen in human TCRs (PRAME), survivin, synovial sarcoma X1 (SSX1), synovial sarcoma X2 (SSX2), synovial sarcoma X3 (SSX3), synovial sarcoma X4 (SSX4), synovial sarcoma X5 (SSX5), synovial sarcoma X8 (SSX8), thyroglobulin, tyrosinase, tyrosinase-related protein (TRP) 1, TRP2, Wilms' tumor protein (WT-1), X antigen family member 1 (XAGE1), and X antigen family member 2 (XAGE2). In some embodiments, the exogenous TCR is an αβ-TCR or a γδ-TCR.

[0028] In some embodiments, the cells further express a CAR, CCR, or flip receptor. In some embodiments, the cells further express a zetakine, an immune cell engager, or a BiTE. In one embodiment, the cells are hematopoietic cells. In some embodiments, the cells are T cells, αβ-T cells, or γδ-T cells. In some embodiments, the cells are CD3 + , CD4 + , and / or CD8 + The non-naturally occurring cell is a cell. In one embodiment, the cell is an immune effector cell. In some embodiments, the cell is a cytotoxic T lymphocyte (CTL), a tumor-infiltrating lymphocyte (TIL), or a helper T cell. In some embodiments, the cell is a natural killer (NK) cell or a natural killer T (NKT) cell. In some embodiments, the source of the cell is a peripheral blood mononuclear cell, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, or a tumor. In one embodiment, the non-naturally occurring cell is an isolated non-naturally occurring cell. In one embodiment, the non-naturally occurring cell is obtained from a subject. In one embodiment, the non-naturally occurring cell is a human cell. In some embodiments, the cell further comprises a polypeptide having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 137. In one embodiment, the cell further comprises the amino acid sequence set forth in SEQ ID NO: 137. In some embodiments, the cell further comprises a polypeptide having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 138. In one embodiment, the cell further comprises the amino acid sequence set forth in SEQ ID NO: 138.

[0029] In one aspect, disclosed herein is a fusion polypeptide comprising a signaling component comprising a first multimerization domain and an actuator domain; a polypeptide cleavage signal; and a targeting component comprising an extracellular domain, a second multimerization domain, and a transmembrane domain. In one embodiment, the first and second multimerization domains are localized extracellularly when the signaling component and the targeting component are expressed.

[0030] In one embodiment, the second multimerization domain and the transmembrane domain are separated by a hinge domain. In some embodiments, the hinge domain is selected from the group consisting of a CD4 hinge, a CD8 hinge, a CD28 hinge, an IgG4 hinge, and any fragment, variant, or combination thereof. In some embodiments, the hinge domain is a CD4 hinge. In some embodiments, the CD4 hinge comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 41. In one embodiment, the CD4 hinge comprises the amino acid sequence set forth in SEQ ID NO: 41. In some embodiments, the hinge domain is a CD28 hinge. In some embodiments, the CD28 hinge comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 42. In one embodiment, the CD28 hinge comprises the amino acid sequence set forth in SEQ ID NO: 42. In some embodiments, the hinge domain is a CD8 hinge. In some embodiments, the CD8 hinge comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 44. In one embodiment, the CD8 hinge comprises the amino acid sequence set forth in SEQ ID NO: 44. In some embodiments, the hinge domain is an IgG4 hinge. In some embodiments, the IgG4 hinge comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 43. In one embodiment, the IgG4 hinge comprises the amino acid sequence set forth in SEQ ID NO: 43.

[0031] In some embodiments, the actuator domain is a CD3 polypeptide, an FcεR1γ polypeptide, an Igα / CD79a polypeptide, an Igβ / CD79b polypeptide, a DAP10 polypeptide, or a DAP12 polypeptide. In some embodiments, the CD3 polypeptide is CD3 epsilon (CD3ε) or a fragment or variant thereof, CD3 gamma (CD3γ) or a fragment or variant thereof, or CD3 delta (CD3δ) or a fragment or variant thereof. In some embodiments, the actuator domain is a CD3ε polypeptide comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 32. In one embodiment, the actuator domain is a CD3ε polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 32. In some embodiments, the actuator domain is a CD3γ polypeptide or a variant thereof. In some embodiments, the actuator domain is a CD3γ polypeptide comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 33. In one embodiment, the actuator domain is a CD3γ polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 33. In some embodiments, the actuator domain is a CD3δ polypeptide or a variant thereof. In some embodiments, the actuator domain is a CD3δ polypeptide comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 34. In one embodiment, the actuator domain is a CD3δ polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 34. In some embodiments, the actuator domain is an FcεR1γ polypeptide or a variant thereof. In some embodiments, the actuator domain is an FcεR1γ polypeptide comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 35. In one embodiment, the actuator domain is an FcεR1γ polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 35.In some embodiments, the actuator domain is an FcεR1γ polypeptide comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 36. In one embodiment, the actuator domain is an FcεR1γ polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 36. In some embodiments, the actuator domain is an Igα / CD79a polypeptide or a variant thereof. In some embodiments, the actuator domain is an Igα / CD79a polypeptide comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 37. In one embodiment, the actuator domain is an Igα / CD79a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 37. In some embodiments, the actuator domain is an Igβ / CD79b polypeptide or a variant thereof. In some embodiments, the actuator domain is an Igβ / CD79b polypeptide comprising an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 38. In one embodiment, the actuator domain is an Igβ / CD79b polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 38. In some embodiments, the actuator domain is a DAP10 polypeptide or a variant thereof. In some embodiments, the actuator domain is a DAP10 polypeptide comprising an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 39. In one embodiment, the actuator domain is a DAP10 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 39. In some embodiments, the actuator domain is a DAP12 polypeptide or a variant thereof. In some embodiments, the actuator domain is a DAP12 polypeptide comprising an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 40. In one embodiment, the actuator domain is a DAP12 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:40.In some embodiments, the actuator domain comprises both an extracellular portion and an intracellular portion.

[0032] In some embodiments, the fusion polypeptide further comprises a signal sequence, optionally wherein the signal sequence has or comprises at least 90%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:95, SEQ ID NO:96, or SEQ ID NO:97.

[0033] In some embodiments, the first and second multimerization domains are different. In some embodiments, the multimerization domains of the signaling and targeting components are associated with a cross-linking agent selected from the group consisting of rapamycin or a rapalog thereof, gibberellin or a derivative thereof, abscisic acid (ABA) or a derivative thereof, methotrexate or a derivative thereof, cyclosporin A or a derivative thereof, FK506 / cyclosporin A or a derivative thereof, trimethoprim (Tmp) synthetic ligand for FK506 binding protein (FKBP) (SLF) or a derivative thereof, wherein the cross-linking agent promotes the formation of a polypeptide complex, and the cross-linking agent is associated with and disposed between the multimerization domains of the signaling component and the targeting component. In some embodiments, the first multimerization domain and the second multimerization domain are a pair selected from the group consisting of FK506-binding protein 1A (FKBP12) and FKBP12-rapamycin-binding (FRB), FKBP12 and calcineurin, FKBP and cyclophilin A or any other member of the peptidyl prolyl cis-trans isomerase (PPIase) family, FKBP and dihydrofolate reductase (DHFR), calcineurin and cyclophilin A or any other member of the peptidyl prolyl cis-trans isomerase (PPIase) family, and PYR1-like 1 (PYL1) and abscisic acid-insensitive 1 (ABI1). In some embodiments, the first multimerization domain comprises a first FRB polypeptide or a variant thereof, and the second multimerization domain comprises a first FKBP12 polypeptide or a variant thereof. In some embodiments, the first multimerization domain comprises a first FKBP12 polypeptide or a variant thereof, and the second multimerization domain comprises a first FRB polypeptide or a variant thereof. In one embodiment, the FRB polypeptide is an FRB T2098L variant. In some embodiments, the FRB polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the FKBP12 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4.

[0034] In some embodiments, the cross-linking agent is AP1903, AP20187, AP21967 (also known as C16-(S)-7-methylindolerapamycin), everolimus, novolimus, pimecrolimus, ridaforolimus, sirolimus, tacrolimus, temsirolimus, umirolimus, zotarolimus, or BPC015. In some embodiments, the first multimerization domain and the second multimerization domain are heterodimerization domains derived from a pair of antibodies. In some embodiments, the first multimerization domain comprises a polypeptide or comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 5; and the second multimerization domain comprises a polypeptide or comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the first multimerization domain comprises a polypeptide or comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 6; and the second multimerization domain comprises a polypeptide or comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the first multimerization domain comprises a polypeptide or comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 7; and the second multimerization domain comprises a polypeptide or comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the first multimerization domain comprises a polypeptide or comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 8; and the second multimerization domain comprises a polypeptide or comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 7.In some embodiments, the first multimerization domain comprises a polypeptide or comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:9; and the second multimerization domain comprises a polypeptide or comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the first multimerization domain comprises a polypeptide or comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 10; and the second multimerization domain comprises a polypeptide or comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:9.

[0035] In some embodiments, the first multimerization domain and the actuator domain are separated by a first polypeptide linker between 2 and 40 amino acids in length. In some embodiments, the first polypeptide linker is selected from the group consisting of GG, GS, SG, SS, GSS, SSG, GSG, SGS, SGG, G4S, 2xG4S, 3xG4S, 4xG4S, 5xG4S, and any combination thereof. In one embodiment, the first polypeptide linker is a 3xG4S linker. In some embodiments, the first polypeptide linker comprises a polypeptide comprising an amino acid sequence set forth as any one of SEQ ID NOs: 16-31. In some embodiments, the extracellular domain and the second multimerization domain are separated by a second polypeptide linker between 2 and 40 amino acids in length. In some embodiments, the second polypeptide linker is selected from the group consisting of GG, GS, SG, SS, GSS, SSG, GSG, SGS, SGG, G4S, 2xG4S, 3xG4S, 4xG4S, and any combination thereof. In one embodiment, the second polypeptide linker is a G4S linker. In some embodiments, the second polypeptide linker comprises a polypeptide comprising an amino acid sequence set forth as any one of SEQ ID NOs: 16-31.

[0036] In some embodiments, the transmembrane domain is a CD4 transmembrane domain. In some embodiments, the CD4 transmembrane domain comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 45. In one embodiment, the CD4 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 45. In some embodiments, the transmembrane domain is a CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 46. In one embodiment, the CD28 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 46. In some embodiments, the transmembrane domain is a CD8 transmembrane domain. In some embodiments, the CD8 transmembrane domain comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 47. In one embodiment, the CD8 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 47.

[0037] In some embodiments, the targeting moiety further comprises an intracellular signaling or costimulatory domain derived from a protein selected from the group consisting of an antigen receptor, a costimulatory receptor, a growth receptor, a cytokine receptor, an adaptor signaling protein, an intracellular signaling protein, or any fragment or variant thereof. In some embodiments, the intracellular signaling or costimulatory domain on the targeting moiety is selected from the group consisting of Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain family member 11 (CARD11), CD2, CD3ε, CD3γ, CD3δ, CD4, CD7, CD8, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), common gamma chain cytokine, DNAX-activation protein 10 (DAP10), linker of activation of T-cell family member 1 (LAT), interleukin-2 receptor (IL-2R), IL-4R, IL-7R, IL-9R, IL-12R, IL-13R, IL-15R, IL-21R, SH2 domain-containing leukocyte protein 76 kD (SLP76), T-cell receptor-associated transmembrane adaptor 1 (TRAT1), TNFR2, TNFRS14, TNFRS18, TNRFS25, and zeta chain of T-cell receptor-associated protein kinase 70 (ZAP70). In some embodiments, the intracellular signaling or costimulatory domain on the targeting moiety is selected from the group consisting of: 4-1BB, CD28, TNFR2, OX40, ICOS, and DAP10 costimulatory domain. In some embodiments, the costimulatory domain on the targeting moiety is a 4-1BB costimulatory domain, optionally wherein the 4-1BB costimulatory domain comprises the amino acid sequence set forth in SEQ ID NO: 98. In some embodiments, the costimulatory domain on the targeting moiety is a CD28 costimulatory domain, optionally wherein the CD28 costimulatory domain comprises the amino acid sequence set forth in SEQ ID NO: 99.

[0038] In some embodiments, the intracellular signaling or costimulatory domain is one or more cytokine receptor intracellular signaling domains. In some embodiments, the one or more cytokine receptor intracellular signaling domains are selected from the group consisting of an IL7Rα intracellular signaling domain, an IL2Rβ intracellular signaling domain, a common gamma chain intracellular signaling domain, and both an IL2Rβ and a common gamma chain intracellular signaling domain. In some embodiments, the IL7Rα intracellular signaling domain comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 103. In one embodiment, the IL7Rα intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 103. In some embodiments, the IL2Rβ intracellular signaling domain comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 104 or SEQ ID NO: 105. In some embodiments, the IL2Rβ intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 104 or SEQ ID NO: 105. In some embodiments, the common gamma chain intracellular signaling domain comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 106. In one embodiment, the common gamma chain intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 106. In some embodiments, the intracellular signaling or costimulatory domain is a LAT domain. In some embodiments, the LAT domain comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 102. In one embodiment, the LAT domain comprises the amino acid sequence set forth in SEQ ID NO: 102.

[0039] In some embodiments, the intracellular signaling or costimulatory domain is a CD4 co-receptor domain. In some embodiments, the CD4 co-receptor domain comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 100. In one embodiment, the CD4 co-receptor domain comprises the amino acid sequence set forth in SEQ ID NO: 100. In some embodiments, the intracellular signaling or costimulatory domain is a CD8 co-receptor domain. In some embodiments, the CD8 co-receptor domain comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 101. In one embodiment, the CD8 co-receptor domain comprises the amino acid sequence set forth in SEQ ID NO: 101.

[0040] In some embodiments, the targeting moiety does not comprise a functional intracellular signaling or costimulatory domain. In some embodiments, the targeting moiety further comprises a truncated intracellular CD4 polypeptide. In some embodiments, the truncated intracellular CD4 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 48 or SEQ ID NO: 49. In some embodiments, the truncated intracellular CD4 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 48 or SEQ ID NO: 49. In some embodiments, the extracellular domain comprises a first targeting domain. In some embodiments, the first targeting domain comprises a single-chain variable fragment (scFv) or a single-domain antibody (sdAb). In some embodiments, the sdAb is a camelid VHH, a nanobody, or a heavy-chain-only antibody (HcAb). In one embodiment, the sdAb is a camelid VHH. In some embodiments, the scFv or sdAb is human or humanized. In some embodiments, the first targeting domain comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 50-83. In some embodiments, the first targeting domain comprises a sequence set forth in any one of SEQ ID NOs: 50-83. In some embodiments, the first targeting domain comprises a PD1 ectodomain, human A-proliferation-inducing ligand (APRIL), trimerized human APRIL, or NKG2D membrane protein. In some embodiments, the PD1 ectodomain comprises a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 84 or SEQ ID NO: 85. In one embodiment, the PD1 ectodomain comprises a sequence set forth in SEQ ID NO: 85. In some embodiments, APRIL comprises a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 86, or trimerized human APRIL comprises a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 87. In some embodiments, APRIL comprises the sequence set forth in SEQ ID NO:86, or the trimerized human APRIL comprises the sequence set forth in SEQ ID NO:87.In some embodiments, the NKG2D membrane protein comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 88. In one embodiment, the NKG2D membrane protein comprises the sequence set forth as SEQ ID NO: 88.

[0041] In some embodiments, the extracellular domain further comprises a second targeting domain. In some embodiments, the second targeting domain comprises a second single-chain variable fragment (scFv) or a second single-domain antibody (sdAb). In some embodiments, the second sdAb is a camelid VHH, a nanobody, or a heavy-chain-only antibody (HcAb). In one embodiment, the second sdAb is a camelid VHH. In some embodiments, the second scFv or second sdAb is human or humanized. In some embodiments, the second targeting domain comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 50-83. In some embodiments, the second targeting domain comprises a sequence set forth in any one of SEQ ID NOs: 50-83. In some embodiments, the second targeting domain comprises a PD1 ectodomain, human A-growth-inducing ligand (APRIL), trimerized human APRIL, or NKG2D membrane protein. In some embodiments, the PD1 ectodomain comprises a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 84 or SEQ ID NO: 85. In one embodiment, the PD1 ectodomain comprises the sequence set forth in SEQ ID NO: 85. In some embodiments, APRIL comprises a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 86, or trimerized human APRIL comprises a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 87. In some embodiments, APRIL comprises the sequence set forth in SEQ ID NO: 86, or trimerized human APRIL comprises the sequence set forth in SEQ ID NO: 87. In some embodiments, the NKG2D membrane protein comprises a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 88. In one embodiment, the NKG2D membrane protein comprises the sequence set forth as SEQ ID NO: 88. In some embodiments, the first targeting domain and the second targeting domain bind to the same antigen or different antigens. In some embodiments, the first targeting domain and the second targeting domain are separated by a third polypeptide linker between 2 and 40 amino acids in length.In some embodiments, the third polypeptide linker is selected from the group consisting of: GG, GS, SG, SS, GSS, SSG, GSG, SGS, SGG, G4S, 2xG4S, 3xG4s, 4xG4S, 5xG4S, any one of SEQ ID NOs: 16-31, and any combination thereof.

[0042] In some embodiments, the polypeptide cleavage signal is a viral auto-cleaving polypeptide. In some embodiments, the polypeptide cleavage signal is a viral auto-cleaving 2A polypeptide. In some embodiments, the polypeptide cleavage signal is a viral auto-cleaving polypeptide selected from the group consisting of a foot-and-mouth disease virus (FMDV) (F2A) peptide, an equine rhinitis A virus (ERAV) (E2A) peptide, a Thosea asigna virus (TaV) (T2A) peptide, a porcine teschovirus-1 (PTV-1) (P2A) peptide, a tylovirus 2A peptide, and an encephalomyocarditis virus 2A peptide.

[0043] In one embodiment, the signaling component further comprises a signal sequence. In one embodiment, the signal sequence is a CD8 signal sequence. In one embodiment, the CD8 signal sequence comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 96. In another embodiment, the CD8 signal sequence comprises the amino acid sequence set forth as SEQ ID NO: 96.

[0044] In another embodiment, the targeting moiety further comprises a signal sequence. In one embodiment, the signal sequence is an IgK signal sequence. In one embodiment, the IgK signal sequence comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 95. In one embodiment, the IgK signal sequence comprises the amino acid sequence set forth as SEQ ID NO: 95.

[0045] In some embodiments, the signaling component comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, or SEQ ID NO: 115. In some embodiments, the signaling component comprises a sequence set forth as SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, or SEQ ID NO: 115.

[0046] In some embodiments, the targeting moiety comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to the sequence set forth as SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, or SEQ ID NO:136. In some embodiments, the targeting moiety comprises the sequence set forth as SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, or SEQ ID NO: 136. In some embodiments, the fusion polypeptide comprises a targeting moiety and a signaling moiety.

[0047] In some embodiments, the fusion polypeptide comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, or SEQ ID NO:170. In some embodiments, the fusion polypeptide comprises the sequence set forth as SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, or SEQ ID NO:170.

[0048] In one aspect, the disclosure provides a nucleic acid molecule encoding any one of the fusion polypeptides disclosed herein.

[0049] In another aspect, the present disclosure provides a cell comprising any one of the fusion polypeptides disclosed herein.

[0050] In another aspect, the present disclosure provides a cell comprising any one of the nucleic acid molecules disclosed herein.

[0051] In some embodiments, the cells further express an exogenous costimulatory factor, an immunomodulatory factor, an agonist for a costimulatory factor, an antagonist for an immunosuppressive factor, an immune cell engager, a flip receptor, or any combination thereof. In some embodiments, the cells further express an exogenous lymphocyte receptor or co-receptor. In some embodiments, the exogenous lymphocyte receptor or co-receptor is selected from the group consisting of TCR alpha (TCRα), TCR beta (TCRβ), TCR gamma (TCRγ), TCR delta (TCRδ), CD4, CD8, pre-T cell receptor alpha (pTα), Fc receptor alpha (FcRα), Fc receptor beta (FcRβ), Fc receptor gamma (FcRγ), natural killer group 2 member D (NKG2D), CD79A, CD79B, and any combination thereof. In one embodiment, the cells further express an exogenous TCR.In some embodiments, the exogenous TCR is selected from the group consisting of alpha-fetoprotein (AFP), B melanoma antigen (BAGE) family members, siblings of regulator of imprinted sites (BORIS), cancer testis antigen, cancer testis antigen 83 (CT-83), carbonic anhydrase IX (CA1X), carcinoembryonic antigen (CEA), cytomegalovirus (CMV) antigen, cytotoxic T cell (CTL)-recognized antigen against melanoma (CAMEL), Epstein-Barr virus (EBV) antigen, G antigen 1 ( GAGE-1), GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, GAGE-8, glycoprotein 100 (GP100), hepatitis B virus (HBV) antigen, hepatitis C virus (HCV) nonstructural protein 3 (NS3), human epidermal growth factor receptor 2 (HER-2), human papillomavirus (HPV)-E6, HPV-E7, human telomerase reverse transcriptase (hTERT), IGF2BP3 / A3, K-Ras, K-Ras G12C, K-Ras G12D, K-Ras G12V, latent membrane protein 2 (LMP2), melanoma antigen family A, 1 (MAGE-A1), MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, melanoma antigen recognized by T cells (MART-1), mesothelin (MSLN), mucin 1 (MUC1), mucin 16 (MUC16), New York esophageal squamous cell carcinoma-1 (NYESO-1), p53, P antigen (PAGE) family member, placenta-specific 1 (PLAC1), melanoma The exogenous TCR binds to a target antigen selected from the group consisting of preferentially expressed antigen in tumor grafts (PRAME), survivin, synovial sarcoma X1 (SSX1), synovial sarcoma X2 (SSX2), synovial sarcoma X3 (SSX3), synovial sarcoma X4 (SSX4), synovial sarcoma X5 (SSX5), synovial sarcoma X8 (SSX8), thyroglobulin, tyrosinase, tyrosinase-related protein (TRP) 1, TRP2, Wilms' tumor protein (WT-1), X antigen family member 1 (XAGE1), and X antigen family member 2 (XAGE2). In some embodiments, the exogenous TCR is an αβ-TCR or a γδ-TCR. In some embodiments, the cells further express a CAR, CCR, or flip receptor.In some embodiments, the cells further express a zetakine, an immune cell engager, or a BiTE.

[0052] In one embodiment, the cell is a hematopoietic cell. In some embodiments, the cell is a T cell, an αβ-T cell, or a γδ-T cell. In some embodiments, the cell is a CD3 + , CD4 + , and / or CD8 + The cell is a cell. In one embodiment, the cell is an immune effector cell. In some embodiments, the cell is a cytotoxic T lymphocyte (CTL), a tumor-infiltrating lymphocyte (TIL), or a helper T cell. In some embodiments, the cell is a natural killer (NK) cell or a natural killer T (NKT) cell. In some embodiments, the source of the cell is a peripheral blood mononuclear cell, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, or a tumor. In one embodiment, the cell is an isolated cell. In one embodiment, the cell is obtained from a subject. In one embodiment, the cell is a human cell.

[0053] In one aspect, disclosed herein is a polypeptide complex comprising a signaling component comprising a first multimerization domain and an actuator domain; and a targeting component comprising an extracellular domain, a second multimerization domain, and a transmembrane domain. In some embodiments, the first and second multimerization domains are localized extracellularly when the signaling component and the targeting component are expressed.

[0054] In some embodiments, the second multimerization domain and the transmembrane domain are separated by a hinge domain. In some embodiments, the hinge domain is selected from the group consisting of a CD4 hinge, a CD8 hinge, a CD28 hinge, an IgG4 hinge, and any fragment, variant, or combination thereof. In some embodiments, the hinge domain is a CD4 hinge. In some embodiments, the CD4 hinge comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 41. In one embodiment, the CD4 hinge comprises the amino acid sequence set forth in SEQ ID NO: 41. In some embodiments, the hinge domain is a CD28 hinge. In some embodiments, the CD28 hinge comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 42. In one embodiment, the CD28 hinge comprises the amino acid sequence set forth in SEQ ID NO: 42. In some embodiments, the hinge domain is a CD8 hinge. In some embodiments, the CD8 hinge comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 44. In one embodiment, the CD8 hinge comprises the amino acid sequence set forth in SEQ ID NO: 44. In some embodiments, the hinge domain is an IgG4 hinge. In some embodiments, the IgG4 hinge comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 43. In one embodiment, the IgG4 hinge comprises the amino acid sequence set forth in SEQ ID NO: 43. In some embodiments, the actuator domain is a CD3 polypeptide, an FcεR1γ polypeptide, an Igα / CD79a polypeptide, an Igβ / CD79b polypeptide, a DAP10 polypeptide, or a DAP12 polypeptide. In some embodiments, the CD3 polypeptide is CD3 epsilon (CD3ε) or a fragment or variant thereof, CD3 gamma (CD3γ) or a fragment or variant thereof, or CD3 delta (CD3δ) or a fragment or variant thereof.In some embodiments, the actuator domain is a CD3ε polypeptide comprising an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 32. In one embodiment, the actuator domain is a CD3ε polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 32. In some embodiments, the actuator domain is a CD3γ polypeptide or a variant thereof. In some embodiments, the actuator domain is a CD3γ polypeptide comprising an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 33. In one embodiment, the actuator domain is a CD3γ polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 33. In some embodiments, the actuator domain is a CD3δ polypeptide or a variant thereof. In some embodiments, the actuator domain is a CD3δ polypeptide comprising an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 34. In one embodiment, the actuator domain is a CD3δ polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 34. In some embodiments, the actuator domain is an FcεR1γ polypeptide or a variant thereof. In some embodiments, the actuator domain is an FcεR1γ polypeptide comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 35. In one embodiment, the actuator domain is an FcεR1γ polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 35. In some embodiments, the actuator domain is an FcεR1γ polypeptide comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 36. In one embodiment, the actuator domain is an FcεR1γ polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 36. In some embodiments, the actuator domain is an Igα / CD79a polypeptide or a variant thereof.In some embodiments, the actuator domain is an Igα / CD79a polypeptide comprising an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 37. In one embodiment, the actuator domain is an Igα / CD79a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 37. In some embodiments, the actuator domain is an Igβ / CD79b polypeptide or a variant thereof. In one embodiment, the actuator domain is an Igβ / CD79b polypeptide comprising an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 38. In one embodiment, the actuator domain is an Igβ / CD79b polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 38. In some embodiments, the actuator domain is a DAP10 polypeptide or a variant thereof. In some embodiments, the actuator domain is a DAP10 polypeptide comprising an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 39. In one embodiment, the actuator domain is a DAP10 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 39. In some embodiments, the actuator domain is a DAP12 polypeptide or a variant thereof. In some embodiments, the actuator domain is a DAP12 polypeptide comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 40. In one embodiment, the actuator domain is a DAP12 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 40. In some embodiments, the actuator domain comprises both an extracellular portion and an intracellular portion.

[0055] In some embodiments, the polypeptide further comprises a signal sequence, optionally wherein the signal sequence has at least 90%, 95%, 96%, 97%, 98%, 99% identity to or comprises SEQ ID NO: 95, SEQ ID NO: 96, or SEQ ID NO:

[0056] In some embodiments, the first and second multimerization domains are different. In some embodiments, the multimerization domains of the signaling and targeting components are associated with a cross-linking agent selected from the group consisting of rapamycin or a rapalog thereof, gibberellin or a derivative thereof, abscisic acid (ABA) or a derivative thereof, methotrexate or a derivative thereof, cyclosporin A or a derivative thereof, FK506 / cyclosporin A or a derivative thereof, trimethoprim (Tmp) synthetic ligand for FK506 binding protein (FKBP) (SLF) or a derivative thereof, wherein the cross-linking agent promotes the formation of a polypeptide complex, and the cross-linking agent is associated with and disposed between the multimerization domains of the signaling component and the targeting component. In some embodiments, the first multimerization domain and the second multimerization domain are a pair selected from the group consisting of FK506-binding protein 1A (FKBP12) and FKBP12-rapamycin-binding (FRB), FKBP12 and calcineurin, FKBP and cyclophilin A or any other member of the peptidyl prolyl cis-trans isomerase (PPIase) family, FKBP and dihydrofolate reductase (DHFR), calcineurin and cyclophilin A or any other member of the peptidyl prolyl cis-trans isomerase (PPIase) family, and PYR1-like 1 (PYL1) and abscisic acid-insensitive 1 (ABI1). In some embodiments, the first multimerization domain comprises a first FRB polypeptide or a variant thereof, and the second multimerization domain comprises a first FKBP12 polypeptide or a variant thereof. In some embodiments, the first multimerization domain comprises a first FKBP12 polypeptide or a variant thereof, and the second multimerization domain comprises a first FRB polypeptide or a variant thereof. In one embodiment, the FRB polypeptide is an FRB T2098L variant. In some embodiments, the FRB polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the FKBP12 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4.

[0057] In some embodiments, the cross-linking agent is AP1903, AP20187, AP21967 (also known as C16-(S)-7-methylindolerapamycin), everolimus, novolimus, pimecrolimus, ridaforolimus, sirolimus, tacrolimus, temsirolimus, umirolimus, zotarolimus, or BPC015. In some embodiments, the first multimerization domain and the second multimerization domain are heterodimerization domains derived from a pair of antibodies. In some embodiments, the first multimerization domain comprises a polypeptide or comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 5; and the second multimerization domain comprises a polypeptide or comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the first multimerization domain comprises a polypeptide or comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 6; and the second multimerization domain comprises a polypeptide or comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the first multimerization domain comprises a polypeptide or comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 7; and the second multimerization domain comprises a polypeptide or comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the first multimerization domain comprises a polypeptide or comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 8; and the second multimerization domain comprises a polypeptide or comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 7.In some embodiments, the first multimerization domain comprises a polypeptide or comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:9; and the second multimerization domain comprises a polypeptide or comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the first multimerization domain comprises a polypeptide or comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 10; and the second multimerization domain comprises a polypeptide or comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO:9.

[0058] In some embodiments, the first multimerization domain and the actuator domain are separated by a first polypeptide linker between 2 and 40 amino acids in length. In some embodiments, the first polypeptide linker is selected from the group consisting of GG, GS, SG, SS, GSS, SSG, GSG, SGS, SGG, G4S, 2xG4S, 3xG4S, 4xG4S, 5xG4S, and any combination thereof. In one embodiment, the first polypeptide linker is a 3xG4S linker. In some embodiments, the first polypeptide linker comprises a polypeptide comprising an amino acid sequence set forth as any one of SEQ ID NOs: 16-31. In some embodiments, the extracellular domain and the second multimerization domain are separated by a second polypeptide linker between 2 and 40 amino acids in length. In some embodiments, the second polypeptide linker is selected from the group consisting of GG, GS, SG, SS, GSS, SSG, GSG, SGS, SGG, G4S, 2xG4S, 3xG4S, 4xG4S, and any combination thereof. In one embodiment, the second polypeptide linker is a G4S linker. In some embodiments, the second polypeptide linker comprises a polypeptide comprising an amino acid sequence set forth as any one of SEQ ID NOs: 16-31.

[0059] In some embodiments, the transmembrane domain is a CD4 transmembrane domain. In some embodiments, the CD4 transmembrane domain comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 45. In one embodiment, the CD4 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 45. In some embodiments, the transmembrane domain is a CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 46. In one embodiment, the CD28 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 46. In some embodiments, the transmembrane domain is a CD8 transmembrane domain. In some embodiments, the CD8 transmembrane domain comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 47. In one embodiment, the CD8 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 47.

[0060] In some embodiments, the targeting moiety further comprises an intracellular signaling or costimulatory domain derived from a protein selected from the group consisting of an antigen receptor, a costimulatory receptor, a growth receptor, a cytokine receptor, an adaptor signaling protein, an intracellular signaling protein, or any fragment or variant thereof. In some embodiments, the intracellular signaling or costimulatory domain on the targeting moiety is selected from the group consisting of Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain family member 11 (CARD11), CD2, CD3ε, CD3γ, CD3δ, CD4, CD7, CD8, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICAM), CD83 (ICAM), CD94 (ICAM), ...83 (ICAM), CD94 (ICAM), CD134 (OX40), CD137 (4-1BB), CD278 (ICAM), CD83 (ICAM), CD83 (ICAM), CD83 (ICAM), CD83 (ICAM), CD83 (ICAM), CD83 (ICAM), CD83 (ICAM), CD COS), common gamma chain cytokine, DNAX-activation protein 10 (DAP10), linker for activation of T-cell family member 1 (LAT), interleukin 2 receptor (IL-2R), IL-4R, IL-7R, IL-9R, IL-12R, IL-13R, IL-15R, IL-21R, SH2 domain-containing leukocyte protein 76 kD (SLP76), T cell receptor-associated transmembrane adaptor 1 (TRAT1), TNFR2, TNFRS14, TNFRS18, TNRFS25, and zeta chain of T cell receptor-associated protein kinase 70 (ZAP70). In some embodiments, the intracellular signaling or costimulatory domain on the targeting moiety is selected from the group consisting of 4-1BB, CD28, TNFR2, OX40, ICOS, and a DAP10 costimulatory domain. In some embodiments, the costimulatory domain on the targeting moiety is a 4-1BB costimulatory domain, optionally wherein the 4-1BB costimulatory domain comprises the amino acid sequence set forth in SEQ ID NO: 98. In some embodiments, the costimulatory domain on the targeting moiety is a CD28 costimulatory domain, optionally wherein the CD28 costimulatory domain comprises the amino acid sequence set forth in SEQ ID NO: 99. In some embodiments, the intracellular signaling or costimulatory domain is one or more cytokine receptor intracellular signaling domains.In some embodiments, the one or more cytokine receptor intracellular signaling domains are selected from the group consisting of an IL7Rα intracellular signaling domain, an IL2Rβ intracellular signaling domain, a common gamma chain intracellular signaling domain, and both an IL2Rβ and a common gamma chain intracellular signaling domain. In some embodiments, the IL7Rα intracellular signaling domain comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 103. In one embodiment, the IL7Rα intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 103. In some embodiments, the IL2Rβ intracellular signaling domain comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 104 or SEQ ID NO: 105. In some embodiments, the IL2Rβ intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 104 or SEQ ID NO: 105. In some embodiments, the common gamma chain intracellular signaling domain comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 106. In one embodiment, the common gamma chain intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 106. In some embodiments, the intracellular signaling or costimulatory domain is a LAT domain. In some embodiments, the LAT domain comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 102. In one embodiment, the LAT domain comprises the amino acid sequence set forth in SEQ ID NO: 102. In some embodiments, the intracellular signaling or costimulatory domain is a CD4 co-receptor domain. In some embodiments, the CD4 co-receptor domain comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 100. In one embodiment, the CD4 co-receptor domain comprises the amino acid sequence set forth in SEQ ID NO: 100. In some embodiments, the intracellular signaling or costimulatory domain is a CD8 co-receptor domain.In some embodiments, the CD8 co-receptor domain comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 101. In one embodiment, the CD8 co-receptor domain comprises the amino acid sequence set forth in SEQ ID NO: 101.

[0061] In some embodiments, the targeting moiety does not comprise a functional intracellular signaling or costimulatory domain. In some embodiments, the targeting moiety further comprises a truncated intracellular CD4 polypeptide. In some embodiments, the truncated intracellular CD4 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:48 or SEQ ID NO:49. In some embodiments, the truncated intracellular CD4 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:48 or SEQ ID NO:49.

[0062] In some embodiments, the extracellular domain comprises a first targeting domain. In some embodiments, the first targeting domain comprises a single-chain variable fragment (scFv) or a single-domain antibody (sdAb). In some embodiments, the sdAb is a camelid VHH, a nanobody, or a heavy-chain-only antibody (HcAb). In one embodiment, the sdAb is a camelid VHH. In some embodiments, the scFv or sdAb is human or humanized. In some embodiments, the first targeting domain comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 50-83. In some embodiments, the first targeting domain comprises a sequence set forth in any one of SEQ ID NOs: 50-83. In some embodiments, the first targeting domain comprises a PD1 ectodomain, human A-growth-inducing ligand (APRIL), trimerized human APRIL, or NKG2D membrane protein. In some embodiments, the PD1 ectodomain comprises a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 84 or SEQ ID NO: 85. In one embodiment, the PD1 ectodomain comprises the sequence set forth in SEQ ID NO: 85. In some embodiments, APRIL comprises a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 86, or trimerized human APRIL comprises a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 87. In some embodiments, APRIL comprises the sequence set forth in SEQ ID NO: 86, or trimerized human APRIL comprises the sequence set forth in SEQ ID NO: 87. In some embodiments, the NKG2D membrane protein comprises a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 88. In some embodiments, the NKG2D membrane protein comprises the sequence shown as SEQ ID NO:88.

[0063] In some embodiments, the extracellular domain further comprises a second targeting domain. In some embodiments, the second targeting domain comprises a second single-chain variable fragment (scFv) or a second single-domain antibody (sdAb). In some embodiments, the second sdAb is a camelid VHH, a nanobody, or a heavy-chain-only antibody (HcAb). In one embodiment, the second sdAb is a camelid VHH. In some embodiments, the second scFv or second sdAb is human or humanized. In some embodiments, the second targeting domain comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 50-83. In some embodiments, the second targeting domain comprises a sequence set forth in any one of SEQ ID NOs: 50-83. In some embodiments, the second targeting domain comprises a PD1 ectodomain, human A-growth-inducing ligand (APRIL), trimerized human APRIL, or NKG2D membrane protein. In some embodiments, the PD1 ectodomain comprises a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 84 or SEQ ID NO: 85. In one embodiment, the PD1 ectodomain comprises the sequence set forth in SEQ ID NO: 85. In some embodiments, APRIL comprises a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 86, or trimerized human APRIL comprises a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 87. In some embodiments, APRIL comprises the sequence set forth in SEQ ID NO: 86, or trimerized human APRIL comprises the sequence set forth in SEQ ID NO: 87. In some embodiments, the NKG2D membrane protein comprises a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 88. In one embodiment, the NKG2D membrane protein comprises the sequence set forth as SEQ ID NO: 88. In some embodiments, the first targeting domain and the second targeting domain bind to the same antigen or different antigens. In some embodiments, the targeting domain and the second targeting domain are separated by a third polypeptide linker between 2 and 40 amino acids in length.In some embodiments, the third polypeptide linker is selected from the group consisting of: GG, GS, SG, SS, GSS, SSG, GSG, SGS, SGG, G4S, 2xG4S, 3xG4s, 4xG4S, 5xG4S, any one of SEQ ID NOs: 16-31, and any combination thereof.

[0064] In one embodiment, the signaling component further comprises a signal sequence. In one embodiment, the signal sequence is a CD8 signal sequence. In one embodiment, the CD8 signal sequence comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 96. In another embodiment, the CD8 signal sequence comprises the amino acid sequence set forth as SEQ ID NO: 96.

