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 USC § 119(e) of U.S. Provisional Application No. 63 / 329,003, filed April 8, 2022, which is incorporated by reference herein 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 in adoptive immunotherapy. [Background technology]

[0003] Description of related fields Worldwide, cancer doubled 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 now, adoptive cell therapy has emerged as a powerful paradigm for delivering complex biological signals to treat cancer. In contrast to small molecule and biopharmaceuticals, adoptive cell therapy has the potential to exert unique therapeutic tasks through a wide variety of sensory and response programs, as well as more clearly defined gene regulatory mechanisms. To realize such therapeutic value, cells need to 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 to target immune cells to specific antigens (e.g., tumor antigens), stimulate T cell activation signaling, and ultimately attack and kill antigen-associated 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: (i) a first multimerization domain comprising an FRB polypeptide or a variant thereof or an FKBP polypeptide or a variant thereof, (ii) a first polypeptide linker, and (iii) a signaling component comprising a CD3ε polypeptide or a variant thereof; and a targeting component comprising: (i) an anti-CLL1 scFv or single domain antibody (sdAb), (ii) an anti-CD33 scFv or single domain antibody (sdAb), (iii) a second polypeptide linker, (iv) a second multimerization domain comprising an FRB polypeptide or a variant thereof or an FKBP polypeptide or a variant thereof, (v) a CD4 hinge polypeptide, (vi) a CD4 transmembrane polypeptide, and (vii) a truncated CD4 intracellular polypeptide.

[0008] In some embodiments, the targeting moiety does not comprise a functional intracellular domain or a costimulatory domain having signaling capability. In some embodiments, the CD4 hinge polypeptide 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 polypeptide comprises the amino acid sequence shown in SEQ ID NO: 41.

[0009] In some embodiments, the CD3ε polypeptide comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 32. In one embodiment, the CD3ε polypeptide comprises the amino acid sequence shown in SEQ ID NO: 32. In some embodiments, the CD3ε polypeptide comprises both an extracellular portion and an intracellular portion.

[0010] In some embodiments, the FRB polypeptide and the FKBP polypeptide are localized outside the cell when the signaling component and the targeting component are expressed. In some embodiments, the first and second multimerization domains are different. In some embodiments, the first multimerization domain comprises an FRB polypeptide or a variant thereof, and the second multimerization domain comprises an FKBP12 polypeptide or a variant thereof. In some embodiments, the first multimerization domain comprises an FKBP12 polypeptide or a variant thereof, and the second multimerization domain comprises an FRB polypeptide or a variant thereof. In one embodiment, the FRB polypeptide is an FRB T2098L variant. In some embodiments, the FRB polypeptide comprises an amino acid sequence that has at least 90%, 95%, 96%, 97%, 98%, 99% identity with or includes the sequence shown as SEQ ID NO:1. In some embodiments, the FRB polypeptide comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to or including the sequence set forth as SEQ ID NO: 2. In some embodiments, the FKBP12 polypeptide comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to or including the sequence set forth as SEQ ID NO: 3. In some embodiments, the FKBP12 polypeptide comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to or including the sequence set forth as SEQ ID NO: 4.

[0011] In some embodiments, the multimerization domains of the signaling component and the targeting component are associated with a cross-linking agent. In some embodiments, the cross-linking agent is 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 wherein 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 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.

[0012] In some embodiments, the first polypeptide linker is a 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 second polypeptide linker is a 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 anti-CLL1 scFv or sdAb and the anti-CD33 scFv or sdAb are separated by a third polypeptide linker. In some embodiments, the third polypeptide linker is a 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, and any combination thereof. In some embodiments, the third polypeptide linker is a G4S linker.

[0013] In some embodiments, 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. In some embodiments, the CD4 transmembrane polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 45. In some embodiments, the truncated intracellular CD4 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 48. In one embodiment, the truncated intracellular CD4 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 49.

[0014] In some embodiments, the anti-CLL1 antibody and the anti-CD33 antibody are each sdAbs. In some embodiments, the sdAbs are Camelid VHHs, nanobodies, or heavy chain only antibodies (HcAbs). In one embodiment, the sdAbs are Camelid VHHs. In some embodiments, the scFvs or sdAbs are human or humanized. In some embodiments, the anti-CLL1 scFvs or sdAbs comprise the CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 92, 93, and 94, respectively. In some embodiments, the anti-CLL1 scFvs or sdAbs comprise a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 75. In some embodiments, the anti-CLL1 scFvs or sdAbs comprise a sequence shown in SEQ ID NO: 75. In some embodiments, the anti-CD33 scFv or sdAb comprises the CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 89, 90, and 91, respectively. In some embodiments, the anti-CD33 scFv or sdAb comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 72. In some embodiments, the anti-CD33 scFv or sdAb comprises the sequence set forth in SEQ ID NO: 72.

[0015] In some embodiments, the signaling moiety further comprises a signal sequence. In some embodiments, the signal sequence is a CD8 signal sequence. In some embodiments, 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 one embodiment, the CD8 signal sequence comprises the amino acid sequence set forth as SEQ ID NO:96. In some embodiments, the targeting moiety further comprises a signal sequence. In some embodiments, the signal sequence is an IgK signal sequence. In some embodiments, 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. In some embodiments, the signaling moiety comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:111. In one embodiment, the signaling moiety comprises the sequence set forth as SEQ ID NO:111. 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: 122. In one embodiment, the targeting moiety comprises the sequence set forth as SEQ ID NO: 122.

[0016] In some embodiments, 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: 151. In one embodiment, the fusion polypeptide comprises the sequence set forth as SEQ ID NO: 151.

[0017] In some embodiments, the cell comprises a first nucleic acid molecule encoding a signaling moiety. In some embodiments, the cell comprises a second nucleic acid molecule encoding a targeting moiety. In some embodiments, the cell comprises nucleic acid molecules encoding both a signaling moiety and a targeting moiety.

[0018] In some embodiments, the cells further express an exogenous costimulator, an immune modulator, an agonist for a costimulator, an antagonist for an immune suppressor, 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 embodiments, 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), in melanoma The exogenous TCR binds to a target antigen selected from the group consisting of preferentially expressed antigen in sarcoma (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.

[0019] In some embodiments, the cell further expresses a CAR, CCR, or flip receptor. In some embodiments, the cell further expresses a zetakine, an immune cell engager, or a BiTE. 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 non-naturally occurring 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 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 some embodiments, the cell further comprises an amino acid sequence set forth in SEQ ID NO: 138.

[0020] In one aspect, disclosed herein is a fusion polypeptide comprising: (i) a first multimerization domain comprising an FRB polypeptide or variant thereof or an FKBP polypeptide or variant thereof, (ii) a first polypeptide linker, and (iii) a CD3ε polypeptide or variant thereof; and a signaling moiety comprising a polypeptide cleavage signal; and a targeting moiety comprising: (i) an anti-CLL1 scFv or single domain antibody (sdAb), (ii) an anti-CD33 scFv or single domain antibody (sdAb), (iii) a second polypeptide linker, (iv) a second multimerization domain comprising an FRB polypeptide or variant thereof or an FKBP polypeptide or variant thereof, (v) a CD4 hinge polypeptide, (vi) a CD4 transmembrane polypeptide, and (vii) a cleaved CD4 intracellular polypeptide.

[0021] In some embodiments, the targeting moiety does not comprise a functional intracellular domain or a costimulatory domain having signaling capability. In some embodiments, the CD4 hinge polypeptide 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 polypeptide comprises the amino acid sequence shown in SEQ ID NO: 41.

[0022] In some embodiments, the CD3ε polypeptide comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 32. In one embodiment, the CD3ε polypeptide comprises the amino acid sequence shown in SEQ ID NO: 32. In some embodiments, the CD3ε polypeptide comprises both an extracellular portion and an intracellular portion.

[0023] In some embodiments, the FRB polypeptide and the FKBP polypeptide are localized outside the cell when the signaling component and the targeting component are expressed. In some embodiments, the first and second multimerization domains are different. In some embodiments, the first multimerization domain comprises an FRB polypeptide or a variant thereof, and the second multimerization domain comprises an FKBP12 polypeptide or a variant thereof. In some embodiments, the first multimerization domain comprises an FKBP12 polypeptide or a variant thereof, and the second multimerization domain comprises an FRB polypeptide or a variant thereof. In one embodiment, the FRB polypeptide is an FRB T2098L variant. In some embodiments, the FRB polypeptide comprises an amino acid sequence that has at least 90%, 95%, 96%, 97%, 98%, 99% identity with or includes the sequence shown as SEQ ID NO:1. In some embodiments, the FRB polypeptide comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to or including the sequence set forth as SEQ ID NO: 2. In some embodiments, the FKBP12 polypeptide comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to or including the sequence set forth as SEQ ID NO: 3. In some embodiments, the FKBP12 polypeptide comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to or including the sequence set forth as SEQ ID NO: 4.

[0024] In some embodiments, the multimerization domains of the signaling component and the targeting component are associated with a cross-linking agent. In some embodiments, the cross-linking agent is 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 wherein 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 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.

[0025] In some embodiments, the first polypeptide linker is a 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 second polypeptide linker is a 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 some embodiments, the second polypeptide linker is a G4S linker. In some embodiments, the anti-CLL1 scFv or sdAb and the anti-CD33 scFv or sdAb are separated by a third polypeptide linker. In some embodiments, the third polypeptide linker is a 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, and any combination thereof. In one embodiment, the third polypeptide linker is a G4S linker.

[0026] In some embodiments, 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. In one embodiment, the CD4 transmembrane polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 45. In one embodiment, the truncated intracellular CD4 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 48. In one embodiment, the truncated intracellular CD4 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 49. In some embodiments, the anti-CLL1 antibody and the anti-CD33 antibody are each sdAbs. In some embodiments, the sdAbs are Camelidae VHHs, nanobodies, or heavy chain only antibodies (HcAbs). In one embodiment, the sdAbs are Camelidae VHHs. In some embodiments, the scFvs or sdAbs are human or humanized. In some embodiments, the anti-CLL1 scFvs or sdAbs comprise the CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 92, 93, and 94, respectively. In some embodiments, the anti-CLL1 scFv or sdAb comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 75. In some embodiments, the anti-CLL1 scFv or sdAb comprises a sequence set forth in SEQ ID NO: 75. In some embodiments, the anti-CD33 scFv or sdAb comprises the CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 89, 90, and 91, respectively. In some embodiments, the anti-CD33 scFv or sdAb comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 72. In some embodiments, the anti-CD33 scFv or sdAb comprises the sequence set forth in SEQ ID NO: 72.

[0027] In some embodiments, the signaling moiety further comprises a signal sequence. In some embodiments, the signal sequence is a CD8 signal sequence. In some embodiments, 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 one embodiment, the CD8 signal sequence comprises the amino acid sequence set forth as SEQ ID NO:96. In some embodiments, the targeting moiety further comprises a signal sequence. In some embodiments, the signal sequence is an IgK signal sequence. In some embodiments, 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. In some embodiments, the signaling moiety comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:111. In one embodiment, the signaling moiety comprises the sequence set forth as SEQ ID NO:111. 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: 122. In one embodiment, the targeting moiety comprises the sequence set forth as SEQ ID NO: 122.

[0028] In some embodiments, the fusion polypeptide 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: 151. In one embodiment, the fusion polypeptide comprises the sequence set forth as SEQ ID NO: 151.

[0029] 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.

[0030] In one aspect, the disclosure provides a nucleic acid molecule encoding any one of the fusion polypeptides disclosed herein.

[0031] In another aspect, the disclosure provides a cell comprising any one of the fusion polypeptides disclosed herein.

[0032] In another aspect, the disclosure provides a cell comprising any one of the nucleic acid molecules disclosed herein.

[0033] In some embodiments, the cells further express an exogenous costimulator, an immune modulator, an agonist for a costimulator, an antagonist for an immune suppressor, 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 embodiments, 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), in melanoma The exogenous TCR binds to a target antigen selected from the group consisting of preferentially expressed antigen in sarcoma (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.

[0034] In some embodiments, the cell further expresses a CAR, CCR, or flip receptor. In some embodiments, the cell further expresses a zetakine, an immune cell engager, or a BiTE. In some embodiments, 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 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.

[0035] In one aspect, disclosed herein is a polypeptide complex comprising: (i) a first multimerization domain comprising an FRB polypeptide or a variant thereof or an FKBP polypeptide or a variant thereof, (ii) a first polypeptide linker, and (iii) a signaling moiety comprising a CD3ε polypeptide or a variant thereof; and a targeting moiety comprising (i) an anti-CLL1 scFv or single domain antibody (sdAb), (ii) an anti-CD33 scFv or single domain antibody (sdAb), (iii) a second polypeptide linker, (iv) a second multimerization domain comprising an FRB polypeptide or a variant thereof or an FKBP polypeptide or a variant thereof, (v) a CD4 hinge polypeptide, (vi) a CD4 transmembrane polypeptide, and (vii) a cleaved CD4 intracellular polypeptide.

[0036] In some embodiments, the targeting moiety does not comprise a functional intracellular domain or a costimulatory domain having signal transduction capability. In some embodiments, the CD4 hinge polypeptide comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 41. In some embodiments, the CD4 hinge polypeptide comprises an amino acid sequence as set forth in SEQ ID NO: 41. In some embodiments, the CD3ε polypeptide comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 32. In one embodiment, the CD3ε polypeptide comprises an amino acid sequence as set forth in SEQ ID NO: 32. In some embodiments, the CD3ε polypeptide comprises both an extracellular portion and an intracellular portion.

[0037] In some embodiments, the FRB polypeptide and the FKBP polypeptide are localized outside the cell when the signaling component and the targeting component are expressed. In some embodiments, the first and second multimerization domains are different. In some embodiments, the first multimerization domain comprises an FRB polypeptide or a variant thereof, and the second multimerization domain comprises an FKBP12 polypeptide or a variant thereof. In some embodiments, the first multimerization domain comprises an FKBP12 polypeptide or a variant thereof, and the second multimerization domain comprises an FRB polypeptide or a variant thereof. In one embodiment, the FRB polypeptide is an FRB T2098L variant. In some embodiments, the FRB polypeptide comprises an amino acid sequence that has at least 90%, 95%, 96%, 97%, 98%, 99% identity with or includes the sequence shown as SEQ ID NO:1. In some embodiments, the FRB polypeptide comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to or including the sequence set forth as SEQ ID NO: 2. In some embodiments, the FKBP12 polypeptide comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to or including the sequence set forth as SEQ ID NO: 3. In some embodiments, the FKBP12 polypeptide comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to or including the sequence set forth as SEQ ID NO: 4.