[0065] In another embodiment, the targeting moiety further comprises a signal sequence. In one embodiment, the signal sequence is an IgK signal sequence. In one embodiment, the IgK signal sequence comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 95. In one embodiment, the IgK signal sequence comprises the amino acid sequence set forth as SEQ ID NO: 95.

[0066] In some embodiments, the signaling component comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, or SEQ ID NO: 115. In some embodiments, the signaling component comprises a sequence set forth as SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, or SEQ ID NO: 115. In some embodiments, the targeting moiety comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to the sequence set forth as SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, or SEQ ID NO:136. In some embodiments, the targeting moiety comprises the sequence set forth as SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, or SEQ ID NO:136.

[0067] In some embodiments, the polypeptide complex comprises a fusion polypeptide comprising a targeting moiety and a signaling moiety. In some embodiments, the polypeptide complex comprises a fusion polypeptide comprising a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, or SEQ ID NO:170. In some embodiments, the polypeptide complex comprises a fusion polypeptide comprising the sequence set forth as SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, or SEQ ID NO:170.

[0068] In one aspect, the present disclosure provides a nucleic acid molecule encoding both the signaling component and the targeting component of any one of the polypeptide complexes disclosed herein. In another aspect, the present disclosure provides a cell comprising any one of the polypeptide complexes disclosed herein. In another aspect, the present disclosure provides a cell comprising any one of the nucleic acid molecules disclosed herein.

[0069] In some embodiments, the cells further express an exogenous costimulator, an immunomodulator, an agonist for a costimulator, an antagonist for an immunosuppressant, an immune cell engager, a flip receptor, or any combination thereof. In some embodiments, the cells further express an exogenous lymphocyte receptor or co-receptor. In some embodiments, the exogenous lymphocyte receptor or co-receptor is selected from the group consisting of TCR alpha (TCRα), TCR beta (TCRβ), TCR gamma (TCRγ), TCR delta (TCRδ), CD4, CD8, pre-T cell receptor alpha (pTα), Fc receptor alpha (FcRα), Fc receptor beta (FcRβ), Fc receptor gamma (FcRγ), natural killer group 2 member D (NKG2D), CD79A, CD79B, and any combination thereof. In some aspects, the cells further express an exogenous TCR.In some embodiments, the exogenous TCR is selected from the group consisting of alpha-fetoprotein (AFP), B melanoma antigen (BAGE) family members, siblings of regulator of imprinted sites (BORIS), cancer testis antigen, cancer testis antigen 83 (CT-83), carbonic anhydrase IX (CA1X), carcinoembryonic antigen (CEA), cytomegalovirus (CMV) antigen, cytotoxic T cell (CTL)-recognized antigen for melanoma (CAMEL), Epstein-Barr virus (EBV) antigen, G antigen 1 (G AGE-1), GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, GAGE-8, glycoprotein 100 (GP100), hepatitis B virus (HBV) antigen, hepatitis C virus (HCV) nonstructural protein 3 (NS3), human epidermal growth factor receptor 2 (HER-2), human papillomavirus (HPV)-E6, HPV-E7, human telomerase reverse transcriptase (hTERT), IGF2BP3 / A3, K-Ras, K-Ras G12C, K-Ras G12D, K-Ras G12V, latent membrane protein 2 (LMP2), melanoma antigen family A, 1 (MAGE-A1), MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, melanoma antigen recognized by T cells (MART-1), mesothelin (MSLN), mucin 1 (MUC1), mucin 16 (MUC16), New York esophageal squamous cell carcinoma-1 (NYESO-1), p53, P antigen (PAGE) family member, placenta-specific 1 (PLAC1), melanoma The exogenous TCR binds to a target antigen selected from the group consisting of preferentially expressed antigen in human TCRs (PRAME), survivin, synovial sarcoma X1 (SSX1), synovial sarcoma X2 (SSX2), synovial sarcoma X3 (SSX3), synovial sarcoma X4 (SSX4), synovial sarcoma X5 (SSX5), synovial sarcoma X8 (SSX8), thyroglobulin, tyrosinase, tyrosinase-related protein (TRP) 1, TRP2, Wilms' tumor protein (WT-1), X antigen family member 1 (XAGE1), and X antigen family member 2 (XAGE2). In some embodiments, the exogenous TCR is an αβ-TCR or a γδ-TCR.

[0070] In some embodiments, the cells further express a CAR, CCR, or flip receptor. In some embodiments, the cells further express a zetakine, an immune cell engager, or a BiTE. In one embodiment, the cells are hematopoietic cells. In some embodiments, the cells are T cells, αβ-T cells, or γδ-T cells. In some embodiments, the cells are CD3 + , CD4 + , and / or CD8 + The cell is a cell. In some embodiments, the cell is an immune effector cell. In some embodiments, the cell is a cytotoxic T lymphocyte (CTL), a tumor-infiltrating lymphocyte (TIL), or a helper T cell. In some embodiments, the cell is a natural killer (NK) cell or a natural killer T (NKT) cell. In some embodiments, the source of the cell is a peripheral blood mononuclear cell, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, or a tumor. In one embodiment, the cell is an isolated cell. In one embodiment, the cell is obtained from a subject. In one embodiment, the cell is a human cell.

[0071] In one aspect, the disclosure provides a polynucleotide encoding the signaling and targeting component of any one of the fusion polypeptides or polypeptide complexes disclosed herein. In another aspect, the disclosure provides a cDNA encoding the signaling and targeting component of any one of the fusion polypeptides or polypeptide complexes disclosed herein. In another aspect, the disclosure provides an RNA encoding the signaling component and targeting component, or the fusion polypeptide, or any one of the polypeptide complexes disclosed herein. In another aspect, the disclosure provides a vector comprising any one of the polynucleotides disclosed herein. In one embodiment, the vector is an expression vector. In one embodiment, the vector is a transposon. In one embodiment, the vector is a piggyBAC transposon or a Sleeping Beauty transposon. In one embodiment, the vector is a viral vector. In some embodiments, the vector is an adenoviral vector, an adeno-associated viral (AAV) vector, a herpes viral vector, a vaccinia viral vector, or a retroviral vector. In some embodiments, the retroviral vector is a lentiviral vector. In some embodiments, the lentiviral vector is selected from the group consisting of human immunodeficiency virus 1 (HIV-1), human immunodeficiency virus 2 (HIV-2), Visna-Maedi virus (VMV); caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and simian immunodeficiency virus (SIV).

[0072] In one aspect, the present disclosure provides a cell comprising any one of the fusion polypeptides, polynucleotides, or vectors disclosed herein. In one embodiment, the cell is a hematopoietic cell. In one embodiment, the cell is an immune effector cell. In some embodiments, the cell is a T cell, an αβ-T cell, or a γδ-T cell. In some embodiments, the cell is a CD3 + , CD4 + , CD8 +or a combination thereof. In some embodiments, the cell is a cytotoxic T lymphocyte (CTL), a tumor-infiltrating lymphocyte (TIL), or a helper T cell. In some embodiments, the cell is a natural killer (NK) cell or a natural killer T (NKT) cell.

[0073] In one aspect, the present disclosure provides a composition comprising any one of the cells or vectors disclosed herein. In one aspect, the present disclosure provides a composition comprising a physiologically acceptable carrier and any one of the cells or vectors disclosed herein.

[0074] In one aspect, the present disclosure provides a method of treating a subject in need thereof, comprising administering to the subject an effective amount of any one of the compositions disclosed herein. In one aspect, the present disclosure provides a method of treating, preventing, or ameliorating at least one symptom of cancer, an infectious disease, an autoimmune disease, an inflammatory disease, and an immune disorder, or a condition associated therewith, comprising administering to a subject an effective amount of any one of the compositions disclosed herein.

[0075] In another aspect, the disclosure provides a method of treating a solid cancer, comprising administering to a subject an effective amount of any one of the compositions disclosed herein. In some embodiments, the solid cancer is selected from the group consisting of lung cancer, squamous cell carcinoma, colorectal cancer, pancreatic cancer, breast cancer, thyroid cancer, bladder cancer, cervical cancer, esophageal cancer, ovarian cancer, gastric cancer, endometrial cancer, or brain cancer. In some embodiments, the solid cancer is non-small cell lung cancer, squamous cell carcinoma of the head and neck, colorectal cancer, pancreatic cancer, breast cancer, thyroid cancer, bladder cancer, cervical cancer, esophageal cancer, ovarian cancer, gastric cancer, endometrial cancer, glioma, glioblastoma, or oligodendroglioma.

[0076] In one aspect, the present disclosure provides a method of treating a hematological malignancy comprising administering to a subject an effective amount of any one of the compositions disclosed herein. In some embodiments, the hematological malignancy is leukemia, lymphoma, or multiple myeloma. In one embodiment, the hematological malignancy is acute myeloid leukemia (AML). [Brief explanation of the drawings]

[0077] [Figure 1A] Figures 1A-1H show the structures of representative engineered immune receptors and fusion constructs. In particular, Figures 1A-1C depict expression constructs and components of several exemplary engineered immune receptors and fusion constructs of the present invention. Figures 1D-1H are cartoons depicting how a representative engineered immune receptor, comprising a signaling component and a targeting component, forms a complex with an immune receptor (e.g., a TCR) to activate receptor signaling in the presence of a target cell expressing a target antigen, and, in some embodiments, in the presence of a cross-linking agent. As one non-limiting example, in Figure 1D, construct SR022 / SR001 comprises a signaling component comprising an FRB multimerization domain, a linker, and a CD3ε actuator domain; and a targeting component comprising a CD4 transmembrane domain, a CD4 hinge, an FKBP multimerization domain, a linker, and a CD33 VHH targeting domain. In the presence of cells expressing CD33, the CD33 VHH targeting domain binds to the target cell and comes into close contact with immune cells, e.g., T cells, expressing the engineered immune receptor. In the presence of a cross-linking agent, the multimerization domains then interact, allowing the signaling moiety to activate the immune receptor (e.g., TCR). [Figure 1B] Same as above. [Figure 1C] Same as above. [Figure 1D] Same as above. [Figure 1E] Same as above. [Figure 1F] Same as above. [Figure 1G] Same as above. [Figure 1H] Same as above. [Figure 2] FIG. 2 shows the percent positive and median fluorescence intensity of SR024 components on T cells as measured by flow cytometry. [Figure 3] FIG. 3 shows IFNγ, IL2, IL4, and TNF secretion in the presence of rapamycin from untransduced and SR024-T cells co-cultured with high CD33-expressing cancer cell lines. [Figure 4] FIG. 4 shows IFNγ secretion from untransduced and SR024-T cells cultured alone, with and without rapamycin. [Figure 5] Figures 5A and 5B show the cytotoxicity and proliferation of control T cells and SR024 T cells, respectively, each co-cultured with a high CD33-expressing cancer cell line. [Figure 6] FIG. 6 shows the percent positive and median fluorescence intensity of SR020, SR022, and SR024 components on T cells as measured by flow cytometry. [Figure 7A] Figure 7A shows IFNγ, IL2, IL4, and TNF secretion from untransduced SR020-T cells, SR022-T cells, and SR024-T cells, each cocultured with a high CD33-expressing cancer cell line, in the presence of rapamycin. [Figure 7B] Figure 7B shows IFNγ secretion from untransduced, SR020-T cells, SR022-T cells, and SR024-T cells, each co-cultured with high, medium, and low CD33-expressing cancer cell lines, in the presence of rapamycin. [Figure 7C] Figure 7C shows IL2 secretion from untransduced, SR020-T cells, SR022-T cells, and SR024-T cells, each co-cultured with high, medium, and low CD33-expressing cancer cell lines, in the presence of rapamycin. [Figure 8] FIG. 8 shows IFNγ secretion from untransduced, SR020-T cells, SR022-T cells, and SR024-T cells alone, with and without rapamycin. [Figure 9] Figures 9A and 9B show the cytotoxicity and proliferation of control, SR020-T cells, SR022-T cells, and SR024-T cells, respectively, each co-cultured with a high CD33-expressing cancer cell line. [Figure 10] FIG. 10 shows the percent positive and median fluorescence intensity of SR021 components on T cells as measured by flow cytometry. [Figure 11]FIG. 11 shows IFNγ, IL2, IL4, and TNF secretion from untransduced and SR021-T cells co-cultured with a high CD33-expressing cancer cell line in the presence of rapamycin. [Figure 12] FIG. 12 shows IFNγ secretion from untransduced and SR021-T cells co-cultured with a high CD33-expressing cancer cell line, with and without rapamycin alone. [Figure 13] Figures 13A and 13B show the cytotoxicity and proliferation, respectively, of control and SR021-T cells co-cultured with high CD33-expressing cancer cell lines. [Figure 14] FIG. 14 shows the percent positive and median fluorescence intensity of SR022 and SR001 components on T cells as measured by flow cytometry. [Figure 15] FIG. 15 shows IFNγ, IL2, and TNF secretion from untransduced, SR022-, and SR001-T cells, each co-cultured with a high CD33-expressing cancer cell line, in the presence of rapamycin. [Figure 16] FIG. 16 shows IFNγ secretion from untransduced, SR022- and SR001-T cells, each co-cultured with a high CD33-expressing cancer cell line, with and without rapamycin. [Figure 17] Figures 17A and 17B show the cytotoxicity and proliferation of control and SR001-T cells, respectively, each co-cultured with a high CD33-expressing cancer cell line. [Figure 18] FIG. 18 shows the percent positive and median fluorescence intensity of SR028 components on T cells as measured by flow cytometry. [Figure 19] FIG. 19 shows IFNγ, IL2, IL4, and TNF secretion from untransduced and SR028-T cells co-cultured with a high CD33-expressing cancer cell line in the presence of rapamycin. [Figure 20] FIG. 20 shows IFNγ secretion from untransduced and SR028-T cells co-cultured with a high CD33-expressing cancer cell line, with and without rapamycin. [Figure 21]Figures 21A and 21B show the cytotoxicity and proliferation of control and SR028-T cells, respectively, each co-cultured with a high CD33-expressing cancer cell line. [Figure 22] FIG. 22 shows the percent positive and median fluorescence intensity of SR004 and SR006 components on T cells as measured by flow cytometry. [Figure 23] FIG. 23 shows IFNγ, IL2, IL4, and TNF secretion from untransduced, SR004- and SR006-T cells, each co-cultured with a high CD33-expressing cancer cell line, in the presence of rapamycin. [Figure 24] FIG. 24 shows IFNγ secretion from non-transduced, SR004- and SR006-T cells, each co-cultured with a high CD33-expressing cancer cell line, with and without rapamycin. [Figure 25] Figures 25A and 25B show the cytotoxicity and proliferation of control, SR004- and SR006-T cells, respectively, each co-cultured with a high CD33-expressing cancer cell line. [Figure 26AB] Figures 26A and 26B show the percent positive and median fluorescence intensity of SR008 and SR030 components on T cells as measured by flow cytometry. [Figure 26CD] Figures 26C and 26D show the phenotypes of CD4+ and CD8+ T cells transduced with vectors encoding the indicated constructs. [Figure 27A] Figures 27A and 27B show IFNγ, IL2, IL4, and TNF secretion from untransduced cells, SR008-T cells, and SR030-T cells, each co-cultured with either a high CD33-expressing cancer cell line or a high CLL1-expressing cancer cell line, in the presence of rapamycin. [Figure 27B] Same as above. [Figure 27C] FIG. 27C shows IFNγ secretion from non-transduced cells, SR008-T cells, and SR030-T cells alone, with and without rapamycin. [Figure 28]Figures 28A and 28B show the cytotoxicity of untransduced cells, SR008-T cells, and SR030-T cells, each co-cultured with a high CD33-expressing or high CLL1-expressing cancer cell line, respectively. [Figure 29] FIG. 29 shows the percent positive and median fluorescence intensity of SR001 and SR001-28 components on T cells as measured by flow cytometry. [Figure 30] FIG. 30 shows IFNγ, IL2, IL4, and TNF secretion from untransduced, SR001- and SR001-28-T cells, each co-cultured with a high CD33-expressing cancer cell line, in the presence of rapamycin. [Figure 31] Figures 31A and 31B show the cytotoxicity and proliferation of control, SR001- and SR001-28-T cells, respectively, each co-cultured with a high CD33-expressing cancer cell line. [Figure 32AB] Figures 32A and 32B show in vivo tumor growth in NSG mouse xenografts and controls following administration of 10E6 SR001±41BB or CD28 costimulatory domain. All tumor controls are rapamycin regulated. [Figure 32CD] Figures 32C and 32D show in vivo tumor growth in NSG mouse xenografts and controls following administration of 3E6 SR001±41BB or CD28 costimulatory domain in the presence of rapamycin. [Figure 33] Figures 33A and 33B show the percent positive and median fluorescence intensity of SR022±CD4 or CD8 co-receptor signaling domains as measured by flow cytometry. [Figure 34] Figure 34 shows IFNγ and IL2 secretion from untransduced cells, SR022-T cells ± CD4 or CD8 co-receptor signaling domains, each co-cultured with a high CD33-expressing cancer cell line, with and without rapamycin. [Figure 35ABC]Figures 35A-35C show vector copy number, percent positive, and geometric mean fluorescence intensity of tetramer and TCR beta chain staining for untransduced TCR, transgenic TCR, SR001, or a combination of transgenic TCR and SR001, as measured by flow cytometry. [Figure 35DE] Figures 35D and 35E show the percent positive and geometric mean fluorescence intensity of anti-VHH staining for untransduced TCR, transgenic TCR, SR001, or the combination of transgenic TCR and SR001, as measured by flow cytometry. [Figure 36A] Figures 36A and 36B show IFNγ, IL2, and TNFα secretion from untransduced, transgenic TCR, SR001, or a combination of transgenic TCR and SR001, each co-cultured with high HLA-A2+TCR epitope+ or CD33+ expressing cancer cell lines in the absence and presence of rapamycin. [Figure 36B] Same as above. [Figure 36C] Figure 36C shows the cytotoxicity of untransduced, transgenic TCR cells, SR001-T cells, or a combination of transgenic TCR and SR001 co-cultured with high HLA-A2+TCR epitope+ or CD33+ expressing cancer cell lines in the absence or presence of rapamycin. [Figure 37] Figures 37A and 37B show IL2 secretion from untransduced or SR001, respectively, co-cultured with very low CD33 or CLL1 expressing cancer cell lines in the presence of rapamycin. [Figure 38] Figure 38 shows in vivo tumor growth in NSG mouse xenografts and controls following administration of 10E6 SR007 or comparative regulatory CAR T cells (targeting either CD33 or CLL1). All tumor controls are regulated by rapamycin. [Figure 39] Figures 39A-39C show vector copy number, percent positive for FRB and VHH staining for expression in the absence or presence of rapamycin. [Figure 40] Figures 40A and 40B show IFNγ secretion of SR10167 or SR10168-T cells alone ± rapamycin compared to regulatory CAR controls. [Figure 41] Figures 41A and 41B show IFNγ secretion of SR10167 or SR10168-T cells ± rapamycin compared to regulatory CAR controls when cultured with CD19+ target cell lines. [Figure 42AB] Figures 42A-42D show IL2 and TNFα secretion of SR10167 or SR10168-T cells ± rapamycin compared to regulatory CAR controls when cultured with a CD19+ target cell line. [Figure 42CD] Same as above. [Figure 43] Figures 43A and 43B show the cytotoxicity of control, SR10167-T cells, and regulatory CAR comparison T cells, each co-cultured with either CD19+ Jeko-1 or Daudi cell lines. All tumor controls are regulated by rapamycin. [Figure 44] FIG. 44 shows a schematic diagram of representative constitutively active (non-regulatable) engineered immune receptor and fusion constructs. [Figure 45] Figures 45A and 45B show the percent positive VHH staining and median fluorescence intensity for expression of SR292, SR293, SR296, SR001, and related comparators including non-regulatory CAR, regulatory CAR, and non-regulatory TCR-based structural comparators. [Figure 46] Figures 46A and 46B show IFNγ and IL2 secretion of SR292, SR293, SR296, SR001 and related comparative molecules including non-regulatory CAR, regulatory CAR, and non-regulatory TCR-based structural counterparts when cultured with CD33+ target cell lines. [Figure 47] Figure 47 shows IFNγ secretion of T cells alone expressing SR292, SR293, SR296, SR001 and related comparison molecules including non-regulatory CAR, regulatory CAR, and non-regulatory TCR-based structural comparisons. [Figure 48]Figures 48A and 48B show the cytotoxicity and T cell proliferation of control, SR292, SR293, SR296, and one non-regulated CAR after co-culture with a CD33+ target line. [Figure 49] FIG. 49 shows the percent positive and median fluorescence intensity of FRB staining for expression of SR001±IL7 receptor alpha, common gamma chain, IL2 receptor beta, or both common gamma chain and IL2 receptor beta. [Figure 50AB] Figures 50A-50D show IFNγ and IL2 secretion by SR001 ± IL7 receptor alpha, common gamma chain, IL2 receptor beta, or both common gamma chain and IL2 receptor beta when cultured with a CD33+ target cell line with and without rapamycin. [Figure 50CD] Same as above. [Figure 51] FIG. 51 shows T cell proliferation of control or SR001±IL7 receptor alpha, common gamma chain, IL2 receptor beta, or both common gamma chain and IL2 receptor beta after co-culture with CD33+ target lines. [Figure 52] Figure 52 shows the median fluorescence intensity of FRB or PD-1 staining of T cells expressing either SR300 (affinity-enhanced PD1) or SR301 (wild-type PD1). [Figure 53AB] Figures 53A-53D show the cytotoxicity of T cells expressing SR300 or SR301 when cultured with a PDL1+ target cell line or the same cell line with knocked-out PDL1 and PDL2, with and without rapamycin. [Figure 53CD] Same as above. [Figure 54] Figure 54 shows the percent positive and median fluorescence intensity of VHH staining for expression of the SR354 construct. [Figure 55AB] Figures 55A and 55B show IFNγ secretion of NK cells expressing SR354 when cultured with a CD33+ target cell line or the same cell line with CD33 knocked out. [Figure 55CD]Figures 55C and 55D show the cytotoxicity of NK cells expressing SR354 when cultured with CD33+ target cell lines or the same cell lines with CD33 knocked out. [Figure 56] FIG. 56 shows the percent positive for SR303 components on T cells as measured by flow cytometry. [Fig. 57AB] Figures 57A and 57B show IFNγ secretion of SR303 cells when cultured with a ROR1+ target cell line with and without rapamycin. [Figure 57C] Figure 57C shows IFNγ secretion by T cells expressing the SR303 molecule alone. DETAILED DESCRIPTION OF THE INVENTION

[0078] Brief description of sequence identifiers SEQ ID NOs: 1-4 show the amino acid sequences of exemplary FRB and FKBP12 polypeptides. SEQ ID NOs: 5 to 10 show the amino acid sequences of exemplary antibody-derived heterodimerization domains. SEQ ID NOs: 11 to 31 show the amino acid sequences of exemplary linkers. SEQ ID NOs: 32 to 40 show the amino acid sequences of exemplary actuator domains. SEQ ID NOs: 41 to 44 show the amino acid sequences of exemplary hinge domains. SEQ ID NOs: 45 to 47 show the amino acid sequences of exemplary transmembrane polypeptides. SEQ ID NOs: 48 and 49 show the amino acid sequences of exemplary truncated intracellular CD4 polypeptides. SEQ ID NOs: 50-94 show the amino acid sequences of exemplary targeting domains. SEQ ID NOs: 95 to 97 show the amino acid sequences of exemplary signal sequences. SEQ ID NOs: 98-106 show the amino acid sequences of exemplary intracellular signaling domains. SEQ ID NOs: 107-115 show the amino acid sequences of exemplary signaling components. SEQ ID NOs: 116-136 show the amino acid sequences of exemplary targeting moieties. SEQ ID NOs: 137 and 138 show the amino acid sequences of exemplary co-signaling components. SEQ ID NOs: 139-170 show the amino acid sequences of exemplary fusion polypeptides. SEQ ID NOs: 171 to 192 show the amino acid sequences of the protease cleavage site and the self-cleaving polypeptide cleavage site. SEQ ID NO: 193 shows the spacer amino acid sequence. SEQ ID NO: 194 shows the furin recognition amino acid sequence. SEQ ID NOs: 195 to 197 represent TEV (tobacco etch virus) protease cleavage sites. SEQ ID NO: 198 shows the Kozak amino acid sequence.

[0079] In the above sequences, X refers to any amino acid, if present, or to the absence of an amino acid.

[0080] A. Overview Cancer is one of the leading causes of death worldwide. Although adoptive cell therapy has been used successfully to treat some hematologic malignancies, treatment of solid tumors with both chimeric antigen receptor (CAR) T cells and T cells expressing T cell receptors (TCRs) against tumor antigens remains largely ineffective.

[0081] In addition, while TCRs are known to be exquisitely sensitive to low levels of target antigens, they lack the ability to distinguish non-MHC-presented antigens. On the other hand, while CARs can be engineered to target almost any extracellular antigen through their antibody-like binding domains, they are generally less sensitive to low levels of target antigens and tend to exhibit sustained and / or antigen-independent signaling. Furthermore, T cells engineered to express CARs or TCRs generally lack spatial and temporal control of T cell activity and / or exhibit insufficient activation of T cell signaling pathways.

[0082] Lack of control over engineered T cell activity can induce a wide range of side effects, many of which begin silently but can rapidly worsen. A particularly serious complication is cytokine release syndrome (CRS) or "cytokine storm," in which CAR T cells induce massive and even fatal cytokine release. CRS can result in dangerously high fever, extreme fatigue, difficulty breathing, and a sudden drop in blood pressure. CRS can also cause a second wave of side effects, which can affect the nervous system, including neurotoxicity, tremors, headache, confusion, loss of balance, difficulty speaking, seizures, and hallucinations. Insufficient activation of T cell signaling pathways can result in failure to eradicate cancer and / or lead to cancer becoming resistant to treatment. The compositions and methods contemplated herein provide solutions to these and other problems plaguing adoptive cell therapy.

[0083] Thus, the present disclosure relates to improved compositions and methods for modulating antigen recognition capacity, sensitivity, and / or spatial and temporal control of adoptive cell therapy by using engineered immune receptor complexes that can bind to selected target antigens and recruit and activate natural or transgenic immune receptor signaling complexes.

[0084] While not wishing to be bound by any particular theory, the engineered immune receptor compositions and methods contemplated herein offer numerous advantages over existing CAR T cell and TCR T cell therapies in the art, including, but not limited to, spatial and temporal control over immune effector cell signaling, binding and signaling activity, and activating signaling pathways without requiring MHC complex recognition. In some embodiments, temporal control primes the engineered immune receptor machinery for signaling through cross-linking agent-mediated association of the targeting moiety with the signaling moiety. In other embodiments, the machinery is primed by association of the multimerization domain without the need for a cross-linking agent. Spatial control engages the signaling machinery through recognition of the target antigen by the extracellular domain or targeting domain of the targeting moiety, while the signaling moiety comprises an actuator domain that forms a complex with a lymphocyte immune receptor. In this manner, immune effector cells activate receptor signaling in the presence of target cells expressing the target antigen, and, in certain embodiments, in the presence of a cross-linking agent.

[0085] In various embodiments, the present disclosure contemplates signaling and targeting components that generate an immune receptor-based response against cells expressing a target antigen without natural immune receptor recognition of the target antigen. In various embodiments, the present disclosure contemplates signaling and targeting components that generate an immune receptor-based anti-cancer response against cancers expressing a target antigen without requiring MHC complex recognition of the target antigen and / or lymphocytic immune receptors, either natural or engineered.

[0086] In certain embodiments, the engineered immune receptor complex comprises a signaling component comprising a multimerization domain polypeptide or variant thereof and an actuator domain or variant thereof (e.g., a CD3ε, CD3δ, CD3γ, FcεR1γ, Igα / CD79a, Igβ / CD79b, DAP10, or DAP12 polypeptide); and a targeting component comprising a targeting domain that binds to a target antigen expressed on a target cell, a multimerization domain polypeptide or variant thereof, a transmembrane domain, and, optionally, an extracellular domain comprising a hinge domain disposed between the multimerization domain and the transmembrane domain. In some embodiments, in the presence of a cross-linking agent, the signaling component and the binding / targeting component associate with each other through the cross-linking agent to form a functionally active immune receptor. In some embodiments, the components associate without the need for a cross-linking agent.

[0087] In certain embodiments, the multimerization domains of the signaling component and the targeting component are located extracellularly. Extracellular location of the multimerization domain offers numerous advantages over intracellular location, including, but not limited to, more efficient positioning of the targeting domain, greater temporal sensitivity to cross-linking agent modulation, and lower toxicity due to the ability to use non-immunosuppressive doses of certain cross-linking agents.

[0088] Contemplated herein are polynucleotides encoding engineered immune receptors, targeting moieties, signaling moieties, protein / polypeptide complexes, and fusion proteins; polypeptides, including engineered immune receptors, targeting moieties, signaling moieties, protein / polypeptide complexes, and fusion proteins; cells containing and / or expressing polynucleotides encoding engineered immune receptors, targeting moieties, and signaling moieties; vectors encoding engineered immune receptors, targeting moieties, signaling moieties, protein / polypeptide complexes, and fusion proteins; and methods of using the same to treat diseases or disorders (e.g., cancer or immune disorders).

[0089] Recombinant (i.e., engineered) DNA, peptide and oligonucleotide synthesis, immunoassays, tissue culture, transformation (e.g., electroporation, lipofection), enzymatic reactions, purification and related techniques and procedures may generally be performed as described in various general and more specific reference works in microbiology, molecular biology, biochemistry, molecular genetics, cell biology, virology and immunology, as cited and discussed throughout this specification. For example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2008); Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Glover, DNA Cloning: A Practical Approach, vol. I & II (IRL Press, Oxford Univ. Press USA, 1985); Current Protocols in Immunology (Edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M.Shevach、Warren Strober 2001 John Wiley & Sons、NY、NY);Real-Time PCR:Current Technology and Applications、Edited by Julie Logan、Kirstin Edwards and Nick Saunders、2009、Caister Academic Press、Norfolk、UK;Anand、Techniques for the Analysis of Complex Genomes、(Academic Press、New York、1992);Guthrie and Fink、Guide to Yeast Genetics and Molecular Biology(Academic Press、New York、1991);Oligonucleotide Synthesis(N.Gait、Ed.、1984);Nucleic Acid The Hybridization(B.Hames & S.Higgins、Eds.、1985);Transcription and Translation(B.Hames & S.Higgins、Eds.、1984);Animal Cell Culture(R.Freshney、Ed.、1986);Perbal、A Practical Guide to Molecular Cloning(1984);Next-Generation Genome Sequencing(Janitz、2008 Wiley-VCH);PCR Protocols(Methods in Molecular Biology)(Park、Ed.、3rd Edition、2010 Humana Press);Immobilized Cells And Enzymes(IRL Press、1986);the treatise、Methods In Enzymology(Academic Press、Inc.、N.Y.);Gene Transfer Vectors For Mammalian Cells(J.H.Miller and M.P.Calos eds., 1987, Cold Spring Harbor Laboratory); Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D.M. Weir and C.C. Blackwell, eds., 1986); Roitt, Essential Immunology, 6th Edition, (Blackwell Scientific Publications, Oxford, 1988); Current Protocols in Immunology (Q.E. Coligigan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach and W. Strober, eds., 1991); Annual Review of Immunology; and research articles in journals such as, for example, Advances in Immunology.

[0090] B. Definition Before describing this disclosure in more detail, it may be helpful to an understanding thereof to provide definitions of certain terms that will be used herein.

[0091] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of particular embodiments, preferred embodiments of the compositions, methods, and materials are described herein. For purposes of this disclosure, the following terms are defined below.

[0092] The articles "a," "an," and "the" are used herein to refer to one or to more than one (i.e., to at least one or to more than one) of the grammatical object of the article. By way of example, "an element" means one element or one or more elements.

[0093] The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination of the alternatives.

[0094] The term "and / or" should be understood to mean either one or both of the alternatives.

[0095] As used herein, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by up to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% relative to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the term "about" or "approximately" refers to a range of ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length relative to the reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0096] In one embodiment, a range, for example, 1 to 5, about 1 to 5, or about 1 to about 5, refers to each number encompassed by the range. For example, in one non-limiting and merely illustrative embodiment, the range "1 to 5" is equivalent to the following expressions: 1, 2, 3, 4, 5; or 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0; or 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0.

[0097] As used herein, the term "substantially" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, "substantially the same" refers to an amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that produces about the same effect, e.g., a physiological effect, as the reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0098] Throughout this specification, unless the context requires otherwise, the words "comprise" and "comprising" should be understood to imply the inclusion of the recited step or element or group of steps or elements, but not the exclusion of any other step or element or group of steps or elements. By "consisting of," it is meant to include and be limited to everything that follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the recited elements are required or mandatory, and that no other elements will be present. By "consisting essentially of," it is meant to include any elements listed after this phrase, and any elements limited to other elements that do not interfere with or contribute to the activity or function specified in this disclosure for the recited elements. Thus, the phrase "consisting essentially of" indicates that the recited elements are required or mandatory, but that there are no other elements that materially affect the activity or function of the recited elements.

[0099] References throughout this specification to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "an embodiment," "an additional embodiment," or "a further embodiment," or combinations thereof, mean that the particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment. As such, the appearances of these phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. Additionally, it should be understood that the affirmative recitation of a feature in an embodiment serves as a basis for the exclusion of that feature in certain embodiments.

[0100] As used herein, the term "TCR complex" refers to the complex formed by the association of CD3 with a TCR. For example, a TCR complex may be composed of a CD3γ chain, a CD3δ chain, two CD3ε chains, a homodimer of a CD3ζ chain, a TCRα chain, and a TCRβ chain. In some embodiments, a TCR complex may be composed of a CD3γ chain, a CD3δ chain, two CD3ε chains, a homodimer of a CD3ζ chain, a TCRγ chain, and a TCRδ chain.

[0101] As used herein, "component of a TCR complex" refers to a TCR chain (i.e., TCRα, TCRβ, TCRγ, or TCRδ), a CD3 chain (i.e., CD3γ, CD3δ, CD3ε, or CD3ζ), or a complex formed by two or more TCR chains or CD3 chains (e.g., a complex of TCRα and TCRβ, a complex of TCRγ and TCRδ, a complex of CD3ε and CD3δ, a complex of CD3γ and CD3ε, or a sub-TCR complex of TCRα, TCRβ, CD3γ, CD3δ, and two CD3ε chains).

[0102] As used herein, "actuator polypeptide," "actuator domain," or "actuator" refers to a polypeptide that associates, integrates, or complexes with a multimeric immune receptor complex, either directly or indirectly, to promote signal transduction, but does not itself contain direct antigen-binding properties. In certain embodiments, an actuator domain is part of a protein or protein complex that signals when bound to a target molecule. An actuator domain can directly contribute to a cellular response if it contains a signaling domain or motif, such as an immunoreceptor tyrosine-based activation motif (ITAM). In other embodiments, an actuator domain signals directly, thus promoting a cellular response indirectly by associating with one or more other proteins that promote the cellular response. Exemplary actuator domains include, for example, a CD3 polypeptide, an FcεR1γ polypeptide, an Igα / CD79a polypeptide, an Igβ / CD79b polypeptide, a DAP10 polypeptide, or a DAP12 polypeptide, or any combination thereof.

[0103] As used herein, a "multimerization domain" or "multimerization domain polypeptide" refers to a polypeptide that preferentially interacts or associates with another distinct polypeptide, either directly or through a bridging molecule, such as a chemically induced dimerizer, where the interaction of the distinct multimerization domains significantly contributes to or effectively promotes multimerization (i.e., the formation of dimers, trimers, or multimerized complexes, which may be homodimers, heterodimers, homotrimers, heterotrimers, homomultimers, or heteromultimers). Multimerization domains may be derived from natural, synthetic, semi-synthetic, or recombinant sources.

[0104] Illustrative examples of multimerization domains suitable for use in certain embodiments contemplated herein include an FK506 binding protein (FKBP) polypeptide or variant thereof, an FKBP-rapamycin binding (FRB) polypeptide or variant thereof, a calcineurin polypeptide or variant thereof, a cyclophilin polypeptide or variant thereof, a bacterial dihydrofolate reductase (DHFR) polypeptide or variant thereof, a PYR1-like 1 (PYL1) polypeptide or variant thereof, an abscisic acid insensitive 1 (ABI1) polypeptide or variant thereof, a GIB1 polypeptide or variant thereof, or a GAI polypeptide or variant thereof.

[0105] As used herein, the term "FKBP-rapamycin binding polypeptide" refers to an FRB polypeptide. In certain embodiments, the FRB polypeptide is an FKBP12-rapamycin binding polypeptide. FRB polypeptides suitable for use in certain embodiments contemplated herein generally contain at least about 85 to about 100 amino acid residues. In certain embodiments, the FRB polypeptide comprises a 93 amino acid sequence from Ile-2021 to Lys-2113 and a T2098L mutation (T82L is the equivalent position in the 93 amino acid FRB polypeptide), as referenced in GenBank Accession No. L34075.1. As used herein, the terms "FRB star," "FRB star," or "FRB*" refer to such an FRB T2098L (T82L) mutant. The FRB polypeptides contemplated herein bind to FKBP polypeptides via cross-linking agents, thereby forming a ternary complex.

[0106] As used herein, the term "FK506-binding protein" refers to an FKBP polypeptide. In certain embodiments, the FKBP polypeptide is an FKBP12 polypeptide or an FKBP12 polypeptide containing the F36V mutation. In certain embodiments, an FKBP domain may also be referred to as a "rapamycin-binding domain." Information regarding the nucleotide sequences, cloning, and other aspects of various FKBP species is known in the art (see, e.g., Staendart et al., Nature 346:671, 1990 (human FKBP12); Kay, Biochem. J. 314:361, 1996). The FKBP polypeptides contemplated herein bind to FRB polypeptides via cross-linking factors, thereby forming ternary complexes.

[0107] A "cross-linking agent" refers to a molecule that associates with and is positioned between two or more multimerization domains. In certain embodiments, a multimerization domain significantly contributes to or efficiently promotes the formation of a polypeptide complex only in the presence of the cross-linking agent. In certain embodiments, a multimerization domain does not contribute to or efficiently promote the formation of a polypeptide complex in the absence of the cross-linking agent. Illustrative examples of cross-linking agents suitable for use in certain embodiments contemplated herein include, but are not limited to, AP21967, rapamycin (sirolimus) or a rapalog thereof, coumermycin or a derivative thereof, gibberellin or a derivative thereof, abscisic acid (ABA) or a derivative thereof, methotrexate or a derivative thereof, cyclosporin A or a derivative thereof, FKCsA or a derivative thereof, trimethoprim (Tmp)-synthetic ligand for FKBP (SLF) or a derivative thereof, or any combination thereof.

[0108] Rapamycin analogs (rapalogs) include, but are not limited to, those disclosed in U.S. Pat. No. 6,649,595. Rapalog structures are incorporated herein by reference in their entirety. In certain embodiments, the cross-linking agent is a rapalog with a substantially reduced immunosuppressive effect compared to rapamycin. In a preferred embodiment, the rapalog is AP21967 (also known as C-16-(S)-7-methylindole rapamycin, IC 50 =10 nM, a chemically modified non-immunosuppressive rapamycin analog). Other exemplary rapalogs suitable for use in certain embodiments contemplated herein include, but are not limited to, AP1903, AP20187, everolimus, novolimus, pimecrolimus, ridaforolimus, sirolimus, tacrolimus, temsirolimus, umirolimus, zotarolimus, and BPC015.

[0109] "Substantially reduced immunosuppressive effect" refers to an immunosuppressive effect that is at least 0.1 to 0.005 times less than that observed or predicted for the same dose, as measured clinically or appropriately in vitro (e.g., inhibition of T-cell proliferation), or as measured in an in vivo surrogate of human immunosuppressive activity.

[0110] A "transmembrane domain" or "TM domain" is a domain that anchors a polypeptide to a cell membrane. TM domains can be derived from any source: natural, synthetic, semi-synthetic, or recombinant.

[0111] As used herein, the term "extracellular domain" refers to a domain or portion of a polypeptide that is located or present outside a cell. The extracellular domain of a targeting component, as disclosed herein, comprises at least one targeting domain, which, when the targeting component is associated with a signaling component, e.g., by a cross-linking agent connecting the multimerization domains of the targeting component and the signaling component, or by association of the multimerization domains of the targeting component and the signaling component without the need for a cross-linking agent, redirects activation of an immune receptor complex, e.g., a TCR, to one or more selected target antigens on a target cell, e.g., a cancer cell.

[0112] The term "effector function" or "effector cell function" refers to the specialized function of an immune effector cell. Effector functions include, but are not limited to, cytotoxic activity, including activation, cytokine production, proliferation, and release of cytotoxic factors, or other cellular responses triggered by antigen binding to a receptor expressed on an immune effector cell. An "intracellular signaling domain" or "endodomain" refers to the portion of a protein that transmits an effector function signal and instructs the cell to carry out a specialized function. While the entire intracellular signaling domain can usually be used, in many cases, it is not necessary to use the entire domain. To the extent that a truncated portion of an intracellular signaling domain is used, such a truncated portion may be used in place of the entire domain, so long as the effector function signal is transmitted. The term intracellular signaling domain is meant to include any truncated portion of the intracellular signaling domain that is necessary or sufficient to transmit the effector function signal.

[0113] It is known that signals generated through the TCR alone are insufficient for full activation of T cells; secondary or costimulatory signals are also required. Thus, T cell activation can be said to be mediated by two distinct classes of intracellular signaling domains: primary signaling domains, which initiate antigen-dependent primary activation through the TCR (e.g., the TCR / CD3 complex), and costimulatory signaling domains, which act in an antigen-independent manner to provide secondary or costimulatory signals.

[0114] As used herein, the term "costimulatory signaling domain" or "costimulatory domain" refers to the intracellular signaling domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule, other than an antigen receptor or an Fc receptor, that, upon binding to an antigen, generates a secondary signal required for efficient activation and function of T lymphocytes. Illustrative examples of costimulatory molecules from which costimulatory domains can be isolated include, but are not limited to, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain family member 11 (CARD11), CD2, CD3ε, CD3γ, CD3δ, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DNAX-activation protein 1 (DNAX-activation protein 1). 0 (DAP10), FYN, linker for activation of T cells family member 1 (LAT), 76 kD SH2 domain-containing leukocyte protein (SLP76), LCK, T cell receptor-associated transmembrane adaptor 1 (TRAT1), TNFR2, TNF receptor superfamily member 14 (TNFRS14; HVEM), TNF receptor superfamily member 18 (TNFRS18; GITR), TNF receptor superfamily member 25 (TNFRS25; DR3), and the zeta chain of T cell receptor-associated protein kinase 70 (ZAP70).

[0115] "Hinge domain" refers to a polypeptide that plays a role in spatially positioning a domain away from the effector cell surface to allow proper cell / cell contact, antigen binding, and activation. In certain embodiments, a polypeptide may comprise one or more hinge domains between the extracellular domain and the transmembrane domain (TM), between the multimerization domain and the transmembrane domain, or between the multimerization domain and the actuator domain. Hinge domains may be derived from any natural, synthetic, semi-synthetic, or recombinant source. Hinge domains may comprise the amino acid sequence of a naturally occurring immunoglobulin hinge region or a modified immunoglobulin hinge region.

[0116] "Linker" or "linker polypeptide" refers to a number of amino acid residues between various polypeptide domains, added for proper spacing and conformation of the molecule. In certain embodiments, the linker is a sequence that connects the variable regions. A "sequence that connects the variable regions" is a sequence that connects the V H and V L and a linker that provides a spacer function compatible with the interaction of the two sub-binding domains, such that the resulting polypeptide retains the same specific binding affinity for the same target molecule as an antibody comprising the same light chain variable region and heavy chain variable region. In certain embodiments, the linker separates one or more heavy or light chain variable domains, hinge domains, multimerization domains, transmembrane domains, costimulatory domains, and / or primary signaling domains.

[0117] Illustrative examples of linkers suitable for use in certain embodiments contemplated herein include, but are not limited to, the following amino acid sequences: GGG; DGGGS (SEQ ID NO: 16); TGEKP (SEQ ID NO: 17) (see, e.g., Liu et al., PNAS 5525-5530 (1997)); GGRR (SEQ ID NO: 18) (Pomerantz et al. 1995, supra); (GGGGS) n, where n=1, 2, 3, 4, or 5 (SEQ ID NOs: 11-15) (Kim et al., PNAS 93, 1156-1160 (1996); EGKSSGSGSESKVD (SEQ ID NO: 19) (Chaudhary et al., 1990, Proc. Natl. Acad. Sci. USA 87:1066-1070); KESGSVSSSEQLAQFRSLD (SEQ ID NO: 20) (Bird et al., 1988, Science 242:423-426), GGRRGGGS (SEQ ID NO: 21); LQRDGERP (SEQ ID NO: 22); LRQKDGGGSERP (SEQ ID NO: 23); LRQKD(GGGS)2ERP (SEQ ID NO: 24). Alternatively, flexible linkers can be rationally designed using computer programs capable of modeling both the DNA binding site and the peptide itself (Desjarlais & Berg, PNAS 90:2256-2260 (1993), PNAS 91:11099-11103 (1994) or by phage display methods. In one embodiment, the linker comprises the following amino acid sequence: GSTSGSGKPGSGEGSTKG (SEQ ID NO: 25) (Cooper et al., Blood, 101(4):1637-1644 (2003)).

[0118] A "spacer domain" refers to a polypeptide that separates two domains. In one embodiment, the spacer domain displaces the targeting domain away from the effector cell surface, allowing for proper cell-cell contact, antigen binding, and activation (Patel et al., Gene Therapy, 1999;6:412-419). In certain embodiments, the spacer domain separates one or more heavy or light chain variable domains, multimerization domains, transmembrane domains, costimulatory domains, and / or primary signaling domains. Spacer domains can be derived from natural, synthetic, semi-synthetic, or recombinant sources. In certain embodiments, the spacer domain is a portion of an immunoglobulin, including one or more heavy chain constant regions, e.g., CH2 and CH3. The spacer domain may comprise the amino acid sequence of a naturally occurring immunoglobulin hinge region or a modified immunoglobulin hinge region.

[0119] "Antigen (Ag)" refers to a compound, composition, or substance that can stimulate antibody production or a T-cell response in an animal, including compositions injected or absorbed into an animal (e.g., compositions containing a cancer-specific protein). Examples of antigens include, but are not limited to, lipids, carbohydrates, polysaccharides, glycoproteins, peptides, or nucleic acids. An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous antigens, such as the disclosed antigens.

[0120] "Target antigen" or "target antigen of interest" refers to a molecule expressed on the cell surface of a target cell to which a binding or targeting domain contemplated herein is designed to bind. In certain embodiments, the target antigen is an epitope of a polypeptide expressed on the surface of a cancer cell.

[0121] As used herein, the term "targeting domain" refers to a domain, e.g., a domain of a targeting moiety, that provides a polypeptide with the ability to specifically bind to a target antigen of interest. A targeting domain can be derived from any natural, synthetic, semi-synthetic, or recombinant source. A targeting domain may comprise, for example, an antibody or antigen-binding fragment thereof, or, for example, an ectodomain.

[0122] As used herein, the terms "specific binding affinity" or "specifically binds" or "specifically bound" or "specific binding" or "specifically targeted" describe the binding of a targeting domain to a target antigen with a binding affinity that is greater than background binding. A targeting domain may be one that binds to a target antigen with a binding affinity that is greater than background binding, for example, about 10 5 M -1 Affinity greater than or equal to K a (i.e., the equilibrium binding constant of a particular binding interaction, with units of 1 / M). In one embodiment, the targeting domain (or a fusion protein comprising the same) binds to the target with a binding affinity of about 10 6 M -1 , 10 7 M -1 , 10 8 M -1 , 10 9 M -1 , 10 10 M -1 , 10 11 M -1 , 10 12 M -1 , or 10 13 M -1 Greater than or equal to K a A "high affinity" targeting domain (or single-chain fusion protein thereof) binds with at least 10 7 M 1 , at least 10 8 M -1 , at least 10 9 M -1 , at least 10 10 M -1 , at least 10 11 M -1, at least 10 12 M -1 , at least 10 13 M -1 or larger K a refers to a targeting domain with

[0123] The terms "selectively bind" or "selectively bound" or "selective binding" or "selectively target" describe the preferential binding of one molecule to a target molecule (on-target binding) in the presence of multiple off-target molecules.

[0124] "Antibody" refers to a binding agent that is a polypeptide comprising at least a light or heavy chain immunoglobulin variable region that specifically recognizes and binds to an epitope of an antigen, such as a lipid, carbohydrate, polysaccharide, glycoprotein, peptide, or nucleic acid, including an antigenic determinant, e.g., one recognized by an immune cell.

[0125] "Epitope" or "antigenic determinant" refers to a region of an antigen to which a binding agent binds.

[0126] Antibodies include antigen-binding fragments such as camel Ig, llama Ig, alpaca Ig, Ig NAR, Fab' fragments, F(ab')2 fragments, bispecific Fab dimers (Fab2), trispecific Fab trimers (Fab3), Fv, single-chain Fv proteins ("scFv"), bis-scFv, (scFv)2, minibodies, diabodies, triabodies, tetrabodies, disulfide-stabilized Fv proteins ("dsFv"), and single-domain antibodies (sdAb, camelid VHH, nanobodies), as well as the portion of a full-length antibody that is responsible for antigen binding. The term also includes genetically engineered forms, such as chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (such as bispecific antibodies), and antigen-binding fragments thereof. See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rd Ed., W.H. Freeman & Co., New York, 1997.

[0127] As used herein, the term "cancer" generally relates to certain diseases or conditions in which abnormal cells divide uncontrollably and can invade nearby tissues.

[0128] As used herein, the term "malignant" refers to a cancer in which tumor cells exhibit one or more of the following: uncontrolled growth (i.e., dividing beyond normal limits), invasion (i.e., invading and destroying adjacent tissues), and metastasis (i.e., spreading to other parts of the body via the lymph or blood). As used herein, the term "metastasizing" refers to the spread of cancer from one part of the body to another. A tumor formed by the spread cells is called a "metastatic tumor" or "metastatic cancer." A metastatic tumor contains cells similar to those in the original (primary) tumor.

[0129] As used herein, the terms "benign" or "non-malignant" refer to tumors that can grow large but do not spread to other parts of the body. Benign tumors are self-limited and often do not invade or metastasize.

[0130] "Cancer cell" refers to an individual cell of a cancerous growth or cancerous tissue. Cancer cells include both solid and liquid cancers. "Tumor" or "tumor cell" generally refers to a lesion formed by the abnormal proliferation of cells or an expansion caused by the abnormal proliferation of cells, which may be benign, precancerous, or malignant. Most cancers form tumors, but liquid cancers, such as leukemia, do not necessarily form tumors. For cancers that form tumors, the terms cancer (cell) and tumor (cell) are used interchangeably. The amount of tumor in an individual is the "tumor burden," which can be measured as the number, volume, or weight of tumors.

[0131] The term "recurrence" refers to the diagnosis of a return of cancer, or the diagnosis of signs and symptoms of a return of cancer, after a period of improvement or remission.

[0132] "Remission," also referred to as "clinical remission," includes both partial and complete remission. In a partial remission, some, but not all, signs and symptoms of cancer disappear. In a complete remission, all signs and symptoms of cancer disappear, although cancer may still be present in the body.

[0133] "Refractory" refers to a cancer that is resistant or unresponsive to treatment with a particular therapeutic agent. A cancer may be refractory as a result of developing resistance to a therapeutic agent either from the start of treatment (i.e., unresponsive to initial exposure to the therapeutic agent), over the course of an initial treatment period, or during subsequent treatment periods.

[0134] "Antigen-negative" refers to cells that do not express the antigen or that express such small amounts of the antigen that they are undetectable. In one embodiment, antigen-negative cells do not bind to the target receptor for the antigen. In one embodiment, antigen-negative cells do not substantially bind to the target receptor for the antigen.

[0135] As used herein, the terms "individual" and "subject" are often used interchangeably and refer to any animal exhibiting symptoms of cancer or other immune disorder that can be treated with the compositions and methods otherwise contemplated herein. Suitable subjects (e.g., patients) include laboratory animals (e.g., mice, rats, rabbits, or guinea pigs), farm animals, domestic animals, or pets (e.g., cats or dogs). Non-human primates, preferably human patients, are also included. Exemplary subjects include human patients who have, have been diagnosed with, or are at risk of having cancer or another immune disorder.

[0136] As used herein, the term "patient" refers to a subject diagnosed with cancer or another immune disorder that can be treated with the compositions and methods elsewhere disclosed herein.

[0137] As used herein, "treatment" or "treating" includes any beneficial or desired effect on the symptoms or pathology of a disease or pathological condition, and may include even a minimal reduction in one or more measurable markers of the disease or condition being treated. Treatment can optionally include a reduction in the disease or condition, or a slowing of the progression of the disease or condition, e.g., a slowing of tumor growth. "Treatment" does not necessarily indicate a complete elimination or cure of the disease or condition, or its associated symptoms.

[0138] As used herein, "prevent" and similar terms, e.g., "prevented," "preventing," refer to an approach aimed at preventing, inhibiting, or reducing the likelihood of occurrence or recurrence of a disease or condition. It also refers to delaying the onset or recurrence of a disease or condition, or delaying the onset or recurrence of symptoms of a disease or condition. As used herein, "prevention" and similar terms also include reducing the intensity, effects, symptoms, and / or burden of a disease or condition prior to the onset or recurrence of the disease or condition.

[0139] As used herein, "ameliorating at least one symptom of" refers to a decrease in one or more symptoms of the disease or condition for which a subject is being treated. In certain embodiments, the disease or condition being treated is cancer, in which the one or more symptoms ameliorated include, but are not limited to, weakness, fatigue, shortness of breath, easy bruising and bleeding, frequent infections, enlarged lymph nodes, abdominal distension or pain (caused by distended abdominal organs), bone or joint pain, broken bones, unexpected weight loss, loss of appetite, night sweats, persistent low-grade fever, and decreased urination (caused by impaired kidney function).

[0140] "Enhance," or "promote," or "increase," or "proliferate" generally refers to the ability of a composition contemplated herein to produce, elicit, or generate a greater physiological response (i.e., downstream effect) compared to the response caused by either a solvent or a control molecule / composition. Measurable physiological responses include, among others, increases in T-cell proliferation, activation, persistence, cytokine secretion, and / or cancer cell killing capacity, as would be apparent from an understanding of the art and the descriptions herein. An "increased" or "enhanced" amount is typically a "statistically significant" amount and may include an increase of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, or more (e.g., 500-fold, 1000-fold) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) over the response produced by a solvent or control composition.

[0141] By "reduce," or "lower," or "lower," or "decreasing," or "attenuating," generally refers to the ability of a composition contemplated herein to produce, induce, or cause a physiological response (i.e., a downstream effect) that is smaller than the response caused by a solvent or control molecule / composition. A "reduction" or "decreased" amount is typically a "statistically significant" amount and can include a decrease of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, or more (e.g., 500-fold, 1000-fold) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) of the response produced by a solvent, a control composition, or a response in a particular cell line.

[0142] "Maintain," or "preserve," or "maintain," or "no change," or "no substantial change," or "no substantial decrease" generally refer to the ability of a composition contemplated herein to produce, elicit, or cause a substantially similar or equivalent physiological response (i.e., downstream effect) in a cell compared to the response caused by either a solvent, a control molecule / composition, or a response in a particular cell line. An equivalent response is a response that is not significantly or measurably different from the reference response.

[0143] Additional definitions are set forth throughout this disclosure.

[0144] C. Engineered immune receptor structures In certain embodiments, one or more engineered immune receptors are contemplated that redirect the cytotoxicity of immune effector cells against cancer cells expressing a target antigen and recruit immune receptor complexes. As used herein, the terms "engineered immune receptor," "engineered immune receptor complex," or "engineered immune receptor system" refer to one or more non-naturally occurring polypeptides that, upon exposure to a predefined or selected target antigen and association / multimerization with an immune receptor complex, facilitate the transduction of immunostimulatory signals in immune effector cells. In some embodiments, a multimerizing agent or cross-linking agent is required to promote multimerization of the non-naturally occurring polypeptides, thereby stimulating immune effector cell activity and function through activation of the immune receptor complex. In a preferred embodiment, the engineered immune receptor or system is a multi-chain chimeric receptor comprising a signaling component that associates with a member of the immune receptor complex and a targeting component that redirects or complements the antigen specificity of the receptor / complex.

[0145] In one embodiment, the signaling component and the targeting component are expressed from the same cell. In another embodiment, the signaling component and the targeting component are expressed from different cells. In another embodiment, the signaling component is expressed from a cell and the targeting component is exogenously provided as a polypeptide. In certain embodiments, the targeting component preloaded with a cross-linking agent is exogenously provided to the cell expressing the signaling component.

[0146] 1. Signal transduction components "Signaling component" refers to a polypeptide comprising one or more multimerization domains or variants thereof and an actuator domain capable of recruiting an immune receptor complex (e.g., a TCR, FcR, BCR, or NKG2D complex). Such actuator domains include, but are not limited to, 1) a CD3 polypeptide, domain, or fragment thereof that associates with or binds to a TCR complex, e.g., CD3ε, CD3γ, or CD3δ; 2) an FcεR1γ polypeptide chain, domain, or fragment thereof that associates with or binds to an Fc receptor complex; 3) an Igα / CD79A polypeptide, domain, or fragment thereof; 4) an Igβ / CD79B polypeptide, domain, or fragment thereof; 5) a DAP10 polypeptide, domain, or fragment thereof; and 6) a DAP12 polypeptide, domain, or fragment thereof, or any combination thereof.

[0147] In one embodiment, the signaling moiety does not include a primary signaling domain (eg, CD3z).

[0148] In various embodiments, the actuator domain comprises a CD3 polypeptide or a functional fragment or variant thereof. In some embodiments, the CD3 polypeptide is a CD3 epsilon (CD3ε) polypeptide, domain, or functional fragment or variant thereof; a CD3 gamma (CD3γ) polypeptide, domain, or functional fragment or variant thereof; or a CD3 delta (CD3δ) polypeptide, domain, or functional fragment or variant thereof. In some embodiments, the CD3 polypeptide comprises the extracellular and intracellular portions of a CD3ε, CD3γ, or CD3δ polypeptide.

[0149] In various embodiments, the CD3 polypeptide comprises a CD3ε peptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 32, or a functional fragment or variant thereof. In some embodiments, the CD3 polypeptide comprises a CD3ε peptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 32, or a functional fragment or variant thereof. In some embodiments, the CD3 polypeptide comprises a CD3ε peptide comprising an amino acid sequence having at least 96% identity to SEQ ID NO: 32, or a functional fragment or variant thereof. In some embodiments, the CD3 polypeptide comprises a CD3ε peptide comprising an amino acid sequence having at least 97% identity to SEQ ID NO: 32, or a functional fragment or variant thereof. In some embodiments, the CD3 polypeptide comprises a CD3ε peptide comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 32, or a functional fragment or variant thereof. In some embodiments, the CD3 polypeptide comprises a CD3ε peptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 32, or a functional fragment or variant thereof. In a specific embodiment, the CD3 polypeptide comprises a CD3ε peptide comprising the amino acid sequence set forth in SEQ ID NO: 32.

[0150] In various embodiments, the CD3 polypeptide comprises a CD3γ peptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 33, or a functional fragment or variant thereof. In some embodiments, the CD3 polypeptide comprises a CD3γ peptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 33, or a functional fragment or variant thereof. In some embodiments, the CD3 polypeptide comprises a CD3γ peptide comprising an amino acid sequence having at least 96% identity to SEQ ID NO: 33, or a functional fragment or variant thereof. In some embodiments, the CD3 polypeptide comprises a CD3γ peptide comprising an amino acid sequence having at least 97% identity to SEQ ID NO: 33, or a functional fragment or variant thereof. In some embodiments, the CD3 polypeptide comprises a CD3γ peptide comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 33, or a functional fragment or variant thereof. In some embodiments, the CD3 polypeptide comprises a CD3γ peptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 33, or a functional fragment or variant thereof. In a specific embodiment, the CD3 polypeptide comprises a CD3γ peptide comprising the amino acid sequence set forth in SEQ ID NO: 33.

[0151] In various embodiments, the CD3 polypeptide comprises a CD3δ peptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 34, or a functional fragment or variant thereof. In some embodiments, the CD3 polypeptide comprises a CD3δ peptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 34, or a functional fragment or variant thereof. In some embodiments, the CD3 polypeptide comprises a CD3δ peptide comprising an amino acid sequence having at least 96% identity to SEQ ID NO: 34, or a functional fragment or variant thereof. In some embodiments, the CD3 polypeptide comprises a CD3δ peptide comprising an amino acid sequence having at least 97% identity to SEQ ID NO: 34, or a functional fragment or variant thereof. In some embodiments, the CD3 polypeptide comprises a CD3δ peptide comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 34, or a functional fragment or variant thereof. In some embodiments, the CD3 polypeptide comprises a CD3δ peptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 34, or a functional fragment or variant thereof. In a specific embodiment, the CD3 polypeptide comprises a CD3δ peptide comprising the amino acid sequence set forth in SEQ ID NO: 34.

[0152] In various embodiments, the CD3 polypeptide is a human CD3 polypeptide.

[0153] In certain embodiments, the signaling component comprises an FRB or FRB T2098L multimerization domain, a linker polypeptide, and a CD3ε polypeptide. In certain embodiments, the signaling component comprises an antibody-derived heterodimerization domain, a linker polypeptide, and a CD3ε polypeptide. In certain embodiments, the signaling component comprises a lock-and-key multimerization domain, a linker polypeptide, and a CD3ε polypeptide. In certain embodiments, the signaling component comprises a DEKK multimerization domain, a linker polypeptide, and a CD3ε polypeptide. In certain embodiments, the signaling component comprises a SEEDbody multimerization domain, a linker polypeptide, and a CD3ε polypeptide.

[0154] In certain embodiments, the signaling component comprises an FRB or FRB T2098L multimerization domain, a linker polypeptide, and a CD3δ polypeptide. In certain embodiments, the signaling component comprises an antibody-derived heterodimerization domain, a linker polypeptide, and a CD3δ polypeptide. In certain embodiments, the signaling component comprises a lock-and-key multimerization domain, a linker polypeptide, and a CD3δ polypeptide. In certain embodiments, the signaling component comprises a DEKK multimerization domain, a linker polypeptide, and a CD3δ polypeptide. In certain embodiments, the signaling component comprises a SEEDbody multimerization domain, a linker polypeptide, and a CD3δ polypeptide.

[0155] In certain embodiments, the signaling component comprises an FRB or FRB T2098L multimerization domain, a linker polypeptide, and a CD3γ polypeptide. In certain embodiments, the signaling component comprises an antibody-derived heterodimerization domain, a linker polypeptide, and a CD3γ polypeptide. In certain embodiments, the signaling component comprises a lock-and-key multimerization domain, a linker polypeptide, and a CD3γ polypeptide. In certain embodiments, the signaling component comprises a DEKK multimerization domain, a linker polypeptide, and a CD3γ polypeptide. In certain embodiments, the signaling component comprises a SEEDbody multimerization domain, a linker polypeptide, and a CD3γ polypeptide.

[0156] In various embodiments, the signaling component comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 107. In one embodiment, the signaling component comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 107. In one embodiment, the signaling component comprises an amino acid sequence having at least 96% identity to SEQ ID NO: 107. In one embodiment, the signaling component comprises an amino acid sequence having at least 97% identity to SEQ ID NO: 107. In one embodiment, the signaling component comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 107. In one embodiment, the signaling component comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 107. In a particular embodiment, the signaling component comprises the amino acid sequence set forth in SEQ ID NO: 107.

[0157] In various embodiments, the signaling component comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 108. In various embodiments, the signaling component comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 108. In various embodiments, the signaling component comprises an amino acid sequence having at least 96% identity to SEQ ID NO: 108. In various embodiments, the signaling component comprises an amino acid sequence having at least 97% identity to SEQ ID NO: 108. In various embodiments, the signaling component comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 108. In various embodiments, the signaling component comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 108. In a particular embodiment, the signaling component comprises the amino acid sequence set forth in SEQ ID NO: 108.

[0158] In various embodiments, the signaling component comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 109. In various embodiments, the signaling component comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 109. In various embodiments, the signaling component comprises an amino acid sequence having at least 96% identity to SEQ ID NO: 109. In various embodiments, the signaling component comprises an amino acid sequence having at least 97% identity to SEQ ID NO: 109. In various embodiments, the signaling component comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 109. In various embodiments, the signaling component comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 109. In a particular embodiment, the signaling component comprises the amino acid sequence set forth in SEQ ID NO: 109.

[0159] In various embodiments, the signaling component comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 110. In various embodiments, the signaling component comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 110. In various embodiments, the signaling component comprises an amino acid sequence having at least 96% identity to SEQ ID NO: 110. In various embodiments, the signaling component comprises an amino acid sequence having at least 97% identity to SEQ ID NO: 110. In various embodiments, the signaling component comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 110. In various embodiments, the signaling component comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 110. In a particular embodiment, the signaling component comprises the amino acid sequence set forth in SEQ ID NO: 110.

[0160] In various embodiments, the signaling component comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 111. In various embodiments, the signaling component comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 111. In various embodiments, the signaling component comprises an amino acid sequence having at least 96% identity to SEQ ID NO: 111. In various embodiments, the signaling component comprises an amino acid sequence having at least 97% identity to SEQ ID NO: 111. In various embodiments, the signaling component comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 111. In various embodiments, the signaling component comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 111. In a particular embodiment, the signaling component comprises the amino acid sequence set forth in SEQ ID NO: 111.

[0161] In various embodiments, the signaling component comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 112. In various embodiments, the signaling component comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 112. In various embodiments, the signaling component comprises an amino acid sequence having at least 96% identity to SEQ ID NO: 112. In various embodiments, the signaling component comprises an amino acid sequence having at least 97% identity to SEQ ID NO: 112. In various embodiments, the signaling component comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 112. In various embodiments, the signaling component comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 112. In a particular embodiment, the signaling component comprises the amino acid sequence set forth in SEQ ID NO: 112.

[0162] In various embodiments, the signaling component comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 113. In various embodiments, the signaling component comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 113. In various embodiments, the signaling component comprises an amino acid sequence having at least 96% identity to SEQ ID NO: 113. In various embodiments, the signaling component comprises an amino acid sequence having at least 97% identity to SEQ ID NO: 113. In various embodiments, the signaling component comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 113. In various embodiments, the signaling component comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 113. In a particular embodiment, the signaling component comprises the amino acid sequence set forth in SEQ ID NO: 113.

[0163] In various embodiments, the signaling component comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 114. In various embodiments, the signaling component comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 114. In various embodiments, the signaling component comprises an amino acid sequence having at least 96% identity to SEQ ID NO: 114. In various embodiments, the signaling component comprises an amino acid sequence having at least 97% identity to SEQ ID NO: 114. In various embodiments, the signaling component comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 114. In various embodiments, the signaling component comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 114. In a particular embodiment, the signaling component comprises the amino acid sequence set forth in SEQ ID NO: 114.

[0164] In various embodiments, the actuator domain comprises an FcεR1γ polypeptide or a functional fragment or variant thereof. In some embodiments, the FcεR1γ polypeptide comprises an extracellular portion and an intracellular portion of an FcεR1γ polypeptide. In various embodiments, the FcεR1γ polypeptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 35. In some embodiments, the FcεR1γ chain polypeptide comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 35. In some embodiments, the FcεR1γ polypeptide comprises an amino acid sequence having at least 96% identity to SEQ ID NO: 35. In some embodiments, the FcεR1γ polypeptide comprises an amino acid sequence having at least 97% identity to SEQ ID NO: 35. In some embodiments, the FcεR1γ polypeptide comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 35. In some embodiments, the FcεR1γ polypeptide comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 35. In a specific embodiment, the FcεR1γ polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 35.

[0165] In various embodiments, the FcεR1 γ polypeptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 36. In some embodiments, the FcεR1 γ chain polypeptide comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 36. In some embodiments, the FcεR1 γ polypeptide comprises an amino acid sequence having at least 96% identity to SEQ ID NO: 36. In some embodiments, the FcεR1 γ polypeptide comprises an amino acid sequence having at least 97% identity to SEQ ID NO: 36. In some embodiments, the FcεR1 γ polypeptide comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 36. In some embodiments, the FcεR1 γ chain polypeptide comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 36. In a specific embodiment, the FcεR1 γ chain polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 36.

[0166] In one embodiment, the signaling component comprises a sequence having at least 90% identity to SEQ ID NO: 115. In one embodiment, the signaling component comprises a sequence having at least 95% identity to SEQ ID NO: 115. In one embodiment, the signaling component comprises a sequence having at least 96% identity to SEQ ID NO: 115. In one embodiment, the signaling component comprises a sequence having at least 97% identity to SEQ ID NO: 115. In one embodiment, the signaling component comprises a sequence having at least 98% identity to SEQ ID NO: 115. In one embodiment, the signaling component comprises a sequence having at least 99% identity to SEQ ID NO: 115. In one embodiment, the signaling component comprises the sequence set forth as SEQ ID NO: 115.

[0167] In certain embodiments, the signaling component comprises an FRB or FRB T2098L multimerization domain, a linker polypeptide, and an FcεR1γ polypeptide. In certain embodiments, the signaling component comprises an antibody-derived heterodimerization domain, a linker polypeptide, and an FcεR1γ polypeptide. In certain embodiments, the signaling component comprises a lock-and-key multimerization domain, a linker polypeptide, and an FcεR1γ polypeptide. In certain embodiments, the signaling component comprises a DEKK multimerization domain, a linker polypeptide, and an FcεR1γ chain polypeptide. In certain embodiments, the signaling component comprises a SEEDbody multimerization domain, a linker polypeptide, and an FcεR1γ polypeptide.

[0168] In various embodiments, the signaling component comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 115. In various embodiments, the signaling component comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 115. In various embodiments, the signaling component comprises an amino acid sequence having at least 96% identity to SEQ ID NO: 115. In various embodiments, the signaling component comprises an amino acid sequence having at least 97% identity to SEQ ID NO: 115. In various embodiments, the signaling component comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 115. In various embodiments, the signaling component comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 115. In a particular embodiment, the signaling component comprises the amino acid sequence set forth in SEQ ID NO: 115.

[0169] In various embodiments, the actuator domain comprises an Igα / CD79A polypeptide, or a functional fragment or variant thereof. In one embodiment, the Igα / CD79A polypeptide comprises a sequence at least 90% identical to SEQ ID NO: 37. In one embodiment, the Igα / CD79A polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 37. In one embodiment, the Igα / CD79A polypeptide comprises a sequence at least 96% identical to SEQ ID NO: 37. In one embodiment, the Igα / CD79A polypeptide comprises a sequence at least 97% identical to SEQ ID NO: 37. In one embodiment, the Igα / CD79A polypeptide comprises a sequence at least 98% identical to SEQ ID NO: 37. In one embodiment, the Igα / CD79A polypeptide comprises a sequence at least 99% identical to SEQ ID NO: 37. In one embodiment, the Igα / CD79A polypeptide comprises the sequence set forth as SEQ ID NO: 37.

[0170] In various embodiments, the actuator domain comprises an Igβ / CD79A polypeptide, or a functional fragment or variant thereof. In one embodiment, the Igβ / CD79A polypeptide comprises a sequence having at least 90% identity to SEQ ID NO: 38. In one embodiment, the Igβ / CD79A polypeptide comprises a sequence having at least 95% identity to SEQ ID NO: 38. In one embodiment, the Igβ / CD79A polypeptide comprises a sequence having at least 96% identity to SEQ ID NO: 38. In one embodiment, the Igβ / CD79A polypeptide comprises a sequence having at least 97% identity to SEQ ID NO: 38. In one embodiment, the Igβ / CD79A polypeptide comprises a sequence having at least 98% identity to SEQ ID NO: 38. In one embodiment, the Igβ / CD79A polypeptide comprises a sequence having at least 99% identity to SEQ ID NO: 38. In one embodiment, the Igβ / CD79A polypeptide comprises the sequence set forth as SEQ ID NO: 38.

[0171] In various embodiments, the actuator domain comprises a DAP10 polypeptide or a functional fragment or variant thereof. In one embodiment, the DAP10 polypeptide comprises a sequence having at least 90% identity to SEQ ID NO: 39. In one embodiment, the DAP10 polypeptide comprises a sequence having at least 95% identity to SEQ ID NO: 39. In one embodiment, the DAP10 polypeptide comprises a sequence having at least 96% identity to SEQ ID NO: 39. In one embodiment, the DAP10 polypeptide comprises a sequence having at least 97% identity to SEQ ID NO: 39. In one embodiment, the DAP10 polypeptide comprises a sequence having at least 98% identity to SEQ ID NO: 39. In one embodiment, the DAP10 polypeptide comprises a sequence having at least 99% identity to SEQ ID NO: 39. In one embodiment, the DAP10 polypeptide comprises the sequence set forth as SEQ ID NO: 39.

[0172] In various embodiments, the actuator domain comprises a DAP12 polypeptide or a functional fragment or variant thereof. In one embodiment, the DAP12 polypeptide comprises a sequence having at least 90% identity to SEQ ID NO: 40. In one embodiment, the DAP12 polypeptide comprises a sequence having at least 95% identity to SEQ ID NO: 40. In one embodiment, the DAP12 polypeptide comprises a sequence having at least 96% identity to SEQ ID NO: 40. In one embodiment, the DAP12 polypeptide comprises a sequence having at least 97% identity to SEQ ID NO: 40. In one embodiment, the DAP12 polypeptide comprises a sequence having at least 98% identity to SEQ ID NO: 40. In one embodiment, the DAP12 polypeptide comprises a sequence having at least 99% identity to SEQ ID NO: 40. In one embodiment, the DAP12 polypeptide comprises the sequence set forth as SEQ ID NO: 40.

[0173] In one embodiment, the signaling component does not comprise the amino acid sequence of SEQ ID NO:111.

[0174] In one embodiment, the signaling component comprises a multimerization domain. In various embodiments, the multimerization domain is localized extracellularly when the signaling component is expressed.

[0175] Illustrative examples of suitable multimerization domains for use in certain signaling components contemplated herein include, but are not limited to, an FKBP polypeptide or variant thereof, an FRB polypeptide or variant thereof, a calcineurin polypeptide or variant thereof, a cyclophilin polypeptide or variant thereof, a DHFR polypeptide or variant thereof, a PYL1 polypeptide or variant thereof, and an ABI1 polypeptide or variant thereof.

[0176] In certain embodiments, the signaling component comprises an FRB polypeptide or a variant thereof. In various embodiments, the signaling component comprises an FRB polypeptide comprising a T2098L (T82L) mutation or a variant thereof. In certain embodiments, the FRB polypeptide comprises an amino acid sequence at least 90% identical to SEQ ID NO: 1. In certain embodiments, the FRB polypeptide comprises an amino acid sequence at least 95% identical to SEQ ID NO: 1. In certain embodiments, the FRB polypeptide comprises an amino acid sequence at least 96% identical to SEQ ID NO: 1. In certain embodiments, the FRB polypeptide comprises an amino acid sequence at least 97% identical to SEQ ID NO: 1. In certain embodiments, the FRB polypeptide comprises an amino acid sequence at least 98% identical to SEQ ID NO: 1. In certain embodiments, the FRB polypeptide comprises an amino acid sequence at least 99% identical to SEQ ID NO: 1. In certain embodiments, the FRB polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, the FRB polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2. In certain embodiments, the FRB polypeptide comprises an amino acid sequence at least 90% identical to SEQ ID NO: 2. In certain embodiments, the FRB polypeptide comprises an amino acid sequence having at least 95% identity to SEQ ID NO:2. In certain embodiments, the FRB polypeptide comprises an amino acid sequence having at least 96% identity to SEQ ID NO:2. In certain embodiments, the FRB polypeptide comprises an amino acid sequence having at least 97% identity to SEQ ID NO:2. In certain embodiments, the FRB polypeptide comprises an amino acid sequence having at least 98% identity to SEQ ID NO:2. In certain embodiments, the FRB polypeptide comprises an amino acid sequence having at least 99% identity to SEQ ID NO:2. In certain embodiments, the FRB polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2.

[0177] In certain embodiments, the signaling component comprises an FKBP12 polypeptide or a variant thereof. In various embodiments, the signaling component comprises an FKBP12 polypeptide comprising an F36V mutation. In one embodiment, the FKBP12 polypeptide comprises an amino acid sequence at least 90% identical to SEQ ID NO:3. In one embodiment, the FKBP12 polypeptide comprises an amino acid sequence at least 95% identical to SEQ ID NO:3. In one embodiment, the FKBP12 polypeptide comprises an amino acid sequence at least 96% identical to SEQ ID NO:3. In one embodiment, the FKBP12 polypeptide comprises an amino acid sequence at least 97% identical to SEQ ID NO:3. In one embodiment, the FKBP12 polypeptide comprises an amino acid sequence at least 98% identical to SEQ ID NO:3. In one embodiment, the FKBP12 polypeptide comprises an amino acid sequence at least 99% identical to SEQ ID NO:3. In certain embodiments, the FKBP12 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:3. In certain embodiments, the FKBP12 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:4. In one embodiment, the FKBP12 polypeptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO:4. In one embodiment, the FKBP12 polypeptide comprises an amino acid sequence having at least 95% identity to SEQ ID NO:4. In one embodiment, the FKBP12 polypeptide comprises an amino acid sequence having at least 96% identity to SEQ ID NO:4. In one embodiment, the FKBP12 polypeptide comprises an amino acid sequence having at least 97% identity to SEQ ID NO:4. In one embodiment, the FKBP12 polypeptide comprises an amino acid sequence having at least 98% identity to SEQ ID NO:4. In one embodiment, the FKBP12 polypeptide comprises an amino acid sequence having at least 99% identity to SEQ ID NO:4. In a specific embodiment, the FKBP12 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:4.