[0038] In some embodiments, the multimerization domains of the signaling component and the targeting component are associated with a cross-linking agent. In some embodiments, the cross-linking agent is 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 wherein 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 cross-linking agent is AP1903, AP20187, AP21967 (also known as C16-(S)-7-methylindole rapamycin), everolimus, novolimus, pimecrolimus, ridaforolimus, sirolimus, tacrolimus, temsirolimus, umirolimus, zotarolimus, or BPC015. In some embodiments, the first polypeptide linker is a 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 second polypeptide linker is a 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 anti-CLL1 scFv or sdAb and the anti-CD33 scFv or sdAb are separated by a third polypeptide linker. In some embodiments, the third polypeptide linker is a 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, and any combination thereof. In one embodiment, the third polypeptide linker is a G4S linker.

[0039] In some embodiments, 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. In one embodiment, the CD4 transmembrane polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 45. In one embodiment, the truncated intracellular CD4 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 48. In one embodiment, the truncated intracellular CD4 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 49.

[0040] In some embodiments, the anti-CLL1 antibody and the anti-CD33 antibody are each sdAbs. In some embodiments, the sdAbs are Camelid VHHs, nanobodies, or heavy chain only antibodies (HcAbs). In one embodiment, the sdAbs are Camelid VHHs. In some embodiments, the scFvs or sdAbs are human or humanized. In some embodiments, the anti-CLL1 scFvs or sdAbs comprise the CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 92, 93, and 94, respectively. In some embodiments, the anti-CLL1 scFvs or sdAbs comprise a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 75. In some embodiments, the anti-CLL1 scFvs or sdAbs comprise a sequence shown in SEQ ID NO: 75. In some embodiments, the anti-CD33 scFv or sdAb comprises the CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 89, 90, and 91, respectively. In some embodiments, the anti-CD33 scFv or sdAb comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 72. In some embodiments, the anti-CD33 scFv or sdAb comprises the sequence set forth in SEQ ID NO: 72.

[0041] In some embodiments, the signaling moiety further comprises a signal sequence. In some embodiments, the signal sequence is a CD8 signal sequence. In some embodiments, 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 one embodiment, the CD8 signal sequence comprises the amino acid sequence set forth as SEQ ID NO:96. In some embodiments, the targeting moiety further comprises a signal sequence. In some embodiments, the signal sequence is an IgK signal sequence. In some embodiments, 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. In some embodiments, the signaling moiety comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:111. In one embodiment, the signaling moiety comprises the sequence set forth as SEQ ID NO:111. 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: 122. In one embodiment, the targeting moiety comprises a sequence set forth as SEQ ID NO: 122. In some embodiments, the polypeptide complex 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: 151. In one embodiment, the fusion polypeptide comprises a sequence set forth as SEQ ID NO: 151.

[0042] In one aspect, the disclosure provides a nucleic acid molecule encoding both the signaling and targeting components of any one of the polypeptide complexes disclosed herein.

[0043] In another aspect, the disclosure provides a cell comprising any one of the polypeptide complexes disclosed herein.

[0044] In another aspect, the disclosure provides a cell comprising any one of the nucleic acid molecules disclosed herein.

[0045] In some embodiments, the cells further express an exogenous costimulator, an immune modulator, an agonist for a costimulator, an antagonist for an immune suppressor, 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 embodiments, 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), in melanoma The exogenous TCR binds to a target antigen selected from the group consisting of preferentially expressed antigen in sarcoma (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.

[0046] In some embodiments, the cell further expresses a CAR, CCR, or flip receptor. In some embodiments, the cell further expresses a zetakine, an immune cell engager, or a BiTE. 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 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.

[0047] In one aspect, the disclosure provides a polynucleotide encoding the signaling component and the targeting component of any one of the fusion polypeptides or polypeptide complexes disclosed herein.

[0048] In another aspect, the disclosure provides a cDNA encoding the signaling component and the targeting component of any one of the fusion polypeptides or polypeptide complexes disclosed herein.

[0049] In another aspect, the disclosure provides an RNA encoding any one of the signaling moieties and targeting moieties, or any one of the fusion polypeptides, or any one of the polypeptide complexes disclosed herein.

[0050] In another aspect, the disclosure provides a vector comprising any one of the polynucleotides disclosed herein.

[0051] In some embodiments, the vector is an expression vector. In one embodiment, the vector is a transposon. In some embodiments, the vector is a piggyBAC transposon or a Sleeping Beauty transposon. In some embodiments, the vector is a viral vector. In some embodiments, the vector is an adenovirus vector, an adeno-associated virus (AAV) vector, a herpes virus vector, a vaccinia virus vector, or a retrovirus vector. In some embodiments, the retrovirus vector is a lentivirus vector. In some embodiments, the lentivirus 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).

[0052] In one aspect, the disclosure provides a cell comprising any one of the fusion polypeptides, polynucleotides, or vectors disclosed herein.

[0053] In one embodiment, the cell is a hematopoietic cell. In some embodiments, 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.

[0054] In one aspect, the disclosure provides a composition comprising any one of the cells or vectors disclosed herein.

[0055] In another aspect, the disclosure provides a composition comprising a physiologically acceptable carrier and any one of the cells or vectors disclosed herein.

[0056] In another 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.

[0057] In another 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.

[0058] In another aspect, the present disclosure provides a method of treating a solid tumor comprising administering to a subject an effective amount of any one of the compositions disclosed herein.

[0059] 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.

[0060] In one aspect, the 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 description of the drawings]

[0061] [Figure 1A]Figures 1A-1H show the structures of representative engineered immune receptors and fusion constructs. In particular, Figures 1A-1C depict the 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, including a signaling component and a targeting component, complexes 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 includes a signaling component including an FRB multimerization domain, a linker, and a CD3ε actuator domain; and a targeting component including a CD4 transmembrane domain, a CD4 hinge, an FKBP multimerization domain, a linker, and a CD33 VHH targeting domain. In the presence of a cell expressing CD33, the CD33 VHH targeting domain binds to the target cell and comes into close contact with an immune cell expressing an engineered immune receptor, e.g., a T cell. In the presence of a cross-linking agent, the multimerization domain then interacts, 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. [Diagram 2] FIG. 2 shows the percent positive and median fluorescence intensity of SR024 components on T cells as measured by flow cytometry. [Diagram 3] FIG. 3 shows IFNγ, IL2, IL4, and TNF secretion in the presence of rapamycin from non-transduced and SR024-T cells co-cultured with high CD33 expressing cancer cell lines. [Figure 4]FIG. 4 shows IFNγ secretion from non-transduced and SR024-T cells cultured alone, with and without rapamycin. [Diagram 5] 5A and 5B show the cytotoxicity and proliferation, respectively, of control T cells and SR024 T cells, 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] FIG. 7A shows IFNγ, IL2, IL4, and TNF secretion from untransduced SR020-T cells, SR022-T cells, and SR024-T cells, each co-cultured with a high CD33-expressing cancer cell line, in the presence of rapamycin. [Figure 7B] FIG. 7B shows IFNγ secretion from non-transduced, 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] FIG. 7C shows IL2 secretion from non-transduced, 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 non-transduced, 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, respectively, of control, SR020-T cells, SR022-T cells, and SR024-T cells, 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 non-transduced and SR021-T cells co-cultured with high CD33 expressing cancer cell lines in the presence of rapamycin. [Figure 12] FIG. 12 shows IFNγ secretion from non-transduced and SR021-T cells co-cultured with high CD33 expressing cancer cell lines, 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 non-transduced, 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 non-transduced, 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, respectively, of control and SR001-T cells, 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 non-transduced and SR028-T cells co-cultured with high CD33 expressing cancer cell lines, in the presence of rapamycin. [Figure 20] FIG. 20 shows IFNγ secretion from non-transduced and SR028-T cells co-cultured with high CD33 expressing cancer cell lines, with and without rapamycin. [Figure 21]Figures 21A and 21B show the cytotoxicity and proliferation, respectively, of control and SR028-T cells, 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. [Diagram 23] FIG. 23 shows IFNγ, IL2, IL4, and TNF secretion from non-transduced, 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. [Diagram 25] Figures 25A and 25B show the cytotoxicity and proliferation, respectively, of control cells, SR004- and SR006-T cells, each co-cultured with a high CD33-expressing cancer cell line. [Fig. 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. [Fig. 26C-D] 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 non-transduced cells, SR008-T cells, and SR030-T cells, each co-cultured with either high CD33-expressing or high CLL1-expressing cancer cell lines, 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 non-transduced cells, SR008-T cells, and SR030-T cells, each co-cultured with high CD33-expressing or high CLL1-expressing cancer cell lines, 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. [Diagram 30] FIG. 30 shows IFNγ, IL2, IL4, and TNF secretion from non-transduced, SR001- and SR001-28-T cells, each co-cultured with a high CD33-expressing cancer cell line, in the presence of rapamycin. [Diagram 31] Figures 31A and 31B show the cytotoxicity and proliferation, respectively, of control cells, SR001- and SR001-28-T cells, each co-cultured with a high CD33-expressing cancer cell line. [Fig. 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 regulated by rapamycin. [Fig. 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. [Diagram 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. [Diagram 34] FIG. 34 shows IFNγ and IL2 secretion from non-transduced 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. [Fig. 35ABC]Figures 35A-35C show vector copy number, percent positive, and geometric mean fluorescence intensity of tetramer and TCR beta chain staining for non-transduced TCR, transgenic TCR, SR001, or a combination of transgenic TCR and SR001, as measured by flow cytometry. [Fig. 35DE] Figures 35D and 35E show the percent positive and geometric mean fluorescence intensity of anti-VHH staining for non-transduced 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 non-transduced, 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 non-transduced, 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 non-transduced 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. [Diagram 40] Figures 40A and 40B show IFNγ secretion in SR10167 or SR10168-T cells alone ± rapamycin compared to regulatory CAR controls. [Diagram 41] Figures 41A and 41B show IFNγ secretion of SR10167 or SR10168-T cells ± rapamycin compared to regulatory CAR control when cultured with a CD19+ target cell line. [Fig. 42AB] Figures 42A-42D show IL2 and TNFα secretion of SR10167 or SR10168-T cells ± rapamycin compared to regulatory CAR controls when cultured with CD19+ target cell lines. [Fig. 42CD] Same as above. [Diagram 43] Figures 43A and 43B show the cytotoxicity of control, SR10167-T cells, and CAR-regulated T cells, each co-cultured with either CD19+ Jeko-1 or Daudi cell lines. All tumor controls are regulated by rapamycin. [Diagram 44] FIG. 44 shows schematics of representative constitutively active (non-regulatable) engineered immune receptor and fusion constructs. [Diagram 45] Figures 45A and 45B show the percent positive and median fluorescence intensity of VHH staining for expression of SR292, SR293, SR296, SR001, and related comparator molecules 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 comparison molecules including non-regulatory CAR, regulatory CAR, and non-regulatory TCR based structural comparisons when cultured with CD33+ target cell lines. [Figure 47] FIG. 47 shows IFNγ secretion from 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 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 SR001±IL7 receptor alpha, common gamma chain, IL2 receptor beta, or both common gamma chain and IL2 receptor beta expression. [Fig. 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. [Fig. 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] FIG. 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). [Fig. 53AB] Figures 53A-53D show the cytotoxicity of T cells expressing SR300 or SR301 when cultured with PDL1+ target cell lines or the same cell lines with knocked-out PDL1 and PDL2, with and without rapamycin. [Fig. 53CD] Same as above. [Figure 54] FIG. 54 shows the percent positive and median fluorescence intensity of VHH staining for expression of the SR354 construct. [Fig. 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. [Fig. 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] FIG. 57C shows IFNγ secretion by T cells alone expressing the SR303 molecule. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0062] 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.

[0063] In the above sequences, X refers to any amino acid, if present, or to the absence of an amino acid.

[0064] A. Overview Cancer is one of the leading causes of death worldwide. Although adoptive cell therapy has been used successfully to treat some hematological malignancies, treatment of solid tumors with both chimeric antigen receptor (CAR) T cells and T cells expressing T cell receptors (TCR) against tumor antigens remains largely ineffective.

[0065] 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 be persistent 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 show poor activation of T cell signaling pathways.

[0066] 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 lethal cytokine release. CRS can result in dangerously high fever, extreme fatigue, difficulty breathing, and a sudden drop in blood pressure. CRS can also produce a second wave of side effects, including neurotoxicity, tremors, headaches, confusion, loss of balance, difficulty speaking, seizures, and hallucinations, in the nervous system. Insufficient activation of T cell signaling pathways can lead to 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 that plague adoptive cell therapy.

[0067] 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.

[0068] Without wishing to be bound by any particular theory, the engineered immune receptor compositions and methods contemplated herein offer numerous advantages over CAR T cell and TCR T cell therapies existing 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, the temporal control primes the engineered immune receptor machinery for signaling through cross-linking agent-mediated association of the targeting moiety to the signaling moiety. In other embodiments, the machinery is primed by association of the multimerization domain without the need for a cross-linking agent. The spatial control engages the signaling machinery through recognition of the target antigen by the extracellular domain or targeting domain of the targeting moiety, whereas the signaling moiety includes an actuator domain that forms a complex with a lymphocyte immune receptor. In this manner, the immune effector cell activates receptor signaling when a target cell expressing the target antigen is present, and in some embodiments, when a cross-linking agent is present.

[0069] 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, of the target antigen.

[0070] 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., 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.

[0071] In certain embodiments, the multimerization domains of the signaling component and the targeting component are located extracellularly. The extracellular location of the multimerization domain offers a number of advantages over an 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.

[0072] 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 that contain and / or express 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 a disease or disorder (e.g., cancer or an immune disorder).

[0073] 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 references 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. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach and W. Strober, eds., 1991); Annual Review of Immunology; as well as research articles in journals such as, for example, Advances in Immunology.

[0074] B. Definition Before describing the present disclosure in more detail, it may be helpful to an understanding thereof to provide definitions of certain terms that will be used herein.

[0075] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled 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.

[0076] 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 one or more) of the grammatical object of the article. By way of example, "an element" means one element or one or more elements.

[0077] The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination of the alternatives.

[0078] The term "and / or" should be understood to mean either one or both of the alternatives.

[0079] 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 a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length relative to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0080] In one embodiment, a range, for example, 1 to 5, about 1 to 5, or about 1 to about 5, refers to every number encompassed by the range. For example, in one non-limiting and merely illustrative embodiment, the range "1 to 5" is equivalent to: 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.

[0081] 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 the quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length of a reference. 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 amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length of a reference.

[0082] Throughout this specification, unless the context requires otherwise, the words "comprise" and "comprising" are to be understood as implying 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 recited after this phrase, and any elements limited to other elements that do not interfere with or contribute to the activity or action 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 action of the recited elements.

[0083] 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 association with this embodiment is included in at least one embodiment. As such, the appearances of such phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the 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 a particular embodiment.