[0178] Other illustrative examples of multimerization domains suitable for use in certain signaling components contemplated herein include antibody-derived heterodimerization domains / pairs. Such antibody heterodimerization domains include, but are not limited to, lock-and-key, DEKK, SEEDbody, DuoBody, dual variable domain immunoglobulin (DVD-Ig), and Fabs-in-tandem immunoglobulin (FIT-Ig) multimerization domains (see, e.g., Ma et al., Front Immunol. 2021 May 5;12:626616).

[0179] In various embodiments, the signaling component comprises a lock and key multimerization domain / polypeptide or variant thereof. In some embodiments, the lock and key multimerization domain / polypeptide or variant thereof comprises the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the lock and key multimerization domain / polypeptide or variant thereof comprises the amino acid sequence set forth in SEQ ID NO: 6.

[0180] In various embodiments, the signaling component comprises a DEKK multimerization domain / polypeptide or variant thereof. In some embodiments, the DEKK multimerization domain / polypeptide or variant thereof comprises the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the DEKK multimerization domain / polypeptide or variant thereof comprises the amino acid sequence set forth in SEQ ID NO: 8.

[0181] In various embodiments, the signaling component comprises a SEEDbody multimerization domain / polypeptide or variant thereof. In some embodiments, the SEEDbody multimerization domain / polypeptide or variant thereof comprises the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the SEEDbody multimerization domain / polypeptide or variant thereof comprises the amino acid sequence set forth in SEQ ID NO: 10.

[0182] Other illustrative examples of suitable multimerization domains for use in certain signaling components contemplated herein include, but are not limited to, leucine zipper multimerization domains, mFos:mJun coil multimerization domains, p53 and MDM2 multimerization domains, VPS36 and VPS28 multimerization domains. Other suitable multimerization or dimerization domains will be known to those of skill in the art.

[0183] In various embodiments, the first multimerization domain and the actuator domain are separated by a first polypeptide linker between 2 and 40 amino acids in length. In various embodiments, a short oligo- or polypeptide linker, preferably between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length, connects one or more domains in the signaling component. Glycine-serine-based linkers provide particularly suitable linkers. In some embodiments, the first polypeptide linker is selected from the group consisting of GG, GS, SG, SS, GSS, SSG, GSG, SGS, SGG, G4S, 2xG4S, 3xG4S, 4xG4S, and combinations thereof. In some embodiments, the first polypeptide linker is a G4S (GGGGS; SEQ ID NO: 11) linker. In some embodiments, the first polypeptide linker is a 2xG4S (GGGGSGGGGS; SEQ ID NO: 12) linker. In some embodiments, the first polypeptide linker is a 3xG4S (GGGGSGGGGSGGGS; SEQ ID NO: 13) linker. In some embodiments, the first polypeptide linker is a 4xG4S (GGGGSGGGGSGGGSGGGGS; SEQ ID NO: 14) linker. In some embodiments, the first polypeptide linker is a 5xG4S (GGGGSGGGGSGGGGSGGGGS; SEQ ID NO: 15) linker. In one embodiment, the linker has the sequence set forth as SEQ ID NO: 16. In one embodiment, the linker has the sequence set forth as SEQ ID NO: 17. In one embodiment, the linker has the sequence set forth as SEQ ID NO: 18. In one embodiment, the linker has the sequence set forth as SEQ ID NO: 19. In one embodiment, the linker has the sequence set forth as SEQ ID NO: 20. In one embodiment, the linker has the sequence set forth as SEQ ID NO: 21. In one embodiment, the linker has the sequence set forth as SEQ ID NO: 22. In one embodiment, the linker has the sequence set forth as SEQ ID NO: 23. In one embodiment, the linker has the sequence set forth as SEQ ID NO: 24. In one embodiment, the linker has the sequence set forth as SEQ ID NO: 25. In one embodiment, the linker has the sequence set forth as SEQ ID NO: 26.In one embodiment, the linker has the sequence set forth as SEQ ID NO: 27. In one embodiment, the linker has the sequence set forth as SEQ ID NO: 28. In one embodiment, the linker has the sequence set forth as SEQ ID NO: 29. In one embodiment, the linker has the sequence set forth as SEQ ID NO: 30. In one embodiment, the linker has the sequence set forth as SEQ ID NO: 31.

[0184] In various embodiments, signaling components contemplated herein include a signal peptide, e.g., a secretory signal polypeptide or signal sequence. Illustrative examples of signal peptides / sequences suitable for use in particular signaling components include, but are not limited to, an IgG1 heavy chain signal polypeptide, an Igκ light chain signal polypeptide, a CD8α signal polypeptide, or a human GM-CSF receptor alpha signal polypeptide. In various embodiments, a signaling component includes an Igκ light chain signal polypeptide. In some embodiments, an Igκ light chain signal polypeptide includes the amino acid sequence set forth in SEQ ID NO: 95. In various embodiments, a signaling component includes a CD8α signal polypeptide. In some embodiments, a CD8α light chain signal polypeptide includes the amino acid sequence set forth in SEQ ID NO: 96. In various embodiments, a signaling component includes a PD1 signal polypeptide. In one embodiment, a PD1 signal polypeptide includes the amino acid sequence set forth as SEQ ID NO: 97.

[0185] 2. Targeting components "Targeting moiety" refers to a polypeptide comprising an extracellular domain, one or more multimerization domains, and a transmembrane domain, which, in conjunction with a signaling moiety, redirects activation of an immune receptor complex to one or more selected target antigens. In some embodiments, the extracellular domain comprises one or more targeting domains that associate with and / or bind to one or more target antigens. In certain embodiments, the targeting moiety comprises a first targeting domain, a second multimerization domain, a hinge domain, and a transmembrane domain.

[0186] In certain embodiments, the targeting moiety comprises an extracellular domain comprising at least one targeting domain, wherein the targeting domain is an antibody or antigen-binding fragment thereof directed against one or more target antigens. Antigen-binding fragments directed against one or more target antigens suitable for use in certain embodiments contemplated herein include those selected from the group consisting of camel Ig, llama Ig, alpaca Ig, Ig NAR, Fab' fragment, F(ab')2 fragment, bispecific Fab dimer (Fab2), trispecific Fab trimer (Fab3), Fv, single-chain Fv protein ("scFv"), bis-scFv, (scFv)2, minibody, diabody, triabody, tetrabody, disulfide-stabilized Fv protein ("dsFv"), and single-domain antibodies (e.g., sdAb, camelid VHH, or nanobody).

[0187] In various embodiments, the targeting domain comprises a single chain variable fragment (scFv) or a single domain antibody (sdAb). In various embodiments, the targeting domain comprises a single chain variable fragment (scFv). In some embodiments, the sdAb is a camelid VHH, a nanobody, or a heavy chain-only antibody (HcAb). In some embodiments, the sdAb is a camelid VHH. In some embodiments, the scFv or sdAb is human or humanized. In certain embodiments, the VHH is humanized.

[0188] The present disclosure provides a platform technology that is applicable to numerous classes of targeting domains and fragments and should not be limited to one particular targeting domain, e.g., antibody, antigen-binding fragment, ectodomain, etc., for targeting a particular target antigen.

[0189] For example, in some embodiments, the target antigen is an antigen expressed on a target cell, including, for example, a cancer cell. In some embodiments, the targeting domain binds to a target antigen selected from the group consisting of: alpha folate receptor (FRα), α vβ6 integrin, B-cell maturation antigen (BCMA), B7-H3 (CD276), B7-H6, carbonic anhydrase IX (CAIX), CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD133, CD138, CD171, carcinoembryonic antigen (CEA), C-type lectin-like molecule 1 (CLL-1), CD2 subset 1 (CS-1), chondroitin sulfate proteoglycan 4 (CSPG4), cutaneous T-cell lymphoma-associated antigen 1 (CTAGE1), epidermal growth factor receptor (EGFR), epidermal growth factor EGFR family including receptor variant III (EGFRvIII), epithelial glycoprotein 2 (EGP2), epithelial glycoprotein 40 (EGP40), epithelial cell adhesion molecule (EPCAM), ephrin type A receptor 2 (EPHA2), fibroblast activation protein (FAP), Fc receptor-like 5 (FCRL5), fetal acetylcholinesterase receptor (AchR), ganglioside G2 (GD2), ganglioside G3 (GD3), glypican 3 (GPC3), ErbB2 (HER2), IGF2BP3 / A3, IL-10Rα, IL-13Rα2, kappa, cancer / testis antigen 2 (LAGE-1A), K-Ras, and K-Ras G12C, K-Ras G12D, K-Ras G12V, lambda, Lewis Y (LeY), L1 cell adhesion molecule (L1-CAM), melanoma antigen gene (MAGE)-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGEA10, melanoma antigen 1 recognized by T cells (MelanA or MART1), mesothelin (MSLN), MUC1, MUC16, MHC class I chain-related protein A (MICA), MHC class I chain-related protein B (MICB), neural cell adhesion molecule (NCAM), cancer / testis antigen 1 (NY-ESO-1), polysialic acid;Placenta-specific 1 (PLAC1), antigen preferentially expressed in melanoma (PRAME), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), synovial sarcoma, X-breakpoint 2 (SSX2), survivin, tumor-associated glycoprotein 72 (TAG72), transmembrane activator and CAML interactor (TACI), tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), trophoblast glycoprotein (TPBG), UL16-binding protein (ULBP) 1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, vascular endothelial growth factor receptor 2 (VEGFR2), and Wilms' tumor 1 (WT-1);

[0190] In certain embodiments, the targeting domain binds to a target antigen selected from the group consisting of: BCMA, B7-H3 (CD276), CD19, CD20, CD22, CD33, CD79a, CD79b, CD123, CLL-1, EGFR, EGFRvIII, MUC16, and PRAME.

[0191] In certain embodiments, the targeting domain binds to CD33 or CLL-1.

[0192] In various embodiments, the targeting moiety comprises an extracellular domain that includes a second targeting domain.

[0193] In various embodiments, the second targeting domain comprises a single chain variable fragment (scFv) or a single domain antibody (sdAb). In various embodiments, the second targeting domain comprises a single chain variable fragment (scFv). In some embodiments, the sdAb is a camelid VHH, a nanobody, or a heavy chain-only antibody (HcAb). In some embodiments, the sdAb is a camelid VHH. In some embodiments, the scFv or sdAb is human or humanized. In certain embodiments, the VHH is humanized.

[0194] In certain embodiments, the extracellular domain contemplated includes a second targeting domain that binds to a target antigen selected from the group consisting of: alpha folate receptor (FRα), α vβ6 integrin, B-cell maturation antigen (BCMA), B7-H3 (CD276), B7-H6, carbonic anhydrase IX (CAIX), CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD133, CD138, CD171, carcinoembryonic antigen (CEA), C-type lectin-like molecule 1 (CLL-1), CD2 subset 1 (CS-1), chondroitin sulfate proteoglycan 4 (CSPG4), cutaneous T-cell lymphoma-associated antigen 1 (CTAGE1), epidermal growth factor receptor (EGFR), epidermal growth factor EGFR family including receptor variant III (EGFRvIII), epithelial glycoprotein 2 (EGP2), epithelial glycoprotein 40 (EGP40), epithelial cell adhesion molecule (EPCAM), ephrin type A receptor 2 (EPHA2), fibroblast activation protein (FAP), Fc receptor-like 5 (FCRL5), fetal acetylcholinesterase receptor (AchR), ganglioside G2 (GD2), ganglioside G3 (GD3), glypican 3 (GPC3), ErbB2 (HER2), IGF2BP3 / A3, IL-10Rα, IL-13Rα2, kappa, cancer / testis antigen 2 (LAGE-1A), K-Ras, and K-Ras G12C, K-Ras G12D, K-Ras G12V, lambda, Lewis Y (LeY), L1 cell adhesion molecule (L1-CAM), melanoma antigen gene (MAGE)-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGEA10, melanoma antigen 1 recognized by T cells (MelanA or MART1), mesothelin (MSLN), MUC1, MUC16, MHC class I chain-related protein A (MICA), MHC class I chain-related protein B (MICB), neural cell adhesion molecule (NCAM), cancer / testis antigen 1 (NY-ESO-1), polysialic acid;Placenta-specific 1 (PLAC1), antigen preferentially expressed in melanoma (PRAME), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), synovial sarcoma, X-breakpoint 2 (SSX2), survivin, tumor-associated glycoprotein 72 (TAG72), transmembrane activator and CAML interactor (TACI), tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), trophoblast glycoprotein (TPBG), UL16-binding protein (ULBP) 1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, vascular endothelial growth factor receptor 2 (VEGFR2), and Wilms' tumor 1 (WT-1);

[0195] In certain embodiments, the second targeting domain binds to a target antigen selected from the group consisting of: BCMA, B7-H3 (CD276), CD19, CD20, CD22, CD33, CD79a, CD79b, CD123, CLL-1, EGFR, EGFRvIII, MUC16, and PRAME.

[0196] In certain embodiments, the second targeting domain binds to CD33 or CLL-1.

[0197] In one embodiment, the targeting domain is an anti-BCMA targeting domain.

[0198] In one embodiment, the targeting domain comprises a sequence having at least 90% identity to SEQ ID NO: 50. In one embodiment, the targeting domain comprises a sequence having at least 95% identity to SEQ ID NO: 50. In one embodiment, the targeting domain comprises a sequence having at least 96% identity to SEQ ID NO: 50. In one embodiment, the targeting domain comprises a sequence having at least 97% identity to SEQ ID NO: 50. In one embodiment, the targeting domain comprises a sequence having at least 98% identity to SEQ ID NO: 50. In one embodiment, the targeting domain comprises a sequence having at least 99% identity to SEQ ID NO: 50. In one embodiment, the targeting domain comprises the sequence set forth as SEQ ID NO: 50.

[0199] In one embodiment, the targeting domain comprises a sequence having at least 90% identity to SEQ ID NO: 51. In one embodiment, the targeting domain comprises a sequence having at least 95% identity to SEQ ID NO: 51. In one embodiment, the targeting domain comprises a sequence having at least 96% identity to SEQ ID NO: 51. In one embodiment, the targeting domain comprises a sequence having at least 97% identity to SEQ ID NO: 51. In one embodiment, the targeting domain comprises a sequence having at least 98% identity to SEQ ID NO: 51. In one embodiment, the targeting domain comprises a sequence having at least 99% identity to SEQ ID NO: 51. In one embodiment, the targeting domain comprises the sequence set forth as SEQ ID NO: 51.

[0200] In one embodiment, the targeting domain comprises a sequence having at least 90% identity to SEQ ID NO: 52. In one embodiment, the targeting domain comprises a sequence having at least 95% identity to SEQ ID NO: 52. In one embodiment, the targeting domain comprises a sequence having at least 96% identity to SEQ ID NO: 52. In one embodiment, the targeting domain comprises a sequence having at least 97% identity to SEQ ID NO: 52. In one embodiment, the targeting domain comprises a sequence having at least 98% identity to SEQ ID NO: 52. In one embodiment, the targeting domain comprises a sequence having at least 99% identity to SEQ ID NO: 52. In one embodiment, the targeting domain comprises the sequence set forth as SEQ ID NO: 52.

[0201] In one embodiment, the targeting domain is a CD19 targeting domain. In one embodiment, the targeting domain comprises a sequence having at least 90% identity to SEQ ID NO: 53. In one embodiment, the targeting domain comprises a sequence having at least 95% identity to SEQ ID NO: 53. In one embodiment, the targeting domain comprises a sequence having at least 96% identity to SEQ ID NO: 53. In one embodiment, the targeting domain comprises a sequence having at least 97% identity to SEQ ID NO: 53. In one embodiment, the targeting domain comprises a sequence having at least 98% identity to SEQ ID NO: 53. In one embodiment, the targeting domain comprises a sequence having at least 99% identity to SEQ ID NO: 53. In one embodiment, the targeting domain comprises the sequence set forth as SEQ ID NO: 53.

[0202] In one embodiment, the targeting domain is a CD20 targeting domain of n.

[0203] In one embodiment, the targeting domain comprises a sequence having at least 90% identity to SEQ ID NO: 54. In one embodiment, the targeting domain comprises a sequence having at least 95% identity to SEQ ID NO: 54. In one embodiment, the targeting domain comprises a sequence having at least 96% identity to SEQ ID NO: 54. In one embodiment, the targeting domain comprises a sequence having at least 97% identity to SEQ ID NO: 54. In one embodiment, the targeting domain comprises a sequence having at least 98% identity to SEQ ID NO: 54. In one embodiment, the targeting domain comprises a sequence having at least 99% identity to SEQ ID NO: 54. In one embodiment, the targeting domain comprises the sequence set forth as SEQ ID NO: 54.

[0204] In one embodiment, the targeting domain comprises a sequence having at least 90% identity to SEQ ID NO: 55. In one embodiment, the targeting domain comprises a sequence having at least 95% identity to SEQ ID NO: 55. In one embodiment, the targeting domain comprises a sequence having at least 96% identity to SEQ ID NO: 55. In one embodiment, the targeting domain comprises a sequence having at least 97% identity to SEQ ID NO: 55. In one embodiment, the targeting domain comprises a sequence having at least 98% identity to SEQ ID NO: 55. In one embodiment, the targeting domain comprises a sequence having at least 99% identity to SEQ ID NO: 55. In one embodiment, the targeting domain comprises the sequence set forth as SEQ ID NO: 55.

[0205] In one embodiment, the targeting domain comprises a sequence having at least 90% identity to SEQ ID NO: 56. In one embodiment, the targeting domain comprises a sequence having at least 95% identity to SEQ ID NO: 56. In one embodiment, the targeting domain comprises a sequence having at least 96% identity to SEQ ID NO: 56. In one embodiment, the targeting domain comprises a sequence having at least 97% identity to SEQ ID NO: 56. In one embodiment, the targeting domain comprises a sequence having at least 98% identity to SEQ ID NO: 56. In one embodiment, the targeting domain comprises a sequence having at least 99% identity to SEQ ID NO: 56. In one embodiment, the targeting domain comprises the sequence set forth as SEQ ID NO: 56.

[0206] In one embodiment, the targeting domain is a CD22 targeting domain. In one embodiment, the targeting domain comprises a sequence having at least 90% identity to SEQ ID NO: 57. In one embodiment, the targeting domain comprises a sequence having at least 95% identity to SEQ ID NO: 57. In one embodiment, the targeting domain comprises a sequence having at least 96% identity to SEQ ID NO: 57. In one embodiment, the targeting domain comprises a sequence having at least 97% identity to SEQ ID NO: 57. In one embodiment, the targeting domain comprises a sequence having at least 98% identity to SEQ ID NO: 57. In one embodiment, the targeting domain comprises a sequence having at least 99% identity to SEQ ID NO: 57. In one embodiment, the targeting domain comprises the sequence set forth as SEQ ID NO: 57.

[0207] In one embodiment, the targeting domain is a CD33 targeting domain. In one embodiment, the targeting domain comprises a sequence having at least 90% identity to SEQ ID NO: 58. In one embodiment, the targeting domain comprises a sequence having at least 95% identity to SEQ ID NO: 58. In one embodiment, the targeting domain comprises a sequence having at least 96% identity to SEQ ID NO: 58. In one embodiment, the targeting domain comprises a sequence having at least 97% identity to SEQ ID NO: 58. In one embodiment, the targeting domain comprises a sequence having at least 98% identity to SEQ ID NO: 58. In one embodiment, the targeting domain comprises a sequence having at least 99% identity to SEQ ID NO: 58. In one embodiment, the targeting domain comprises the sequence set forth as SEQ ID NO: 58.

[0208] In one embodiment, the targeting domain is a CD79A targeting domain. In one embodiment, the targeting domain comprises a sequence having at least 90% identity to SEQ ID NO: 59. In one embodiment, the targeting domain comprises a sequence having at least 95% identity to SEQ ID NO: 59. In one embodiment, the targeting domain comprises a sequence having at least 96% identity to SEQ ID NO: 59. In one embodiment, the targeting domain comprises a sequence having at least 97% identity to SEQ ID NO: 59. In one embodiment, the targeting domain comprises a sequence having at least 98% identity to SEQ ID NO: 59. In one embodiment, the targeting domain comprises a sequence having at least 99% identity to SEQ ID NO: 59. In one embodiment, the targeting domain comprises the sequence set forth as SEQ ID NO: 59.

[0209] In one embodiment, the targeting domain is an anti-CD79B targeting domain. In one embodiment, the targeting domain comprises a sequence having at least 90% identity to SEQ ID NO: 60. In one embodiment, the targeting domain comprises a sequence having at least 95% identity to SEQ ID NO: 60. In one embodiment, the targeting domain comprises a sequence having at least 96% identity to SEQ ID NO: 60. In one embodiment, the targeting domain comprises a sequence having at least 97% identity to SEQ ID NO: 60. In one embodiment, the targeting domain comprises a sequence having at least 98% identity to SEQ ID NO: 60. In one embodiment, the targeting domain comprises a sequence having at least 99% identity to SEQ ID NO: 60. In one embodiment, the targeting domain comprises the sequence set forth as SEQ ID NO: 60.

[0210] In one embodiment, the targeting domain comprises a sequence having at least 90% identity to SEQ ID NO: 61. In one embodiment, the targeting domain comprises a sequence having at least 95% identity to SEQ ID NO: 61. In one embodiment, the targeting domain comprises a sequence having at least 96% identity to SEQ ID NO: 61. In one embodiment, the targeting domain comprises a sequence having at least 97% identity to SEQ ID NO: 61. In one embodiment, the targeting domain comprises a sequence having at least 98% identity to SEQ ID NO: 61. In one embodiment, the targeting domain comprises a sequence having at least 99% identity to SEQ ID NO: 61. In one embodiment, the targeting domain comprises the sequence set forth as SEQ ID NO: 61.

[0211] In one embodiment, the targeting domain is a B7H3 targeting domain. In one embodiment, the targeting domain comprises a sequence having at least 90% identity to SEQ ID NO: 62. In one embodiment, the targeting domain comprises a sequence having at least 95% identity to SEQ ID NO: 62. In one embodiment, the targeting domain comprises a sequence having at least 96% identity to SEQ ID NO: 62. In one embodiment, the targeting domain comprises a sequence having at least 97% identity to SEQ ID NO: 62. In one embodiment, the targeting domain comprises a sequence having at least 98% identity to SEQ ID NO: 62. In one embodiment, the targeting domain comprises a sequence having at least 99% identity to SEQ ID NO: 62. In one embodiment, the targeting domain comprises the sequence set forth as SEQ ID NO: 62.

[0212] In one embodiment, the targeting domain is a MUC16 targeting domain. In one embodiment, the targeting domain comprises a sequence having at least 90% identity to SEQ ID NO: 63. In one embodiment, the targeting domain comprises a sequence having at least 95% identity to SEQ ID NO: 63. In one embodiment, the targeting domain comprises a sequence having at least 96% identity to SEQ ID NO: 63. In one embodiment, the targeting domain comprises a sequence having at least 97% identity to SEQ ID NO: 63. In one embodiment, the targeting domain comprises a sequence having at least 98% identity to SEQ ID NO: 63. In one embodiment, the targeting domain comprises a sequence having at least 99% identity to SEQ ID NO: 63. In one embodiment, the targeting domain comprises the sequence set forth as SEQ ID NO: 63.

[0213] In one embodiment, the targeting domain is a HER2 targeting domain. In one embodiment, the targeting domain comprises a sequence having at least 90% identity to SEQ ID NO: 64. In one embodiment, the targeting domain comprises a sequence having at least 95% identity to SEQ ID NO: 64. In one embodiment, the targeting domain comprises a sequence having at least 96% identity to SEQ ID NO: 64. In one embodiment, the targeting domain comprises a sequence having at least 97% identity to SEQ ID NO: 64. In one embodiment, the targeting domain comprises a sequence having at least 98% identity to SEQ ID NO: 64. In one embodiment, the targeting domain comprises a sequence having at least 99% identity to SEQ ID NO: 64. In one embodiment, the targeting domain comprises the sequence set forth as SEQ ID NO: 64.

[0214] In one embodiment, the targeting domain is an EGFR targeting domain. In one embodiment, the targeting domain comprises a sequence having at least 90% identity to SEQ ID NO: 65. In one embodiment, the targeting domain comprises a sequence having at least 95% identity to SEQ ID NO: 65. In one embodiment, the targeting domain comprises a sequence having at least 96% identity to SEQ ID NO: 65. In one embodiment, the targeting domain comprises a sequence having at least 97% identity to SEQ ID NO: 65. In one embodiment, the targeting domain comprises a sequence having at least 98% identity to SEQ ID NO: 65. In one embodiment, the targeting domain comprises a sequence having at least 99% identity to SEQ ID NO: 65. In one embodiment, the targeting domain comprises the sequence set forth as SEQ ID NO: 65.

[0215] In one embodiment, the targeting domain is an FN-EDB targeting domain. In one embodiment, the targeting domain comprises a sequence having at least 90% identity to SEQ ID NO: 66. In one embodiment, the targeting domain comprises a sequence having at least 95% identity to SEQ ID NO: 66. In one embodiment, the targeting domain comprises a sequence having at least 96% identity to SEQ ID NO: 66. In one embodiment, the targeting domain comprises a sequence having at least 97% identity to SEQ ID NO: 66. In one embodiment, the targeting domain comprises a sequence having at least 98% identity to SEQ ID NO: 66. In one embodiment, the targeting domain comprises a sequence having at least 99% identity to SEQ ID NO: 66. In one embodiment, the targeting domain comprises the sequence set forth as SEQ ID NO: 66.

[0216] In one embodiment, the targeting domain is a CLDN18.2 targeting domain. In one embodiment, the targeting domain comprises a sequence having at least 90% identity to SEQ ID NO: 67. In one embodiment, the targeting domain comprises a sequence having at least 95% identity to SEQ ID NO: 67. In one embodiment, the targeting domain comprises a sequence having at least 96% identity to SEQ ID NO: 67. In one embodiment, the targeting domain comprises a sequence having at least 97% identity to SEQ ID NO: 67. In one embodiment, the targeting domain comprises a sequence having at least 98% identity to SEQ ID NO: 67. In one embodiment, the targeting domain comprises a sequence having at least 99% identity to SEQ ID NO: 67. In one embodiment, the targeting domain comprises the sequence set forth as SEQ ID NO: 67.

[0217] In one embodiment, the targeting domain is a DLL3 targeting domain. In one embodiment, the targeting domain comprises a sequence having at least 90% identity to SEQ ID NO: 68. In one embodiment, the targeting domain comprises a sequence having at least 95% identity to SEQ ID NO: 68. In one embodiment, the targeting domain comprises a sequence having at least 96% identity to SEQ ID NO: 68. In one embodiment, the targeting domain comprises a sequence having at least 97% identity to SEQ ID NO: 68. In one embodiment, the targeting domain comprises a sequence having at least 98% identity to SEQ ID NO: 68. In one embodiment, the targeting domain comprises a sequence having at least 99% identity to SEQ ID NO: 68. In one embodiment, the targeting domain comprises the sequence set forth as SEQ ID NO: 68.

[0218] In one embodiment, the targeting domain is a FLT3 targeting domain. In one embodiment, the targeting domain comprises a sequence having at least 90% identity to SEQ ID NO: 69. In one embodiment, the targeting domain comprises a sequence having at least 95% identity to SEQ ID NO: 69. In one embodiment, the targeting domain comprises a sequence having at least 96% identity to SEQ ID NO: 69. In one embodiment, the targeting domain comprises a sequence having at least 97% identity to SEQ ID NO: 69. In one embodiment, the targeting domain comprises a sequence having at least 98% identity to SEQ ID NO: 69. In one embodiment, the targeting domain comprises a sequence having at least 99% identity to SEQ ID NO: 69. In one embodiment, the targeting domain comprises the sequence set forth as SEQ ID NO: 69.

[0219] In one embodiment, the targeting domain comprises a sequence having at least 90% identity to SEQ ID NO: 70. In one embodiment, the targeting domain comprises a sequence having at least 95% identity to SEQ ID NO: 70. In one embodiment, the targeting domain comprises a sequence having at least 96% identity to SEQ ID NO: 70. In one embodiment, the targeting domain comprises a sequence having at least 97% identity to SEQ ID NO: 70. In one embodiment, the targeting domain comprises a sequence having at least 98% identity to SEQ ID NO: 70. In one embodiment, the targeting domain comprises a sequence having at least 99% identity to SEQ ID NO: 70. In one embodiment, the targeting domain comprises the sequence set forth as SEQ ID NO: 70.

[0220] In one embodiment, the targeting domain is a ROR1 targeting domain. In one embodiment, the targeting domain comprises a sequence having at least 90% identity to SEQ ID NO: 71. In one embodiment, the targeting domain comprises a sequence having at least 95% identity to SEQ ID NO: 71. In one embodiment, the targeting domain comprises a sequence having at least 96% identity to SEQ ID NO: 71. In one embodiment, the targeting domain comprises a sequence having at least 97% identity to SEQ ID NO: 71. In one embodiment, the targeting domain comprises a sequence having at least 98% identity to SEQ ID NO: 71. In one embodiment, the targeting domain comprises a sequence having at least 99% identity to SEQ ID NO: 71. In one embodiment, the targeting domain comprises the sequence set forth as SEQ ID NO: 71.

[0221] In one embodiment, the targeting domain is a ROR1 targeting domain. In one embodiment, the targeting domain comprises a sequence having at least 90% identity to SEQ ID NO: 71. In one embodiment, the targeting domain comprises a sequence having at least 95% identity to SEQ ID NO: 71. In one embodiment, the targeting domain comprises a sequence having at least 96% identity to SEQ ID NO: 71. In one embodiment, the targeting domain comprises a sequence having at least 97% identity to SEQ ID NO: 71. In one embodiment, the targeting domain comprises a sequence having at least 98% identity to SEQ ID NO: 71. In one embodiment, the targeting domain comprises a sequence having at least 99% identity to SEQ ID NO: 71. In one embodiment, the targeting domain comprises the sequence set forth as SEQ ID NO: 71.

[0222] In one embodiment, the targeting domain is a CD33 targeting domain. In one embodiment, the targeting domain comprises a sequence having at least 90% identity to SEQ ID NO: 72. In one embodiment, the targeting domain comprises a sequence having at least 95% identity to SEQ ID NO: 72. In one embodiment, the targeting domain comprises a sequence having at least 96% identity to SEQ ID NO: 72. In one embodiment, the targeting domain comprises a sequence having at least 97% identity to SEQ ID NO: 72. In one embodiment, the targeting domain comprises a sequence having at least 98% identity to SEQ ID NO: 72. In one embodiment, the targeting domain comprises a sequence having at least 99% identity to SEQ ID NO: 72. In one embodiment, the targeting domain comprises the sequence set forth as SEQ ID NO: 72.

[0223] In one embodiment, the targeting domain comprises a sequence having at least 90% identity to SEQ ID NO: 73. In one embodiment, the targeting domain comprises a sequence having at least 95% identity to SEQ ID NO: 73. In one embodiment, the targeting domain comprises a sequence having at least 96% identity to SEQ ID NO: 73. In one embodiment, the targeting domain comprises a sequence having at least 97% identity to SEQ ID NO: 73. In one embodiment, the targeting domain comprises a sequence having at least 98% identity to SEQ ID NO: 73. In one embodiment, the targeting domain comprises a sequence having at least 99% identity to SEQ ID NO: 73. In one embodiment, the targeting domain comprises the sequence set forth as SEQ ID NO: 73.

[0224] In one embodiment, the targeting domain comprises a sequence having at least 90% identity to SEQ ID NO: 74. In one embodiment, the targeting domain comprises a sequence having at least 95% identity to SEQ ID NO: 74. In one embodiment, the targeting domain comprises a sequence having at least 96% identity to SEQ ID NO: 74. In one embodiment, the targeting domain comprises a sequence having at least 97% identity to SEQ ID NO: 74. In one embodiment, the targeting domain comprises a sequence having at least 98% identity to SEQ ID NO: 74. In one embodiment, the targeting domain comprises a sequence having at least 99% identity to SEQ ID NO: 74. In one embodiment, the targeting domain comprises the sequence set forth as SEQ ID NO: 74.

[0225] In one embodiment, the targeting domain is a CLL1 targeting domain. In one embodiment, the targeting domain comprises a sequence having at least 90% identity to SEQ ID NO: 75. In one embodiment, the targeting domain comprises a sequence having at least 95% identity to SEQ ID NO: 75. In one embodiment, the targeting domain comprises a sequence having at least 96% identity to SEQ ID NO: 75. In one embodiment, the targeting domain comprises a sequence having at least 97% identity to SEQ ID NO: 75. In one embodiment, the targeting domain comprises a sequence having at least 98% identity to SEQ ID NO: 75. In one embodiment, the targeting domain comprises a sequence having at least 99% identity to SEQ ID NO: 75. In one embodiment, the targeting domain comprises the sequence set forth as SEQ ID NO: 75.

[0226] In one embodiment, the targeting domain comprises a sequence having at least 90% identity to SEQ ID NO: 76. In one embodiment, the targeting domain comprises a sequence having at least 95% identity to SEQ ID NO: 76. In one embodiment, the targeting domain comprises a sequence having at least 96% identity to SEQ ID NO: 76. In one embodiment, the targeting domain comprises a sequence having at least 97% identity to SEQ ID NO: 76. In one embodiment, the targeting domain comprises a sequence having at least 98% identity to SEQ ID NO: 76. In one embodiment, the targeting domain comprises a sequence having at least 99% identity to SEQ ID NO: 76. In one embodiment, the targeting domain comprises the sequence set forth as SEQ ID NO: 76.

[0227] In one embodiment, the targeting domain is a CD123 targeting domain. In one embodiment, the targeting domain comprises a sequence having at least 90% identity to SEQ ID NO: 77. In one embodiment, the targeting domain comprises a sequence having at least 95% identity to SEQ ID NO: 77. In one embodiment, the targeting domain comprises a sequence having at least 96% identity to SEQ ID NO: 77. In one embodiment, the targeting domain comprises a sequence having at least 97% identity to SEQ ID NO: 77. In one embodiment, the targeting domain comprises a sequence having at least 98% identity to SEQ ID NO: 77. In one embodiment, the targeting domain comprises a sequence having at least 99% identity to SEQ ID NO: 77. In one embodiment, the targeting domain comprises the sequence set forth as SEQ ID NO: 77.

[0228] In one embodiment, the targeting domain is a CD123 targeting domain. In one embodiment, the targeting domain comprises a sequence having at least 90% identity to SEQ ID NO: 78. In one embodiment, the targeting domain comprises a sequence having at least 95% identity to SEQ ID NO: 78. In one embodiment, the targeting domain comprises a sequence having at least 96% identity to SEQ ID NO: 78. In one embodiment, the targeting domain comprises a sequence having at least 97% identity to SEQ ID NO: 78. In one embodiment, the targeting domain comprises a sequence having at least 98% identity to SEQ ID NO: 78. In one embodiment, the targeting domain comprises a sequence having at least 99% identity to SEQ ID NO: 78. In one embodiment, the targeting domain comprises the sequence set forth as SEQ ID NO: 78.

[0229] In one embodiment, the targeting domain is a CD20 targeting domain. In one embodiment, the targeting domain comprises a sequence having at least 90% identity to SEQ ID NO: 79. In one embodiment, the targeting domain comprises a sequence having at least 95% identity to SEQ ID NO: 79. In one embodiment, the targeting domain comprises a sequence having at least 96% identity to SEQ ID NO: 79. In one embodiment, the targeting domain comprises a sequence having at least 97% identity to SEQ ID NO: 79. In one embodiment, the targeting domain comprises a sequence having at least 98% identity to SEQ ID NO: 79. In one embodiment, the targeting domain comprises a sequence having at least 99% identity to SEQ ID NO: 79. In one embodiment, the targeting domain comprises the sequence set forth as SEQ ID NO: 79.

[0230] In one embodiment, the targeting domain is an EGFR targeting domain. In one embodiment, the targeting domain comprises a sequence having at least 90% identity to SEQ ID NO: 80. In one embodiment, the targeting domain comprises a sequence having at least 95% identity to SEQ ID NO: 80. In one embodiment, the targeting domain comprises a sequence having at least 96% identity to SEQ ID NO: 80. In one embodiment, the targeting domain comprises a sequence having at least 97% identity to SEQ ID NO: 80. In one embodiment, the targeting domain comprises a sequence having at least 98% identity to SEQ ID NO: 80. In one embodiment, the targeting domain comprises a sequence having at least 99% identity to SEQ ID NO: 80. In one embodiment, the targeting domain comprises the sequence set forth as SEQ ID NO: 80.

[0231] In one embodiment, the targeting domain is a BCMA targeting domain. In one embodiment, the targeting domain comprises a sequence having at least 90% identity to SEQ ID NO: 81. In one embodiment, the targeting domain comprises a sequence having at least 95% identity to SEQ ID NO: 81. In one embodiment, the targeting domain comprises a sequence having at least 96% identity to SEQ ID NO: 81. In one embodiment, the targeting domain comprises a sequence having at least 97% identity to SEQ ID NO: 81. In one embodiment, the targeting domain comprises a sequence having at least 98% identity to SEQ ID NO: 81. In one embodiment, the targeting domain comprises a sequence having at least 99% identity to SEQ ID NO: 81. In one embodiment, the targeting domain comprises the sequence set forth as SEQ ID NO: 81.

[0232] In one embodiment, the targeting domain is a BCMA targeting domain. In one embodiment, the targeting domain comprises a sequence having at least 90% identity to SEQ ID NO: 82. In one embodiment, the targeting domain comprises a sequence having at least 95% identity to SEQ ID NO: 82. In one embodiment, the targeting domain comprises a sequence having at least 96% identity to SEQ ID NO: 82. In one embodiment, the targeting domain comprises a sequence having at least 97% identity to SEQ ID NO: 82. In one embodiment, the targeting domain comprises a sequence having at least 98% identity to SEQ ID NO: 82. In one embodiment, the targeting domain comprises a sequence having at least 99% identity to SEQ ID NO: 82. In one embodiment, the targeting domain comprises the sequence set forth as SEQ ID NO: 82.