[0084] As used herein, the term "TCR complex" refers to a complex formed by the association of CD3 with a TCR. For example, a TCR complex may be composed of a CD3 gamma chain, a CD3 delta chain, two CD3 epsilon chains, a homodimer of a CD3 zeta chain, a TCR alpha chain, and a TCR beta chain. In some embodiments, a TCR complex may be composed of a CD3 gamma chain, a CD3 delta chain, two CD3 epsilon chains, a homodimer of a CD3 zeta chain, a TCR gamma chain, and a TCR delta chain.

[0085] As used herein, a "component of a TCR complex" refers to a complex formed by a TCR chain (i.e., TCRα, TCRβ, TCRγ, or TCRδ), a CD3 chain (i.e., CD3γ, CD3δ, CD3ε, or CD3ζ), or 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).

[0086] As used herein, "actuator polypeptide," "actuator domain," or "actuator" refers to a polypeptide that associates, integrates, or complexes, either directly or indirectly, with a multimeric immune receptor complex to promote signaling, and does not itself contain direct antigen binding properties. In certain embodiments, the actuator domain is part of a protein or protein complex that signals when bound to a target molecule. The actuator domain may 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, the actuator domain directly signals, 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.

[0087] 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 dimerizing agent, where the interaction of the distinct multimerization domains significantly contributes to or efficiently drives 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 any source, natural, synthetic, semi-synthetic, or recombinant.

[0088] Illustrative examples of multimerization domains suitable for use in certain embodiments contemplated herein include an FK506 binding protein (FKBP) polypeptide or a variant thereof, an FKBP-rapamycin binding (FRB) polypeptide or a variant thereof, a calcineurin polypeptide or a variant thereof, a cyclophilin polypeptide or a variant thereof, a bacterial dihydrofolate reductase (DHFR) polypeptide or a variant thereof, a PYR1-like 1 (PYL1) polypeptide or a variant thereof, an abscisic acid insensitive 1 (ABI1) polypeptide or a variant thereof, a GIB1 polypeptide or a variant thereof, or a GAI polypeptide or a variant thereof.

[0089] 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 comprise 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, with reference to GenBank Accession No. L34075.1, and a T2098L mutation (T82L is the equivalent position in the 93 amino acid FRB polypeptide). As used herein, the term "FRB star", FRB star", or "FRB*" refers to such an FRB T2098L (T82L) mutant. The FRB polypeptides contemplated herein bind to FKBP polypeptides via a cross-linking agent, thereby forming a ternary complex.

[0090] 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 an F36V mutation. In some embodiments, the 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 agents, thereby forming a ternary complex.

[0091] A "cross-linking agent" refers to a molecule that associates with and is disposed between two or more multimerization domains. In certain embodiments, the multimerization domains contribute significantly or efficiently promote the formation of a polypeptide complex only in the presence of the cross-linking agent. In certain embodiments, the multimerization domains do not contribute significantly 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, synthetic ligand for trimethoprim (Tmp)-FKBP (SLF) or a derivative thereof, or any combination thereof.

[0092] 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 substantially reduced immunosuppressive effects compared to rapamycin. In a preferred embodiment, the rapalog is AP21967 (also known as C-16-(S)-7-methylindolerapamycin, IC 50 =10 nM, a chemically modified non-immunosuppressive rapamycin analogue). Other illustrative 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.

[0093] "Substantially reduced immunosuppressive effect" refers to an immunosuppressive effect that is at least 0.1 to 0.005 times less than the observed or predicted immunosuppressive effect 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.

[0094] 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.

[0095] 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 moiety, as disclosed herein, comprises at least one targeting domain, which, when the targeting moiety is associated with a signaling moiety, for example, by a bridging agent connecting the multimerization domains of the targeting moiety and the signaling moiety, or by association of the multimerization domains of the targeting moiety and the signaling moiety without the need for a bridging agent, redirects the 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.

[0096] The term "effector function" or "effector cell function" refers to a 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. "Intracellular signaling domain" or "endodomain" refers to a protein portion that transmits an effector function signal, instructing a 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.

[0097] It is known that signals generated through the TCR alone are insufficient for full activation of T cells, and that 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 that initiate antigen-dependent primary activation through the TCR (e.g., the TCR / CD3 complex), and costimulatory signaling domains that act in an antigen-independent manner to provide secondary or costimulatory signals.

[0098] 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 0 (DAP10), FYN, linker for activation of T cells family member 1 (LAT), SH2 domain-containing leukocyte protein of 76 kD (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 zeta chain of T cell receptor-associated protein kinase 70 (ZAP70).

[0099] "Hinge domain" refers to a polypeptide that plays a role in spatially positioning a domain away from the effector cell surface to allow for proper cell / cell contact, antigen binding and activation. In certain embodiments, a polypeptide may include 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. The hinge domain may be derived from any source, natural, synthetic, semi-synthetic, or recombinant. The hinge domain may include the amino acid sequence of a naturally occurring immunoglobulin hinge region or a modified immunoglobulin hinge region.

[0100] "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 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.

[0101] 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 NO: 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); KESGSVSSEQLAQFRSLD (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)).

[0102] "Spacer domain" refers to a polypeptide that separates two domains. In one embodiment, the spacer domain moves the targeting domain away from the effector cell surface to allow 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 any source, natural, synthetic, semi-synthetic, or recombinant. In certain embodiments, the spacer domain is a portion of an immunoglobulin that includes one or more heavy chain constant regions, e.g., CH2 and CH3. The spacer domain may include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an engineered immunoglobulin hinge region.

[0103] "Antigen (Ag)" refers to a compound, composition, or substance that can stimulate antibody production or a T-cell response in an animal, including compositions that are injected or absorbed into an animal (e.g., compositions that include a cancer-specific protein, etc.). 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.

[0104] "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.

[0105] 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 source, natural, synthetic, semi-synthetic, or recombinant. A targeting domain may include, for example, an antibody, or an antigen-binding fragment thereof, or, for example, an ectodomain.

[0106] 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 is specifically targeted to a target antigen, e.g., when it is about 10 5 M -1 Affinity greater than or equal to K a (i.e., the equilibrium binding constant for a particular binding interaction, with units of 1 / M). In one embodiment, a targeting domain (or a fusion protein comprising the same) binds to a target with a specific 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 greater a It refers to a targeting domain with

[0107] 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.

[0108] "Antibody" refers to a binding agent that is a polypeptide comprising at least a light or heavy 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 antigenic determinants, such as those recognized by immune cells.

[0109] "Epitope" or "antigenic determinant" refers to a region of an antigen to which a binding agent binds.

[0110] 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 involved in 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.

[0111] As used herein, the term "cancer" generally relates to certain diseases or conditions in which abnormal cells divide uncontrollably and can invade nearby tissues.

[0112] As used herein, the term "malignant" refers to a cancer in which tumor cells exhibit one or more of the following: uncontrolled proliferation (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 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 referred to as a "metastatic tumor" or "metastatic cancer." A metastatic tumor contains cells similar to those in the original (primary) tumor.

[0113] As used herein, the terms "benign" or "non-malignant" refer to tumors that may grow large but do not spread to other parts of the body. Benign tumors are self-limited and often do not invade or metastasize.

[0114] "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 swelling or lesion formed by 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" and can be measured as the number, volume, or weight of tumors.

[0115] The term "recurrence" refers to the diagnosis of a return of cancer, or the diagnosis of returning signs and symptoms of cancer, after a period of improvement or remission.

[0116] "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, but cancer may still be present in the body.

[0117] "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 initiation of treatment (i.e., unresponsive to initial exposure to the therapeutic agent), or over the course of an initial treatment period, or during a subsequent treatment period.

[0118] "Antigen negative" refers to cells that do not express an antigen or that express an antigen in such small amounts that it cannot be detected. In one embodiment, an antigen negative cell does not bind to the target receptor for the antigen. In one embodiment, an antigen negative cell does not substantially bind to the target receptor for the antigen.

[0119] 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 having, diagnosed with, or at risk of having cancer or another immune disorder.

[0120] 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.

[0121] 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 may 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.

[0122] 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 occurrence or recurrence of a disease or condition, or delaying the occurrence 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 occurrence or recurrence of the disease or condition.

[0123] 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 that are 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, bone fractures, unexpected weight loss, loss of appetite, night sweats, persistent low-grade fever, and decreased urination (caused by impaired kidney function).

[0124] "Enhance" or "promote" or "increase" or "proliferate" generally refers to the ability of the compositions 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, increased T cell proliferation, activation, persistence, cytokine secretion, and / or cancer cell killing capacity, as will be apparent from an understanding of the art and the description 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 decimals 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.

[0125] By "reduce" or "lower" or "lower" or "reduce" or "attenuate" generally refers to the ability of the compositions contemplated herein to produce, induce, or cause a physiological response (i.e., downstream effect) that is smaller than the response caused by a solvent or control molecule / composition. The amount of "reduction" or "reduction" is typically a "statistically significant" amount and may 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 decimals 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 lineage.

[0126] "Maintain" or "preserve" or "maintain" or "no change" or "no substantial change" or "no substantial decrease" generally refers to the ability of the compositions 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 lineage. An equivalent response is one that is not significantly or measurably different from the reference response.

[0127] Additional definitions are set forth throughout this disclosure.

[0128] 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 term "engineered immune receptor," "engineered immune receptor complex," or "engineered immune receptor system" refers 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 immune stimulatory 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 that includes 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.

[0129] 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.

[0130] 1. Signal transduction components "Signaling component" refers to a polypeptide comprising one or more multimerization domains (e.g., an FRB polypeptide or an FKBP polypeptide) or a variant thereof, and a CD3ε domain / polypeptide, or a functional fragment or variant thereof, capable of recruiting an immune receptor complex (e.g., a TCR complex).

[0131] In various embodiments, the CD3ε peptide comprises 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 CD3ε peptide comprises 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 CD3ε peptide comprises 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 CD3ε peptide comprises 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 CD3ε peptide comprises 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 CD3ε peptide comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 32, or a functional fragment or variant thereof. In certain embodiments, the CD3ε peptide comprises the amino acid sequence shown in SEQ ID NO: 32.

[0132] In various embodiments, the CD3ε polypeptide is a human CD3ε polypeptide.

[0133] In certain embodiments, the signaling component comprises an FRB or FRB T2098L multimerization domain / polypeptide, a linker polypeptide, and a CD3ε polypeptide.

[0134] 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 certain embodiments, the signaling component comprises the amino acid sequence set forth in SEQ ID NO: 111.

[0135] In various embodiments, the multimerization domain (eg, FRB or FKBP) is localized extracellularly when the signaling component is expressed.

[0136] 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 having at least 90% identity to SEQ ID NO:1. In certain embodiments, the FRB polypeptide comprises an amino acid sequence having at least 95% identity to SEQ ID NO:1. In certain embodiments, the FRB polypeptide comprises an amino acid sequence having at least 96% identity to SEQ ID NO:1. In certain embodiments, the FRB polypeptide comprises an amino acid sequence having at least 97% identity to SEQ ID NO:1. In certain embodiments, the FRB polypeptide comprises an amino acid sequence having at least 98% identity to SEQ ID NO:1. In certain embodiments, the FRB polypeptide comprises an amino acid sequence having at least 99% identity to SEQ ID NO:1. In certain embodiments, the FRB polypeptide comprises an amino acid sequence set forth in SEQ ID NO:1. In certain embodiments, the FRB polypeptide comprises an amino acid sequence set forth in SEQ ID NO:2. In certain embodiments, the FRB polypeptide comprises an amino acid sequence having at least 90% identity 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 shown in SEQ ID NO:2.

[0137] 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 having at least 90% identity to SEQ ID NO:3. In one embodiment, the FKBP12 polypeptide comprises an amino acid sequence having at least 95% identity to SEQ ID NO:3. In one embodiment, the FKBP12 polypeptide comprises an amino acid sequence having at least 96% identity to SEQ ID NO:3. In one embodiment, the FKBP12 polypeptide comprises an amino acid sequence having at least 97% identity to SEQ ID NO:3. In one embodiment, the FKBP12 polypeptide comprises an amino acid sequence having at least 98% identity to SEQ ID NO:3. In one embodiment, the FKBP12 polypeptide comprises an amino acid sequence having at least 99% identity 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. 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 particular embodiment, the FKBP12 polypeptide comprises the amino acid sequence shown in SEQ ID NO:4.

[0138] In various embodiments, the first multimerization domain (e.g., FRB or FKBP) and the CD3ε domain / polypeptide 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, links 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 a sequence set forth as SEQ ID NO: 16. In one embodiment, the linker has a sequence set forth as SEQ ID NO: 17. In one embodiment, the linker has a sequence set forth as SEQ ID NO: 18. In one embodiment, the linker has a sequence set forth as SEQ ID NO: 19. In one embodiment, the linker has a sequence set forth as SEQ ID NO: 20. In one embodiment, the linker has a sequence set forth as SEQ ID NO: 21. In one embodiment, the linker has a sequence set forth as SEQ ID NO: 22. In one embodiment, the linker has the sequence shown as SEQ ID NO: 23. In one embodiment, the linker has the sequence shown as SEQ ID NO: 24. In one embodiment, the linker has the sequence shown as SEQ ID NO: 25.In one embodiment, the linker has the sequence shown as SEQ ID NO: 26. In one embodiment, the linker has the sequence shown as SEQ ID NO: 27. In one embodiment, the linker has the sequence shown as SEQ ID NO: 28. In one embodiment, the linker has the sequence shown as SEQ ID NO: 29. In one embodiment, the linker has the sequence shown as SEQ ID NO: 30. In one embodiment, the linker has the sequence shown as SEQ ID NO: 31.

[0139] In various embodiments, the signaling components contemplated herein include a signal peptide, e.g., a secretory signal polypeptide or signal sequence. Illustrative examples of suitable signal peptides / sequences for use in a particular signaling component 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, the signaling component includes an Igκ light chain signal polypeptide. In some embodiments, the Igκ light chain signal polypeptide includes the amino acid sequence set forth in SEQ ID NO:95. In various embodiments, the signaling component includes a CD8α signal polypeptide. In some embodiments, the CD8α light chain signal polypeptide includes the amino acid sequence set forth in SEQ ID NO:96. In various embodiments, the signaling component includes a PD1 signal polypeptide. In one embodiment, the PD1 signal polypeptide includes the amino acid sequence set forth as SEQ ID NO:97.

[0140] In certain embodiments, the signaling component comprises a FRB polypeptide or variant thereof, a 3xG4S linker, and a CD3ε polypeptide or variant thereof. In certain embodiments, the signaling component comprises a signaling sequence, a FRB polypeptide or variant thereof, a 3xG4S linker, and a CD3ε polypeptide or variant thereof. In certain embodiments, the signaling component comprises a CD8 signaling sequence, a FRB* polypeptide or variant thereof, a 3xG4S linker, and a CD3ε polypeptide or variant thereof. In certain embodiments, the signaling component comprises a CD8 signaling sequence (e.g., as shown in SEQ ID NO:96), a FRB* polypeptide or variant thereof (e.g., as shown in SEQ ID NO:1), a 3xG4S linker (e.g., as shown in SEQ ID NO:13), and a CD3ε polypeptide (e.g., as shown in SEQ ID NO:32).

[0141] 2. Targeting component "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.

[0142] In certain embodiments, the targeting moiety comprises an extracellular domain comprising at least one targeting domain, where 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' 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 (e.g., sdAb, camelid VHH, or nanobodies).

[0143] 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.

[0144] The present disclosure provides a platform technology that is applicable to multiple classes and fragments of targeting domains and should not be limited to one particular targeting domain, e.g., antibodies, antigen-binding fragments, ectodomains, etc., for targeting a particular target antigen.

[0145] 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 CD33 target antigen and / or a C-type lectin-like molecule-1 (CLL-1) target antigen. In various embodiments, the targeting moiety comprises an extracellular domain that includes a second targeting domain.

[0146] 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.

[0147] In certain embodiments, the extracellular domain contemplated comprises a second targeting domain that binds to a CD33 target antigen and / or a C-type lectin-like molecule-1 (CLL-1) target antigen. 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. 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 shown as SEQ ID NO:72.

[0148] In one embodiment, the targeting domain is a CLL1 targeting domain. 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. 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 a sequence set forth as SEQ ID NO: 75.

[0149] 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.

[0150] 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.

[0151] In one embodiment, the second 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 first 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.

[0152] 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, 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, and any combination thereof.

[0153] In certain embodiments, the targeting moiety comprises one or more multimerization domains.

[0154] 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 a variant thereof, an FKBP-rapamycin binding (FRB) polypeptide or a variant thereof, a calcineurin polypeptide or a variant thereof, a cyclophilin polypeptide or a variant thereof, a bacterial dihydrofolate reductase (DHFR) polypeptide or a variant thereof, a PYR1-like 1 (PYL1) polypeptide or a variant thereof, and an abscisic acid insensitive 1 (ABI1) polypeptide or a variant thereof.

[0155] 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 shown in SEQ ID NO:1. In certain embodiments, the FRB polypeptide comprises the amino acid sequence shown in SEQ ID NO:2.

[0156] 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 shown in SEQ ID NO:3. In certain embodiments, the FKBP12 polypeptide comprises the amino acid sequence shown in SEQ ID NO:4.

[0157] Other illustrative examples of suitable multimerization domains for use in certain targeting moieties contemplated herein include antibody-derived heterodimerization domains. Such antibody heterodimerization domains include, but are not limited to, lock-and-lock, 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).

[0158] In various embodiments, the extracellular domain or the 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, links 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.

[0159] In various embodiments, the second multimerization domain and the transmembrane domain are separated by a hinge domain. 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% identity to SEQ ID NO: 41. In some embodiments, the CD4 hinge comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 41. In some embodiments, the CD4 hinge comprises an amino acid sequence having at least 96% identity to SEQ ID NO: 41. In some embodiments, the CD4 hinge comprises an amino acid sequence having at least 97% identity to SEQ ID NO: 41. In some embodiments, the CD4 hinge comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 41. In some embodiments, the CD4 hinge comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 41. In some embodiments, the CD4 hinge comprises the amino acid sequence set forth in SEQ ID NO: 41.

[0160] In some embodiments, the targeting moiety comprises a linker comprising the GGR amino acid sequence. In some embodiments, the targeting moiety comprises a linker comprising the amino acid sequence shown in SEQ ID NO:13 or SEQ ID NO:14.

[0161] In certain embodiments, the targeting moiety comprises a transmembrane domain.

[0162] Illustrative examples of suitable transmembrane domains 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, CD 16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD71, CD80, CD86, CD 134, CD137, CD152, CD 154, 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.

[0163] 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 shown in SEQ ID NO: 45.

[0164] 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 shown 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: 49. 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 shown in SEQ ID NO: 49.

[0165] In certain embodiments, the targeting moiety does not comprise a primary signaling domain (e.g., CD3z). In various embodiments, the targeting moiety does not comprise an intracellular signaling or costimulatory domain.

[0166] 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 shown in SEQ ID NO: 48. In some embodiments, the truncated intracellular CD4 polypeptide comprises the amino acid sequence shown in SEQ ID NO: 49.

[0167] In a preferred embodiment, the targeting moiety comprises a first targeting domain comprising a VHH or scFv that binds CLL1, a linker polypeptide, a second targeting domain comprising a VHH or scFv that binds CD33, a linker polypeptide, a FRB or FKBP12 multimerization domain, a CD4 hinge, a CD4 transmembrane domain, and an extracellular domain comprising a truncated CD4ic domain. In a preferred embodiment, the targeting moiety comprises a first targeting domain comprising a VHH or scFv that binds CD33, a linker polypeptide, a second targeting domain comprising a VHH or scFv that binds CLL1, a linker polypeptide, a FRB or FKBP12 multimerization domain, a CD4 hinge, a CD4 transmembrane domain, and an extracellular domain comprising a truncated CD4ic domain.

[0168] 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.

[0169] In various embodiments, the targeting moieties contemplated herein include a signal peptide, e.g., a secretory signal polypeptide or sequence. Illustrative examples of suitable signal polypeptides / sequences for use in a particular targeting moiety 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.

[0170] In various preferred embodiments, the targeting moiety comprises an Ig κ light chain signal polypeptide. In some embodiments, the Ig κ light chain signal polypeptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO:95. In some embodiments, the Ig κ light chain signal polypeptide comprises an amino acid sequence having at least 95% identity to SEQ ID NO:95. In some embodiments, the Ig κ light chain signal polypeptide comprises an amino acid sequence having at least 96% identity to SEQ ID NO:95. In some embodiments, the Ig κ light chain signal polypeptide comprises an amino acid sequence having at least 97% identity to SEQ ID NO:95. In some embodiments, the Ig κ light chain signal polypeptide comprises an amino acid sequence having at least 98% identity to SEQ ID NO:95. In some embodiments, the Ig κ light chain signal polypeptide comprises an amino acid sequence having at least 99% identity to SEQ ID NO:95. In some embodiments, the Ig κ light chain signal polypeptide comprises the amino acid sequence set forth in SEQ ID NO:95.

[0171] In certain embodiments, the targeting moiety comprises a CLL1 targeting domain, a 3xG4S linker, a CD33 targeting domain, a 3xG4S linker, an FKBP polypeptide or variant thereof, a CD4 hinge domain, a CD4 transmembrane domain, and a truncated intracellular CD4 polypeptide. In certain embodiments, the targeting moiety comprises a signaling sequence, a CLL1 targeting domain, a 3xG4S linker, a CD33 targeting domain, a 3xG4S linker, an FKBP polypeptide or variant thereof, a CD4 hinge domain, a CD4 transmembrane domain, and a truncated intracellular CD4 polypeptide. In certain embodiments, the targeting moiety comprises an IgK signaling sequence, a CLL1 VHH targeting domain, a 3xG4S linker, a CD33 VHH targeting domain, a 3xG4S linker, an FKBP polypeptide or variant thereof, a CD4 hinge domain, a CD4 transmembrane domain, and a truncated intracellular CD4 polypeptide. In certain embodiments, the targeting moiety comprises an IgK signaling sequence (e.g., as shown in SEQ ID NO: 95), a CLL1 VHH targeting domain (e.g., comprising a CDR1 represented as SEQ ID NO: 92, a CDR2 represented as SEQ ID NO: 93, and a CDR2 represented as SEQ ID NO: 94; and / or a VHH represented as SEQ ID NO: 75), a 3xG4S linker (e.g., as shown in SEQ ID NO: 13), a CD33 VHH targeting domain (e.g., comprising a CDR1 represented as SEQ ID NO: 89, a CDR2 represented as SEQ ID NO: 90, a CDR3 represented as SEQ ID NO: 91; and / or a VHH comprising SEQ ID NO: 72), a 3xG4S linker (e.g., as shown in SEQ ID NO: 13), an FKBP polypeptide or a variant thereof (e.g., as shown in SEQ ID NO: 3), a CD4 hinge domain (e.g., as shown in SEQ ID NO: 41), a CD4 transmembrane domain (e.g., as shown in SEQ ID NO: 45), and a truncated intracellular CD4 polypeptide (e.g., as shown in SEQ ID NO: 48 or SEQ ID NO: 49).

[0172] 3. Crosslinking factor The cross-linking agent contemplated in certain embodiments herein 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 disposed 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 initiate immune effector cell activity against the target cell when the targeting component is bound to a target antigen on the target cell. In the absence of the cross-linking agent, the targeting component does not associate with the signaling component, does not recruit the immune receptor complex, and the engineered receptor is inactive against the target antigen of the targeting component.

[0173] 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).

[0174] In certain 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.

[0175] In certain embodiments, the signaling moiety and the targeting moiety comprise one or more FRB and / or FKBP multimerization domains or variants thereof. In certain embodiments, the signaling moiety comprises an FKBP12 multimerization domain or variants thereof, and the targeting moiety comprises an FRB multimerization domain or variants thereof. In certain embodiments, the signaling moiety comprises an FRB multimerization domain or variants thereof, and the targeting moiety comprises an FKBP12 multimerization domain or variants thereof. In particularly preferred embodiments, the signaling moiety comprises an FKBP12 or FKBP12 F36V multimerization domain or variants thereof, and the targeting moiety comprises an FRB T2098L multimerization domain or variants 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.

[0176] 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).

[0177] D. Antigen Receptors and Binding Molecules In certain embodiments, the immune effector cells contemplated herein further express an exogenous lymphocyte receptor or an engineered antigen receptor, and one or more components of the 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 zetakines, or a flip receptor. In some embodiments, the immune effector cells contemplated herein further express an exogenous costimulator, an immune modulator, an agonist for a costimulator, an antagonist for an immune suppressor, an immune cell engager, or any combination thereof. In some embodiments, the immune effector cells contemplated herein further express a BiTE.

[0178] In various embodiments, the exogenous lymphocyte receptor or engineered antigen receptor is an engineered αβ or γδ T cell receptor (TCR), a chimeric antigen receptor (CAR), or a 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, the immune effector cells contemplated herein comprise engineered / exogenous TCRs and engineered immune receptor components as 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 subunits) 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.

[0179] In one embodiment, T cells are engineered by introducing a polynucleotide or vector encoding an engineered antigen receptor (e.g., TCR, CAR, or CCR) and one or more components of an 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 an 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.

[0180] Natural T cell receptors contain two subunits, alpha and beta chain subunits (αβTCR) or gamma and delta chain subunits (γδTCR), each of which is a unique protein produced by a recombination event in the genome of each T cell. Libraries of TCRs may be screened for selectivity against a particular target antigen. In this way, natural TCRs with high avidity and reactivity against the target antigen are selected, cloned, and then introduced into a T cell population to be used for adoptive immunotherapy. In one embodiment, the TCR is an αβ TCR. In one embodiment, the TCR is a γδ TCR.

[0181] In one embodiment, T cells are modified 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 the 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 directed to target cells, and participates in immunologically relevant cytokine signaling. The engineered TCR also preferably binds with high avidity to target cells displaying a relevant peptide (e.g., a tumor-associated peptide), and optionally mediates efficient killing of target cells presenting the relevant peptide in vivo.

[0182] The nucleic acids encoding the engineered TCRs are preferably isolated from their natural content in the (naturally occurring) chromosomes of the 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 a cell, preferably a T cell in certain embodiments. The modified T cell can then express one or more chains of the TCR encoded by the transduced nucleic acid. In a preferred embodiment, the TCR is an exogenous TCR, since 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 to target antigens in an MHC-independent manner.

[0183] TCRs may be expressed with additional polypeptides attached to the amino- or carboxyl-terminal portions of the TCR alpha or beta chains, or the TCR gamma or delta chains, so long as the attached additional polypeptides do not interfere with the ability of the alpha or beta chains to form a functional T cell receptor and MHC-dependent antigen recognition.

[0184] Antigens recognized by the TCR contemplated in certain embodiments include cancer antigens, including, but not limited to, antigens on both hematological cancers and solid tumors. Illustrative antigens include, but are not limited to, alpha-fetoprotein (AFP), B-type melanoma antigen (BAGE) family members, brothers 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 (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) ), preferentially expressed antigen in melanoma (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).

[0185] In other embodiments, the immune effector cells contemplated herein comprise a CAR and an engineered immune receptor component as described herein. Chimeric antigen receptors (CARs) are molecules that combine antibody-based specificity for a target antigen (e.g., tumor antigen) with a T cell receptor activating intracellular domain to generate a chimeric protein that exhibits specific anti-tumor cell-mediated immune activity. As used herein, the term "chimeric" describes a molecule that is composed of different protein or DNA portions from different sources.

[0186] In other embodiments, immune effector cells contemplated herein comprise a CCR and an engineered immune receptor component as described herein. Unlike CARs, chimeric costimulatory receptors (CCRs) are molecules that combine antibody-based specificity for a desired antigen with a T cell receptor costimulatory domain, but lack a primary signaling domain (see, e.g., WO2020 / 252110 and WO2021 / 211948).

[0187] In other embodiments, immune effector cells contemplated herein express bispecific T cell engagers (BiTEs) and engineered immune receptor components as described herein. BiTEs are molecules that contain two different antibody fragments (e.g., single-chain variable fragments (scFvs)) connected by a small linker peptide, in which one antibody fragment binds to a component of the T cell receptor (TCR) complex (e.g., the CD3 chain) and the other antibody fragment binds to another antigen (e.g., a tumor-associated antigen).

[0188] In other embodiments, the immune effector cells contemplated herein comprise flip receptors and engineered immune receptor components described herein. Flip receptors comprise the extracellular portion of a receptor, the transmembrane domain, and the intracellular signaling portion of a different receptor. In this way, flip receptors "convert" the binding properties of one receptor type into the intracellular signaling events of another receptor type. See, e.g., WO2018 / 094244, WO2016 / 122738, WO2014 / 172584, and WO2012 / 138858.

[0189] In one embodiment, the CAR, CCR, immune cell engager, BiTE, or flip receptor 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 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 recognized by T cells 1 (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 CAR or CCR binds to a target antigen selected from the group consisting of: BCMA, CD33, CD20, CD79a, CD79b, CLL-1, IGF2BP3 / A3, MUC16, NY-ESO, PRAME, TACI, and TP53.

[0191] In various embodiments, immune effector cells contemplated herein comprise one or more chains of the zetakine receptors described herein and engineered immune receptor components. Zetakines are chimeric transmembrane immune receptors that comprise an extracellular domain that comprises a soluble receptor ligand linked to a support region capable of linking the extracellular domain to the cell surface, a transmembrane region, and an intracellular signaling domain. When zetakines are expressed on the surface of T lymphocytes, they direct T cell activity to cells expressing receptors specific for the soluble receptor ligand. Zetakine chimeric immune receptors redirect the antigen specificity of T cells, particularly via the autocrine / paracrine cytokine system utilized by human malignancies, with applications in the treatment of various cancers.