[0233] In one embodiment, the targeting domain is a CD19 targeting domain. In one embodiment, the targeting domain comprises a sequence having at least 90% identity to SEQ ID NO: 83. In one embodiment, the targeting domain comprises a sequence having at least 95% identity to SEQ ID NO: 83. In one embodiment, the targeting domain comprises a sequence having at least 96% identity to SEQ ID NO: 83. In one embodiment, the targeting domain comprises a sequence having at least 97% identity to SEQ ID NO: 83. In one embodiment, the targeting domain comprises a sequence having at least 98% identity to SEQ ID NO: 83. In one embodiment, the targeting domain comprises a sequence having at least 99% identity to SEQ ID NO: 83. In one embodiment, the targeting domain comprises the sequence set forth as SEQ ID NO: 83.

[0234] In one embodiment, the targeting domain comprises a CD33 targeting domain. In one embodiment, the CD33 targeting domain is a VHH. In one embodiment, the CD33 VHH comprises a CDR1 comprising SEQ ID NO: 89, a CDR2 comprising SEQ ID NO: 90, and a CDR3 comprising SEQ ID NO: 91.

[0235] In one embodiment, the targeting domain comprises a CLL1 targeting domain. In one embodiment, the CLL1 targeting domain is a VHH. In one embodiment, the CD33 VHH comprises a CDR1 comprising SEQ ID NO: 92, a CDR2 comprising SEQ ID NO: 93, and a CDR3 comprising SEQ ID NO: 94.

[0236] In one embodiment, the targeting moiety comprises a first targeting domain and a second targeting domain. In one embodiment, the first targeting domain is a CD33 targeting domain, and the second targeting domain is a CLL1 targeting domain. In another embodiment, the first targeting domain is a CLL1 targeting domain, and the second targeting domain is a CD33 targeting domain. In one embodiment, the first targeting domain is a CD33 VHH, wherein the CD33 VHH comprises a CDR1 comprising SEQ ID NO: 89, a CDR2 comprising SEQ ID NO: 90, and a CDR3 comprising SEQ ID NO: 91; and the second targeting domain is a CLL1 VHH, wherein the CLL1 VHH comprises a CDR1 comprising SEQ ID NO: 92, a CDR2 comprising SEQ ID NO: 93, and a CDR3 comprising SEQ ID NO: 94.

[0237] In various embodiments, the first targeting domain and / or the second targeting domain comprises a receptor ectodomain, hi some embodiments, the ectodomain is derived from a receptor selected from the group consisting of PD1 or high-affinity PD1.

[0238] In various embodiments, the ectodomain is a PD1 ectodomain. In some embodiments, the PD1 ectodomain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 84. In some embodiments, the PD1 ectodomain comprises an amino acid sequence at least 95% identical to SEQ ID NO: 84. In some embodiments, the PD1 ectodomain comprises an amino acid sequence at least 96% identical to SEQ ID NO: 84. In some embodiments, the PD1 ectodomain comprises an amino acid sequence at least 97% identical to SEQ ID NO: 84. In some embodiments, the PD1 ectodomain comprises an amino acid sequence at least 98% identical to SEQ ID NO: 84. In some embodiments, the PD1 ectodomain comprises an amino acid sequence at least 99% identical to SEQ ID NO: 84. In specific embodiments, the PD1 ectodomain comprises the amino acid sequence set forth in SEQ ID NO: 84.

[0239] In various embodiments, the ectodomain is a high-affinity PD1 ectodomain. See, e.g., US20220378873A1. In some embodiments, the high-affinity PD1 ectodomain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 85. In some embodiments, the high-affinity PD1 ectodomain comprises an amino acid sequence at least 95% identical to SEQ ID NO: 85. In some embodiments, the high-affinity PD1 ectodomain comprises an amino acid sequence at least 96% identical to SEQ ID NO: 85. In some embodiments, the high-affinity PD1 ectodomain comprises an amino acid sequence at least 97% identical to SEQ ID NO: 85. In some embodiments, the high-affinity PD1 ectodomain comprises an amino acid sequence at least 98% identical to SEQ ID NO: 85. In some embodiments, the high-affinity PD1 ectodomain comprises an amino acid sequence at least 99% identical to SEQ ID NO: 85. In certain embodiments, the high-affinity PD1 ectodomain comprises the amino acid sequence set forth in SEQ ID NO: 85.

[0240] In various embodiments, the first targeting domain and / or the second targeting domain comprises human A proliferation-inducing ligand (APRIL), trimerized human APRIL, or NKG2D membrane protein.

[0241] In various embodiments, the first targeting domain and / or the second targeting domain comprises APRIL. In some embodiments, APRIL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 86. In some embodiments, APRIL comprises an amino acid sequence at least 95% identical to SEQ ID NO: 86. In some embodiments, APRIL comprises an amino acid sequence at least 96% identical to SEQ ID NO: 86. In some embodiments, APRIL comprises an amino acid sequence at least 97% identical to SEQ ID NO: 86. In some embodiments, APRIL comprises an amino acid sequence at least 98% identical to SEQ ID NO: 86. In some embodiments, APRIL comprises an amino acid sequence at least 99% identical to SEQ ID NO: 86. In some embodiments, APRIL comprises the amino acid sequence set forth as SEQ ID NO: 86.

[0242] In various embodiments, the first targeting domain and / or the second targeting domain comprises trimerized human APRIL. In some embodiments, APRIL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 87. In some embodiments, APRIL comprises an amino acid sequence at least 95% identical to SEQ ID NO: 87. In some embodiments, APRIL comprises an amino acid sequence at least 96% identical to SEQ ID NO: 87. In some embodiments, APRIL comprises an amino acid sequence at least 97% identical to SEQ ID NO: 87. In some embodiments, APRIL comprises an amino acid sequence at least 98% identical to SEQ ID NO: 87. In some embodiments, APRIL comprises an amino acid sequence at least 99% identical to SEQ ID NO: 87. In some embodiments, APRIL comprises the amino acid sequence set forth as SEQ ID NO: 87.

[0243] In various embodiments, the first targeting domain and / or the second targeting domain comprises an NKG2D membrane protein. In some embodiments, the NKG2D comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 88. In some embodiments, the NKG2D comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 88. In some embodiments, the NKG2D comprises an amino acid sequence having at least 96% identity to SEQ ID NO: 88. In some embodiments, the NKG2D comprises an amino acid sequence having at least 97% identity to SEQ ID NO: 88. In some embodiments, the NKG2D comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 88. In some embodiments, the NKG2D comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 88. In some embodiments, the NKG2D comprises the amino acid sequence set forth as SEQ ID NO: 88.

[0244] In various embodiments, the first targeting domain and the second targeting domain associate with or bind to the same or different target antigens.

[0245] In various embodiments, the first targeting domain and the second targeting domain are separated by a third polypeptide linker between 2 and 40 amino acids in length, hi some embodiments, the third polypeptide linker is selected from the group consisting of GG, GS, SG, SS, GSS, SSG, GSG, SGS, SGG, G4S, 2xG4S, 3xG4s, 4xG4S, and any combination thereof.

[0246] In certain embodiments, the targeting moiety comprises one or more multimerization domains.

[0247] Illustrative examples of suitable multimerization domains for use in certain targeting moieties contemplated herein include, but are not limited to, an FK506 binding protein (FKBP) polypeptide or variant thereof, an FKBP-rapamycin binding (FRB) polypeptide or variant thereof, a calcineurin polypeptide or variant thereof, a cyclophilin polypeptide or variant thereof, a bacterial dihydrofolate reductase (DHFR) polypeptide or variant thereof, a PYR1-like 1 (PYL1) polypeptide or variant thereof, and an abscisic acid insensitive 1 (ABI1) polypeptide or variant thereof.

[0248] In various embodiments, the targeting moiety comprises an FRB polypeptide. In various embodiments, the targeting moiety comprises an FRB polypeptide comprising a T2098L (T82L) mutation or a variant thereof. In certain embodiments, the FRB polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, the FRB polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2.

[0249] In various embodiments, the targeting moiety comprises an FKBP12 polypeptide or a variant thereof. In various embodiments, the targeting moiety comprises an FKBP12 polypeptide comprising an F36V mutation. In certain embodiments, the FKBP12 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 3. In certain embodiments, the FKBP12 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 4.

[0250] Other illustrative examples of multimerization domains suitable for use in certain targeting moieties contemplated herein include antibody-derived heterodimerization domains, including, but not limited to, lock-and-key, DEKK, SEEDbody, DuoBody, dual variable domain immunoglobulin (DVD-Ig), and Fabs-in-tandem immunoglobulin (FIT-Ig) multimerization domains (see, e.g., Ma et al., Front Immunol. 2021 May 5;12:626616).

[0251] In various embodiments, the targeting moiety comprises a lock and lock multimerization domain / polypeptide or variant thereof. In some embodiments, the lock and lock multimerization domain / polypeptide or variant thereof comprises an amino acid sequence at least 90% identical to SEQ ID NO:5. In one embodiment, the lock and lock multimerization domain / polypeptide or variant thereof comprises an amino acid sequence at least 95% identical to SEQ ID NO:5. In one embodiment, the lock and lock multimerization domain / polypeptide or variant thereof comprises an amino acid sequence at least 96% identical to SEQ ID NO:5. In one embodiment, the lock and lock multimerization domain / polypeptide or variant thereof comprises an amino acid sequence at least 97% identical to SEQ ID NO:5. In one embodiment, the lock and lock multimerization domain / polypeptide or variant thereof comprises an amino acid sequence at least 98% identical to SEQ ID NO:5. In one embodiment, the lock and lock multimerization domain / polypeptide or variant thereof comprises an amino acid sequence at least 99% identical to SEQ ID NO:5. In one embodiment, the lock and lock multimerization domain / polypeptide or variant thereof comprises the amino acid sequence set forth in SEQ ID NO:5. In some embodiments, the lock and lock multimerization domain / polypeptide or variant thereof comprises an amino acid sequence having at least 90% identity to SEQ ID NO:6. In one embodiment, the lock and lock multimerization domain / polypeptide or variant thereof comprises an amino acid sequence having at least 95% identity to SEQ ID NO:6. In one embodiment, the lock and lock multimerization domain / polypeptide or variant thereof comprises an amino acid sequence having at least 96% identity to SEQ ID NO:6. In one embodiment, the lock and lock multimerization domain / polypeptide or variant thereof comprises an amino acid sequence having at least 97% identity to SEQ ID NO:6. In one embodiment, the lock and lock multimerization domain / polypeptide or variant thereof comprises an amino acid sequence having at least 98% identity to SEQ ID NO:6. In one embodiment, the lock and lock multimerization domain / polypeptide or variant thereof comprises an amino acid sequence having at least 99% identity to SEQ ID NO:6.In some embodiments, the lock and key multimerization domain / polypeptide or variant thereof comprises the amino acid sequence shown in SEQ ID NO:6.

[0252] In various embodiments, the targeting moiety comprises a DEKK multimerization domain / polypeptide or variant thereof. In some embodiments, the DEKK multimerization domain / polypeptide or variant thereof comprises an amino acid sequence at least 90% identical to SEQ ID NO:7. In some embodiments, the DEKK multimerization domain / polypeptide or variant thereof comprises an amino acid sequence at least 95% identical to SEQ ID NO:7. In some embodiments, the DEKK multimerization domain / polypeptide or variant thereof comprises an amino acid sequence at least 96% identical to SEQ ID NO:7. In some embodiments, the DEKK multimerization domain / polypeptide or variant thereof comprises an amino acid sequence at least 97% identical to SEQ ID NO:7. In some embodiments, the DEKK multimerization domain / polypeptide or variant thereof comprises an amino acid sequence at least 98% identical to SEQ ID NO:7. In some embodiments, the DEKK multimerization domain / polypeptide or variant thereof comprises an amino acid sequence at least 99% identical to SEQ ID NO:7. In some embodiments, the DEKK multimerization domain / polypeptide or variant thereof comprises the amino acid sequence set forth in SEQ ID NO:7. In some embodiments, the DEKK multimerization domain / polypeptide or variant thereof comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 8. In some embodiments, the DEKK multimerization domain / polypeptide or variant thereof comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 8. In some embodiments, the DEKK multimerization domain / polypeptide or variant thereof comprises an amino acid sequence having at least 96% identity to SEQ ID NO: 8. In some embodiments, the DEKK multimerization domain / polypeptide or variant thereof comprises an amino acid sequence having at least 97% identity to SEQ ID NO: 8. In some embodiments, the DEKK multimerization domain / polypeptide or variant thereof comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 8. In some embodiments, the DEKK multimerization domain / polypeptide or variant thereof comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 8.In some embodiments, the DEKK multimerization domain / polypeptide or variant thereof comprises the amino acid sequence shown in SEQ ID NO:8.

[0253] In various embodiments, the targeting moiety comprises a SEEDbody multimerization domain / polypeptide or variant thereof. In some embodiments, the SEEDbody multimerization domain / polypeptide or variant thereof comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 9. In some embodiments, the SEEDbody multimerization domain / polypeptide or variant thereof comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 9. In some embodiments, the SEEDbody multimerization domain / polypeptide or variant thereof comprises an amino acid sequence having at least 96% identity to SEQ ID NO: 9. In some embodiments, the SEEDbody multimerization domain / polypeptide or variant thereof comprises an amino acid sequence having at least 97% identity to SEQ ID NO: 9. In some embodiments, the SEEDbody multimerization domain / polypeptide or variant thereof comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 9. In some embodiments, the SEEDbody multimerization domain / polypeptide or variant thereof comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 9. In some embodiments, the SEEDbody multimerization domain / polypeptide or variant thereof comprises the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the SEEDbody multimerization domain / polypeptide or variant thereof comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 10. In some embodiments, the SEEDbody multimerization domain / polypeptide or variant thereof comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 10. In some embodiments, the SEEDbody multimerization domain / polypeptide or variant thereof comprises an amino acid sequence having at least 96% identity to SEQ ID NO: 10. In some embodiments, the SEEDbody multimerization domain / polypeptide or variant thereof comprises an amino acid sequence having at least 97% identity to SEQ ID NO: 10. In some embodiments, the SEEDbody multimerization domain / polypeptide or variant thereof comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 10.In some embodiments, the SEEDbody multimerization domain / polypeptide or variant thereof comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 10. In some embodiments, the SEEDbody multimerization domain / polypeptide or variant thereof comprises the amino acid sequence shown in SEQ ID NO: 10.

[0254] Other illustrative examples of suitable multimerization domains for use in certain targeting moieties contemplated herein include, but are not limited to, leucine zipper multimerization domains, mFos:mJun coil multimerization domains, p53 and MDM2 multimerization domains, VPS36 and VPS28 multimerization domains. Other suitable multimerization or dimerization domains will be known to those of skill in the art.

[0255] In various embodiments, the extracellular domain or first targeting domain and the second multimerization domain are separated by a second polypeptide linker between 2 and 40 amino acids in length. In various embodiments, a short oligo- or polypeptide linker, preferably between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length, connects one or more domains in the targeting moiety. Glycine-serine-based linkers provide particularly suitable linkers. In some embodiments, the second polypeptide linker is selected from the group consisting of GG, GS, SG, SS, GSS, SSG, GSG, SGS, SGG, G4S, 2xG4S, 3xG4S, 4xG4S, and combinations thereof. In some embodiments, the second polypeptide linker is a G4S (GGGGS; SEQ ID NO: 11) linker. In some embodiments, the second polypeptide linker is a 2xG4S (GGGGSGGGS; SEQ ID NO: 12) linker. In some embodiments, the second polypeptide linker is a 3xG4S (GGGGSGGGGSGGGS; SEQ ID NO: 13) linker. In some embodiments, the second polypeptide linker is a 4xG4S (GGGGSGGGGSGGGSGGGGS; SEQ ID NO: 14) linker. In some embodiments, the second polypeptide linker is a 5xG4S (GGGGSGGGGSGGGGSGGGGS; SEQ ID NO: 15) linker. In some embodiments, the second polypeptide linker comprises an amino acid sequence set forth in any one or more of SEQ ID NOs: 16-31.

[0256] In various embodiments, the second multimerization domain and the transmembrane domain are separated by a hinge domain.

[0257] In some embodiments, the hinge domain is selected from the group consisting of: a CD4 hinge, a CD8 hinge, a CD28 hinge, an IgG4 hinge, and any fragment, variant, or combination thereof. In some embodiments, the hinge domain is a CD4 hinge. In some embodiments, the CD4 hinge comprises an amino acid sequence at least 90% identical to SEQ ID NO: 41. In some embodiments, the CD4 hinge comprises an amino acid sequence at least 95% identical to SEQ ID NO: 41. In some embodiments, the CD4 hinge comprises an amino acid sequence at least 96% identical to SEQ ID NO: 41. In some embodiments, the CD4 hinge comprises an amino acid sequence at least 97% identical to SEQ ID NO: 41. In some embodiments, the CD4 hinge comprises an amino acid sequence at least 98% identical to SEQ ID NO: 41. In some embodiments, the CD4 hinge comprises an amino acid sequence at least 99% identical to SEQ ID NO: 41. In some embodiments, the CD4 hinge comprises the amino acid sequence set forth in SEQ ID NO: 41.

[0258] In some embodiments, the hinge domain is a CD28 hinge. In some embodiments, the CD28 hinge comprises an amino acid sequence at least 90% identical to SEQ ID NO: 42. In some embodiments, the CD28 hinge comprises an amino acid sequence at least 95% identical to SEQ ID NO: 42. In some embodiments, the CD28 hinge comprises an amino acid sequence at least 96% identical to SEQ ID NO: 42. In some embodiments, the CD28 hinge comprises an amino acid sequence at least 97% identical to SEQ ID NO: 1042. In some embodiments, the CD28 hinge comprises an amino acid sequence at least 98% identical to SEQ ID NO: 42. In some embodiments, the CD28 hinge comprises an amino acid sequence at least 99% identical to SEQ ID NO: 42. In some embodiments, the CD28 hinge comprises the amino acid sequence set forth in SEQ ID NO: 42.

[0259] In some embodiments, the hinge domain is an IgG4 hinge. In one embodiment, the IgG4 hinge comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 43. In one embodiment, the IgG4 hinge comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 43. In one embodiment, the IgG4 hinge comprises an amino acid sequence having at least 96% identity to SEQ ID NO: 43. In one embodiment, the IgG4 hinge comprises an amino acid sequence having at least 97% identity to SEQ ID NO: 43. In one embodiment, the IgG4 hinge comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 43. In one embodiment, the IgG4 hinge comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 43. In one embodiment, the IgG4 hinge comprises the amino acid sequence set forth as SEQ ID NO: 43.

[0260] In one embodiment, the hinge domain comprises a CD8 hinge. In one embodiment, the CD8 hinge comprises an amino acid sequence at least 90% identical to SEQ ID NO: 44. In one embodiment, the CD8 hinge comprises an amino acid sequence at least 95% identical to SEQ ID NO: 44. In one embodiment, the CD8 hinge comprises an amino acid sequence at least 96% identical to SEQ ID NO: 44. In one embodiment, the CD8 hinge comprises an amino acid sequence at least 97% identical to SEQ ID NO: 44. In one embodiment, the CD8 hinge comprises an amino acid sequence at least 98% identical to SEQ ID NO: 44. In one embodiment, the CD8 hinge comprises an amino acid sequence at least 99% identical to SEQ ID NO: 44. In one embodiment, the CD8 hinge comprises the amino acid sequence set forth as SEQ ID NO: 44.

[0261] In some embodiments, the targeting moiety does not include a hinge domain. In some embodiments, the targeting moiety includes a linker comprising a GGR amino acid sequence. In some embodiments, the targeting moiety includes a linker comprising the amino acid sequence set forth in SEQ ID NO: 13 or 14.

[0262] In certain embodiments, the targeting moiety comprises a transmembrane domain.

[0263] Illustrative examples of transmembrane domains suitable for use in certain targeting moieties contemplated herein include, but are not limited to, the transmembrane regions of the alpha, beta, gamma, or delta chains of the T-cell receptor, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD71, CD80, CD86, CD134, CD137, CD152, CD154, amnionless (AMN), and programmed cell death 1 (PDCD1). In various embodiments, the targeting moiety comprises a CD4 transmembrane domain, a CD28 transmembrane domain, or a CD8α transmembrane domain.

[0264] In certain embodiments, the targeting moiety comprises a CD4 transmembrane domain. In some embodiments, the CD4 transmembrane domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 45. In some embodiments, the CD4 transmembrane domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 45. In some embodiments, the CD4 transmembrane domain comprises an amino acid sequence having at least 96% identity to SEQ ID NO: 45. In some embodiments, the CD4 transmembrane domain comprises an amino acid sequence having at least 97% identity to SEQ ID NO: 45. In some embodiments, the CD4 transmembrane domain comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 45. In some embodiments, the CD4 transmembrane domain comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 45. In some embodiments, the CD4 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 45.

[0265] In certain embodiments, the targeting moiety comprises a CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 46. In some embodiments, the CD28 transmembrane domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 46. In some embodiments, the CD28 transmembrane domain comprises an amino acid sequence having at least 96% identity to SEQ ID NO: 46. In some embodiments, the CD28 transmembrane domain comprises an amino acid sequence having at least 97% identity to SEQ ID NO: 46. In some embodiments, the CD28 transmembrane domain comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 46. In some embodiments, the CD28 transmembrane domain comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 46. In some embodiments, the CD28 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 46.

[0266] In certain embodiments, the targeting moiety comprises a CD8 transmembrane domain. In some embodiments, the CD28 transmembrane domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 47. In some embodiments, the CD8 transmembrane domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 47. In some embodiments, the CD8 transmembrane domain comprises an amino acid sequence having at least 96% identity to SEQ ID NO: 47. In some embodiments, the CD8 transmembrane domain comprises an amino acid sequence having at least 97% identity to SEQ ID NO: 47. In some embodiments, the CD8 transmembrane domain comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 47. In some embodiments, the CD8 transmembrane domain comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 47. In some embodiments, the CD8 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 47.

[0267] In certain embodiments, the targeting moiety comprises a truncated CD4 intracellular (CD4ic) domain. In one embodiment, the truncated CD4ic domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 48. In one embodiment, the truncated CD4ic domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 48. In one embodiment, the truncated CD4ic domain comprises an amino acid sequence having at least 96% identity to SEQ ID NO: 48. In one embodiment, the truncated CD4ic domain comprises an amino acid sequence having at least 97% identity to SEQ ID NO: 48. In one embodiment, the truncated CD4ic domain comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 48. In one embodiment, the truncated CD4ic domain comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 48. In various embodiments, the truncated CD4ic domain comprises the amino acid sequence set forth in SEQ ID NO: 48. In one embodiment, the truncated CD4ic domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 49. In one embodiment, the truncated CD4ic domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 48. In one embodiment, the truncated CD4ic domain comprises an amino acid sequence having at least 96% identity to SEQ ID NO: 49. In one embodiment, the truncated CD4ic domain comprises an amino acid sequence having at least 97% identity to SEQ ID NO: 49. In one embodiment, the truncated CD4ic domain comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 49. In one embodiment, the truncated CD4ic domain comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 49. In various embodiments, the truncated CD4ic domain comprises the amino acid sequence set forth in SEQ ID NO: 49.

[0268] In certain embodiments, targeting moieties contemplated herein comprise one or more functional intracellular signaling or costimulatory domains (i.e., have intracellular or costimulatory signaling capability). In various embodiments, the intracellular signaling or costimulatory domain is derived from a protein selected from the group consisting of an antigen receptor, a co-receptor, a costimulatory receptor, a growth receptor, or a cytokine receptor, an adaptor signaling protein, an intracellular signaling protein, or any fragment or variant thereof.

[0269] In various embodiments, the targeting moiety comprises one or more intracellular signaling or costimulatory domains and, optionally, a CD3ζ primary signaling domain. The primary signaling domain and one or more intracellular signaling or costimulatory domains can be linked in tandem, in any order, to the carboxyl terminus of the transmembrane domain. In certain embodiments, the targeting moiety does not comprise a primary signaling domain (e.g., CD3z).

[0270] In various embodiments, the one or more intracellular signaling or costimulatory domains are selected from CD3ε, CD3γ, CD3δ, CD4, or CD8.

[0271] In various embodiments, the co-receptor domain is a CD4 co-receptor domain. In some embodiments, the CD4 co-receptor domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 100. In some embodiments, the CD4 co-receptor domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 100. In some embodiments, the CD4 co-receptor domain comprises an amino acid sequence having at least 96% identity to SEQ ID NO: 100. In some embodiments, the CD4 co-receptor domain comprises an amino acid sequence having at least 97% identity to SEQ ID NO: 100. In some embodiments, the CD4 co-receptor domain comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 100. In some embodiments, the CD4 co-receptor domain comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 100. In some embodiments, the CD4 co-receptor domain comprises the amino acid sequence set forth in SEQ ID NO: 100.

[0272] In various embodiments, the co-receptor domain is a CD8 co-receptor domain. In some embodiments, the CD8 co-receptor domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 101. In some embodiments, the CD8 co-receptor domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 101. In some embodiments, the CD8 co-receptor domain comprises an amino acid sequence having at least 96% identity to SEQ ID NO: 101. In some embodiments, the CD8 co-receptor domain comprises an amino acid sequence having at least 97% identity to SEQ ID NO: 101. In some embodiments, the CD8 co-receptor domain comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 101. In some embodiments, the CD8 co-receptor domain comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 101. In some embodiments, the CD8 co-receptor domain comprises the amino acid sequence set forth in SEQ ID NO: 101.

[0273] In various embodiments, the one or more intracellular signaling or costimulatory domains comprise an immunoreceptor tyrosine-based activation motif (ITAM). Illustrative examples of ITAMs comprising primary signaling domains suitable for use in particular signaling components contemplated herein include, but are not limited to, those derived from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

[0274] Illustrative examples of intracellular signaling or costimulatory domains suitable for use in certain targeting moieties contemplated herein include, but are not limited to, domains isolated from the following costimulatory molecules: Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain family member 11 (CARD11), CD2, CD3ε, CD3γ, CD3δ, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4 -1BB), CD278 (ICOS), common gamma chain cytokine, DNAX-activation protein 10 (DAP10), FYN, linker for activation of T-cell family member 1 (LAT), LCK, interleukin 2 receptor (IL-2R), IL-4R, IL-7R, IL-9R, IL-12R, IL-13R, IL-15R, IL-21R, SH2 domain-containing leukocyte protein 76 kD (SLP76), T-cell receptor-associated transmembrane adaptor 1 (TRAT1), TNFR2, TNFRS14, TNFRS18, TNRFS25, and zeta chain of T-cell receptor-associated protein kinase 70 (ZAP70).

[0275] In various embodiments, the costimulatory domain is selected from the group consisting of: 4-1BB, CD28, TNFR2, OX40, ICOS, and DAP10 costimulatory domains, hi some embodiments, the costimulatory domain is a 4-1BB costimulatory domain.

[0276] In some embodiments, the 4-1BB costimulatory domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 98. In some embodiments, the 4-1BB costimulatory domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 98. In some embodiments, the 4-1BB costimulatory domain comprises an amino acid sequence having at least 96% identity to SEQ ID NO: 98. In some embodiments, the 4-1BB costimulatory domain comprises an amino acid sequence having at least 97% identity to SEQ ID NO: 98. In some embodiments, the 4-1BB costimulatory domain comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 98. In some embodiments, the 4-1BB costimulatory domain comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 98. In some embodiments, the 4-1BB costimulatory domain comprises the amino acid sequence set forth in SEQ ID NO: 98. In some embodiments, the costimulatory domain is a CD28 costimulatory domain. In some embodiments, the CD28 costimulatory domain comprises the amino acid sequence set forth in SEQ ID NO: 99. In some embodiments, the CD28 costimulatory domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 99. In some embodiments, the CD28 costimulatory domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 99. In some embodiments, the CD28 costimulatory domain comprises an amino acid sequence having at least 96% identity to SEQ ID NO: 99. In some embodiments, the CD28 costimulatory domain comprises an amino acid sequence having at least 97% identity to SEQ ID NO: 99. In some embodiments, the CD28 costimulatory domain comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 99. In some embodiments, the CD28 costimulatory domain comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 99.

[0277] In various embodiments, the intracellular signaling or costimulatory domain is a LAT domain. In one embodiment, the LAT domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 102. In one embodiment, the LAT domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 102. In one embodiment, the LAT domain comprises an amino acid sequence having at least 96% identity to SEQ ID NO: 102. In one embodiment, the LAT domain comprises an amino acid sequence having at least 97% identity to SEQ ID NO: 102. In one embodiment, the LAT domain comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 102. In one embodiment, the LAT domain comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 102. In one embodiment, the LAT domain comprises the amino acid sequence set forth as SEQ ID NO: 102.

[0278] In various embodiments, the intracellular signaling or costimulatory domain is one or more cytokine receptor intracellular signaling domains, hi some embodiments, the one or more cytokine receptor intracellular signaling domains are selected from the group consisting of an IL7Rα intracellular signaling domain, an IL2Rβ intracellular signaling domain, a common gamma chain intracellular signaling domain, and both an IL2Rβ and a common gamma chain intracellular signaling domain.

[0279] In various embodiments, the IL7Rα intracellular signaling domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 103. In some embodiments, the IL7Rα intracellular signaling domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 103. In some embodiments, the IL7Rα intracellular signaling domain comprises an amino acid sequence having at least 96% identity to SEQ ID NO: 103. In some embodiments, the IL7Rα intracellular signaling domain comprises an amino acid sequence having at least 97% identity to SEQ ID NO: 103. In some embodiments, the IL7Rα intracellular signaling domain comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 103. In some embodiments, the IL7Rα intracellular signaling domain comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 103. In some embodiments, the IL7Rα intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 103.

[0280] In various embodiments, the IL2Rβ intracellular signaling domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 104. In some embodiments, the IL2Rβ intracellular signaling domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 104. In some embodiments, the IL2Rβ intracellular signaling domain comprises an amino acid sequence having at least 96% identity to SEQ ID NO: 104. In some embodiments, the IL2Rβ intracellular signaling domain comprises an amino acid sequence having at least 97% identity to SEQ ID NO: 104. In some embodiments, the IL2Rβ intracellular signaling domain comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 104. In some embodiments, the IL2Rβ intracellular signaling domain comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 104. In some embodiments, the IL2Rβ intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 104.

[0281] In various embodiments, the IL2Rβ intracellular signaling domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 105. In some embodiments, the IL2Rβ intracellular signaling domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 105. In some embodiments, the IL2Rβ intracellular signaling domain comprises an amino acid sequence having at least 96% identity to SEQ ID NO: 105. In some embodiments, the IL2Rβ intracellular signaling domain comprises an amino acid sequence having at least 97% identity to SEQ ID NO: 105. In some embodiments, the IL2Rβ intracellular signaling domain comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 105. In some embodiments, the IL2Rβ intracellular signaling domain comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 105. In some embodiments, the IL2Rβ intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 105.

[0282] In various embodiments, the common gamma chain intracellular signaling domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 106. In some embodiments, the common gamma chain intracellular signaling domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 106. In some embodiments, the common gamma chain intracellular signaling domain comprises an amino acid sequence having at least 96% identity to SEQ ID NO: 106. In some embodiments, the common gamma chain intracellular signaling domain comprises an amino acid sequence having at least 97% identity to SEQ ID NO: 106. In some embodiments, the common gamma chain intracellular signaling domain comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 106. In some embodiments, the common gamma chain intracellular signaling domain comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 106. In some embodiments, the common gamma chain intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 106.

[0283] In various embodiments, the targeting moiety does not comprise an intracellular signaling or costimulatory domain.

[0284] In various embodiments, the targeting moiety further comprises a truncated intracellular CD4 polypeptide. In some embodiments, the truncated intracellular CD4 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 48. In some embodiments, the truncated intracellular CD4 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 49.

[0285] In preferred embodiments, the targeting moiety comprises a first targeting domain comprising a VHH or scFv that binds to the target antigen, a linker polypeptide, an FRB or FKBP12 multimerization domain, a CD4 hinge, and an extracellular domain comprising a CD4 transmembrane domain. In various embodiments, the targeting moiety comprises a first targeting domain comprising a VHH or scFv that binds to the target antigen, a linker polypeptide, an FRB or FKBP12 multimerization domain, a CD4 hinge, a CD4 transmembrane domain, and an extracellular domain comprising a truncated CD4 domain. In various embodiments, the targeting moiety comprises a first targeting domain comprising a VHH or scFv that binds to the target antigen, a linker polypeptide, an FRB or FKBP12 multimerization domain, a CD4 hinge, a CD4 transmembrane domain, a truncated CD4 domain, and a 4-1BB costimulatory domain. In various embodiments, the targeting moiety comprises a first targeting domain comprising a VHH or scFv that binds to the target antigen, a linker polypeptide, an FRB or FKBP12 multimerization domain, an extracellular domain comprising a CD4 hinge, a CD4 transmembrane domain, a truncated CD4 domain, and does not comprise an intracellular signaling or costimulatory domain.

[0286] In preferred embodiments, the targeting moiety comprises a first targeting domain comprising a VHH or scFv that binds to the target antigen, a linker polypeptide, an antibody-derived heterodimerization domain, a CD4 hinge, and an extracellular domain comprising a CD4 transmembrane domain. In various embodiments, the targeting moiety comprises a first targeting domain comprising a VHH or scFv that binds to the target antigen, a linker polypeptide, an antibody-derived heterodimerization domain, a CD4 hinge, a CD4 transmembrane domain, and an extracellular domain comprising a truncated CD4 domain. In various embodiments, the targeting moiety comprises a first targeting domain comprising a VHH or scFv that binds to the target antigen, a linker polypeptide, an antibody-derived heterodimerization domain, a CD4 hinge, a CD4 transmembrane domain, a truncated CD4 domain, and an extracellular domain comprising a 4-1BB costimulatory domain. In various embodiments, the targeting moiety comprises a first targeting domain comprising a VHH or scFv that binds to the target antigen, a linker polypeptide, an antibody-derived heterodimerization domain, an extracellular domain comprising a CD4 hinge, a CD4 transmembrane domain, a truncated CD4 domain, and no intracellular signaling or costimulatory domain.

[0287] In preferred embodiments, the targeting moiety comprises a first targeting domain comprising a VHH or scFv that binds to the target antigen, a linker polypeptide, a lock-and-lock multimerization domain, a CD4 hinge, and an extracellular domain comprising a CD4 transmembrane domain. In various embodiments, the targeting moiety comprises a first targeting domain comprising a VHH or scFv that binds to the target antigen, a linker polypeptide, a lock-and-lock multimerization domain, a CD4 hinge, a CD4 transmembrane domain, and an extracellular domain comprising a truncated CD4 domain. In various embodiments, the targeting moiety comprises a first targeting domain comprising a VHH or scFv that binds to the target antigen, a linker polypeptide, a lock-and-lock multimerization domain, a CD4 hinge, a CD4 transmembrane domain, a truncated CD4 domain, and a 4-1BB costimulatory domain. In various embodiments, the targeting moiety comprises a first targeting domain comprising a VHH or scFv that binds to the target antigen, a linker polypeptide, a lock-and-key multimerization domain, an extracellular domain comprising a CD4 hinge, a CD4 transmembrane domain, a truncated CD4 domain, and no intracellular signaling or costimulatory domain.

[0288] In preferred embodiments, the targeting moiety comprises a first targeting domain comprising a VHH or scFv that binds to the target antigen, a linker polypeptide, a DEFK multimerization domain, a CD4 hinge, and an extracellular domain comprising a CD4 transmembrane domain. In various embodiments, the targeting moiety comprises a first targeting domain comprising a VHH or scFv that binds to the target antigen, a linker polypeptide, a DEFK multimerization domain, a CD4 hinge, a CD4 transmembrane domain, and an extracellular domain comprising a truncated CD4 domain. In various embodiments, the targeting moiety comprises a first targeting domain comprising a VHH or scFv that binds to the target antigen, a linker polypeptide, a DEFK multimerization domain, a CD4 hinge, a CD4 transmembrane domain, a truncated CD4 domain, and an extracellular domain comprising a 4-1BB costimulatory domain. In various embodiments, the targeting moiety comprises a first targeting domain comprising a VHH or scFv that binds to the target antigen, a linker polypeptide, a DEVK multimerization domain, an extracellular domain comprising a CD4 hinge, a CD4 transmembrane domain, a truncated CD4 domain, and does not comprise an intracellular signaling or costimulatory domain.

[0289] In preferred embodiments, the targeting moiety comprises a first targeting domain comprising a VHH or scFv that binds to the target antigen, a linker polypeptide, a SEEDbody multimerization domain, a CD4 hinge, and an extracellular domain comprising a CD4 transmembrane domain. In various embodiments, the targeting moiety comprises a first targeting domain comprising a VHH or scFv that binds to the target antigen, a linker polypeptide, a SEEDbody multimerization domain, a CD4 hinge, a CD4 transmembrane domain, and an extracellular domain comprising a truncated CD4 domain. In various embodiments, the targeting moiety comprises a first targeting domain comprising a VHH or scFv that binds to the target antigen, a linker polypeptide, a SEEDbody multimerization domain, a CD4 hinge, a CD4 transmembrane domain, a truncated CD4 domain, and a 4-1BB costimulatory domain. In various embodiments, the targeting moiety comprises a first targeting domain comprising a VHH or scFv that binds to the target antigen, a linker polypeptide, a SEEDbody multimerization domain, an extracellular domain comprising a CD4 hinge, a CD4 transmembrane domain, a truncated CD4 domain, and does not comprise an intracellular signaling or costimulatory domain.

[0290] In one embodiment, the targeting moiety comprises a sequence having at least 90% identity to SEQ ID NO: 116. In one embodiment, the targeting moiety comprises a sequence having at least 95% identity to SEQ ID NO: 116. In one embodiment, the targeting moiety comprises a sequence having at least 96% identity to SEQ ID NO: 116. In one embodiment, the targeting moiety comprises a sequence having at least 97% identity to SEQ ID NO: 116. In one embodiment, the targeting moiety comprises a sequence having at least 98% identity to SEQ ID NO: 116. In one embodiment, the targeting moiety comprises a sequence having at least 99% identity to SEQ ID NO: 116. In one embodiment, the targeting moiety comprises the sequence set forth as SEQ ID NO: 116.

[0291] In one embodiment, the targeting moiety comprises a sequence having at least 90% identity to SEQ ID NO: 117. In one embodiment, the targeting moiety comprises a sequence having at least 95% identity to SEQ ID NO: 117. In one embodiment, the targeting moiety comprises a sequence having at least 96% identity to SEQ ID NO: 117. In one embodiment, the targeting moiety comprises a sequence having at least 97% identity to SEQ ID NO: 117. In one embodiment, the targeting moiety comprises a sequence having at least 98% identity to SEQ ID NO: 117. In one embodiment, the targeting moiety comprises a sequence having at least 99% identity to SEQ ID NO: 117. In one embodiment, the targeting moiety comprises the sequence set forth as SEQ ID NO: 117.