[0192] In other embodiments, immune effector cells are modified by introducing an Fc receptor (FcR) subunit capable of binding to a fragment crystallizable (Fc) portion / region of an antibody and an engineered immune receptor component as described herein. In some embodiments, the FcR subunit can be from an Fc gamma receptor, an Fc alpha receptor, or an Fc epsilon receptor. See, e.g., Mkaddem et al., Front Immunol. 2019 Apr 12;10:811.

[0193] In other embodiments, immune effector cells are modified by introducing an NKG2D receptor and an engineered immune receptor component as described herein.

[0194] E. Polypeptides A variety of polypeptides are contemplated herein, including, but not limited to, engineered immune receptor systems, targeting moieties, signaling moieties, immune receptors and their associated subunits or adapter molecules, exogenous lymphocyte receptors, engineered antigen receptors, TCRs, FcRs, CARs, CCRs, BiTEs, zetakines, flip receptors, exogenous costimulators, immune modulators, agonists for costimulators, antagonists for immune suppressors, immune cell engagers, or any combination thereof. Also contemplated herein are fusion proteins comprising the aforementioned polypeptides, and fragments thereof. In a preferred embodiment, the polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 107-115, 116-138, or 139-170.

[0195] "Polypeptide", "peptide" and "protein" are used interchangeably and follow their conventional meaning, i.e., as an amino acid sequence, unless otherwise specified to the contrary. In one embodiment, "polypeptide" includes fusion polypeptides and other variants. Polypeptides may be prepared using any of a variety of well-known recombinant and / or synthetic techniques. Polypeptides are not limited to a particular length, e.g., polypeptides may include full-length protein sequences, fragments of full-length proteins, or fusion proteins, and may include post-translational modifications of the polypeptide, such as, for example, glycosylation, acetylation, phosphorylation, and other modifications, both naturally occurring and non-naturally occurring, known in the art. In a particularly preferred embodiment, fusion polypeptides, polypeptides, fragments and other variants thereof are prepared, obtained, or isolated from one or more human polypeptides.

[0196] "Isolated peptide" or "isolated polypeptide" and the like, as used herein, refers to the in vitro isolation and / or purification of a peptide or polypeptide molecule from the cellular environment and from association with other components of a cell, i.e., it is not significantly associated with in vivo substances. In certain embodiments, an isolated polypeptide is a synthetic polypeptide, a semi-synthetic polypeptide, or a polypeptide obtained or derived from a recombinant source.

[0197] Polypeptides include "polypeptide variants." Polypeptide variants may differ from naturally occurring polypeptides by one or more substitutions, deletions, additions, and / or insertions. Such variants may be natural or may be synthetically produced, for example, by modifying one or more of the above polypeptide sequences. For example, in certain embodiments, it may be desirable to improve the binding affinity and / or other biological properties of the polypeptide by introducing one or more substitutions, deletions, additions, and / or insertions into the polypeptide. In certain embodiments, the polypeptides include those having at least about 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 86%, 97%, 98%, or 99% amino acid identity with any of the reference sequences contemplated herein, and typically the variants maintain at least one biological activity of the reference sequence. In certain embodiments, the biological activity is binding affinity. In certain embodiments, the biological activity is enzymatic activity.

[0198] In some embodiments, the engineered immunoreceptor component described herein recruits a multimeric immunoreceptor complex, comprising (i) a signaling component, e.g., a first fusion polypeptide, having a first multimerization domain and an actuator domain, and (ii) a targeting component, e.g., a second fusion polypeptide, having a second multimerization domain, an extracellular domain, and a transmembrane domain. In certain embodiments, the multimerization domains are the same; in some embodiments, the first multimerization domain is different from the second multimerization domain. The first and second multimerization domains substantially contribute to or efficiently promote the formation of a polypeptide complex, e.g., in the presence of a cross-linking agent. The interaction between the first multimerization domain and the second multimerization domain significantly contributes to or efficiently promotes the multimerization of the first fusion polypeptide and the second fusion polypeptide if, in the absence of the first multimerization domain, the second multimerization domain, or the cross-linking agent, the association between the first fusion polypeptide and the second fusion polypeptide is statistically significantly reduced. In certain embodiments, when the first fusion polypeptide and the second fusion polypeptide are co-expressed, at least about 60%, e.g., at least about 60% to about 70%, at least about 70% to about 80%, at least about 80% to about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, and at least about 90% to about 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, of the first and second single chain polypeptides form multimers with each other in the presence of a cross-linking agent.

[0199] Polypeptide variants include biologically active "polypeptide fragments." Illustrative examples of biologically active polypeptide fragments include binding domains, intracellular signaling domains, and the like. As used herein, the term "biologically active fragment" or "minimal biologically active fragment" refers to a polypeptide fragment that retains at least 100%, at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% of the naturally occurring polypeptide activity. In certain embodiments, a polypeptide fragment may comprise an amino acid chain of at least 5 to about 1700 amino acids in length. In an embodiment, the fragment comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, It is contemplated that the nucleic acid may be 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700 or more amino acids in length.

[0200] In certain embodiments, the polypeptides described herein may comprise one or more amino acids designated as "X." "X," when present in an amino acid SEQ ID NO:, refers to any one or more amino acids. In certain embodiments, a SEQ ID NO: representing a fusion protein comprises a sequence of consecutive X residues that cumulatively represent any amino acid sequence.

[0201] As mentioned above, a polypeptide may be modified in various ways, including amino acid substitution, deletion, truncation, and insertion. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of a reference polypeptide may be generated by mutations in DNA. Methods for mutagenesis and nucleotide sequence modification are known in the art. See, for example, Kunkel (1985, Proc. Natl. Acad. Sci. USA. 82:488-492), Kunkel et al., (1987, Methods in Enzymol, 154:367-382), U.S. Patent No. 4,873,192, Watson, JD et al., (Molecular Biology of the Gene, Fourth Edition, Benjamin / Cummings, Menlo Park, Calif., 1987), and references cited therein. Guidance regarding appropriate amino acid substitutions that do not affect the biological activity of the protein of interest can be found in the model of Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC).

[0202] In some embodiments, the polypeptide variants contain one or more conservative substitutions. A "conservative substitution" is one in which an amino acid is replaced with another amino acid having similar properties, and one skilled in the art of peptide chemistry would expect the secondary structure and hydrophobicity of the polypeptide to be substantially unchanged. Modifications may be made in the polynucleotide and polypeptide structures contemplated in certain embodiments, and still obtain functional molecules encoding variant or derivative polypeptides with desirable characteristics. When it is desired to change the amino acid sequence of a polypeptide to generate an equivalent or improved variant polypeptide, one skilled in the art may change one or more of the codons of the encoding DNA sequence, for example, according to Table 1. [Table 1-1] [Table 1-2]

[0203] Guidance for determining which amino acid residues may be substituted, inserted or deleted without abolishing biological activity can be found using computer programs well known in the art, such as DNASTAR, DNA Strider, Geneious, Mac Vector, or Vector NTI software. Preferably, the amino acid changes in the protein variants disclosed herein are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes include substitutions of one of a family of amino acids that are related at their side chains. Naturally occurring amino acids are generally divided into four families: acidic amino acids (aspartic acid, glutamic acid), basic amino acids (lysine, arginine, histidine), nonpolar amino acids (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar amino acids (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan and tyrosine are sometimes classified jointly as aromatic amino acids. In peptides or proteins, suitable conservative substitutions of amino acids are known to those skilled in the art and can generally be made without altering the biological activity of the resulting molecule. Those skilled in the art generally recognize that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p.224).

[0204] In one embodiment, when expression of more than one polypeptide is desired, the polynucleotide sequences encoding them may be separated by an IRES sequence.

[0205] In certain embodiments, contemplated polypeptides include fusion polypeptides. In certain embodiments, fusion polypeptides and polynucleotides encoding fusion polypeptides are provided. Fusion polypeptides and fusion proteins refer to polypeptides having at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more polypeptide segments. In preferred embodiments, the fusion polypeptide comprises one or more of the components described herein. In other preferred embodiments, the fusion polypeptide comprises a targeting component and a signaling component.

[0206] In certain embodiments, two or more engineered immunoreceptor components and / or other polypeptides may be expressed as a fusion protein that includes one or more self-cleaving peptide sequences between the polypeptides, as disclosed elsewhere herein.

[0207] In certain embodiments, a fusion polypeptide comprises a targeting moiety and one or more signaling moieties.

[0208] Fusion polypeptides may include one or more polypeptide domains or segments, including, but not limited to, signal peptides, cell penetrating peptide domains (CPPs), binding domains, signaling domains, etc., epitope tags (e.g., maltose binding protein ("MBP"), glutathione S-transferase (GST), HIS6, MYC, FLAG, V5, VSV-G, and HA), polypeptide linkers, and polypeptide cleavage signals. Fusion polypeptides are typically linked C-terminus to N-terminus, but they may also be linked C-terminus to C-terminus, N-terminus to N-terminus, or N-terminus to C-terminus. In certain embodiments, the polypeptides of the fusion protein may be in any order. Fusion polypeptides or fusion proteins may include conservatively modified variants, polymorphic variants, alleles, mutants, subsequences, and interspecies homologs, provided that the desired activity of the fusion polypeptide is preserved. Fusion polypeptides may be made by chemical synthesis or by chemical conjugation between two moieties, or may be generally prepared using other standard methods. The ligated DNA sequence comprising the fusion polypeptide is operably linked to suitable transcriptional or translational control elements, as disclosed elsewhere herein.

[0209] A fusion polypeptide may optionally include one or more linkers that can be used to link one or more polypeptides or domains within a polypeptide. Peptide linker sequences can be utilized to separate any two or more polypeptide components by a distance sufficient to ensure that each polypeptide folds into its appropriate secondary and tertiary structure so that the polypeptide domains can perform their desired functions. Such peptide linker sequences are incorporated into the fusion polypeptide using standard techniques in the art. Suitable peptide linker sequences can be selected based on the following factors: (1) their ability to adopt flexible extended conformations, (2) their inability to adopt secondary structures that may interact with functional epitopes on the signaling and targeting components, and (3) the lack of hydrophobic or charged residues that may react with the polypeptide functional epitopes. In certain embodiments, preferred peptide linker sequences include Gly, Asn, and Ser residues. Other near-neutral amino acids, such as Thr and Ala, can also be used in the linker sequence. Amino acid sequences that may be usefully employed as linkers include those disclosed in Maratea et al., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258-8262, 1986, U.S. Pat. No. 4,935,233, and U.S. Pat. No. 4,751,180. Linker sequences are not required if the particular fusion polypeptide segment contains a non-essential N-terminal amino acid region that can be used to separate functional domains and prevent steric interference. In certain embodiments, preferred linkers are typically flexible amino acid subsequences that are synthesized as part of the recombinant fusion protein. Linker polypeptides may be 1-200 amino acids long, 1-100 amino acids long, or 1-50 amino acids long, including all integer values ​​between these values.

[0210] Exemplary polypeptide cleavage signals include polypeptide cleavage recognition sites, such as protease cleavage sites, nuclease cleavage sites (e.g., rare restriction enzyme recognition sites, self-cleaving ribozyme recognition sites), and self-cleaving viral oligopeptides (see deFelipe and Ryan, 2004. Traffic, 5(8);616-26).

[0211] Suitable protease cleavage sites and autocleaving peptides are known to those of skill in the art (see, e.g., Ryan et al., 1997. J. Gener. Virol. 78, 699-722; Scymczak et al. (2004) Nature Biotech. 5, 589-594). Exemplary protease cleavage sites include, but are not limited to, cleavage sites for potyvirus NIa protease (e.g., tobacco etch virus protease), potyvirus HC protease, potyvirus P1 (P35) protease, byovirus NIa protease, biovirus RNA-2-encoded protease, aphthovirus L protease, enterovirus 2A protease, rhinovirus 2A protease, picorna 3C protease, comovirus 24K protease, nepovirus 24K protease, RTSV (Waika virus) 3C-like protease, PYVF (Parsnip yellow mottle virus) 3C-like protease, heparin, thrombin, factor Xa, and enterokinase. Due to its high cleavage stringency, a TEV (Tobacco Etch Virus) protease cleavage site is preferred in one embodiment, e.g., EXXYXQ(G / S) (SEQ ID NO: 195), e.g., ENLYFQG (SEQ ID NO: 196) and ENLYFQS (SEQ ID NO: 197), where X represents any amino acid (cleavage by TEV occurs between Q and G or Q and S).

[0212] In certain embodiments, the polypeptide cleavage signal is a viral autocleaving peptide or a ribosomal skipping sequence.

[0213] Illustrative examples of ribosomal skipping sequences include, but are not limited to, 2A or 2A-like sites, sequences or domains (see Donnelly et al., 2001. J. Gen. Virol. 82:1027-1041). In certain embodiments, the viral 2A peptide is an aphthovirus 2A peptide, a potyvirus 2A peptide, or a cardiovirus 2A peptide.

[0214] In one embodiment, the viral 2A peptide is selected from the group consisting of a foot and mouth disease virus (FMDV) 2A peptide, an equine rhinitis A virus (ERAV) 2A peptide, a zosea signa virus (TaV) 2A peptide, a porcine teschovirus-1 (PTV-1) 2A peptide, a tylovirus 2A peptide, and an encephalomyocarditis virus 2A peptide.

[0215] Illustrative examples of 2A sites are provided in Table 2. [Table 2-1] [Table 2-2]

[0216] In certain embodiments, the self-cleaving polypeptide (e.g., the 2A peptide) comprises an SGSG (SEQ ID NO: 193) spacer sequence. In certain embodiments, the self-cleaving polypeptide (e.g., the 2A peptide) comprises a furin recognition site, e.g., an RAKR (SEQ ID NO: 194) spacer sequence.

[0217] In a preferred embodiment, the polypeptide or fusion polypeptide comprises one or more engineered immune receptors or components. In a preferred embodiment, the fusion polypeptide comprises one or more engineered immune receptors or components separated by one or more self-cleaving polypeptides.

[0218] In certain embodiments, the fusion polypeptide comprises a first multimerization domain, and an actuator domain that forms a complex with and recruits an immune receptor complex (e.g., a CD3ε, CD3δ or CD3γ domain; a viral autocleaving 2A polypeptide; and a targeting moiety (comprising at least a first targeting domain (e.g., an antibody or antigen-binding fragment thereof that binds to a target antigen)) that comprises an extracellular domain, a second multimerization domain, and a signaling moiety that comprises a transmembrane domain.

[0219] In certain embodiments, the fusion polypeptide comprises a first multimerization domain, and an actuator domain that complexes with and recruits an immune receptor complex (e.g., a CD3ε, CD3δ or CD3γ domain; a viral autocleaving 2A polypeptide; and a targeting moiety (comprising at least a first targeting domain (e.g., an antibody or antigen-binding fragment thereof that binds to a target antigen)) that comprises an extracellular domain, a second multimerization domain, a CD4 hinge domain, and a CD4 transmembrane domain.

[0220] In certain embodiments, the fusion polypeptide comprises a first multimerization domain, and an actuator domain that complexes with and recruits an immune receptor complex (e.g., a CD3ε, CD3δ or CD3γ domain; a viral autocleaving 2A polypeptide; and a targeting moiety (comprising at least a first targeting domain (e.g., an antibody or antigen-binding fragment thereof that binds to a target antigen)) that comprises an extracellular domain, a second multimerization domain, a CD4 hinge domain, a CD4 transmembrane domain, and a signaling moiety that comprises a cleaved CD4 domain.

[0221] In certain embodiments, the fusion polypeptide comprises an FRB or FKBP12 multimerization domain, and an actuator domain that complexes with and recruits an immune receptor complex (e.g., a CD3ε, CD3δ or CD3γ domain; a signaling moiety comprising a viral autocleaving 2A polypeptide; and a targeting moiety comprising an extracellular domain comprising a VHH or scFv that binds to a target antigen, an FRB or FKBP12 multimerization domain, a CD4 hinge, a CD4 transmembrane domain, and a cleaved CD4 domain.

[0222] In certain embodiments, the fusion polypeptide comprises an antibody-derived heterodimerization domain, a multimerization domain, and an immune receptor complex (e.g., CD3ε, CD3δ, or CD3γ, domain; a signaling moiety comprising an actuator domain that complexes with and recruits a viral autocleavage 2A polypeptide; and a targeting moiety comprising a VHH or scFv that binds to a target antigen, an antibody-derived heterodimerization domain, a CD4 hinge, a CD4 transmembrane domain, a cleaved CD4 domain, and, optionally, a costimulatory or cytokine receptor intracellular signaling domain.

[0223] In certain embodiments, the fusion polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOs:139-170.

[0224] In certain embodiments, the fusion polypeptide comprises a signaling moiety comprising a first multimerization domain and an actuator domain that complexes with and recruits an Fc receptor complex (e.g., an Fcγ domain / polypeptide); a viral auto-cleaving 2A polypeptide; and an extracellular domain (comprising at least a first targeting domain (e.g., an antibody or antigen-binding fragment thereof that binds to a target antigen)), a second multimerization domain, optionally a hinge domain, and a transmembrane domain.

[0225] In certain embodiments, the fusion polypeptide comprises a signaling moiety comprising a first multimerization domain, and an actuator domain that complexes with and recruits an Fc receptor complex (e.g., an Fcγ domain / polypeptide); a viral auto-cleaving 2A polypeptide; and an extracellular domain (comprising at least a first targeting domain (e.g., an antibody or antigen-binding fragment thereof that binds to a target antigen)), a second multimerization domain, a CD4 hinge, and a CD4 transmembrane domain.

[0226] In certain embodiments, the fusion polypeptide comprises a signaling moiety comprising a first multimerization domain, and an actuator domain that complexes with and recruits an Fc receptor complex (e.g., an Fcγ domain / polypeptide); a viral auto-cleaving 2A polypeptide; and an extracellular domain (comprising at least a first targeting domain (e.g., an antibody or antigen-binding fragment thereof that binds to a target antigen)), a second multimerization domain, a CD4 hinge, a CD4 transmembrane domain, and a cleaved CD4ic domain.

[0227] In certain embodiments, the fusion polypeptide comprises a signaling moiety comprising an FRB or FKBP12 multimerization domain, and an actuator domain that complexes with and recruits an Fc receptor complex (e.g., an Fcγ domain / polypeptide); a viral autocleaving 2A polypeptide; and a targeting moiety comprising an extracellular domain comprising a VHH or scFv that binds to a target antigen, an FRB or FKBP12 multimerization domain, a CD4 hinge, a CD4 transmembrane domain, and a cleaved CD4ic domain.

[0228] In certain embodiments, the fusion polypeptide comprises a signaling moiety comprising an antibody-derived heterodimerization domain and an actuator domain that complexes with and recruits an Fc receptor complex (e.g., an Fcγ domain / polypeptide); a viral autocleaving 2A polypeptide; and a targeting moiety comprising an extracellular domain comprising a VHH or scFv that binds to a target antigen, an antibody-derived heterodimerization domain, a CD4 hinge, a CD4 transmembrane domain, a cleaved CD4 domain, and, optionally, a costimulatory or cytokine receptor intracellular signaling domain.

[0229] In certain embodiments, the fusion polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOs:139-170.

[0230] In one embodiment, the fusion polypeptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 151. In one embodiment, the fusion polypeptide comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 151. In one embodiment, the fusion polypeptide comprises an amino acid sequence having at least 96% identity to SEQ ID NO: 151. In one embodiment, the fusion polypeptide comprises an amino acid sequence having at least 97% identity to SEQ ID NO: 151. In one embodiment, the fusion polypeptide comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 151. In one embodiment, the fusion polypeptide comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 151. In one embodiment, the fusion polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 151.

[0231] In one embodiment, the fusion polypeptide comprises a sequence having at least 90% identity to SEQ ID NO: 137. In one embodiment, the fusion polypeptide comprises a sequence having at least 95% identity to SEQ ID NO: 137. In one embodiment, the fusion polypeptide comprises a sequence having at least 96% identity to SEQ ID NO: 137. In one embodiment, the fusion polypeptide comprises a sequence having at least 97% identity to SEQ ID NO: 137. In one embodiment, the fusion polypeptide comprises a sequence having at least 98% identity to SEQ ID NO: 137. In one embodiment, the fusion polypeptide comprises a sequence having at least 99% identity to SEQ ID NO: 137. In one embodiment, the fusion polypeptide comprises the sequence set forth as SEQ ID NO: 137.

[0232] In one embodiment, the fusion polypeptide comprises a sequence having at least 90% identity to SEQ ID NO: 138. In one embodiment, the fusion polypeptide comprises a sequence having at least 95% identity to SEQ ID NO: 138. In one embodiment, the fusion polypeptide comprises a sequence having at least 96% identity to SEQ ID NO: 138. In one embodiment, the fusion polypeptide comprises a sequence having at least 97% identity to SEQ ID NO: 138. In one embodiment, the fusion polypeptide comprises a sequence having at least 98% identity to SEQ ID NO: 138. In one embodiment, the fusion polypeptide comprises a sequence having at least 99% identity to SEQ ID NO: 138. In one embodiment, the fusion polypeptide comprises the sequence set forth as SEQ ID NO: 138.

[0233] F. Polynucleotides In certain embodiments, the polypeptides include polynucleotides encoding engineered immune receptor systems, one or more engineered immune receptor components, signaling components, targeting components, exogenous lymphocyte receptors, engineered TCRs, CARs, CCRs, BiTEs, zetakines, flip receptors, exogenous costimulators, immune modulators, agonists for costimulators, antagonists for immune suppressors, immune cell engagers, fusion proteins comprising the aforementioned polypeptides, and fragments thereof.

[0234] As used herein, the term "polynucleotide" or "nucleic acid" refers to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and DNA / RNA hybrids. Polynucleotides may be single-stranded or double-stranded, and may be either recombinant, synthetic, or isolated. Polynucleotides include, but are not limited to, pre-messenger RNA (pre-mRNA), messenger RNA (mRNA), RNA, small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozyme, genomic RNA (gRNA), positive strand RNA (RNA(+)), negative strand RNA (RNA(-)), tracrRNA, crRNA, single guide RNA (sgRNA), synthetic RNA, synthetic mRNA, genomic DNA (gDNA), PCR amplified DNA, complementary DNA (cDNA), synthetic DNA, or recombinant DNA. Polynucleotide refers to a polymeric form of nucleotides of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 1000, at least 5000, at least 10000, or at least 15000 or more nucleotides in length, either ribonucleotides or deoxyribonucleotides, or modified forms of either type of nucleotide, as well as all intermediate lengths. In this context, "intermediate length" will be readily understood to mean any length between the recited values, e.g., 6, 7, 8, 9, etc., 101, 102, 103, etc., 151, 152, 153, etc., 201, 202, 203, etc. In certain embodiments, a polynucleotide or variant has at least or about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a reference sequence.

[0235] As used herein, an "isolated polynucleotide" refers to a polynucleotide that has been purified from sequences that naturally flank it, e.g., a DNA fragment that has been removed from sequences that normally flank the fragment. In certain embodiments, an "isolated polynucleotide" also refers to a complementary DNA (cDNA), recombinant DNA, or other polynucleotide that is not found in nature and has been produced by man. In certain embodiments, an isolated polynucleotide is a synthetic polynucleotide, a semi-synthetic polynucleotide, or a polynucleotide obtained or derived from a recombinant source.

[0236] In various embodiments, the polynucleotide comprises an mRNA that encodes a polypeptide contemplated herein, hi some embodiments, the mRNA comprises a cap, one or more nucleotides, and a poly(A) tail.

[0237] In certain embodiments, the polynucleotides described herein, including polynucleotides encoding one or more engineered immune receptor components, may be codon optimized. As used herein, the term "codon optimization" refers to the substitution of codons in a polynucleotide encoding a polypeptide to increase expression, stability, and / or activity of the polypeptide. Factors that influence codon optimization include, but are not limited to, one or more of the following: (i) variation in codon bias between two or more organisms or genes or a synthetically constructed bias table; (ii) variation in the degree of codon bias within an organism, gene or set of genes; (iii) systematic variation of a codon with its context; (iv) variation of a codon with its decoding tRNA; (v) variation of a codon with the GC % either in the entire triplet or in one position of the triplet; (vi) variation in similarity to a reference sequence, such as a natural sequence; (vii) variation in the codon frequency cutoff; (viii) structural properties of the mRNA transcribed from the DNA sequence; (ix) prior knowledge of the function of the DNA sequence on which the design of the codon substitution set is based; (x) synthetic variation of the codon set for each amino acid; and / or (xi) isolated removal of incorrect translation start positions.

[0238] As used herein, the term "nucleotide" refers to a heterocyclic nitrogenous base in N-glycosidic linkage with a phosphorylated sugar. Nucleotides are understood to include natural bases and a wide variety of modified bases recognized in the art. Such bases are generally located at the 1' position of the nucleotide sugar moiety. Nucleotides generally comprise a base, a sugar, and a phosphate group. In ribonucleic acid (RNA), the sugar is ribose, and in deoxyribonucleic acid (DNA), the sugar is deoxyribose, i.e., deoxyribose is a sugar lacking the hydroxyl group present in ribose.

[0239] Illustrative examples of polynucleotides include, but are not limited to, polynucleotides that encode the polypeptides set forth in SEQ ID NOs:107-170.

[0240] In various illustrative embodiments, polynucleotides contemplated herein include, but are not limited to, polynucleotides encoding one or more engineered immune receptor components, engineered antigen receptors, exogenous lymphocyte receptors, fusion polypeptides, as well as expression vectors, viral vectors, and transfer plasmids comprising the polynucleotides contemplated herein.

[0241] As used herein, the terms "polynucleotide variant" and "variant" and the like refer to a polynucleotide that exhibits substantial sequence identity with a reference polynucleotide sequence or hybridizes to a reference sequence under stringent conditions as defined hereinafter. These terms also encompass polynucleotides that are distinguished from a reference polynucleotide by the addition, deletion, substitution, or modification of at least one nucleotide. Thus, the terms "polynucleotide variant" and "variant" include polynucleotides in which one or more nucleotides have been added or deleted, or modified, or replaced with another nucleotide. In this regard, it is well understood in the art that certain modifications, including mutations, additions, deletions, and substitutions, can be made to a reference polynucleotide, and the modified polynucleotide can retain the biological function or activity of the reference polynucleotide.

[0242] "Sequence identity," or phrases such as "a sequence 50% identical to," as used herein, refers to the degree to which sequences are identical nucleotide-by-nucleotide or amino acid-by-amino acid over a comparison window. Thus, a "percentage of sequence identity" may be calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions at which identical nucleobases (e.g., A, T, C, G, I) or identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) occur in both sequences to yield the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying this result by 100 to calculate the percentage of sequence identity. Included are nucleotides and polypeptides having at least about 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 86%, 97%, 98%, or 99% sequence identity to any of the reference sequences described herein.

[0243] As used herein, the term "nucleic acid cassette" or "expression cassette" refers to a genetic sequence in a vector capable of expressing an RNA and subsequently expressing a polypeptide. In one embodiment, the nucleic acid cassette comprises a gene of interest, e.g., a polynucleotide of interest. In another embodiment, the nucleic acid cassette comprises one or more expression control sequences, e.g., a promoter, an enhancer, a poly(A) sequence, and a gene of interest, e.g., a polynucleotide of interest. A vector may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleic acid cassettes. The nucleic acid cassettes are positionally and sequentially oriented within the vector, such that the nucleic acid in the cassette can be transcribed into RNA, translated into a protein or polypeptide, if necessary, subjected to appropriate post-translational modifications required for activity in the transformed cell, targeted to an appropriate intracellular compartment, translocated to an appropriate compartment for biological activity, or secreted into an extracellular compartment. The cassette preferably has a 3' and 5' end adapted for immediate insertion into the vector, e.g., a restriction endonuclease site at each end. The cassette can be excised and inserted as a single unit into a plasmid or viral vector.

[0244] Polynucleotides include polynucleotides of interest. As used herein, the term "polynucleotide of interest" refers to a polynucleotide that encodes a polypeptide or fusion polypeptide, or serves as a template for transcription of an inhibitory polynucleotide, as contemplated herein.

[0245] The polynucleotides contemplated herein, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters and / or enhancers, untranslated regions (UTRs), signal sequences, Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosome entry sites (IRES), recombinase recognition sites (e.g., LoxP, FRT, and Att sites), stop codons, transcription termination signals, and polynucleotides encoding self-cleaving polypeptides, epitope tags, and the like, as disclosed elsewhere herein or known in the art, so that their overall length may vary considerably. It is therefore contemplated that polynucleotide fragments of almost any length may be used, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.

[0246] Polynucleotides may be prepared, manipulated, expressed, and / or delivered using any of a variety of established techniques known and available in the art. To express a desired polypeptide, a nucleotide sequence encoding the polypeptide may be inserted into a suitable vector.

[0247] Illustrative examples of vectors include, but are not limited to, plasmids, autonomously replicating sequences, and transposable elements, e.g., Sleeping Beauty, PiggyBac.

[0248] Additional illustrative examples of vectors include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages, such as lambda phage or M13 phage, and animal viruses.

[0249] Illustrative examples of viruses useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), pox viruses, baculoviruses, papilloma viruses, and papova viruses (e.g., SV40).