[0292] In one embodiment, the targeting moiety comprises a sequence having at least 90% identity to SEQ ID NO: 118. In one embodiment, the targeting moiety comprises a sequence having at least 95% identity to SEQ ID NO: 118. In one embodiment, the targeting moiety comprises a sequence having at least 96% identity to SEQ ID NO: 118. In one embodiment, the targeting moiety comprises a sequence having at least 97% identity to SEQ ID NO: 118. In one embodiment, the targeting moiety comprises a sequence having at least 98% identity to SEQ ID NO: 118. In one embodiment, the targeting moiety comprises a sequence having at least 99% identity to SEQ ID NO: 118. In one embodiment, the targeting moiety comprises the sequence set forth as SEQ ID NO: 118.

[0293] In one embodiment, the targeting moiety comprises a sequence having at least 90% identity to SEQ ID NO: 119. In one embodiment, the targeting moiety comprises a sequence having at least 95% identity to SEQ ID NO: 119. In one embodiment, the targeting moiety comprises a sequence having at least 96% identity to SEQ ID NO: 119. In one embodiment, the targeting moiety comprises a sequence having at least 97% identity to SEQ ID NO: 119. In one embodiment, the targeting moiety comprises a sequence having at least 98% identity to SEQ ID NO: 119. In one embodiment, the targeting moiety comprises a sequence having at least 99% identity to SEQ ID NO: 119. In one embodiment, the targeting moiety comprises the sequence set forth as SEQ ID NO: 119.

[0294] In one embodiment, the targeting moiety comprises a sequence having at least 90% identity to SEQ ID NO: 120. In one embodiment, the targeting moiety comprises a sequence having at least 95% identity to SEQ ID NO: 120. In one embodiment, the targeting moiety comprises a sequence having at least 96% identity to SEQ ID NO: 120. In one embodiment, the targeting moiety comprises a sequence having at least 97% identity to SEQ ID NO: 120. In one embodiment, the targeting moiety comprises a sequence having at least 98% identity to SEQ ID NO: 120. In one embodiment, the targeting moiety comprises a sequence having at least 99% identity to SEQ ID NO: 120. In one embodiment, the targeting moiety comprises the sequence set forth as SEQ ID NO: 120.

[0295] In one embodiment, the targeting moiety comprises a sequence having at least 90% identity to SEQ ID NO: 121. In one embodiment, the targeting moiety comprises a sequence having at least 95% identity to SEQ ID NO: 121. In one embodiment, the targeting moiety comprises a sequence having at least 96% identity to SEQ ID NO: 121. In one embodiment, the targeting moiety comprises a sequence having at least 97% identity to SEQ ID NO: 121. In one embodiment, the targeting moiety comprises a sequence having at least 98% identity to SEQ ID NO: 121. In one embodiment, the targeting moiety comprises a sequence having at least 99% identity to SEQ ID NO: 121. In one embodiment, the targeting moiety comprises the sequence set forth as SEQ ID NO: 121.

[0296] In one embodiment, the targeting moiety comprises a sequence having at least 90% identity to SEQ ID NO: 122. In one embodiment, the targeting moiety comprises a sequence having at least 95% identity to SEQ ID NO: 122. In one embodiment, the targeting moiety comprises a sequence having at least 96% identity to SEQ ID NO: 122. In one embodiment, the targeting moiety comprises a sequence having at least 97% identity to SEQ ID NO: 122. In one embodiment, the targeting moiety comprises a sequence having at least 98% identity to SEQ ID NO: 122. In one embodiment, the targeting moiety comprises a sequence having at least 99% identity to SEQ ID NO: 122. In one embodiment, the targeting moiety comprises the sequence set forth as SEQ ID NO: 122.

[0297] In one embodiment, the targeting moiety comprises a sequence having at least 90% identity to SEQ ID NO: 123. In one embodiment, the targeting moiety comprises a sequence having at least 95% identity to SEQ ID NO: 123. In one embodiment, the targeting moiety comprises a sequence having at least 96% identity to SEQ ID NO: 123. In one embodiment, the targeting moiety comprises a sequence having at least 97% identity to SEQ ID NO: 123. In one embodiment, the targeting moiety comprises a sequence having at least 98% identity to SEQ ID NO: 123. In one embodiment, the targeting moiety comprises a sequence having at least 99% identity to SEQ ID NO: 123. In one embodiment, the targeting moiety comprises the sequence set forth as SEQ ID NO: 123.

[0298] In one embodiment, the targeting moiety comprises a sequence having at least 90% identity to SEQ ID NO: 124. In one embodiment, the targeting moiety comprises a sequence having at least 95% identity to SEQ ID NO: 124. In one embodiment, the targeting moiety comprises a sequence having at least 96% identity to SEQ ID NO: 124. In one embodiment, the targeting moiety comprises a sequence having at least 97% identity to SEQ ID NO: 124. In one embodiment, the targeting moiety comprises a sequence having at least 98% identity to SEQ ID NO: 124. In one embodiment, the targeting moiety comprises a sequence having at least 99% identity to SEQ ID NO: 124. In one embodiment, the targeting moiety comprises the sequence set forth as SEQ ID NO: 124.

[0299] In one embodiment, the targeting moiety comprises a sequence having at least 90% identity to SEQ ID NO: 125. In one embodiment, the targeting moiety comprises a sequence having at least 95% identity to SEQ ID NO: 125. In one embodiment, the targeting moiety comprises a sequence having at least 96% identity to SEQ ID NO: 125. In one embodiment, the targeting moiety comprises a sequence having at least 97% identity to SEQ ID NO: 125. In one embodiment, the targeting moiety comprises a sequence having at least 98% identity to SEQ ID NO: 125. In one embodiment, the targeting moiety comprises a sequence having at least 99% identity to SEQ ID NO: 125. In one embodiment, the targeting moiety comprises the sequence set forth as SEQ ID NO: 125.

[0300] In one embodiment, the targeting moiety comprises a sequence having at least 90% identity to SEQ ID NO: 126. In one embodiment, the targeting moiety comprises a sequence having at least 95% identity to SEQ ID NO: 126. In one embodiment, the targeting moiety comprises a sequence having at least 96% identity to SEQ ID NO: 126. In one embodiment, the targeting moiety comprises a sequence having at least 97% identity to SEQ ID NO: 126. In one embodiment, the targeting moiety comprises a sequence having at least 98% identity to SEQ ID NO: 126. In one embodiment, the targeting moiety comprises a sequence having at least 99% identity to SEQ ID NO: 126. In one embodiment, the targeting moiety comprises the sequence set forth as SEQ ID NO: 126.

[0301] In one embodiment, the targeting moiety comprises a sequence having at least 90% identity to SEQ ID NO: 127. In one embodiment, the targeting moiety comprises a sequence having at least 95% identity to SEQ ID NO: 127. In one embodiment, the targeting moiety comprises a sequence having at least 96% identity to SEQ ID NO: 127. In one embodiment, the targeting moiety comprises a sequence having at least 97% identity to SEQ ID NO: 127. In one embodiment, the targeting moiety comprises a sequence having at least 98% identity to SEQ ID NO: 127. In one embodiment, the targeting moiety comprises a sequence having at least 99% identity to SEQ ID NO: 127. In one embodiment, the targeting moiety comprises the sequence set forth as SEQ ID NO: 127.

[0302] In one embodiment, the targeting moiety comprises a sequence having at least 90% identity to SEQ ID NO: 128. In one embodiment, the targeting moiety comprises a sequence having at least 95% identity to SEQ ID NO: 128. In one embodiment, the targeting moiety comprises a sequence having at least 96% identity to SEQ ID NO: 128. In one embodiment, the targeting moiety comprises a sequence having at least 97% identity to SEQ ID NO: 128. In one embodiment, the targeting moiety comprises a sequence having at least 98% identity to SEQ ID NO: 128. In one embodiment, the targeting moiety comprises a sequence having at least 99% identity to SEQ ID NO: 128. In one embodiment, the targeting moiety comprises the sequence set forth as SEQ ID NO: 128.

[0303] In one embodiment, the targeting moiety comprises a sequence having at least 90% identity to SEQ ID NO: 129. In one embodiment, the targeting moiety comprises a sequence having at least 95% identity to SEQ ID NO: 129. In one embodiment, the targeting moiety comprises a sequence having at least 96% identity to SEQ ID NO: 129. In one embodiment, the targeting moiety comprises a sequence having at least 97% identity to SEQ ID NO: 129. In one embodiment, the targeting moiety comprises a sequence having at least 98% identity to SEQ ID NO: 129. In one embodiment, the targeting moiety comprises a sequence having at least 99% identity to SEQ ID NO: 129. In one embodiment, the targeting moiety comprises the sequence set forth as SEQ ID NO: 129.

[0304] In one embodiment, the targeting moiety comprises a sequence having at least 90% identity to SEQ ID NO: 130. In one embodiment, the targeting moiety comprises a sequence having at least 95% identity to SEQ ID NO: 130. In one embodiment, the targeting moiety comprises a sequence having at least 96% identity to SEQ ID NO: 130. In one embodiment, the targeting moiety comprises a sequence having at least 97% identity to SEQ ID NO: 130. In one embodiment, the targeting moiety comprises a sequence having at least 98% identity to SEQ ID NO: 130. In one embodiment, the targeting moiety comprises a sequence having at least 99% identity to SEQ ID NO: 130. In one embodiment, the targeting moiety comprises the sequence set forth as SEQ ID NO: 130.

[0305] In one embodiment, the targeting moiety comprises a sequence having at least 90% identity to SEQ ID NO: 131. In one embodiment, the targeting moiety comprises a sequence having at least 95% identity to SEQ ID NO: 131. In one embodiment, the targeting moiety comprises a sequence having at least 96% identity to SEQ ID NO: 131. In one embodiment, the targeting moiety comprises a sequence having at least 97% identity to SEQ ID NO: 131. In one embodiment, the targeting moiety comprises a sequence having at least 98% identity to SEQ ID NO: 131. In one embodiment, the targeting moiety comprises a sequence having at least 99% identity to SEQ ID NO: 131. In one embodiment, the targeting moiety comprises the sequence set forth as SEQ ID NO: 131.

[0306] In one embodiment, the targeting moiety comprises a sequence having at least 90% identity to SEQ ID NO: 132. In one embodiment, the targeting moiety comprises a sequence having at least 95% identity to SEQ ID NO: 132. In one embodiment, the targeting moiety comprises a sequence having at least 96% identity to SEQ ID NO: 132. In one embodiment, the targeting moiety comprises a sequence having at least 97% identity to SEQ ID NO: 132. In one embodiment, the targeting moiety comprises a sequence having at least 98% identity to SEQ ID NO: 132. In one embodiment, the targeting moiety comprises a sequence having at least 99% identity to SEQ ID NO: 132. In one embodiment, the targeting moiety comprises the sequence set forth as SEQ ID NO: 132.

[0307] In one embodiment, the targeting moiety comprises a sequence having at least 90% identity to SEQ ID NO: 133. In one embodiment, the targeting moiety comprises a sequence having at least 95% identity to SEQ ID NO: 133. In one embodiment, the targeting moiety comprises a sequence having at least 96% identity to SEQ ID NO: 133. In one embodiment, the targeting moiety comprises a sequence having at least 97% identity to SEQ ID NO: 133. In one embodiment, the targeting moiety comprises a sequence having at least 98% identity to SEQ ID NO: 133. In one embodiment, the targeting moiety comprises a sequence having at least 99% identity to SEQ ID NO: 133. In one embodiment, the targeting moiety comprises the sequence set forth as SEQ ID NO: 133.

[0308] In one embodiment, the targeting moiety comprises a sequence having at least 90% identity to SEQ ID NO: 134. In one embodiment, the targeting moiety comprises a sequence having at least 95% identity to SEQ ID NO: 134. In one embodiment, the targeting moiety comprises a sequence having at least 96% identity to SEQ ID NO: 134. In one embodiment, the targeting moiety comprises a sequence having at least 97% identity to SEQ ID NO: 134. In one embodiment, the targeting moiety comprises a sequence having at least 98% identity to SEQ ID NO: 134. In one embodiment, the targeting moiety comprises a sequence having at least 99% identity to SEQ ID NO: 134. In one embodiment, the targeting moiety comprises the sequence set forth as SEQ ID NO: 134.

[0309] In one embodiment, the targeting moiety comprises a sequence having at least 90% identity to SEQ ID NO: 135. In one embodiment, the targeting moiety comprises a sequence having at least 95% identity to SEQ ID NO: 135. In one embodiment, the targeting moiety comprises a sequence having at least 96% identity to SEQ ID NO: 135. In one embodiment, the targeting moiety comprises a sequence having at least 97% identity to SEQ ID NO: 135. In one embodiment, the targeting moiety comprises a sequence having at least 98% identity to SEQ ID NO: 135. In one embodiment, the targeting moiety comprises a sequence having at least 99% identity to SEQ ID NO: 135. In one embodiment, the targeting moiety comprises the sequence set forth as SEQ ID NO: 135.

[0310] In one embodiment, the targeting moiety comprises a sequence having at least 90% identity to SEQ ID NO: 136. In one embodiment, the targeting moiety comprises a sequence having at least 95% identity to SEQ ID NO: 136. In one embodiment, the targeting moiety comprises a sequence having at least 96% identity to SEQ ID NO: 136. In one embodiment, the targeting moiety comprises a sequence having at least 97% identity to SEQ ID NO: 136. In one embodiment, the targeting moiety comprises a sequence having at least 98% identity to SEQ ID NO: 136. In one embodiment, the targeting moiety comprises a sequence having at least 99% identity to SEQ ID NO: 136. In one embodiment, the targeting moiety comprises the sequence set forth as SEQ ID NO: 136.

[0311] In one embodiment, the targeting moiety does not comprise the sequence shown as SEQ ID NO:122.

[0312] In various embodiments, targeting moieties contemplated herein comprise a signal peptide, e.g., a secretory signal polypeptide or sequence. Illustrative examples of signal polypeptides / sequences suitable for use in particular targeting moieties include, but are not limited to, an IgG1 heavy chain signal polypeptide, an Igκ light chain signal polypeptide, a CD8α signal polypeptide, or a human GM-CSF receptor alpha signal polypeptide. In various preferred embodiments, a targeting moiety comprises an Igκ light chain signal polypeptide. In some embodiments, an Igκ light chain signal polypeptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 95. In some embodiments, an Igκ light chain signal polypeptide comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 95. In some embodiments, an Igκ light chain signal polypeptide comprises an amino acid sequence having at least 96% identity to SEQ ID NO: 95. In some embodiments, an Igκ light chain signal polypeptide comprises an amino acid sequence having at least 97% identity to SEQ ID NO: 95. In some embodiments, an Igκ light chain signal polypeptide comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 95. In some embodiments, the Igκ light chain signal polypeptide comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 95. In some embodiments, the Igκ light chain signal polypeptide comprises the amino acid sequence set forth in SEQ ID NO:95.

[0313] In various embodiments, the signaling component comprises a CD8α signal polypeptide. In some embodiments, the CD8α light chain signal polypeptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 96. In some embodiments, the CD8α light chain signal polypeptide comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 96. In some embodiments, the CD8α light chain signal polypeptide comprises an amino acid sequence having at least 96% identity to SEQ ID NO: 96. In some embodiments, the CD8α light chain signal polypeptide comprises an amino acid sequence having at least 97% identity to SEQ ID NO: 96. In some embodiments, the CD8α light chain signal polypeptide comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 96. In some embodiments, the CD8α light chain signal polypeptide comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 96. In some embodiments, the CD8α light chain signal polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 96.

[0314] In some embodiments, the signal polypeptide is a PD1 signal polypeptide. In one embodiment, the PD1 signal polypeptide comprises an amino acid sequence at least 90% identical to SEQ ID NO: 97. In one embodiment, the PD1 signal polypeptide comprises an amino acid sequence at least 95% identical to SEQ ID NO: 97. In one embodiment, the PD1 signal polypeptide comprises an amino acid sequence at least 96% identical to SEQ ID NO: 97. In one embodiment, the PD1 signal polypeptide comprises an amino acid sequence at least 97% identical to SEQ ID NO: 97. In one embodiment, the PD1 signal polypeptide comprises an amino acid sequence at least 98% identical to SEQ ID NO: 97. In one embodiment, the PD1 signal polypeptide comprises an amino acid sequence at least 99% identical to SEQ ID NO: 97. In one embodiment, the PD1 signal polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 97.

[0315] 3. Crosslinking factor In certain embodiments contemplated herein, a cross-linking agent mediates or promotes the association of one or more signaling components with one or more targeting components through the multimerization domains of each component. The cross-linking agent associates with the multimerization domains and is positioned between the multimerization domains, promoting the association of the signaling component and the targeting component. In the presence of the cross-linking agent, the targeting component and the signaling component associate with an immune receptor complex, and when the targeting component binds to a target antigen on the target cell, it initiates immune effector cell activity against the target cell. In the absence of the cross-linking agent, the targeting component does not associate with the signaling component and does not recruit the immune receptor complex, and the engineered receptor is inactive against the target antigen of the targeting component.

[0316] In certain embodiments, the signaling component and the targeting component comprise a cognate pair of multimerization domains selected from the group consisting of: FKBP and FKBP12 rapamycin-binding (FRB), FKBP and calcineurin, FKBP and cyclophilin, FKBP and bacterial dihydrofolate reductase (DHFR), calcineurin and cyclophilin, and PYR1-like 1 (PYL1) and abscisic acid-insensitive 1 (ABI1).

[0317] In some embodiments, the multimerization domains of the signaling component and the targeting component are associated with a cross-linking agent selected from the group consisting of rapamycin or a rapalog thereof, coumermycin or a derivative thereof, gibberellin or a derivative thereof, abscisic acid (ABA) or a derivative thereof, methotrexate or a derivative thereof, cyclosporin A or a derivative thereof, FK506 / cyclosporin A (FKCsA) or a derivative thereof, and trimethoprim (Tmp) synthetic ligand (SLF) for FK506 binding protein (FKBP) or a derivative thereof.

[0318] In certain embodiments, the signaling component and the targeting component comprise one or more FRB and / or FKBP multimerization domains or variants thereof. In some embodiments, the signaling component comprises an FKBP12 multimerization domain or variant thereof, and the targeting component comprises an FRB multimerization domain or variant thereof. In some embodiments, the signaling component comprises an FRB multimerization domain or variant thereof, and the targeting component comprises an FKBP12 multimerization domain or variant thereof. In particularly preferred embodiments, the signaling component comprises an FKBP12 or FKBP12 F36V multimerization domain or variant thereof, and the targeting component comprises an FRB T2098L multimerization domain or variant thereof. In particularly preferred embodiments, the CD3 signaling component comprises an FRB T2098L (T82L) multimerization domain or a variant thereof, and the targeting component comprises an FKBP12 or FKBP12 F36V multimerization domain or a variant thereof.

[0319] Illustrative examples of suitable cross-linking agents for use in certain embodiments contemplated herein include, but are not limited to, AP1903, AP20187, AP21967 (also known as C-16-(S)-7-methylindole rapamycin), everolimus, novolimus, pimecrolimus, ridaforolimus, sirolimus, tacrolimus, temsirolimus, umirolimus, zotarolimus, and BPC015. In certain preferred embodiments, the cross-linking agent is AP21967. In certain preferred embodiments, the cross-linking agent is a non-immunosuppressive dose of sirolimus (rapamycin).

[0320] D. Antigen Receptors and Binding Molecules In certain embodiments, immune effector cells contemplated herein further express an exogenous lymphocyte receptor or engineered antigen receptor, and one or more components of an engineered immune receptor complex or system described herein. In some embodiments, the exogenous lymphocyte receptor or engineered antigen receptor is a chimeric antigen receptor (CAR), a chimeric costimulatory receptor (CCR), a T cell receptor (TCR), an αβ T cell receptor (αβ-TCR), a γδ T cell receptor (γδ-TCR), a zetakine, or a flip receptor. In some embodiments, immune effector cells contemplated herein further express an exogenous costimulator, an immunomodulator, an agonist for a costimulator, an antagonist for an immunosuppressant, an immune cell engager, or any combination thereof. In some embodiments, immune effector cells contemplated herein further express a BiTE.

[0321] In various embodiments, the exogenous lymphocyte receptor or engineered antigen receptor is an engineered αβ or γδ T cell receptor (TCR), chimeric antigen receptor (CAR), or chimeric costimulatory receptor (CCR). In certain embodiments, the exogenous lymphocyte receptor is an engineered αβ or γδ T cell receptor. In certain embodiments, the engineered antigen receptor is a chimeric antigen receptor (CAR). In certain embodiments, the engineered antigen receptor is a chimeric costimulatory receptor (CCR). In various embodiments, immune effector cells contemplated herein comprise the engineered / exogenous TCR and engineered immune receptor components described herein. Without wishing to be limited to any particular theory, the signaling components of the engineered immune receptor components / systems contemplated herein comprise a multimerization domain fused to an actuator domain (e.g., CD3ε, CD3δ, CD3γ, or Fcγ subunit) that associates with a multimeric immune receptor complex, and thus, in the presence of a cross-linking agent, recruits the immune receptor complex by multimerizing with a targeting component that comprises a second multimerization domain.

[0322] In one embodiment, T cells are engineered by introducing a polynucleotide or vector encoding an engineered antigen receptor (e.g., a TCR, a CAR, or a CCR) and one or more components of the engineered immune receptor system separated by one or more polypeptide cleavage signals. In one embodiment, T cells are engineered by introducing a polynucleotide or vector encoding the engineered antigen receptor and a polynucleotide or vector encoding one or more components of the immune receptor system. In one embodiment, T cells engineered to express an engineered immune receptor are further engineered by introducing a polynucleotide or vector encoding one or more components of the immune receptor system.

[0323] Natural T cell receptors comprise two subunits, alpha and beta chain subunits (αβ TCR) or gamma and delta chain subunits (γδ TCR), each of which is a unique protein produced by recombination events in the genome of each T cell. Libraries of TCRs may be screened for selectivity for a particular target antigen. In this way, natural TCRs with high avidity and reactivity to the target antigen are selected, cloned, and then introduced into a T cell population used for adoptive immunotherapy. In one embodiment, the TCR is an αβ TCR. In one embodiment, the TCR is a γδ TCR.

[0324] In one embodiment, T cells are engineered by introducing a TCR subunit capable of forming a TCR that confers specificity to the T cell for cells expressing a target antigen. In certain embodiments, the subunit has one or more amino acid substitutions, deletions, insertions, or modifications compared to a naturally occurring subunit, so long as the subunit retains the ability to form a TCR, confers the ability of the transfected T cell to be targeted to target cells, and participates in immunologically relevant cytokine signaling. The engineered TCR also preferably binds with high avidity to target cells presenting a relevant peptide (e.g., a tumor-associated peptide), and optionally mediates efficient killing of target cells presenting the relevant peptide in vivo.

[0325] Nucleic acids encoding engineered TCRs are preferably isolated from their natural context in the (naturally occurring) chromosomes of T cells and can be incorporated into a suitable vector as described elsewhere herein. Both the nucleic acid and the vector containing it can be introduced into cells, preferably T cells in certain embodiments. The engineered T cells can then express one or more chains of the TCR encoded by the transduced nucleic acid. In preferred embodiments, the TCR is an exogenous TCR because it is introduced into a T cell that does not normally express a particular TCR. In certain embodiments, an essential aspect of a TCR is that it has high avidity for antigens presented by the major histocompatibility complex (MHC) or similar immunological components. In contrast to TCRs, CARs are engineered to bind target antigens in an MHC-independent manner.

[0326] TCR...

Claims

1. A non-native cell, (a) a signaling component comprising a first multimerization domain and an actuator domain; and (b) A non-native cell comprising a targeting moiety comprising an extracellular domain, a second multimerization domain, and a transmembrane domain.

2. The non-naturally occurring cell of claim 1 , wherein the second multimerization domain and the transmembrane domain are separated by a hinge domain.

3. 3. The non-naturally occurring cell of claim 2, wherein the hinge domain is selected from the group consisting of a CD4 hinge, a CD8 hinge, a CD28 hinge, an IgG4 hinge, and any fragment or variant or combination thereof.

4. 4. The non-natural cell of claim 2 or claim 3, wherein the hinge domain is a CD4 hinge.

5. 5. The non-naturally occurring cell of claim 4, wherein the CD4 hinge comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

41.

6. The non-naturally occurring cell of claim 5, wherein the CD4 hinge comprises the amino acid sequence set forth in SEQ ID NO:

41.

7. The non-natural cell of claim 2 or claim 3, wherein the hinge domain is a CD28 hinge.

8. The non-naturally occurring cell of claim 7, wherein the CD28 hinge comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

42.

9. The non-naturally occurring cell of claim 8, wherein the CD28 hinge comprises the amino acid sequence set forth in SEQ ID NO:

42.

10. The non-natural cell of claim 1 or claim 2, wherein the hinge domain is a CD8 hinge.

11. The non-naturally occurring cell of claim 10, wherein the CD8 hinge comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

44.

12. The non-naturally occurring cell of claim 11 , wherein the CD8 hinge comprises the amino acid sequence set forth in SEQ ID NO:

44.

13. 3. The non-natural cell of claim 1 or claim 2, wherein the hinge domain is an IgG4 hinge.

14. The non-naturally occurring cell of claim 13, wherein the IgG4 hinge comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

43.

15. The non-naturally occurring cell of claim 14, wherein the IgG4 hinge comprises the amino acid sequence set forth in SEQ ID NO:

43.

16. 16. The non-naturally occurring cell of any one of claims 1 to 15, wherein the actuator domain is a CD3 polypeptide, an FcεR1γ polypeptide, an Igα / CD79a polypeptide, an Igβ / CD79b polypeptide, a DAP10 polypeptide, or a DAP12 polypeptide.

17. 17. The non-naturally occurring cell of any one of claims 1 to 16, wherein the CD3 polypeptide is a CD3 epsilon (CD3ε) polypeptide or a variant thereof, a CD3 gamma (CD3γ) or a variant thereof, or a CD3 delta (CD3δ) or a variant thereof.

18. The non-naturally occurring cell of any one of claims 1 to 17, wherein the actuator domain is a CD3ε polypeptide or a variant thereof.

19. 19. The non-naturally occurring cell of any one of claims 1 to 18, wherein the actuator domain is a CD3ε polypeptide comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

32.

20. 20. The non-naturally occurring cell of any one of claims 1 to 19, wherein the actuator domain is a CD3ε polypeptide comprising the amino acid sequence set forth in SEQ ID NO:

32.

21. The non-naturally occurring cell of any one of claims 1 to 17, wherein the actuator domain is a CD3γ polypeptide or a variant thereof.

22. 22. The non-naturally occurring cell of any one of claims 1 to 17 or 21, wherein the actuator domain is a CD3γ polypeptide comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

33.

23. 23. The non-naturally occurring cell of any one of claims 1 to 17, 21 or 22, wherein the actuator domain is a CD3γ polypeptide comprising the amino acid sequence set forth in SEQ ID NO:

33.

24. The non-naturally occurring cell of any one of claims 1 to 17, wherein the actuator domain is a CD3δ polypeptide or a variant thereof.

25. 25. The non-naturally occurring cell of any one of claims 1 to 17 or 24, wherein the actuator domain is a CD3δ polypeptide comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

34.

26. 26. The non-naturally occurring cell of any one of claims 1 to 7, 24, or 25, wherein the actuator domain is a CD3δ polypeptide comprising the amino acid sequence set forth in SEQ ID NO:

34.

27. The non-naturally occurring cell of any one of claims 1 to 16, wherein the actuator domain is an FcεR1γ polypeptide or a variant thereof.

28. 28. The non-naturally occurring cell of any one of claims 1 to 16 or claim 27, wherein the actuator domain is an FcεR1γ polypeptide comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:

35.

29. 29. The non-naturally occurring cell of any one of claims 1 to 16, 27, or 28, wherein the actuator domain is an FcεR1γ polypeptide comprising the amino acid sequence set forth in SEQ ID NO:

35.

30. 28. The non-naturally occurring cell of any one of claims 1 to 16 or claim 27, wherein the actuator domain is an FcεR1γ polypeptide comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

36.

31. 31. The non-naturally occurring cell of any one of claims 1 to 16, 27, or 30, wherein the actuator domain is an FcεR1γ polypeptide comprising the amino acid sequence set forth in SEQ ID NO:

36.

32. The non-naturally occurring cell of any one of claims 1 to 16, wherein the actuator domain is an Igα / CD79a polypeptide or a variant thereof.

33. 33. The non-naturally occurring cell of any one of claims 1 to 16 or 32, wherein the actuator domain is an Igα / CD79a polypeptide comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

37.

34. 34. The non-naturally occurring cell of any one of claims 1 to 16, 32 or 33, wherein the actuator domain is an Igα / CD79a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:

37.

35. The non-naturally occurring cell of any one of claims 1 to 16, wherein the actuator domain is an Igβ / CD79b polypeptide or a variant thereof.

36. 36. The non-naturally occurring cell of any one of claims 1-16 or 35, wherein the actuator domain is an Igβ / CD79b polypeptide comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

38.

37. 37. The non-naturally occurring cell of any one of claims 1 to 16, 35, or 36, wherein the actuator domain is an Igβ / CD79b polypeptide comprising the amino acid sequence set forth in SEQ ID NO:

38.

38. The non-natural cell of any one of claims 1 to 16, wherein the actuator domain is a DAP10 polypeptide or a variant thereof.

39. 39. The non-naturally occurring cell of any one of claims 1 to 16 or 38, wherein the actuator domain is a DAP10 polypeptide comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

39.

40. 40. The non-naturally occurring cell of any one of claims 1 to 16, 38, or 39, wherein the actuator domain is a DAP10 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:

39.

41. The non-naturally occurring cell of any one of claims 1 to 16, wherein the actuator domain is a DAP12 polypeptide or a variant thereof.

42. 42. The non-naturally occurring cell of any one of claims 1 to 16 or 41, wherein the actuator domain is a DAP12 polypeptide comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

40.

43. 43. The non-naturally occurring cell of any one of claims 1 to 16, 41, or 42, wherein the actuator domain is a DAP12 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:

40.

44. 44. The non-naturally occurring cell of any one of claims 1 to 43, wherein the actuator domain comprises both an extracellular portion and an intracellular portion.

45. 45. The non-naturally occurring cell of any one of claims 1 to 44, wherein the first and second multimerization domains are localized extracellularly when the signaling component and targeting component are expressed.

46. 46. ​​The non-naturally occurring cell of any one of claims 1 to 45, wherein the first and second multimerization domains are different.

47. 47. The non-naturally occurring cell of any one of claims 1-46, wherein the multimerization domains of the signaling component and the targeting component are associated with a cross-linking agent selected from the group consisting of rapamycin or a rapalog thereof, gibberellin or a derivative thereof, abscisic acid (ABA) or a derivative thereof, methotrexate or a derivative thereof, cyclosporin A or a derivative thereof, FK506 / cyclosporin A or a derivative thereof, trimethoprim (Tmp) synthetic ligand (SLF) for FK506 binding protein (FKBP) or a derivative thereof, wherein the cross-linking agent promotes the formation of a polypeptide complex, and wherein the cross-linking agent is associated with and disposed between the multimerization domains of the signaling component and the targeting component.

48. 48. The non-naturally occurring cell of any one of claims 1 to 47, wherein the first multimerization domain and the second multimerization domain are a pair selected from the group consisting of FK506-binding protein 1A (FKBP12) and FKBP12-rapamycin-binding (FRB), FKBP12 and calcineurin, FKBP and cyclophilin A or any other member of the peptidyl-prolyl cis-trans-isomerase (PPIase) family, FKBP and dihydrofolate reductase (DHFR), calcineurin and cyclophilin A or any other member of the peptidyl-prolyl cis-trans-isomerase (PPIase) family, and PYR1-like 1 (PYL1) and abscisic acid-insensitive 1 (ABI1).

49. 49. The non-naturally occurring cell of any one of claims 1 to 48, wherein the first multimerization domain comprises a first FRB polypeptide or a variant thereof, and the second multimerization domain comprises a first FKBP12 polypeptide or a variant thereof.

50. 49. The non-naturally occurring cell of any one of claims 1 to 48, wherein the first multimerization domain comprises a first FKBP12 polypeptide or a variant thereof, and the second multimerization domain comprises a first FRB polypeptide or a variant thereof.

51. 51. The non-naturally occurring cell of any one of claims 48-50, wherein the FRB polypeptide is an FRB T2098L variant.

52. 52. The non-naturally occurring cell of any one of claims 48-51, wherein the FRB polypeptide comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to, or including, the sequence set forth in SEQ ID NO:1 or SEQ ID NO:

2.

53. 53. The non-naturally occurring cell of any one of claims 48 to 52, wherein the FKBP12 polypeptide comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to, or includes, the sequence set forth in SEQ ID NO:3 or SEQ ID NO:

4.

54. 54. The non-naturally occurring cell of any one of claims 1 to 53, wherein the cross-linking agent is AP1903, AP20187, AP21967 (also known as C16-(S)-7-methylindolerapamycin), everolimus, novolimus, pimecrolimus, ridaforolimus, sirolimus, tacrolimus, temsirolimus, umirolimus, zotarolimus, or BPC015.

55. 47. The non-natural cell of any one of claims 1 to 46, wherein the first multimerization domain and the second multimerization domain are a pair of heterodimerization domains derived from an antibody.

56. 56. The non-naturally occurring cell of any one of claims 1-46 or 55, wherein the first multimerization domain comprises a polypeptide having at least 90%, 95%, 96%, 97%, 98%, 99% identity to, or comprises an amino acid sequence set forth in SEQ ID NO:5; and the second multimerization domain comprises a polypeptide having at least 90%, 95%, 96%, 97%, 98%, 99% identity to, or comprises an amino acid sequence set forth in SEQ ID NO:

6.

57. 56. The non-naturally occurring cell of any one of claims 1 to 46 or 55, wherein the first multimerization domain comprises a polypeptide having at least 90%, 95%, 96%, 97%, 98%, 99% identity to, or comprises an amino acid sequence set forth in SEQ ID NO:6; and the second multimerization domain comprises a polypeptide having at least 90%, 95%, 96%, 97%, 98%, 99% identity to, or comprises an amino acid sequence set forth in SEQ ID NO:

5.

58. 56. The non-naturally occurring cell of any one of claims 1-46 or 55, wherein the first multimerization domain comprises a polypeptide having at least 90%, 95%, 96%, 97%, 98%, 99% identity to, or comprises an amino acid sequence set forth in SEQ ID NO:7; and the second multimerization domain comprises a polypeptide having at least 90%, 95%, 96%, 97%, 98%, 99% identity to, or comprises an amino acid sequence set forth in SEQ ID NO:

8.

59. 56. The non-naturally occurring cell of any one of claims 1-46 or 55, wherein the first multimerization domain comprises a polypeptide having at least 90%, 95%, 96%, 97%, 98%, 99% identity to, or comprises an amino acid sequence set forth in SEQ ID NO:8; and the second multimerization domain comprises a polypeptide having at least 90%, 95%, 96%, 97%, 98%, 99% identity to, or comprises an amino acid sequence set forth in SEQ ID NO:

7.

60. 56. The non-naturally occurring cell of any one of claims 1-46 or 55, wherein the first multimerization domain comprises a polypeptide having at least 90%, 95%, 96%, 97%, 98%, 99% identity to, or comprises an amino acid sequence set forth in SEQ ID NO:9; and the second multimerization domain comprises a polypeptide having at least 90%, 95%, 96%, 97%, 98%, 99% identity to, or comprises an amino acid sequence set forth in SEQ ID NO:

10.

61. 56. The non-naturally occurring cell of any one of claims 1-46 or 55, wherein the first multimerization domain comprises a polypeptide having at least 90%, 95%, 96%, 97%, 98%, 99% identity to, or comprises an amino acid sequence set forth in SEQ ID NO: 10; and the second multimerization domain comprises a polypeptide having at least 90%, 95%, 96%, 97%, 98%, 99% identity to, or comprises an amino acid sequence set forth in SEQ ID NO:

9.

62. 62. The non-naturally occurring cell of any one of claims 1 to 61, wherein the first multimerization domain and the actuator domain are separated by a first polypeptide linker of 2 to 40 amino acids in length.

63. 63. The non-naturally occurring cell of claim 62, wherein the first polypeptide linker is selected from the group consisting of GG, GS, SG, SS, GSS, SSG, GSG, SGS, SGG, G4S, 2xG4S, 3xG4S, 4xG4S, 5xG4S, and any combination thereof.

64. 64. The non-naturally occurring cell of claim 63, wherein the first polypeptide linker is a 3xG4S linker.

65. 63. The non-naturally occurring cell of claim 62, wherein the first polypeptide linker comprises a polypeptide comprising an amino acid sequence set forth as any one of SEQ ID NOs: 16-31.

66. 66. The non-naturally occurring cell of any one of claims 1 to 65, wherein the extracellular domain and the second multimerization domain are separated by a second polypeptide linker of 2 to 40 amino acids in length.

67. 67. The non-naturally occurring cell of claim 66, wherein the second polypeptide linker is selected from the group consisting of GG, GS, SG, SS, GSS, SSG, GSG, SGS, SGG, G4S, 2xG4S, 3xG4s, 4xG4S, and any combination thereof.

68. 68. The non-naturally occurring cell of claim 67, wherein the second polypeptide linker is a G4S linker.

69. 67. The non-naturally occurring cell of claim 66, wherein the second polypeptide linker comprises a polypeptide comprising an amino acid sequence set forth as any one of SEQ ID NOs: 16-31.

70. 70. The non-naturally occurring cell of any one of claims 1 to 69, wherein the transmembrane domain is a CD4 transmembrane domain.

71. 71. The non-naturally occurring cell of claim 70, wherein the CD4 transmembrane domain comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

45.

72. 72. The non-naturally occurring cell of claim 71, wherein the CD4 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:

45.

73. 70. The non-naturally occurring cell of any one of claims 1 to 69, wherein the transmembrane domain is a CD28 transmembrane domain.

74. 74. The non-naturally occurring cell of claim 73, wherein the CD28 transmembrane domain comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

46.

75. The non-naturally occurring cell of claim 74, wherein the CD28 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:

46.

76. 70. The non-naturally occurring cell of any one of claims 1 to 69, wherein the transmembrane domain is a CD8 transmembrane domain.

77. 77. The non-naturally occurring cell of claim 76, wherein the CD8 transmembrane domain comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

47.

78. 78. The non-naturally occurring cell of claim 77, wherein the CD8 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:

47.

79. 79. The non-naturally occurring cell of any one of claims 1-78, wherein the targeting moiety further comprises an intracellular signaling or costimulatory domain derived from a protein selected from the group consisting of an antigen receptor, a costimulatory receptor, a growth receptor, a cytokine receptor, an adaptor signaling protein, an intracellular signaling protein, or any fragment or variant thereof.