[0250] Illustrative examples of expression vectors include, but are not limited to, pClneo vectors (Promega) for expression in mammalian cells, pLenti4 / V5-DEST™, pLenti6 / V5-DEST™, and pLenti6.2 / V5-GW / lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. In certain embodiments, coding sequences for the polypeptides disclosed herein may be ligated into such expression vectors for expression of the polypeptides in mammalian cells.

[0251] In certain embodiments, the vector is an episomal vector, or a vector that is maintained extrachromosomally. As used herein, the term "episomal" refers to a vector that can replicate without integration into host chromosomal DNA, and without gradual loss from dividing host cells, which also means that the vector replicates extrachromosomally, or episomally.

[0252] "Expression control sequences," "control elements," or "regulatory sequences" present in an expression vector are non-translated regions of the vector and include origins of replication, selection cassettes, promoters, enhancers, translation initiation signals (Shine Dalgarno or Kozak sequences), introns, polyadenylation sequences, 5' and 3' non-translated regions, all of which interact with host cellular proteins to effect transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation factors may be used, including ubiquitous and inducible promoters.

[0253] In certain embodiments, the polynucleotide comprises a vector, including but not limited to expression vectors and viral vectors. A vector may comprise one or more exogenous, endogenous, or heterologous control sequences, such as promoters and / or enhancers. An "endogenous control sequence" is a sequence that is naturally linked to a given gene in the genome. An "exogenous control sequence" is a sequence that is placed in juxtaposition to a gene by genetic engineering means (i.e., molecular biology techniques), whose transcription is induced by its linked enhancer / promoter. A "heterologous control sequence" is an exogenous sequence that originates from a different species than the cell being genetically engineered. A "synthetic" control sequence may comprise one or more endogenous and / or exogenous sequence elements, and / or in vitro or in silico determined sequence elements, that provide optimal promoter and / or enhancer activity for a particular therapy.

[0254] As used herein, the term "promoter" refers to a recognition site in a polynucleotide (DNA or RNA) to which an RNA polymerase binds. The RNA polymerase initiates and transcribes a polynucleotide operably linked to the promoter. In certain embodiments, a promoter that operates in a mammalian cell contains an AT-rich region located approximately 25-30 bases upstream from the site where transcription is initiated, and / or another sequence, a CNCAAT region, located 70-80 bases upstream from the transcription start site, where N can be any nucleotide.

[0255] The term "enhancer" refers to a DNA segment that contains a sequence capable of providing enhanced transcription and, in some instances, can function regardless of orientation relative to another control sequence. Enhancers can function cooperatively or additively with promoters and / or other enhancer elements. The term "promoter / enhancer" refers to a DNA segment that contains a sequence that can provide both promoter and enhancer functions.

[0256] The term "operably linked" refers to a juxtaposition where the described components are in a relationship permitting them to function in their intended manner. In one embodiment, the term refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter and / or enhancer) and a second polynucleotide sequence, e.g., a polynucleotide of interest, where the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.

[0257] As used herein, the term "structural expression control sequence" refers to a promoter, enhancer, or promoter / enhancer that allows for continuous or sequential transcription of an operably linked sequence. A structural expression control sequence may be a "ubiquitous" promoter, enhancer, or promoter / enhancer that allows for expression in a variety of cell and tissue types, or it may be a "cell-specific," "cell type-specific," "cell line-specific," or "tissue-specific" promoter, enhancer, or promoter / enhancer that allows for expression in restricted cell and tissue types, respectively.

[0258] Illustrative ubiquitous expression control sequences suitable for use in certain embodiments include, but are not limited to, the cytomegalovirus (CMV) immediate early promoter, the viral Simian Virus 40 (SV40) (e.g., early or late), Moloney Murine Leukemia Virus (MoMLV) LTR promoter, Rous Sarcoma Virus (RSV) LTR, herpes simplex virus (HSV) (thymidine kinase) promoter, the H5, P7.5 and P11 promoters from vaccinia virus, elongation factor 1 alpha (EF1a) promoter, early growth response protein 1 (EGR1), ferritin H (FerH), ferritin L (FerL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70 kDa protein 5 (HSPA5), heat shock protein 90 kDa beta, member 1 (HSP90B1), heat shock protein 70 kDa (HSP70), beta-kinesin (β-KIN), human ROSA 26 locus (Irions et al., Nature Biotechnology 25, 1477-1482 (2007)), ubiquitin C promoter (UBC), phosphoglycerate kinase-1 (PGK) promoter, cytomegalovirus enhancer / chicken beta-actin (CAG) promoter, beta-actin promoter and myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer binding site substitution (MND) U3 promoter (Haas et al. Journal of Virology. 2003; 77(17):9439-9450).

[0259] In one embodiment, the vector comprises the MNDU3 promoter.

[0260] In one embodiment, the vector comprises the EF1a promoter including the first intron of the human EF1a gene.

[0261] In one embodiment, the vector comprises the EF1a promoter lacking the first intron of the human EF1a gene.

[0262] In certain embodiments, it will be desirable to use cell-, cell type-, cell lineage-, or tissue-specific expression control sequences to achieve cell type-, cell lineage-, or tissue-specific expression of a desired polynucleotide sequence (e.g., expressing a particular nucleic acid encoding a polypeptide in only a subset of cell types, cell lineages, or tissues, or during a particular stage of development).

[0263] In certain embodiments, it may be desirable to express a polynucleotide that is a T cell specific promoter.

[0264] As used herein, "conditional expression" may refer to any type of conditional expression, including, but not limited to, inducible expression, repressible expression, expression in cells or tissues that are in a particular physiological, biological, or disease state. This definition is not intended to exclude cell type-specific or tissue-specific expression. Certain embodiments provide for conditional expression of a polynucleotide of interest, e.g., expression is controlled by exposing a cell, tissue, or organism to a treatment or condition that results in expression of the polynucleotide, or that increases or decreases expression of a polynucleotide encoded by the polynucleotide of interest.

[0265] Illustrative examples of inducible promoters / systems include, but are not limited to, steroid-inducible promoters, such as promoters of genes encoding glucocorticoid receptors or estrogen receptors (inducible by treatment with the corresponding hormones), metallothionein promoters (inducible by treatment with various heavy metals), MX-1 promoters (inducible by interferon), the "GeneSwitch" mifepristone-regulatable system (Sirin et al., 2003, Gene, 323:67), cumate-inducible gene switches (WO2002 / 088346), tetracycline-dependent regulatory systems, etc. Inducing agents include, but are not limited to, glucocorticoids, estrogens, mifepristone (RU486), metals, interferons, small molecules, cumate, tetracycline, doxycycline, and variants thereof.

[0266] As used herein, "internal ribosome entry site" or "IRES" refers to an element that facilitates direct entry of an internal ribosome into the initiation codon, such as ATG, of a cistron (protein coding region), leading to cap-independent translation of a gene. See, e.g., Jackson et al., 1990. Trends Biochem Sci 15(12):477-83) and Jackson and Kaminski. 1995. RNA 1(10):985-1000. Examples of IRES commonly used by those of skill in the art include those described in U.S. Patent No. 6,692,736. Further examples of "IRES" known in the art include, but are not limited to, IRES obtainable from picornaviruses (Jackson et al., 1990) and IRES obtainable from viral or cellular mRNA sources, such as immunoglobulin heavy chain binding protein (BiP), vascular endothelial growth factor (VEGF) (Huez et al. 1998. Mol. Cell. Biol. 18(11):6178-6190), fibroblast growth factor 2 (FGF-2), and insulin-like growth factor (IGFII), translation initiation factor eIF4G, and yeast transcription factors TFIID and HAP4, encephalomyocarditis virus (EMCV) commercially available from Novagen (Duke et al., 1992. J. Virol 66(3):1602-9) and the VEGF IRES (Huez et al., 1998. Mol. Cell. Biol. 18(11):6178-6190). 18(11):6178-90). IRESs have also been reported in the viral genomes of species from the Picornaviridae, Dicistroviridae, and Flaviviridae families, as well as in HCV, Friend murine leukemia virus (FrMLV), and Moloney murine leukemia virus (MoMLV).

[0267] In one embodiment, the IRES used in the polynucleotides contemplated herein is the EMCV IRES.

[0268] In certain embodiments, the polynucleotide is a consensus Kozak sequence. As used herein, the term "Kozak sequence" refers to a short nucleotide sequence that greatly promotes initial binding of mRNA to the small ribosomal subunit and increases translation. The consensus Kozak sequence is (GCC)RCCATGG (SEQ ID NO: 198), where R is a purine (A or G) (Kozak, 1986. Cell. 44(2):283-92, and Kozak, 1987. Nucleic Acids Res. 15(20):8125-48).

[0269] Factors that induce efficient termination and polyadenylation of heterologous nucleic acid transcripts increase expression of heterologous genes. Transcription termination signals are generally found downstream of polyadenylation signals. In certain embodiments, the vector includes a polyadenylation sequence 3' of the polynucleotide encoding the expressed polypeptide. As used herein, the term "polyA site" or "polyA sequence" refers to a DNA sequence that directs both the termination and polyadenylation of the nascent RNA transcript by RNA polymerase II. Polyadenylation sequences can promote mRNA stability by adding a polyA tail to the 3' end of the coding sequence, thus contributing to increased translation efficiency. Cleavage and polyadenylation are directed by poly(A) sequences in the RNA. The core poly(A) sequence of mammalian pre-mRNA has two recognition elements flanking the cleavage polyadenylation site. Typically, a nearly invariant AAUAAA hexamer is located 20-50 nucleotides upstream of a more variable element rich in U or GU residues. Cleavage of the nascent transcript occurs between these two elements and is coupled with the addition of up to 250 adenosines to the 5' cleavage product. In certain embodiments, the core poly(A) sequence is a polyA sequence of choice (e.g., AATAAA, ATTAAA, AGTAAA). In certain embodiments, the poly(A) sequence is the SV40 polyA sequence, bovine growth hormone polyA sequence (BGHpA), rabbit β-globin polyA sequence (rβgpA), variants thereof, or another suitable heterologous or endogenous polyA sequence known in the art. In certain embodiments, the poly(A) sequence is synthetic.

[0270] In certain embodiments, polynucleotides encoding one or more polypeptides or fusion polypeptides may be introduced into immune effector cells, such as T cells, by both non-viral and viral methods. In certain embodiments, delivery of one or more polynucleotides may be provided by the same or different methods and / or on the same or different vectors.

[0271] The term "vector" is used herein to refer to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule. The transferred nucleic acid is generally linked to, e.g., inserted into, the vector nucleic acid molecule. The vector may contain sequences that direct autonomous replication in a cell, or may contain sequences sufficient to allow integration into the host cell DNA. In certain embodiments, non-viral vectors are used to deliver one or more polynucleotides contemplated herein to T cells.

[0272] Illustrative examples of non-viral vectors include, but are not limited to, plasmids (eg, DNA plasmids or RNA plasmids), transposons, cosmids, and bacterial artificial chromosomes.

[0273] Illustrative methods of non-viral delivery of polynucleotides contemplated in certain embodiments include, but are not limited to, electroporation, sonoporation, lipofection, microinjection, biolistec, virosomes, liposomes, immunoliposomes, nanoparticles, polycation or lipid:nucleic acid complexes, naked DNA, artificial virions, DEAE-dextran mediated transfer, gene guns, and heat shock.

[0274] Illustrative examples of polynucleotide delivery systems suitable for use in certain contemplated embodiments include, but are not limited to, systems provided by Amaxa Biosystems, Maxcyte, Inc., BTX Molecular Delivery Systems, and Copernicus Therapeutics Inc. Lipofection reagents are commercially available (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids suitable for efficient receptor-recognition lipofection of polynucleotides have been described in the literature. See, for example, Liu et al. (2003) Gene Therapy. 10:180-187; and Balazs et al. (2011) Journal of Drug Delivery. 2011:1-12. Antibody-targeted delivery, bacteria-guided delivery, and non-biological nanocell-based delivery are also contemplated in certain embodiments.

[0275] As will be apparent to those skilled in the art, the term "viral vector" is used broadly to refer to either a nucleic acid molecule (e.g., a transfer plasmid) that typically contains nucleic acid elements derived from a virus that facilitate the transfer or integration of the nucleic acid molecule into the genome of a cell, or a viral particle that mediates nucleic acid transfer. In addition to the nucleic acid, a viral particle typically contains various viral components, and sometimes also host cell components. The term "viral vector" or "lentiviral vector" can refer to a virus or viral particle capable of transferring a nucleic acid into a cell, or the transferred nucleic acid itself. Viral vectors and transfer plasmids contain structural and / or functional genetic elements that are primarily derived from viruses.

[0276] In certain embodiments, viral vectors comprising contemplated polynucleotides may be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous or intracranial infusion) or local application, as described below. Alternatively, vectors may be delivered ex vivo to cells, such as cells explanted from an individual patient (e.g., mobilized peripheral blood, lymphocytes, bone marrow aspirates, tissue biopsies, etc.) or hematopoietic stem cells of a universal donor, which may then be reimplanted into the patient.

[0277] In one embodiment, a viral vector comprising a polynucleotide as contemplated herein is administered directly to an organism or subject, or is used for transduction of cells in vivo. Alternatively, naked DNA may be administered. Administration is by any route that is normally used to introduce molecules into final contact with blood or tissue cells, including, but not limited to, injection, infusion, topical application, and electroporation. Suitable methods for administering such nucleic acids are available and well known to those skilled in the art, and although a particular composition can be administered using more than one route, a particular route can often provide a more immediate and more effective response than another route.

[0278] Illustrative examples of viral vector systems suitable for use in certain contemplated embodiments include, but are not limited to, adeno-associated viral (AAV), retroviral, herpes simplex viral, adenoviral, and vaccinia viral vectors.

[0279] In various embodiments, one or more polynucleotides encoding one or more engineered immune receptor components and / or other polypeptides contemplated herein are introduced into an immune effector cell, e.g., a T cell, by transducing the cell with a recombinant adeno-associated virus (rAAV) containing the one or more polynucleotides.

[0280] AAV is a small (about 26 nm), replication-deficient, mostly episomal, non-enveloped virus. AAV can infect both dividing and non-dividing cells and can integrate its genome into the genome of a host cell. Recombinant AAV (rAAV) typically consists of, at a minimum, a transgene and its regulatory sequences, as well as 5' and 3' AAV inverted terminal repeats (ITRs). The ITR sequences are about 145 bp long. In certain embodiments, rAAV comprises ITRs and capsid sequences isolated from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10.