80. The intracellular signaling or costimulatory domain on the targeting moiety is selected from the group consisting of Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain family member 11 (CARD11), CD2, CD3ε, CD3γ, CD3δ, CD4, CD7, CD8, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), common gamma chain cytokine, DNAX activation protein, 80. The non-naturally occurring cell of claim 79, wherein the target protein is selected from the group consisting of: IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18, IL-21, IL-22, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, IL-39, IL-40, IL-41, IL-42, IL-43, IL-44, IL-45, IL-46, IL-47, IL-48, IL-49, IL-50, IL-51, IL-52, IL-53, IL-54, IL-55, IL-56, IL-57, IL-58, IL-59 ...9, IL-59, IL-59, IL-59, IL-59, IL-59, IL-59, IL-59, IL-59

81. 81. The non-naturally occurring cell of claim 79 or claim 80, wherein the intracellular signaling or costimulatory domain on the targeting moiety is selected from the group consisting of 4-1BB, CD28, TNFR2, OX40, ICOS, and DAP10 costimulatory domains.

82. 82. The non-naturally occurring cell of any one of claims 79-81, wherein the costimulatory domain on the targeting moiety is a 4-1BB costimulatory domain, and optionally the 4-1BB costimulatory domain comprises the amino acid sequence set forth in SEQ ID NO:

98.

83. 82. The non-naturally occurring cell of any one of claims 79-81, wherein the costimulatory domain on the targeting moiety is a CD28 costimulatory domain, and optionally the CD28 costimulatory domain comprises the amino acid sequence set forth in SEQ ID NO:

99.

84. 80. The non-naturally occurring cell of claim 79, wherein the intracellular signaling or costimulatory domain is one or more cytokine receptor intracellular signaling domains.

85. 85. The non-naturally occurring cell of claim 84, wherein the one or more cytokine receptor intracellular signaling domains are selected from the group consisting of an IL7Rα intracellular signaling domain, an IL2Rβ intracellular signaling domain, a common gamma chain intracellular signaling domain, and both an IL2Rβ and a common gamma chain intracellular signaling domain.

86. 86. The non-naturally occurring cell of claim 85, wherein the IL7Rα intracellular signaling domain comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

103.

87. 87. The non-naturally occurring cell of claim 86, wherein the IL7Rα intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO:

103.

88. 86. The non-naturally occurring cell of claim 85, wherein the IL2Rβ intracellular signaling domain comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 104 or SEQ ID NO:

105.

89. 89. The non-naturally occurring cell of claim 88, wherein the IL2Rβ intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 104 or SEQ ID NO:

105.

90. 86. The non-naturally occurring cell of claim 85, wherein the common gamma chain intracellular signaling domain comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

106.

91. 91. The non-naturally occurring cell of claim 90, wherein the common gamma chain intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO:

106.

92. 80. The non-naturally occurring cell of claim 79, wherein the intracellular signaling or costimulatory domain is a LAT domain.

93. 93. The non-naturally occurring cell of claim 92, wherein the LAT domain comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:

102.

94. 94. The non-naturally occurring cell of claim 93, wherein the LAT domain comprises the amino acid sequence set forth in SEQ ID NO:

102.

95. 80. The non-naturally occurring cell of claim 79, wherein the intracellular signaling or costimulatory domain is a CD4 co-receptor domain.

96. 96. The non-naturally occurring cell of claim 95, wherein the CD4 co-receptor domain comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

100.

97. 97. The non-naturally occurring cell of claim 96, wherein the CD4 co-receptor domain comprises the amino acid sequence set forth in SEQ ID NO:

100.

98. 80. The non-naturally occurring cell of claim 79, wherein the intracellular signaling or costimulatory domain is a CD8 co-receptor domain.

99. 99. The non-naturally occurring cell of claim 98, wherein the CD8 co-receptor domain comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

101.

100. 100. The non-naturally occurring cell of claim 99, wherein the CD8 co-receptor domain comprises the amino acid sequence set forth in SEQ ID NO:

101.

101. 79. The non-naturally occurring cell of any one of claims 1-78, wherein the targeting moiety does not comprise a functional intracellular signaling or costimulatory domain.

102. 102. The non-naturally occurring cell of any one of claims 1-101, wherein the targeting moiety further comprises a truncated intracellular CD4 polypeptide.

103. The non-naturally occurring cell of claim 102, wherein the truncated intracellular CD4 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:48 or SEQ ID NO:

49.

104. The non-naturally occurring cell of claim 103, wherein the truncated intracellular CD4 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:48 or SEQ ID NO:

49.

105. The non-naturally occurring cell of any one of claims 1 to 104, wherein the extracellular domain comprises a first targeting domain.

106. 106. The non-natural cell of any one of claims 1 to 105, wherein the first targeting domain comprises a single chain variable fragment (scFv) or a single domain antibody (sdAb).

107. 107. The non-natural cell of claim 106, wherein the sdAb is a camelid VHH, a nanobody, or a heavy chain-only antibody (HcAb).

108. The non-naturally occurring cell of claim 107, wherein the sdAb is a Camelidae VHH.

109. 109. The non-naturally occurring cell of any one of claims 106 to 108, wherein the scFv or sdAb is human or humanized.

110. 110. The non-naturally occurring cell of any one of claims 105-109, wherein the first targeting domain comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 50-83.

111. The non-naturally occurring cell of claim 110, wherein the first targeting domain comprises a sequence set forth in any one of SEQ ID NOs: 50-83.

112. 106. The non-naturally occurring cell of claim 105, wherein the first targeting domain comprises a PD1 ectodomain, human A-proliferation-inducing ligand (APRIL), trimerized human APRIL, or NKG2D membrane protein.

113. The non-naturally occurring cell of claim 112, wherein the PD1 ectodomain comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:84 or SEQ ID NO:

85.

114. The non-naturally occurring cell of claim 113, wherein the PD1 ectodomain comprises the sequence set forth in SEQ ID NO:

85.

115. The non-naturally occurring cell of claim 112, wherein the APRIL comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 86, or the trimerized human APRIL comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

87.

116. The non-naturally occurring cell of claim 115, wherein said APRIL comprises the sequence set forth in SEQ ID NO: 86 or said trimerized human APRIL comprises the sequence set forth in SEQ ID NO:

87.

117. 113. The non-naturally occurring cell of claim 112, wherein the NKG2D membrane protein comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

88.

118. The non-naturally occurring cell of claim 117, wherein the NKG2D membrane protein comprises the sequence shown as SEQ ID NO:

88.

119. The non-naturally occurring cell of any one of claims 105-118, wherein the extracellular domain further comprises a second targeting domain.

120. 120. The non-natural cell of claim 119, wherein the second targeting domain comprises a second single chain variable fragment (scFv) or a second single domain antibody (sdAb).

121. 121. The non-naturally occurring cell of claim 120, wherein the second sdAb is a camelid VHH, a nanobody, or a heavy chain-only antibody (HcAb).

122. The non-naturally occurring cell of claim 121, wherein the second sdAb is a Camelidae VHH.

123. 123. The non-naturally occurring cell of any one of claims 119 to 122, wherein the second scFv or second sdAb is human or humanized.

124. 124. The non-naturally occurring cell of any one of claims 119-123, wherein the second targeting domain comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 50-83.

125. The non-naturally occurring cell of claim 124, wherein the second targeting domain comprises a sequence set forth in any one of SEQ ID NOs: 50-83.

126. 120. The non-naturally occurring cell of claim 119, wherein the second targeting domain comprises a PD1 ectodomain, human A-proliferation-inducing ligand (APRIL), trimerized human APRIL, or NKG2D membrane protein.

127. The non-naturally occurring cell of claim 126, wherein the PD1 ectodomain comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:84 or SEQ ID NO:

85.

128. The non-naturally occurring cell of claim 127, wherein the PD1 ectodomain comprises the sequence set forth in SEQ ID NO:

85.

129. The non-naturally occurring cell of claim 126, wherein the APRIL comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 86, or the trimerized human APRIL comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

87.

130. 130. The non-naturally occurring cell of claim 129, wherein said APRIL comprises the sequence set forth in SEQ ID NO: 86, or said trimerized human APRIL comprises the sequence set forth in SEQ ID NO:

87.

131. 127. The non-naturally occurring cell of claim 126, wherein the NKG2D membrane protein comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

88.

132. The non-naturally occurring cell of claim 131, wherein the NKG2D membrane protein comprises the sequence shown as SEQ ID NO:

88.

133. 133. The non-naturally occurring cell of any one of claims 119-132, wherein the targeting domain and the second targeting domain bind to the same antigen or different antigens.

134. 133. The non-naturally occurring cell of any one of claims 119-132, wherein the targeting domain and the second targeting domain are separated by a third polypeptide linker between 2 and 40 amino acids in length.

135. 135. The non-naturally occurring cell of claim 134, wherein the third polypeptide linker is selected from the group consisting of GG, GS, SG, SS, GSS, SSG, GSG, SGS, SGG, G4S, 2xG4S, 3xG4s, 4xG4S, 5xG4S, any one of SEQ ID NOs: 16-31, and any combination thereof.

136. 136. The non-naturally occurring cell of any one of claims 1-135, wherein the signaling component comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, or SEQ ID NO:

115.

137. 137. The non-naturally occurring cell of claim 136, wherein the signaling component comprises the sequence set forth as SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, or SEQ ID NO:

115.

138. 138. The non-naturally occurring cell of any one of claims 1 to 137, wherein the targeting moiety comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to the sequence set forth as SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, or SEQ ID NO:

136.

139. 139. The non-naturally occurring cell of claim 138, wherein the targeting moiety comprises the sequence set forth as SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, or SEQ ID NO:

136.

140. 140. The non-naturally occurring cell of any one of claims 1 to 139, comprising a fusion polypeptide comprising said targeting moiety and said signaling moiety.

141. 141. The non-naturally occurring cell of claim 140, wherein the fusion polypeptide comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, or SEQ ID NO:

170.

142. 142. The non-naturally occurring cell of claim 141, wherein the fusion polypeptide comprises the sequence set forth as SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, or SEQ ID NO:

170.

143. 143. The non-naturally occurring cell of any one of claims 1-142, wherein said cell comprises a first nucleic acid molecule encoding said signaling component.

144. 144. The non-naturally occurring cell of any one of claims 1-143, wherein the cell comprises a second nucleic acid molecule encoding the targeting moiety.

145. 145. The non-naturally occurring cell of any one of claims 1-144, wherein said cell comprises a nucleic acid molecule encoding both said signaling component and said targeting component.

146. 146. The non-naturally occurring cell of any one of claims 1-145, wherein the cell further expresses an exogenous costimulatory factor, an immunomodulatory factor, an agonist for a costimulatory factor, an antagonist for an immunosuppressive factor, an immune cell engager, a flip receptor, or any combination thereof.

147. 147. The non-naturally occurring cell of any one of claims 1 to 146, wherein the cell further expresses an exogenous lymphocyte receptor or co-receptor.

148. 148. The non-naturally occurring cell of claim 147, wherein the exogenous lymphocyte receptor or co-receptor is selected from the group consisting of TCR alpha (TCRα), TCR beta (TCRβ), TCR gamma (TCRγ), TCR delta (TCRδ), CD4, CD8, pre-T cell receptor alpha (pTα), Fc receptor alpha (FcRα), Fc receptor beta (FcRβ), Fc receptor gamma (FcRγ), natural killer group 2 member D (NKG2D), CD79A, CD79B, and any combination thereof.

149. The non-naturally occurring cell of any one of claims 1 to 148, wherein the cell further expresses an exogenous TCR.

150. The exogenous TCR is capable of detecting alpha-fetoprotein (AFP), B melanoma antigen (BAGE) family members, sibling of regulator of imprinted sites (BORIS), cancer testis antigen, cancer testis antigen 83 (CT-83), carbonic anhydrase IX (CA1X), carcinoembryonic antigen (CEA), cytomegalovirus (CMV) antigen, melanoma cytotoxic T cell (CTL)-recognized antigen (CAMEL), Epstein-Barr virus (EBV) antigen, G antigen 1 (GAGE), -1), GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, GAGE-8, glycoprotein 100 (GP100), hepatitis B virus (HBV) antigen, hepatitis C virus (HCV) nonstructural protein 3 (NS3), human epidermal growth factor receptor 2 (HER-2), human papillomavirus (HPV)-E6, HPV-E7, human telomerase reverse transcriptase (hTERT), IGF2BP3 / A3, K-Ras, K-Ras G12C, K-Ras G12D, K-Ras G12V, latent membrane protein 2 (LMP2), melanoma antigen family A, 1 (MAGE-A1), MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, melanoma antigen recognized by T cells (MART-1), mesothelin (MSLN), mucin 1 (MUC1), mucin 16 (MUC16), New York esophageal squamous cell carcinoma-1 (NYESO-1), p53, P antigen (PAGE) family member, placenta-specific 1 (PLAC1), preferentially expressed in melanoma 150. The non-naturally occurring cell of claim 149, wherein the non-naturally occurring cell binds to a target antigen selected from the group consisting of an antigen associated with thyroid cancer (PRAME), survivin, synovial sarcoma X1 (SSX1), synovial sarcoma X2 (SSX2), synovial sarcoma X3 (SSX3), synovial sarcoma X4 (SSX4), synovial sarcoma X5 (SSX5), synovial sarcoma X8 (SSX8), thyroglobulin, tyrosinase, tyrosinase-related protein (TRP) 1, TRP2, Wilms' tumor protein (WT-1), X antigen family member 1 (XAGE1), and X antigen family member 2 (XAGE2).

151. The non-naturally occurring cell of claim 149 or claim 150, wherein the exogenous TCR is an αβ-TCR or a γδ-TCR.

152. The non-naturally occurring cell of any one of claims 1 to 151, wherein the cell further expresses a CAR, CCR, or flip receptor.

153. The non-naturally occurring cell of any one of claims 1-152, wherein the cell further expresses a zetakine, an immune cell engager, or a BiTE.

154. The non-naturally occurring cell of any one of claims 1 to 153, wherein the cell is a hematopoietic cell.

155. 155. The non-naturally occurring cell of any one of claims 1 to 154, wherein the cell is a T cell, an αβ-T cell, or a γδ-T cell.

156. The cells are CD3 ＋ , CD4 ＋ , and / or CD8 ＋ 156. The non-naturally occurring cell of any one of claims 1 to 155, which is a cell.

157. The non-naturally occurring cell of any one of claims 1 to 156, wherein the cell is an immune effector cell.

158. 158. The non-naturally occurring cell of any one of claims 1 to 157, wherein the cell is a cytotoxic T lymphocyte (CTL), a tumor infiltrating lymphocyte (TIL), or a helper T cell.

159. The non-naturally occurring cell of any one of claims 1 to 158, wherein the cell is a natural killer (NK) cell or a natural killer T (NKT) cell.

160. 160. The non-naturally occurring cell of any one of claims 1-159, wherein the source of the cell is peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, or a tumor.

161. 161. The non-naturally occurring cell of any one of claims 1 to 160, wherein the non-naturally occurring cell is an isolated non-naturally occurring cell.

162. The non-naturally occurring cell of any one of claims 1 to 161, wherein the non-naturally occurring cell is obtained from a subject.

163. The non-naturally occurring cell of any one of claims 1 to 162, wherein the non-naturally occurring cell is a human cell.

164. A fusion polypeptide comprising: (a) a signaling component comprising a first multimerization domain and an actuator domain; (b) a polypeptide cleavage signal; and (c) a fusion polypeptide comprising an extracellular domain, a second multimerization domain, and a targeting moiety comprising a transmembrane domain.

165. 165. The fusion polypeptide of claim 164, wherein the first and second multimerization domains are localized extracellularly when the signaling component and the targeting component are expressed.

166. 166. The fusion polypeptide of claim 164 or claim 165, wherein the second multimerization domain and the transmembrane domain are separated by a hinge domain.

167. 167. The fusion polypeptide of claim 166, wherein the hinge domain is selected from the group consisting of a CD4 hinge, a CD8 hinge, a CD28 hinge, an IgG4 hinge, and any fragment or variant or combination thereof.

168. 168. The fusion polypeptide of claim 166 or 167, wherein the hinge domain is a CD4 hinge.

169. The fusion polypeptide cell of claim 168, wherein the CD4 hinge comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

41.

170. The fusion polypeptide of claim 169, wherein the CD4 hinge comprises the amino acid sequence set forth in SEQ ID NO:

41.

171. A fusion polypeptide described in claim 166 or claim 167, wherein the hinge domain is a CD28 hinge.

172. The fusion polypeptide of claim 171, wherein the CD28 hinge comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

42.

173. The fusion polypeptide of claim 172, wherein the CD28 hinge comprises the amino acid sequence shown in SEQ ID NO:

42.

174. 168. The fusion polypeptide of claim 166 or 167, wherein the hinge domain is a CD8 hinge.

175. The fusion polypeptide of claim 174, wherein the CD8 hinge comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

44.

176. The fusion polypeptide of claim 175, wherein the CD8 hinge comprises the amino acid sequence set forth in SEQ ID NO:

44.

177. 168. The fusion polypeptide of claim 166 or claim 167, wherein the hinge domain is an IgG4 hinge.

178. The fusion polypeptide of claim 177, wherein the IgG4 hinge comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

43.

179. The fusion polypeptide of claim 178, wherein the IgG4 hinge comprises the amino acid sequence set forth in SEQ ID NO:

43.

180. 180. The fusion polypeptide of any one of claims 164 to 179, wherein the actuator domain is a CD3 polypeptide, an FcεR1γ polypeptide, an Igα / CD79a polypeptide, an Igβ / CD79b polypeptide, a DAP10 polypeptide, or a DAP12 polypeptide.

181. 181. The fusion polypeptide of any one of claims 164 to 180, wherein the CD3 polypeptide is CD3 epsilon (CD3ε) or a fragment or variant thereof, CD3 gamma (CD3γ) or a fragment or variant thereof, or CD3 delta (CD3δ) or a fragment or variant thereof.

182. 182. The fusion polypeptide of any one of claims 164-181, wherein the actuator domain is a CD3ε polypeptide comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

32.

183. The fusion polypeptide of any one of claims 164 to 182, wherein the actuator domain is a CD3ε polypeptide comprising the amino acid sequence set forth in SEQ ID NO:

32.

184. 182. The fusion polypeptide of any one of claims 164 to 181, wherein the actuator domain is a CD3γ polypeptide or a variant thereof.

185. 185. The fusion polypeptide of any one of claims 164-181 or 184, wherein the actuator domain is a CD3γ polypeptide comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

33.

186. 186. The fusion polypeptide of any one of claims 164-181, 184, or 185, wherein the actuator domain is a CD3γ polypeptide comprising the amino acid sequence set forth in SEQ ID NO:

33.

187. The fusion polypeptide of any one of claims 164 to 181, wherein the actuator domain is a CD3δ polypeptide or a variant thereof.

188. 188. The fusion polypeptide of any one of claims 164-181 or 187, wherein the actuator domain is a CD3δ polypeptide comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

34.

189. 189. The fusion polypeptide of any one of claims 164-181, 187, or 188, wherein the actuator domain is a CD3δ polypeptide comprising the amino acid sequence set forth in SEQ ID NO:

34.

190. 190. The fusion polypeptide of any one of claims 164-181 or 189, wherein the actuator domain is an FcεR1γ polypeptide or a variant thereof.

191. 191. The fusion polypeptide of any one of claims 164-181 or claim 190, wherein the actuator domain is an FcεR1γ polypeptide comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:

35.

192. 192. The fusion polypeptide of any one of claims 164-181, 190, or 191, wherein the actuator domain is an FcεR1γ polypeptide comprising the amino acid sequence set forth in SEQ ID NO:

35.

193. 191. The fusion polypeptide of any one of claims 164-181 or 190, wherein the actuator domain is an FcεR1γ polypeptide comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

36.

194. 194. The fusion polypeptide of any one of claims 164-181, 190 or 193, wherein the actuator domain is an FcεR1γ polypeptide comprising the amino acid sequence set forth in SEQ ID NO:

36.

195. 181. The fusion polypeptide of any one of claims 164 to 180, wherein the actuator domain is an Igα / CD79a polypeptide or a variant thereof.

196. 196. The fusion polypeptide of any one of claims 164-180 or 195, wherein the actuator domain is an Igα / CD79a polypeptide comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

37.

197. 200. The fusion polypeptide of any one of claims 164-180, 195, or 196, wherein the actuator domain is an Igα / CD79a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:

37.

198. 181. The fusion polypeptide of any one of claims 164 to 180, wherein the actuator domain is an Igβ / CD79b polypeptide or a variant thereof.

199. 200. The fusion polypeptide of any one of claims 164-180 or 198, wherein the actuator domain is an Igβ / CD79b polypeptide comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

38.

200. 200. The fusion polypeptide of any one of claims 164-180, 198, or 199, wherein the actuator domain is an Igβ / CD79b polypeptide comprising the amino acid sequence set forth in SEQ ID NO:

38.

201. The fusion polypeptide of any one of claims 164 to 180, wherein the actuator domain is a DAP10 polypeptide or a variant thereof.

202. 202. The fusion polypeptide of any one of claims 164-180 or 201, wherein the actuator domain is a DAP10 polypeptide comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

39.

203. 203. The fusion polypeptide of any one of claims 164-180, 201, or 202, wherein the actuator domain is a DAP10 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:

39.

204. The fusion polypeptide of any one of claims 164 to 180, wherein the actuator domain is a DAP12 polypeptide or a variant thereof.

205. 205. The fusion polypeptide of any one of claims 164-180 or 204, wherein the actuator domain is a DAP12 polypeptide comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

40.

206. 206. The fusion polypeptide of any one of claims 164-180, 204 or 205, wherein the actuator domain is a DAP12 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:

40.

207. The fusion polypeptide of any one of claims 164 to 206, wherein the actuator domain comprises both an extracellular portion and an intracellular portion.

208. 208. The fusion polypeptide of any one of claims 164 to 207, further comprising a signal sequence, optionally wherein the signal sequence has at least 90%, 95%, 96%, 97%, 98%, 99% identity to or comprises SEQ ID NO:95, SEQ ID NO:96, or SEQ ID NO:

97.

209. 209. The fusion polypeptide of any one of claims 164 to 208, wherein the first and second multimerization domains are different.

210. 210. The fusion polypeptide of any one of claims 164-209, wherein the multimerization domains of the signaling and targeting components are associated with a cross-linking agent selected from the group consisting of rapamycin or a rapalog thereof, gibberellin or a derivative thereof, abscisic acid (ABA) or a derivative thereof, methotrexate or a derivative thereof, cyclosporin A or a derivative thereof, FK506 / cyclosporin A or a derivative thereof, trimethoprim (Tmp) synthetic ligand (SLF) for FK506 binding protein (FKBP) or a derivative thereof, wherein the cross-linking agent promotes the formation of a polypeptide complex, and wherein the cross-linking agent is associated with and disposed between the multimerization domains of the signaling component and the targeting component.

211. 211. The fusion polypeptide of any one of claims 164 to 210, wherein the first multimerization domain and the second multimerization domain are a pair selected from the group consisting of FK506-binding protein 1A (FKBP12) and FKBP12-rapamycin-binding (FRB), FKBP12 and calcineurin, FKBP and cyclophilin A or any other member of the peptidyl-prolyl cis-trans-isomerase (PPIase) family, FKBP and dihydrofolate reductase (DHFR), calcineurin and cyclophilin A or any other member of the peptidyl-prolyl cis-trans-isomerase (PPIase) family, and PYR1-like 1 (PYL1) and abscisic acid-insensitive 1 (ABI1).

212. 212. The fusion polypeptide of any one of claims 164-211, wherein the first multimerization domain comprises a first FRB polypeptide or a variant thereof, and the second multimerization domain comprises a first FKBP12 polypeptide or a variant thereof.

213. 212. The fusion polypeptide of any one of claims 164 to 211, wherein the first multimerization domain comprises a first FKBP12 polypeptide or a variant thereof, and the second multimerization domain comprises a first FRB polypeptide or a variant thereof.

214. 214. The fusion polypeptide of any one of claims 211-213, wherein the FRB polypeptide is an FRB T2098L variant.

215. The fusion polypeptide of any one of claims 211 to 214, wherein the FRB polypeptide comprises the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:

2.

216. The fusion polypeptide of any one of claims 211 to 215, wherein the FKBP12 polypeptide comprises the amino acid sequence shown in SEQ ID NO:3 or SEQ ID NO:

4.

217. 217. The fusion polypeptide of any one of claims 164 to 216, wherein the cross-linking agent is AP1903, AP20187, AP21967 (also known as C16-(S)-7-methylindorerapamycin), everolimus, novolimus, pimecrolimus, ridaforolimus, sirolimus, tacrolimus, temsirolimus, umirolimus, zotarolimus, or BPC015.

218. 210. The fusion polypeptide of any one of claims 164 to 209, wherein the first multimerization domain and the second multimerization domain are a pair of antibody-derived heterodimerization domains.

219. 219. The fusion polypeptide of any one of claims 164 to 209 or 218, wherein the first multimerization domain comprises a polypeptide having at least 90%, 95%, 96%, 97%, 98%, 99% identity to, or comprises an amino acid sequence as set forth in SEQ ID NO:5; and the second multimerization domain comprises a polypeptide having at least 90%, 95%, 96%, 97%, 98%, 99% identity to, or comprises an amino acid sequence as set forth in SEQ ID NO:

6.

220. 219. The fusion polypeptide of any one of claims 164 to 209 or 218, wherein the first multimerization domain comprises a polypeptide having at least 90%, 95%, 96%, 97%, 98%, 99% identity to, or comprises an amino acid sequence as set forth in SEQ ID NO:6; and the second multimerization domain comprises a polypeptide having at least 90%, 95%, 96%, 97%, 98%, 99% identity to, or comprises an amino acid sequence as set forth in SEQ ID NO:

5.

221. 219. The fusion polypeptide of any one of claims 164 to 209 or 218, wherein the first multimerization domain comprises a polypeptide having at least 90%, 95%, 96%, 97%, 98%, 99% identity to, or comprises an amino acid sequence as set forth in SEQ ID NO:7; and the second multimerization domain comprises a polypeptide having at least 90%, 95%, 96%, 97%, 98%, 99% identity to, or comprises an amino acid sequence as set forth in SEQ ID NO:

8.

222. 219. The fusion polypeptide of any one of claims 164 to 209 or 218, wherein the first multimerization domain comprises a polypeptide having at least 90%, 95%, 96%, 97%, 98%, 99% identity to, or comprises an amino acid sequence as set forth in SEQ ID NO:8; and the second multimerization domain comprises a polypeptide having at least 90%, 95%, 96%, 97%, 98%, 99% identity to, or comprises an amino acid sequence as set forth in SEQ ID NO:

7.

223. 219. The fusion polypeptide of any one of claims 164 to 209 or 218, wherein the first multimerization domain comprises a polypeptide having at least 90%, 95%, 96%, 97%, 98%, 99% identity to, or comprises an amino acid sequence as set forth in SEQ ID NO:9; and the second multimerization domain comprises a polypeptide having at least 90%, 95%, 96%, 97%, 98%, 99% identity to, or comprises an amino acid sequence as set forth in SEQ ID NO:

10.

224. 219. The fusion polypeptide of any one of claims 164 to 209 or 218, wherein the first multimerization domain comprises a polypeptide having at least 90%, 95%, 96%, 97%, 98%, 99% identity to, or comprises an amino acid sequence as set forth in SEQ ID NO: 10; and the second multimerization domain comprises a polypeptide having at least 90%, 95%, 96%, 97%, 98%, 99% identity to, or comprises an amino acid sequence as set forth in SEQ ID NO:

9.

225. 225. The fusion polypeptide of any one of claims 164 to 224, wherein the first multimerization domain and the actuator domain are separated by a first polypeptide linker of 2 to 40 amino acids in length.

226. 226. The fusion polypeptide of claim 225, wherein the first polypeptide linker is selected from the group consisting of GG, GS, SG, SS, GSS, SSG, GSG, SGS, SGG, G4S, 2xG4S, 3xG4S, 4xG4S, 5xG4S, and any combination thereof.

227. 227. The fusion polypeptide of claim 226, wherein the first polypeptide linker is a 3xG4S linker.

228. 227. The fusion polypeptide of claim 226, wherein the first polypeptide linker comprises a polypeptide comprising an amino acid sequence set forth as any one of SEQ ID NOs: 16-31.

229. 229. The fusion polypeptide of any one of claims 164 to 228, wherein the extracellular domain and the second multimerization domain are separated by a second polypeptide linker of 2 to 40 amino acids in length.

230. 230. The fusion polypeptide of claim 229, wherein the second polypeptide linker is selected from the group consisting of GG, GS, SG, SS, GSS, SSG, GSG, SGS, SGG, G4S, 2xG4S, 3xG4s, 4xG4S, and any combination thereof.

231. The fusion polypeptide of claim 230, wherein the second polypeptide linker is a G4S linker.

232. 231. The fusion polypeptide of claim 230, wherein the second polypeptide linker comprises a polypeptide comprising an amino acid sequence set forth as any one of SEQ ID NOs: 16-31.

233. The fusion polypeptide of any one of claims 164 to 232, wherein the transmembrane domain is a CD4 transmembrane domain.

234. The fusion polypeptide of claim 233, wherein the CD4 transmembrane domain comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

45.

235. The fusion polypeptide of claim 234, wherein the CD4 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:

45.

236. The fusion polypeptide of any one of claims 164 to 232, wherein the transmembrane domain is a CD28 transmembrane domain.

237. The fusion polypeptide of claim 236, wherein the CD28 transmembrane domain comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

46.

238. The fusion polypeptide of claim 237, wherein the CD28 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:

46.

239. The fusion polypeptide of any one of claims 164 to 232, wherein the transmembrane domain is a CD8 transmembrane domain.

240. The fusion polypeptide of claim 239, wherein the CD8 transmembrane domain comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

47.

241. The fusion polypeptide of claim 240, wherein the CD8 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:

47.

242. 242. The fusion polypeptide of any one of Claims 164-241, wherein the targeting moiety further comprises an intracellular signaling or costimulatory domain derived from a protein selected from the group consisting of an antigen receptor, a costimulatory receptor, a growth receptor, a cytokine receptor, an adaptor signaling protein, an intracellular signaling protein, or any fragment or variant thereof.

243. The intracellular signaling or costimulatory domain on the targeting moiety is selected from the group consisting of Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain family member 11 (CARD11), CD2, CD3ε, CD3γ, CD3δ, CD4, CD7, CD8, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), common gamma chain cytokine, DNAX activating protein, 243. The fusion polypeptide of claim 242, wherein the fusion polypeptide is selected from the group consisting of: protein 10 (DAP10), linker for activation of T-cell family member 1 (LAT), interleukin-2 receptor (IL-2R), IL-4R, IL-7R, IL-9R, IL-12R, IL-13R, IL-15R, IL-21R, SH2 domain-containing leukocyte protein 76 kD (SLP76), T-cell receptor-associated transmembrane adaptor 1 (TRAT1), TNFR2, TNFRS14, TNFRS18, TNRFS25, and the zeta chain of T-cell receptor-associated protein kinase 70 (ZAP70).

244. The fusion polypeptide of claim 242 or claim 243, wherein the intracellular signaling or costimulatory domain on the targeting moiety is selected from the group consisting of 4-1BB, CD28, TNFR2, OX40, ICOS, and DAP10 costimulatory domains.

245. The fusion polypeptide of claim 242, wherein the costimulatory domain on the targeting moiety is a 4-1BB costimulatory domain, and optionally the 4-1BB costimulatory domain comprises the amino acid sequence set forth in SEQ ID NO:

98.

246. The fusion polypeptide of claim 242, wherein the costimulatory domain on the targeting moiety is a CD28 costimulatory domain, and optionally the CD28 costimulatory domain comprises the amino acid sequence set forth in SEQ ID NO:

99.

247. 243. The fusion polypeptide of claim 242, wherein the intracellular signaling or costimulatory domain is one or more cytokine receptor intracellular signaling domains.

248. The fusion polypeptide of claim 247, wherein the one or more cytokine receptor intracellular signaling domains are selected from the group consisting of an IL7Rα intracellular signaling domain, an IL2Rβ intracellular signaling domain, a common gamma chain intracellular signaling domain, and both an IL2Rβ and a common gamma chain intracellular signaling domain.

249. The fusion polypeptide of claim 248, wherein the IL7Rα intracellular signaling domain comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

103.

250. The fusion polypeptide of claim 249, wherein the IL7Rα intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO:

103.

251. The fusion polypeptide of claim 248, wherein the IL2Rβ intracellular signaling domain comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 104 or SEQ ID NO:

105.

252. The fusion polypeptide of claim 251, wherein the IL2Rβ intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 104 or SEQ ID NO:

105.

253. The fusion polypeptide of claim 248, wherein the common gamma chain intracellular signaling domain comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

106.

254. The fusion polypeptide of claim 253, wherein the common gamma chain intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO:

106.

255. 243. The fusion polypeptide of claim 242, wherein the intracellular signaling or costimulatory domain is a LAT domain.

256. The fusion polypeptide of claim 255, wherein the LAT domain comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:

102.

257. The fusion polypeptide of claim 256, wherein the LAT domain comprises the amino acid sequence set forth in SEQ ID NO:

102.

258. The fusion polypeptide of claim 242, wherein the intracellular signaling or costimulatory domain is a CD4 co-receptor domain.

259. The fusion polypeptide of claim 258, wherein the CD4 co-receptor domain comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

100.

260. The fusion polypeptide of claim 259, wherein the CD4 co-receptor domain comprises the amino acid sequence set forth in SEQ ID NO:

100.

261. The fusion polypeptide of claim 242, wherein the intracellular signaling or costimulatory domain is a CD8 co-receptor domain.

262. The fusion polypeptide of claim 261, wherein the CD8 co-receptor domain comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

101.

263. The fusion polypeptide of claim 262, wherein the CD8 co-receptor domain comprises the amino acid sequence set forth in SEQ ID NO:

101.

264. The fusion polypeptide of any one of claims 164-241, wherein the targeting moiety does not comprise a functional intracellular signaling or costimulatory domain.

265. The fusion polypeptide of any one of claims 164 to 264, wherein the targeting moiety further comprises a cleaved intracellular CD4 polypeptide.

266. The fusion polypeptide of claim 265, wherein the cleaved intracellular CD4 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:48 or SEQ ID NO:

49.

267. The fusion polypeptide of claim 266, wherein the cleaved intracellular CD4 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:48 or SEQ ID NO:

49.

268. The fusion polypeptide of any one of claims 164 to 267, wherein the extracellular domain comprises a first targeting domain.

269. 269. The fusion polypeptide of any one of claims 164 to 268, wherein the first targeting domain comprises a single chain variable fragment (scFv) or a single domain antibody (sdAb).

270. 270. The fusion polypeptide of claim 269, wherein the sdAb is a camelid VHH, nanobody, or heavy chain-only antibody (HcAb).

271. 271. The fusion polypeptide of claim 270, wherein the sdAb is a Camelidae VHH.

272. 272. The fusion polypeptide of any one of claims 270 or 271, wherein the scFv or sdAb is human or humanized.

273. The fusion polypeptide of any one of claims 268-272, wherein the first targeting domain comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 50-83.

274. The fusion polypeptide of claim 273, wherein the first targeting domain comprises a sequence set forth in any one of SEQ ID NOs: 50-83.

275. 275. The fusion polypeptide of claim 274, wherein the first targeting domain comprises a PD1 ectodomain, human A-proliferation-inducing ligand (APRIL), trimerized human APRIL, or NKG2D membrane protein.

276. The fusion polypeptide of claim 275, wherein the PD1 ectodomain comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:84 or SEQ ID NO:

85.

277. The fusion polypeptide of claim 276, wherein the PD1 ectodomain comprises the sequence set forth in SEQ ID NO:

85.

278. The fusion polypeptide of claim 275, wherein the APRIL comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 86, or the trimerized human APRIL comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

87.

279. The fusion polypeptide of claim 276, wherein the APRIL comprises the sequence shown in SEQ ID NO: 86 or the trimerized human APRIL comprises the sequence shown in SEQ ID NO:

87.

280. 276. The fusion polypeptide of claim 275, wherein the NKG2D membrane protein comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

88.

281. The fusion polypeptide of claim 280, wherein the NKG2D membrane protein comprises the sequence shown as SEQ ID NO:

88.

282. 282. The fusion polypeptide of any one of claims 268-281, wherein the extracellular domain further comprises a second targeting domain.

283. 283. The fusion polypeptide of claim 282, wherein the second targeting domain comprises a second single-chain variable fragment (scFv) or a second single-domain antibody (sdAb).

284. 284. The fusion polypeptide of claim 283, wherein the second sdAb is a camelid VHH, a nanobody, or a heavy chain-only antibody (HcAb).

285. 285. The fusion polypeptide of claim 284, wherein the second sdAb is a Camelidae VHH.

286. 286. The fusion polypeptide of any one of claims 282 to 285, wherein the second scFv or second sdAb is human or humanized.

287. The fusion polypeptide of any one of claims 282-286, wherein the second targeting domain comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 50-83.

288. The fusion polypeptide of claim 287, wherein the second targeting domain comprises a sequence set forth in any one of SEQ ID NOs: 50-83.

289. 283. The fusion polypeptide of claim 282, wherein the second targeting domain comprises a PD1 ectodomain, human A-proliferation-inducing ligand (APRIL), trimerized human APRIL, or NKG2D membrane protein.

290. The fusion polypeptide of claim 289, wherein the PD1 ectodomain comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:84 or SEQ ID NO:

85.

291. The fusion polypeptide of claim 290, wherein the PD1 ectodomain comprises the sequence set forth in SEQ ID NO:

85.

292. The fusion polypeptide of claim 289, wherein the APRIL comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 86, or the trimerized human APRIL comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

87.

293. 293. The fusion polypeptide of claim 292, wherein the APRIL comprises the sequence shown in SEQ ID NO: 86, or the trimerized human APRIL comprises the sequence shown in SEQ ID NO:

87.

294. 290. The fusion polypeptide of claim 289, wherein the NKG2D membrane protein comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

88.

295. 295. The fusion polypeptide of claim 294, wherein the NKG2D membrane protein comprises the sequence shown as SEQ ID NO:

88.

296. 296. The fusion polypeptide of any one of claims 268-295, wherein the first targeting domain and the second targeting domain bind to the same antigen or different antigens.

297. 300. The fusion polypeptide of any one of claims 268-296, wherein the first targeting domain and the second targeting domain are separated by a third polypeptide linker of 2 to 40 amino acids in length.

298. 300. The fusion polypeptide of claim 297, wherein the third polypeptide linker is selected from the group consisting of GG, GS, SG, SS, GSS, SSG, GSG, SGS, SGG, G4S, 2xG4S, 3xG4s, 4xG4S, 5xG4S, any one of SEQ ID NOs: 16-31, and any combination thereof.

299. The fusion polypeptide of any one of claims 164 to 298, wherein the polypeptide cleavage signal is a viral self-cleaving polypeptide.

300. 300. The fusion polypeptide of any one of claims 164 to 299, wherein the polypeptide cleavage signal is a viral self-cleaving 2A polypeptide.

301. 300. The fusion polypeptide of any one of claims 164 to 299, wherein the polypeptide cleavage signal is a viral self-cleaving polypeptide selected from the group consisting of a foot-and-mouth disease virus (FMDV) (F2A) peptide, an equine rhinitis A virus (ERAV) (E2A) peptide, a tosea agave virus (TaV) (T2A) peptide, a porcine teschovirus-1 (PTV-1) (P2A) peptide, a tylovirus 2A peptide, and an encephalomyocarditis virus 2A peptide.

302. 302. The fusion polypeptide of any one of claims 164-301, wherein the signaling component comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, or SEQ ID NO:

115.

303. 303. The fusion polypeptide of claim 302, wherein the signaling component comprises the sequence set forth as SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, or SEQ ID NO:

115.

304. 304. The fusion polypeptide of any one of claims 164 to 303, wherein the targeting moiety comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to the sequence set forth as SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, or SEQ ID NO:

136.

305. The fusion polypeptide of claim 304, wherein the targeting moiety comprises the sequence set forth as SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, or SEQ ID NO:

136.

306. 306. The fusion polypeptide of any one of claims 164 to 305, comprising a fusion polypeptide comprising said targeting moiety and said signaling moiety.

307. 307. The fusion polypeptide of claim 306, wherein the fusion polypeptide comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, or SEQ ID NO:

170.

308. 308. The fusion polypeptide of claim 307, wherein the fusion polypeptide comprises the sequence set forth as SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, or SEQ ID NO:

170.

309. A nucleic acid molecule encoding the fusion polypeptide of any one of claims 164 to 308.

310. A cell comprising the fusion polypeptide of any one of claims 164 to 308.

311. A cell comprising the nucleic acid molecule of claim 309.

312. The cell of claim 310 or claim 311, further expressing an exogenous costimulatory factor, an immunomodulatory factor, an agonist for a costimulatory factor, an antagonist for an immunosuppressive factor, an immune cell engager, a flip receptor, or any combination thereof.

313. 312. The cell of any one of claims 310 to 311, wherein the cell further expresses an exogenous lymphocyte receptor or co-receptor.

314. The cell of claim 313, wherein the exogenous lymphocyte receptor or co-receptor is selected from the group consisting of TCR alpha (TCRα), TCR beta (TCRβ), TCR gamma (TCRγ), TCR delta (TCRδ), CD4, CD8, pre-T cell receptor alpha (pTα), Fc receptor alpha (FcRα), Fc receptor beta (FcRβ), Fc receptor gamma (FcRγ), natural killer group 2 member D (NKG2D), CD79A, CD79B, and any combination thereof.

315. The cell of any one of claims 310 to 314, wherein the cell further expresses an exogenous TCR.

316. The exogenous TCR is capable of detecting alpha-fetoprotein (AFP), B melanoma antigen (BAGE) family members, sibling of regulator of imprinted sites (BORIS), cancer testis antigen, cancer testis antigen 83 (CT-83), carbonic anhydrase IX (CA1X), carcinoembryonic antigen (CEA), cytomegalovirus (CMV) antigen, melanoma cytotoxic T cell (CTL)-recognized antigen (CAMEL), Epstein-Barr virus (EBV) antigen, G antigen 1 (GAGE), -1), GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, GAGE-8, glycoprotein 100 (GP100), hepatitis B virus (HBV) antigen, hepatitis C virus (HCV) nonstructural protein 3 (NS3), human epidermal growth factor receptor 2 (HER-2), human papillomavirus (HPV)-E6, HPV-E7, human telomerase reverse transcriptase (hTERT), IGF2BP3 / A3, K-Ras, K-Ras G12C, K-Ras G12D, K-Ras G12V, latent membrane protein 2 (LMP2), melanoma antigen family A, 1 (MAGE-A1), MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, melanoma antigen recognized by T cells (MART-1), mesothelin (MSLN), mucin 1 (MUC1), mucin 16 (MUC16), New York esophageal squamous cell carcinoma-1 (NYESO-1), P53, P antigen (PAGE) family member, placenta-specific 1 (PLAC1), preferentially in melanoma 316. The cell of claim 315, which binds to a target antigen selected from the group consisting of expressed antigen (PRAME), survivin, synovial sarcoma X1 (SSX1), synovial sarcoma X2 (SSX2), synovial sarcoma X3 (SSX3), synovial sarcoma X4 (SSX4), synovial sarcoma X5 (SSX5), synovial sarcoma X8 (SSX8), thyroglobulin, tyrosinase, tyrosinase-related protein (TRP) 1, TRP2, Wilms' tumor protein (WT-1), X antigen family member 1 (XAGE1), and X antigen family member 2 (XAGE2).

317. The cell of claim 315 or claim 316, wherein the exogenous TCR is an αβ-TCR or a γδ-TCR.

318. The cell of any one of claims 310 to 317, wherein the cell further expresses a CAR, CCR, or flip receptor.

319. The cell of any one of claims 310 to 318, wherein the cell further expresses a zetakine, an immune cell engager, or a BiTE.

320. The cell of any one of claims 310 to 319, wherein the cell is a hematopoietic cell.

321. The cell of any one of claims 310 to 320, wherein the cell is a T cell, an αβ-T cell, or a γδ-T cell.

322. The cells are CD3 ＋ , CD4 ＋ , and / or CD8 ＋ A cell according to any one of claims 310 to 321, which is a cell.

323. The cell of any one of claims 310 to 322, wherein the cell is an immune effector cell.

324. The cell of any one of claims 310 to 323, wherein the cell is a cytotoxic T lymphocyte (CTL), a tumor-infiltrating lymphocyte (TIL), or a helper T cell.

325. The cell of any one of claims 164 to 324, wherein the cell is a natural killer (NK) cell or a natural killer T (NKT) cell.

326. 326. The cell of any one of claims 310 to 325, wherein the source of the cell is peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, or a tumor.

327. The cell of any one of claims 310 to 326, wherein the cell is an isolated cell.

328. The cell of any one of claims 310 to 327, wherein the cell is obtained from a subject.

329. The cell of any one of claims 310 to 328, wherein the cell is a human cell.

330. 1. A polypeptide complex comprising: a signaling component comprising a first multimerization domain and an actuator domain; and A polypeptide complex comprising an extracellular domain, a second multimerization domain, and a targeting moiety comprising a transmembrane domain.

331. 331. The polypeptide complex of claim 330, wherein the first and second multimerization domains are localized extracellularly when the signaling component and the targeting component are expressed.

332. 332. The polypeptide complex of claim 330 or claim 331, wherein the second multimerization domain and the transmembrane domain are separated by a hinge domain.

333. 333. The polypeptide complex of claim 332, wherein the hinge domain is selected from the group consisting of a CD4 hinge, a CD8 hinge, a CD28 hinge, an IgG4 hinge, and any fragment or variant or combination thereof.

334. The polypeptide complex of claim 332 or claim 333, wherein the hinge domain is a CD4 hinge.

335. The polypeptide complex cell of claim 334, wherein the CD4 hinge comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

41.

336. The polypeptide complex of claim 335, wherein the CD4 hinge comprises the amino acid sequence set forth in SEQ ID NO:

41.

337. The polypeptide complex of claim 332 or claim 333, wherein the hinge domain is a CD28 hinge.

338. The polypeptide complex of claim 337, wherein the CD28 hinge comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

42.

339. The polypeptide complex of claim 338, wherein the CD28 hinge comprises the amino acid sequence set forth in SEQ ID NO:

42.

340. The polypeptide complex of claim 332 or claim 333, wherein the hinge domain is a CD8 hinge.

341. The polypeptide complex of claim 340, wherein the CD8 hinge comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

44.

342. The polypeptide complex of claim 341, wherein the CD8 hinge comprises the amino acid sequence set forth in SEQ ID NO:

44.

343. The polypeptide complex of claim 332 or claim 333, wherein the hinge domain is an IgG4 hinge.

344. The polypeptide complex of claim 343, wherein the IgG4 hinge comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

43.

345. The polypeptide complex of claim 355, wherein the IgG4 hinge comprises the amino acid sequence set forth in SEQ ID NO:

43.

346. 346. The polypeptide complex of any one of claims 330 to 345, wherein the actuator domain is a CD3 polypeptide, an FcεR1γ polypeptide, an Igα / CD79a polypeptide, an Igβ / CD79b polypeptide, a DAP10 polypeptide, or a DAP12 polypeptide.

347. 347. The polypeptide complex of any one of claims 330 to 346, wherein the CD3 polypeptide is CD3 epsilon (CD3ε) or a fragment or variant thereof, CD3 gamma (CD3γ) or a fragment or variant thereof, or CD3 delta (CD3δ) or a fragment or variant thereof.

348. The polypeptide complex of any one of claims 330-347, wherein the actuator domain is a CD3ε polypeptide comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

32.

349. The polypeptide complex of any one of claims 330 to 348, wherein the actuator domain is a CD3ε polypeptide comprising the amino acid sequence set forth in SEQ ID NO:

32.

350. The polypeptide complex of any one of claims 330 to 347, wherein the actuator domain is a CD3γ polypeptide or a variant thereof.

351. 351. The polypeptide complex of any one of claims 330-347 or 350, wherein the actuator domain is a CD3γ polypeptide comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

33.

352. 352. The polypeptide complex of any one of claims 330-347, 350, or 351, wherein the actuator domain is a CD3γ polypeptide comprising the amino acid sequence set forth in SEQ ID NO:

33.

353. The polypeptide complex of any one of claims 330 to 347, wherein the actuator domain is a CD3δ polypeptide or a variant thereof.

354. 354. The polypeptide complex of any one of claims 330-347 or 353, wherein the actuator domain is a CD3δ polypeptide comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

34.

355. 355. The polypeptide complex of any one of claims 330-347, 353, or 354, wherein the actuator domain is a CD3δ polypeptide comprising the amino acid sequence set forth in SEQ ID NO:

34.

356. The polypeptide complex of any one of claims 330 to 346, wherein the actuator domain is an FcεR1γ polypeptide or a variant thereof.

357. 357. The polypeptide complex of any one of claims 330-346 or claim 356, wherein the actuator domain is an FcεR1γ polypeptide comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:

35.

358. 358. The polypeptide complex of any one of claims 330-346, 356, or 357, wherein the actuator domain is an FcεR1γ polypeptide comprising the amino acid sequence set forth in SEQ ID NO:

35.

359. 357. The polypeptide complex of any one of claims 330-346 or 356, wherein the actuator domain is an FcεR1γ polypeptide comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

36.

360. 360. The polypeptide complex of any one of claims 330-346, 356, or 359, wherein the actuator domain is an FcεR1γ polypeptide comprising the amino acid sequence set forth in SEQ ID NO:

36.

361. 347. The polypeptide complex of any one of claims 330 to 346, wherein the actuator domain is an Igα / CD79a polypeptide or a variant thereof.

362. 362. The polypeptide complex of any one of claims 330-346 or 361, wherein the actuator domain is an Igα / CD79a polypeptide comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

37.

363. 363. The polypeptide complex of any one of claims 330-346, 361, or 362, wherein the actuator domain is an Igα / CD79a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:

37.

364. The polypeptide complex of any one of claims 330 to 346, wherein the actuator domain is an Igβ / CD79b polypeptide or a variant thereof.

365. 365. The polypeptide complex of any one of claims 330-346 or 364, wherein the actuator domain is an Igβ / CD79b polypeptide comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

38.

366. 366. The polypeptide complex of any one of claims 330-346, 364, or 365, wherein the actuator domain is an Igβ / CD79b polypeptide comprising the amino acid sequence set forth in SEQ ID NO:

38.

367. The polypeptide complex of any one of claims 330 to 346, wherein the actuator domain is a DAP10 polypeptide or a variant thereof.

368. 368. The polypeptide complex of any one of claims 330-346 or 367, wherein the actuator domain is a DAP10 polypeptide comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

39.

369. 369. The polypeptide complex of any one of claims 330-346, 367, or 368, wherein the actuator domain is a DAP10 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:

39.

370. The polypeptide complex of any one of claims 330 to 346, wherein the actuator domain is a DAP12 polypeptide or a variant thereof.

371. 371. The polypeptide complex of any one of claims 330-346 or 370, wherein the actuator domain is a DAP12 polypeptide comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

40.

372. 372. The polypeptide complex of any one of claims 330-346, 370, or 371, wherein the actuator domain is a DAP12 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:

40.

373. The polypeptide complex of any one of claims 330-372, wherein the actuator domain comprises both an extracellular portion and an intracellular portion.

374. 374. The polypeptide complex of any one of claims 330-373, further comprising a signal sequence, optionally wherein the signal sequence has at least 90%, 95%, 96%, 97%, 98%, 99% identity to or comprises SEQ ID NO:95, SEQ ID NO:96, or SEQ ID NO:

97.

375. 375. The polypeptide complex of any one of claims 330-374, wherein the first and second multimerization domains are different.

376. 376. The polypeptide complex of any one of claims 330-375, wherein the multimerization domains of the signaling and targeting components are associated with a cross-linking agent selected from the group consisting of rapamycin or a rapalog thereof, gibberellin or a derivative thereof, abscisic acid (ABA) or a derivative thereof, methotrexate or a derivative thereof, cyclosporin A or a derivative thereof, FK506 / cyclosporin A or a derivative thereof, trimethoprim (Tmp) synthetic ligand (SLF) for FK506 binding protein (FKBP) or a derivative thereof, wherein the cross-linking agent promotes the formation of a polypeptide complex, and wherein the cross-linking agent is associated with and disposed between the multimerization domains of the signaling component and the targeting component.

377. The polypeptide complex of any one of claims 330 to 376, wherein the first multimerization domain and the second multimerization domain are a pair selected from the group consisting of FK506-binding protein 1A (FKBP12) and FKBP12-rapamycin binding (FRB), FKBP12 and calcineurin, FKBP and cyclophilin A or any other member of the peptidyl-prolyl cis-trans-isomerase (PPIase) family, FKBP and dihydrofolate reductase (DHFR), calcineurin and cyclophilin A or any other member of the peptidyl-prolyl cis-trans-isomerase (PPIase) family, and PYR1-like 1 (PYL1) and abscisic acid-insensitive 1 (ABI1).

378. 378. The polypeptide complex of any one of claims 330 to 377, wherein the first multimerization domain comprises a first FRB polypeptide or a variant thereof, and the second multimerization domain comprises a first FKBP12 polypeptide or a variant thereof.

379. 378. The polypeptide complex of any one of claims 330 to 377, wherein the first multimerization domain comprises a first FKBP12 polypeptide or a variant thereof, and the second multimerization domain comprises a first FRB polypeptide or a variant thereof.

380. 380. The polypeptide complex of any one of claims 377-379, wherein the FRB polypeptide is an FRB T2098L variant.

381. The polypeptide complex of any one of claims 377 to 380, wherein the FRB polypeptide comprises the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:

2.

382. The polypeptide complex of any one of claims 377 to 381, wherein the FKBP12 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:

4.

383. 383. The polypeptide conjugate of any one of claims 376 to 382, ​​wherein the cross-linking agent is AP1903, AP20187, AP21967 (also known as C16-(S)-7-methylindolerapamycin), everolimus, novolimus, pimecrolimus, ridaforolimus, sirolimus, tacrolimus, temsirolimus, umirolimus, zotarolimus, or BPC015.

384. 376. The polypeptide complex of any one of claims 330 to 375, wherein the first multimerization domain and the second multimerization domain are a pair of antibody-derived heterodimerization domains.

385. 385. The polypeptide complex of any one of claims 330-375 or 384, wherein the first multimerization domain comprises a polypeptide having at least 90%, 95%, 96%, 97%, 98%, 99% identity to, or comprises an amino acid sequence as set forth in SEQ ID NO:5; and the second multimerization domain comprises a polypeptide having at least 90%, 95%, 96%, 97%, 98%, 99% identity to, or comprises an amino acid sequence as set forth in SEQ ID NO:

6.

386. 385. The polypeptide complex of any one of claims 330-375 or 384, wherein the first multimerization domain comprises a polypeptide having at least 90%, 95%, 96%, 97%, 98%, 99% identity to, or comprises an amino acid sequence as set forth in SEQ ID NO:6; and the second multimerization domain comprises a polypeptide having at least 90%, 95%, 96%, 97%, 98%, 99% identity to, or comprises an amino acid sequence as set forth in SEQ ID NO:

5.

387. 385. The polypeptide complex of any one of claims 330-375 or 384, wherein the first multimerization domain comprises a polypeptide having at least 90%, 95%, 96%, 97%, 98%, 99% identity to, or comprises an amino acid sequence as set forth in SEQ ID NO:7; and the second multimerization domain comprises a polypeptide having at least 90%, 95%, 96%, 97%, 98%, 99% identity to, or comprises an amino acid sequence as set forth in SEQ ID NO:

8.

388. 385. The polypeptide complex of any one of claims 330-375 or 384, wherein the first multimerization domain comprises a polypeptide having at least 90%, 95%, 96%, 97%, 98%, 99% identity to, or comprises an amino acid sequence as set forth in SEQ ID NO:8; and the second multimerization domain comprises a polypeptide having at least 90%, 95%, 96%, 97%, 98%, 99% identity to, or comprises an amino acid sequence as set forth in SEQ ID NO:

7.

389. 385. The polypeptide complex of any one of claims 330-375 or 384, wherein the first multimerization domain comprises a polypeptide having at least 90%, 95%, 96%, 97%, 98%, 99% identity to, or comprises an amino acid sequence as set forth in SEQ ID NO:9; and the second multimerization domain comprises a polypeptide having at least 90%, 95%, 96%, 97%, 98%, 99% identity to, or comprises an amino acid sequence as set forth in SEQ ID NO:

10.

390. 385. The polypeptide complex of any one of claims 330-375 or 384, wherein the first multimerization domain comprises a polypeptide having at least 90%, 95%, 96%, 97%, 98%, 99% identity to, or comprises an amino acid sequence as set forth in SEQ ID NO: 10; and the second multimerization domain comprises a polypeptide having at least 90%, 95%, 96%, 97%, 98%, 99% identity to, or comprises an amino acid sequence as set forth in SEQ ID NO:

9.

391. 391. The polypeptide complex of any one of claims 330 to 390, wherein the first multimerization domain and the actuator domain are separated by a first polypeptide linker of 2 to 40 amino acids in length.

392. The polypeptide complex of claim 391, wherein the first polypeptide linker is selected from the group consisting of GG, GS, SG, SS, GSS, SSG, GSG, SGS, SGG, G4S, 2xG4S, 3xG4S, 4xG4S, 5xG4S, and any combination thereof.

393. The polypeptide complex of claim 392, wherein the first polypeptide linker is a 3xG4S linker.

394. 392. The polypeptide complex of claim 391, wherein the first polypeptide linker comprises a polypeptide comprising an amino acid sequence set forth as any one of SEQ ID NOs: 16-31.

395. 395. The polypeptide complex of any one of claims 330-394, wherein the extracellular domain and the second multimerization domain are separated by a second polypeptide linker of 2 to 40 amino acids in length.

396. The polypeptide complex of claim 395, wherein the second polypeptide linker is selected from the group consisting of GG, GS, SG, SS, GSS, SSG, GSG, SGS, SGG, G4S, 2xG4S, 3xG4s, 4xG4S, and any combination thereof.

397. The polypeptide complex of claim 396, wherein the second polypeptide linker is a G4S linker.

398. 396. The polypeptide complex of claim 395, wherein the second polypeptide linker comprises a polypeptide comprising an amino acid sequence set forth as any one of SEQ ID NOs: 16-31.

399. 399. The polypeptide complex of any one of claims 330 to 398, wherein the transmembrane domain is a CD4 transmembrane domain.

400. 400. The polypeptide complex of claim 399, wherein the CD4 transmembrane domain comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

45.

401. The polypeptide complex of claim 400, wherein the CD4 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:

45.

402. 399. The polypeptide complex of any one of claims 330 to 398, wherein the transmembrane domain is a CD28 transmembrane domain.

403. The polypeptide complex of claim 402, wherein the CD28 transmembrane domain comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

46.

404. The polypeptide complex of claim 403, wherein the CD28 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:

46.

405. 399. The polypeptide complex of any one of claims 330 to 398, wherein the transmembrane domain is a CD8 transmembrane domain.

406. The polypeptide complex of claim 405, wherein the CD8 transmembrane domain comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

47.

407. The polypeptide complex of claim 406, wherein the CD8 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:

47.

408. 408. The polypeptide complex of any one of claims 330-407, wherein said targeting moiety further comprises an intracellular signaling or costimulatory domain derived from a protein selected from the group consisting of an antigen receptor, a costimulatory receptor, a growth receptor, a cytokine receptor, an adaptor signaling protein, an intracellular signaling protein, or any fragment or variant thereof.

409. The intracellular signaling or costimulatory domain on the targeting moiety is selected from the group consisting of Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain family member 11 (CARD11), CD2, CD3ε, CD3γ, CD3δ, CD4, CD7, CD8, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), common gamma chain cytokine, DNAX activating protein, 409. The polypeptide complex of claim 408, wherein the polypeptide complex is selected from the group consisting of: protein 10 (DAP10), linker for activation of T-cell family member 1 (LAT), interleukin-2 receptor (IL-2R), IL-4R, IL-7R, IL-9R, IL-12R, IL-13R, IL-15R, IL-21R, SH2 domain-containing leukocyte protein 76 kD (SLP76), T-cell receptor-associated transmembrane adaptor 1 (TRAT1), TNFR2, TNFRS14, TNFRS18, TNRFS25, and the zeta chain of T-cell receptor-associated protein kinase 70 (ZAP70).

410. The polypeptide complex of claim 408 or claim 409, wherein said intracellular signaling or costimulatory domain on said targeting moiety is selected from the group consisting of 4-1BB, CD28, TNFR2, OX40, ICOS, and DAP10 costimulatory domains.

411. The polypeptide complex of claim 410, wherein the costimulatory domain on the targeting moiety is a 4-1BB costimulatory domain, and optionally the 4-1BB costimulatory domain comprises the amino acid sequence set forth in SEQ ID NO:

98.

412. The polypeptide complex of claim 410, wherein the costimulatory domain on the targeting component is a CD28 costimulatory domain, and optionally the CD28 costimulatory domain comprises the amino acid sequence set forth in SEQ ID NO:

99.

413. The polypeptide complex of claim 408, wherein the intracellular signaling or costimulatory domain is one or more cytokine receptor intracellular signaling domains.

414. The polypeptide complex of claim 413, wherein the one or more cytokine receptor intracellular signaling domains are selected from the group consisting of an IL7Rα intracellular signaling domain, an IL2Rβ intracellular signaling domain, a common gamma chain intracellular signaling domain, and both an IL2Rβ and a common gamma chain intracellular signaling domain.

415. The polypeptide complex of claim 414, wherein the IL7Rα intracellular signaling domain comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

103.

416. The polypeptide complex of claim 415, wherein the IL7Rα intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO:

103.

417. The polypeptide complex of claim 414, wherein the IL2Rβ intracellular signaling domain comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 104 or SEQ ID NO:

105.

418. The polypeptide complex of claim 417, wherein the IL2Rβ intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 104 or SEQ ID NO:

105.

419. The polypeptide complex of claim 414, wherein the common gamma chain intracellular signaling domain comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

106.

420. The polypeptide complex of claim 419, wherein the common gamma chain intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO:

106.

421. The polypeptide complex of claim 408, wherein the intracellular signaling or costimulatory domain is a LAT domain.

422. The polypeptide complex of claim 421, wherein the LAT domain comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:

102.

423. The polypeptide complex of claim 422, wherein the LAT domain comprises the amino acid sequence set forth in SEQ ID NO:

102.

424. The polypeptide complex of claim 408, wherein the intracellular signaling or costimulatory domain is a CD4 co-receptor domain.

425. The polypeptide complex of claim 424, wherein the CD4 co-receptor domain comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

100.

426. The polypeptide complex of claim 425, wherein the CD4 co-receptor domain comprises the amino acid sequence set forth in SEQ ID NO:

100.

427. The polypeptide complex of claim 408, wherein the intracellular signaling or costimulatory domain is a CD8 co-receptor domain.

428. The polypeptide complex of claim 427, wherein the CD8 co-receptor domain comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

101.

429. The polypeptide complex of claim 428, wherein the CD8 co-receptor domain comprises the amino acid sequence set forth in SEQ ID NO:

101.

430. The polypeptide complex of any one of claims 330-407, wherein said targeting moiety does not comprise a functional intracellular signaling or costimulatory domain.

431. The polypeptide complex of any one of claims 330-430, wherein the targeting moiety further comprises a cleaved intracellular CD4 polypeptide.

432. The polypeptide complex of claim 431, wherein the cleaved intracellular CD4 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:48 or SEQ ID NO:

49.

433. The polypeptide complex of claim 432, wherein the cleaved intracellular CD4 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:48 or SEQ ID NO:

49.

434. The polypeptide complex of any one of claims 330-433, wherein the extracellular domain comprises a first targeting domain.

435. 435. The polypeptide complex of any one of claims 330-434, wherein the first targeting domain comprises a single chain variable fragment (scFv) or a single domain antibody (sdAb).

436. 436. The polypeptide complex of claim 435, wherein the sdAb is a camelid VHH, a nanobody, or a heavy chain-only antibody (HcAb).

437. 437. The polypeptide complex of claim 436, wherein the sdAb is a Camelidae VHH.

438. 438. The polypeptide complex of claim 436 or claim 437, wherein the scFv or sdAb is human or humanized.

439. The polypeptide complex of any one of claims 434 to 438, wherein the first targeting domain comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 50 to 83.

440. The polypeptide complex of claim 439, wherein the first targeting domain comprises a sequence set forth in any one of SEQ ID NOs: 50-83.

441. The polypeptide complex of claims 434-438, wherein the first targeting domain comprises a PD1 ectodomain, human A-proliferation-inducing ligand (APRIL), trimerized human APRIL, or NKG2D membrane protein.

442. The polypeptide complex of claim 441, wherein the PD1 ectodomain comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:84 or SEQ ID NO:

85.

443. The polypeptide complex of claim 442, wherein the PD1 ectodomain comprises the sequence set forth in SEQ ID NO:

85.

444. The polypeptide complex of claim 441, wherein the APRIL comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 86, or the trimerized human APRIL comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

87.

445. The polypeptide complex of claim 444, wherein the APRIL comprises the sequence shown in SEQ ID NO: 86, or the trimerized human APRIL comprises the sequence shown in SEQ ID NO:

87.

446. The polypeptide complex of claim 441, wherein the NKG2D membrane protein comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

88.

447. The polypeptide complex of claim 446, wherein the NKG2D membrane protein comprises the sequence shown as SEQ ID NO:

88.

448. The polypeptide complex of any one of claims 434 to 447, wherein the extracellular domain further comprises a second targeting domain.

449. The polypeptide complex of claim 448, wherein the second targeting domain comprises a second single-chain variable fragment (scFv) or a second single-domain antibody (sdAb).

450. 450. The polypeptide complex of claim 449, wherein the second sdAb is a camelid VHH, a nanobody, or a heavy chain-only antibody (HcAb).

451. 451. The polypeptide complex of claim 450, wherein the second sdAb is a Camelidae VHH.

452. 452. The polypeptide complex of any one of claims 449-451, wherein the second scFv or second sdAb is human or humanized.

453. The polypeptide complex of any one of claims 448-453, wherein the second targeting domain comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 50-83.

454. The polypeptide complex of claim 454, wherein the second targeting domain comprises a sequence set forth in any one of SEQ ID NOs: 50-83.

455. The polypeptide complex of claim 448, wherein the second targeting domain comprises a PD1 ectodomain, human A-proliferation-inducing ligand (APRIL), trimerized human APRIL, or NKG2D membrane protein.

456. The polypeptide complex of claim 455, wherein the PD1 ectodomain comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:84 or SEQ ID NO:

85.

457. The polypeptide complex of claim 456, wherein the PD1 ectodomain comprises the sequence set forth in SEQ ID NO:

85.

458. The polypeptide complex of claim 455, wherein the APRIL comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 86, or the trimerized human APRIL comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

87.

459. The polypeptide complex of claim 458, wherein the APRIL comprises the sequence shown in SEQ ID NO: 86, or the trimerized human APRIL comprises the sequence shown in SEQ ID NO:

87.

460. The polypeptide complex of claim 455, wherein the NKG2D membrane protein comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

88.

461. The polypeptide complex of claim 460, wherein the NKG2D membrane protein comprises the sequence shown as SEQ ID NO:

88.

462. The polypeptide complex of any one of claims 448-461, wherein the first targeting domain and the second targeting domain bind to the same antigen or different antigens.

463. The polypeptide complex of any one of claims 448 to 462, wherein the targeting domain and the second targeting domain are separated by a third polypeptide linker of 2 to 40 amino acids in length.

464. The polypeptide complex of claim 463, wherein the third polypeptide linker is selected from the group consisting of GG, GS, SG, SS, GSS, SSG, GSG, SGS, SGG, G4S, 2xG4S, 3xG4s, 4xG4S, 5xG4S, any one of SEQ ID NOs: 16-31, and any combination thereof.

465. 465. The polypeptide complex of any one of claims 330-464, wherein the signaling component comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, or SEQ ID NO:

115.

466. 166. The polypeptide complex of claim 165, wherein the signaling component comprises the sequence set forth as SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, or SEQ ID NO:

115.

467. 467. The polypeptide complex of any one of claims 330-466, wherein the targeting moiety comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to the sequence set forth as SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, or SEQ ID NO:

136.

468. The polypeptide complex of claim 467, wherein the targeting component comprises the sequence set forth as SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, or SEQ ID NO:

136.

469. 10. The polypeptide complex of any one of claims 330-468, comprising a fusion polypeptide comprising said targeting moiety and said signaling moiety.

470. 470. The polypeptide complex of claim 469, wherein the fusion polypeptide comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, or SEQ ID NO:

170.

471. 471. The polypeptide complex of claim 470, wherein the fusion polypeptide comprises the sequence set forth as SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, or SEQ ID NO:

170.

472. A nucleic acid molecule encoding both the signaling component and the targeting component of the polypeptide complex of any one of claims 330-471.

473. A cell comprising the polypeptide complex of any one of claims 330-471.

474. A cell comprising the nucleic acid molecule of claim 472.

475. The cell of any one of claims 473 or 474, wherein the cell further expresses an exogenous costimulatory factor, an immunomodulatory factor, an agonist for a costimulatory factor, an antagonist for an immunosuppressive factor, an immune cell engager, a flip receptor, or any combination thereof.

476. The cell of any one of claims 473 to 475, wherein the cell further expresses an exogenous lymphocyte receptor or co-receptor.

477. The cell of claim 476, wherein the exogenous lymphocyte receptor or co-receptor is selected from the group consisting of TCR alpha (TCRα), TCR beta (TCRβ), TCR gamma (TCRγ), TCR delta (TCRδ), CD4, CD8, pre-T cell receptor alpha (pTα), Fc receptor alpha (FcRα), Fc receptor beta (FcRβ), Fc receptor gamma (FcRγ), natural killer group 2 member D (NKG2D), CD79A, CD79B, and any combination thereof.

478. The cell of any one of claims 473 to 477, wherein the cell further expresses an exogenous TCR.

479. The exogenous TCR is capable of detecting alpha-fetoprotein (AFP), B melanoma antigen (BAGE) family members, sibling of regulator of imprinted sites (BORIS), cancer testis antigen, cancer testis antigen 83 (CT-83), carbonic anhydrase IX (CA1X), carcinoembryonic antigen (CEA), cytomegalovirus (CMV) antigen, melanoma cytotoxic T cell (CTL)-recognized antigen (CAMEL), Epstein-Barr virus (EBV) antigen, G antigen 1 (GAGE), -1), GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, GAGE-8, glycoprotein 100 (GP100), hepatitis B virus (HBV) antigen, hepatitis C virus (HCV) nonstructural protein 3 (NS3), human epidermal growth factor receptor 2 (HER-2), human papillomavirus (HPV)-E6, HPV-E7, human telomerase reverse transcriptase (hTERT), IGF2BP3 / A3, K-Ras, K-Ras G12C, K-Ras G12D, K-Ras G12V, latent membrane protein 2 (LMP2), melanoma antigen family A, 1 (MAGE-A1), MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, melanoma antigen recognized by T cells (MART-1), mesothelin (MSLN), mucin 1 (MUC1), mucin 16 (MUC16), New York esophageal squamous cell carcinoma-1 (NYESO-1), P53, P antigen (PAGE) family member, placenta-specific 1 (PLAC1), preferentially in melanoma 479. The cell of claim 478, which binds to a target antigen selected from the group consisting of expressed antigen (PRAME), survivin, synovial sarcoma X1 (SSX1), synovial sarcoma X2 (SSX2), synovial sarcoma X3 (SSX3), synovial sarcoma X4 (SSX4), synovial sarcoma X5 (SSX5), synovial sarcoma X8 (SSX8), thyroglobulin, tyrosinase, tyrosinase-related protein (TRP) 1, TRP2, Wilms' tumor protein (WT-1), X antigen family member 1 (XAGE1), and X antigen family member 2 (XAGE2).

480. The cell of claim 478 or claim 479, wherein the exogenous TCR is an αβ-TCR or a γδ-TCR.

481. The cell of any one of claims 473 to 480, wherein the cell further expresses a CAR, CCR, or flip receptor.

482. The cell of any one of claims 473-481, wherein the cell further expresses a zetakine, an immune cell engager, or a BiTE.

483. The cell of any one of claims 473 to 482, wherein the cell is a hematopoietic cell.

484. The cell of any one of claims 473 to 483, wherein the cell is a T cell, an αβ-T cell, or a γδ-T cell.

485. The cells are CD3 ＋ , CD4 ＋ , and / or CD8 ＋ The cell of any one of claims 473 to 484, which is a cell.

486. The cell of any one of claims 473 to 485, wherein the cell is an immune effector cell.

487. The cell of any one of claims 473 to 486, wherein the cell is a cytotoxic T lymphocyte (CTL), a tumor-infiltrating lymphocyte (TIL), or a helper T cell.

488. The cell of any one of claims 473 to 487, wherein the cell is a natural killer (NK) cell or a natural killer T (NKT) cell.

489. The cell of any one of claims 473-488, wherein the source of the cell is peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, or a tumor.

490. The cell of any one of claims 473 to 489, wherein the cell is an isolated cell.

491. The cell of any one of claims 473 to 490, wherein the cell is obtained from a subject.

492. The cell of any one of claims 473 to 491, wherein the cell is a human cell.

493. 472. A polynucleotide encoding the signaling and targeting component of the fusion polypeptide of any one of claims 164-308 or the polypeptide complex of any one of claims 330-471.

494. A cDNA encoding the signaling and targeting component of the fusion polypeptide of any one of claims 164-308 or the polypeptide complex of any one of claims 330-471.

495. 10. An RNA encoding a signalling and targeting component of any one of claims 1 to 81 and 144 to 203, or a fusion polypeptide of any one of claims 82 to 143, a fusion polypeptide of any one of claims 164 to 308, or a polypeptide complex of any one of claims 330 to 471.

496. A vector comprising the polynucleotide of claim 493.

497. The vector of claim 496, wherein the vector is an expression vector.

498. The vector of claim 496, wherein the vector is a transposon.

499. The vector of claim 493, wherein the vector is a piggyBAC transposon or a Sleeping Beauty transposon.

500. The vector of claim 496, wherein the vector is a viral vector.

501. 501. The vector of claim 500, wherein the vector is an adenovirus vector, an adeno-associated virus (AAV) vector, a herpes virus vector, a vaccinia virus vector, or a retrovirus vector.

502. The vector of claim 501, wherein the retroviral vector is a lentiviral vector.

503. The vector of claim 502, wherein the lentiviral vector is selected from the group consisting of human immunodeficiency virus 1 (HIV-1); human immunodeficiency virus 2 (HIV-2), Visna-Maedi virus (VMV); caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV).

504. 503. A cell comprising the fusion polypeptide of any one of claims 164-308, the polynucleotide of claim 493, or the vector of any one of claims 496-503.

505. The cell of claim 504, wherein the cell is a hematopoietic cell.

506. The cell of claim 504 or claim 505, wherein the cell is an immune effector cell.

507. The cell of any one of claims 504 to 506, wherein the cell is a T cell, an αβ T cell, or a γδ T cell.

508. The cells are CD3 ＋ , CD4 ＋ , CD8 ＋ , or a combination thereof.

509. The cell of any one of claims 504 to 508, wherein the cell is a cytotoxic T lymphocyte (CTL), a tumor infiltrating lymphocyte (TIL), or a helper T cell.

510. The cell of any one of claims 504 to 508, wherein the cell is a natural killer (NK) cell or a natural killer T (NKT) cell.

511. 504-510, or a vector according to any one of claims 496-503.

512. 504-510, or a vector according to any one of claims 496-503.

513. A method of treating a subject in need thereof, comprising administering to the subject an effective amount of the composition of claim 511 or claim 512.

514. A method for treating, preventing, or ameliorating at least one symptom of cancer, infectious disease, autoimmune disease, inflammatory disease, and immune deficiency, or a condition associated therewith, comprising administering to the subject an effective amount of the composition of claim 511 or claim 512.

515. A method of treating a solid tumor comprising administering to the subject an effective amount of the composition of claim 513 or claim 514.

516. 516. The method of claim 515, wherein the solid cancer is selected from the group consisting of lung cancer, squamous cell carcinoma, colorectal cancer, pancreatic cancer, breast cancer, thyroid cancer, bladder cancer, cervical cancer, esophageal cancer, ovarian cancer, gastric cancer, endometrial cancer, or brain cancer.

517. The method of claim 515 or claim 516, wherein the solid cancer is non-small cell lung cancer, head and neck squamous cell carcinoma, colorectal cancer, pancreatic cancer, breast cancer, thyroid cancer, bladder cancer, cervical cancer, esophageal cancer, ovarian cancer, gastric cancer, endometrial cancer, glioma, glioblastoma, or oligodendroglioma.

518. A method of treating a hematological malignancy comprising administering to the subject an effective amount of the composition of claim 515 or claim 516.

519. 519. The method of claim 518, wherein the hematological malignancy is leukemia, lymphoma, or multiple myeloma.

520. The method of claim 518, wherein the hematological malignancy is acute myeloid leukemia (AML).

521. 164. The non-naturally occurring cell of any one of claims 1-163, wherein the cell further comprises a polypeptide having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

137.

522. 164. The non-naturally occurring cell of any one of claims 1 to 163, wherein the cell further comprises the amino acid sequence set forth in SEQ ID NO:

137.

523. 164. The non-naturally occurring cell of any one of claims 1-163, wherein the cell further comprises a polypeptide having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

138.

524. The non-naturally occurring cell of any one of claims 1 to 163, wherein the cell further comprises the amino acid sequence set forth in SEQ ID NO: 138.

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