[0281] In some embodiments, chimeric rAAVs are used. The ITR sequences are isolated from one AAV serotype, and the capsid sequences are isolated from another AAV serotype. For example, a rAAV that includes ITR sequences from AAV2 and capsid sequences from AAV6 is referred to as AAV2 / AAV6. In certain embodiments, a rAAV vector can include ITRs from AAV2 and capsid proteins from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10. In a preferred embodiment, the rAAV includes ITR sequences from AAV2 and capsid sequences from AAV6. In a preferred embodiment, the rAAV includes ITR sequences from AAV2 and capsid sequences from AAV2.

[0282] In some embodiments, methods of engineering and selection may be performed on AAV capsids to increase the probability that they will transduce cells of interest.

[0283] The construction, production, and purification of rAAV vectors are disclosed, for example, in U.S. Patent Nos. 9,169,494, 9,169,492, 9,012,224, 8,889,641, 8,809,058, and 8,784,799, which are incorporated by reference herein in their entireties.

[0284] In various embodiments, one or more polynucleotides encoding one or more engineered immune receptor components and / or other polypeptides contemplated herein are introduced into an immune effector cell, e.g., a T cell, by transducing the cell with a retrovirus, e.g., a lentivirus, containing the one or more polynucleotides.

[0285] As used herein, the term "retrovirus" refers to an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy and then covalently integrates its genomic DNA into the host genome. Illustrative examples of retroviruses suitable for use in certain embodiments include, but are not limited to, Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemia virus, murine stem cell virus (MSCV), and Rous sarcoma virus (RSV), and lentivirus.

[0286] As used herein, the term "lentivirus" refers to a group (or genus) of complex retroviruses. Illustrative examples of lentiviruses include, but are not limited to, HIV (including human immunodeficiency virus, HIV type 1 and HIV type 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). In one embodiment, an HIV-based vector backbone (i.e., HIV cis-acting sequence elements) is preferred.

[0287] In various embodiments, lentiviral vectors contemplated herein include one or more LTRs and one or more or all of the following accessory elements: cPPT / FLAP, Psi (Ψ) packaging signal, transport element, poly(A) sequence, and optionally, a WPRE or HPRE, an insulator element, a selectable marker, and a cell suicide gene, as discussed elsewhere herein.

[0288] In certain embodiments, the lentivirus vector contemplated herein may be an integrative, non-integrative, or integration-defective lentivirus.As used herein, the term "integration-defective lentivirus" or "IDLV" refers to a lentivirus that has an integrase that is deficient in integrating viral genome into the genome of a host cell.Integration-incompetent viral vectors are described in patent application WO2006 / 010834, which is incorporated herein by reference in its entirety.

[0289] Illustrative mutations in the HIV-1 pol gene suitable for reducing integrase activity include, but are not limited to, the following: H12N, H12C, H16C, H16V, S81 R, D41A, K42A, H51A, Q53C, D55V, D64E, D64V, E69A, K71A, E85A, E87A, D116N, D1161, D116A, N120G, N1201, N120E, E152G, E152A, D35E, K156E, K156A, E157A, K159E, K159A, K160A, R166A, D1 67A, E170A, H171A, K173A, K186Q, K186T, K188T, E198A, R199c, R199T, R199A, D202A, K211A, Q214L, Q216L, Q221L, W235F, W235E, K236S, K236A, K246A, G247W, D253A, R262A, R263A and K264H.

[0290] The term "long terminal repeat (LTR)" refers to domains of base pairs located at the ends of retroviral DNA, which in the context of their natural sequence are direct repeats and include the U3, R and U5 regions.

[0291] As used herein, the term "FLAP element" or "cPPT / FLAP" refers to a nucleic acid whose sequence comprises the central polypurine tract and central termination sequences (cPPT and CTS) of a retrovirus, e.g., HIV-1 or HIV-2. Suitable FLAP elements are described in U.S. Patent No. 6,682,907 and in Zennou, et al., 2000, Cell, 101:173.

[0292] As used herein, the term "packaging signal" or "packaging sequence" refers to the psi [Ψ] sequence located in the retroviral genome and is required for the insertion of viral RNA into the viral capsid or viral particle. See, e.g., Clever et al., 1995. J. of Virology, Vol. 69, No. 4; pp. 2101-2109.

[0293] The term "export element" refers to a cis-acting post-transcriptional regulatory element that regulates the transport of an RNA transcript from the nucleus to the cytoplasm of a cell. Examples of RNA export elements include, but are not limited to, the rev response element (RRE) of human immunodeficiency virus (HIV) (see, e.g., Cullen et al., 1991. J. Virol. 65:1053; and Cullen et al., 1991. Cell 58:423) and the post-transcriptional regulatory element (HPRE) of hepatitis B virus.

[0294] In certain embodiments, the expression of heterologous sequences in viral vectors is increased by incorporating post-transcriptional regulators, efficient polyadenylation sites, and optionally transcription termination signals into the vector.Various post-transcriptional regulator elements can increase the expression of heterologous nucleic acids in proteins, such as the post-transcriptional regulator of woodchuck hepatitis virus (WPRE; Zufferey et al., 1999, J. Virol., 73:2886); the post-transcriptional regulator element (HPRE) present in hepatitis B virus (Huang et al., Mol. Cell. Biol., 5:3864); and the like (Liu et al., 1995, Genes Dev., 9:1766).

[0295] Lentiviral vectors preferably include several safety enhancements as a result of modifying the LTR. A "self-inactivating" (SIN) vector refers to a vector lacking replication capacity, e.g., a retroviral or lentiviral vector in which the right (3') LTR enhancer-promoter region, known as the U3 region, is modified (e.g., by deletion or substitution) to prevent viral transcription beyond the first round of viral replication. Self-inactivation is preferably achieved through the introduction of a deletion in the U3 region of the 3'LTR of the vector DNA, i.e., the DNA used to produce the vector RNA. This deletion is then transferred to the 5'LTR of the proviral DNA during reverse transcription. In certain embodiments, it is desirable to remove the U3 sequence sufficiently to greatly reduce or completely abolish the transcriptional activity of the LTR, thereby greatly reducing or abolishing the production of full-length vector RNA in transduced cells. In the case of HIV-based lentivectors, it has been found that such vectors can tolerate large U3 deletions, including removal of the LTR TATA box (e.g., deletion of -418 to -18), without significant loss of vector titer.

[0296] Additional safety enhancement is provided by replacing the U3 region of the 5'LTR with a heterologous promoter that directs transcription of the viral genome during production of viral particles. Examples of heterologous promoters that can be used include, for example, promoters of simian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV) (e.g., immediate early), Moloney murine leukemia virus (MoMLV), Rous sarcoma virus (RSV), and herpes simplex virus (HSV) (thymidine kinase).

[0297] As used herein, the term "pseudotype" or "pseudotyping" refers to a virus whose viral envelope proteins have been replaced with the envelope of another virus that has favorable properties. For example, the HIV envelope protein (encoded by the env gene) is normally encoded by the CD4 + Although targeting the virus to presentation cells, pseudotyping HIV with the vesicular stomatitis virus G-protein (VSV-G) envelope protein allows HIV to infect a broad range of cells.

[0298] In one embodiment, lentiviral vectors are produced by known methods, see, e.g., Kutner et al., BMC Biotechnol. 2009;9:10.doi:10.1186 / 1472-6750-9-10; Kutner et al. Nat. Protoc. 2009;4(4):495-505.doi:10.1038 / nprot.2009.22.

[0299] According to certain embodiments contemplated herein, most or all of the viral vector backbone sequences are derived from lentiviruses, e.g., HIV-1. However, it should be understood that many different sources of retroviral and / or lentiviral sequences can be used or combined, and numerous substitutions and modifications in a particular lentiviral sequence can be accommodated without compromising the ability of the transfer vector to perform the functions described herein. Furthermore, a variety of lentiviral vectors are known in the art. See Naldini et al., (1996a, 1996b, and 1998); Zufferey et al., (1997); Dull et al., 1998, U.S. Patent Nos. 6,013,516 and 5,994,136. Many of them can be adapted to produce the viral vectors or transfer plasmids contemplated herein.

[0300] In various embodiments, one or more polynucleotides encoding one or more engineered immune receptor components and / or other polypeptides contemplated herein are introduced into immune effector cells by transducing the cells with an adenovirus containing the one or more polynucleotides.

[0301] Adenovirus-based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. High titers and high levels of expression have been obtained using such vectors. The vectors can be produced in large quantities in a relatively simple system. Most adenovirus vectors are engineered such that the transgene replaces the Ad E1a, E1b, and / or E3 genes; the replication-defective vector is then amplified in human 293 cells, which supply the missing gene function in trans. Ad vectors can transduce multiple types of tissues in vivo, including non-dividing cells, differentiated cells, such as those present in the liver, kidney, and muscle. Traditional Ad vectors have a large carrying capacity.

[0302] The creation and amplification of current replication-deficient adenoviral vectors may utilize a unique helper cell line called 293. This cell line was transformed with Ad5 DNA fragments from human embryonic kidney cells and constitutively expresses E1 protein (Graham et al., 1977). Because the E3 region is dispensable for the adenoviral genome (Jones & Shenk, 1978), current adenoviral vectors utilize 293 cells to deliver foreign DNA in either the E1, D3, or both regions (Graham & Prevec, 1991). Adenoviral vectors have been used for eukaryotic gene expression (Levrero et al., 1991; Gomez-Foix et al., 1992) and vaccine development (Grunhaus & Horwitz, 1992; Graham & Prevec, 1992). Tests of recombinant adenoviruses administered to various tissues include tracheal instillation (Rosenfeld et al., 1991; Rosenfeld et al., 1992), intramuscular injection (Ragot et al., 1993), peripheral intravenous injection (Herz & Gerard, 1993), and stereotactic inoculation into the brain (Le Gal La Salle et al., 1993). An example of the use of Ad vectors in clinical trials included polynucleotide therapy for antitumor immunization using intramuscular injection (Sterman et al., Hum. Gene Ther. 7:1083-9 (1998)).

[0303] In various embodiments, one or more polynucleotides encoding one or more engineered immune receptor components and / or other polypeptides contemplated herein are introduced into immune effector cells by transducing the cells with a herpes simplex virus, e.g., HSV-1, HSV-2, containing the one or more polynucleotides.

[0304] Mature HSV virions consist of an enveloped icosahedral capsid containing a viral genome consisting of a linear double-stranded DNA molecule that is 152 kb. In one embodiment, the HSV-based viral vector is defective in one or more essential or non-essential HSV genes. In one embodiment, the HSV-based viral vector is replication-defective. Most replication-defective HSV vectors contain deletions to remove one or more immediate-early, early, or late HSV genes to prevent replication. For example, the HSV vector may be defective in an immediate-early gene selected from the group consisting of: ICP4, ICP22, ICP27, ICP47, and combinations thereof. The advantages of HSV vectors are their ability to enter latency, which can result in long-term DNA expression, and their large viral DNA genome, which can accommodate up to 25 kb of exogenous DNA. HSV-based vectors are described, for example, in U.S. Pat. Nos. 5,837,532, 5,846,782, and 5,804,413, and International Patent Applications WO91 / 02788, WO96 / 04394, WO98 / 15637, and WO99 / 06583, which are incorporated by reference herein in their entireties.

[0305] G. Genetically Modified Cells In various embodiments, the cells are modified to express an engineered immune receptor system, one or more engineered immune receptor components, signaling components, targeting components, engineered TCRs, CARs, CCRs, BiTEs, zetakines, flip receptors, exogenous costimulators, immune modulators, agonists for costimulators, antagonists for immune suppressors, immune cell engagers, and / or fusion proteins as contemplated herein. In certain embodiments, the cells are for use in the treatment of cancer. The cells may be non-genetically modified to express one or more of the polypeptides as contemplated herein, or in certain preferred embodiments, the cells may be genetically modified to express one or more of the polypeptides as contemplated herein. As used herein, the term "genetically engineered" or "genetically modified" refers to the addition of additional genetic material in the form of DNA or RNA to the total genetic material in a cell. The terms "genetically modified cells", "modified cells", and "redirected cells" are used interchangeably in certain embodiments.

[0306] In certain embodiments, one or more engineered immune receptor components that recruit immune receptor complexes as contemplated herein are introduced and expressed in immune effector cells to improve the efficacy of the immune effector cells. In certain embodiments, one or more engineered immune receptor components that recruit immune receptor complexes are introduced and expressed in immune effector cells that are redirected to target cells by co-expressing an exogenous lymphocyte receptor or an engineered antigen receptor, e.g., an engineered TCR or CAR, in the cells.

[0307] In certain embodiments, dual-targeted immune effector cells are contemplated, where the target cell expresses a target antigen recognized by an engineered immune receptor and an MHC-antigen complex recognized by a TCR, e.g., an engineered / exogenous TCR.

[0308] In certain embodiments, dual-targeted immune effector cells are contemplated, where the target cell expresses CD33, CD123, CLL1, B7-H3, BCMA, CD19, CD20, CD22, CD79A, CD79B, EGFR, EGFRvIII, or NKG2D ligands recognized by an engineered immune receptor, and a target antigen recognized by an exogenous lymphocyte receptor or engineered antigen receptor, e.g., an engineered / exogenous TCR or CAR.

[0309] An "immune effector cell" is any cell of the immune system that has one or more effector functions (e.g., cytotoxic cell killing activity, cytokine secretion, induction of ADCC and / or CDC, etc.). Illustrative immune effector cells contemplated herein are T lymphocytes, including, but not limited to, cytotoxic T cells (CTL; ​​CD8+ T cells), TILs, and helper T cells (HTL; CD4+ T cells. In certain embodiments, the cells comprise αβ T cells. In certain embodiments, the cells comprise γδ T cells. In one embodiment, the immune effector cells comprise natural killer (NK) cells. In one embodiment, the immune effector cells comprise natural killer T (NKT) cells.

[0310] Immune effector cells may be self or non-self (e.g., allogeneic, syngeneic, or xenogeneic). As used herein, "autologous" refers to cells derived from the same subject. As used herein, "allogeneic" refers to cells of the same species that are genetically different from the compared cells. As used herein, "syngeneic" refers to cells of a different subject that are genetically identical to the compared cells. As used herein, "xenogeneic" refers to cells of a different species from the compared cells. In a preferred embodiment, the cells are human autoimmune effector cells.

[0311] Exemplary immune effector cells suitable for introducing one or more engineered immune receptors or components contemplated herein include T lymphocytes. The term "T cell" or "T lymphocyte" is art-recognized and is intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. T cells can be T helper (Th) cells, e.g., T helper 1 (Th1) or T helper 2 (Th2) cells. T cells can be T helper cells (HT1; CD4 + T cell) CD4 + T cells, cytotoxic T cells (CTL:CD8 + T cells), CD4 + CD8 + T cells, CD4 - CD8 - The population of T cells may be T cells, or any other subset of T cells. Other illustrative T cell populations suitable for use in certain embodiments include naive T cells and memory T cells. For example, a population of T cells suitable for use in certain embodiments includes naive T cells (T N ), T memory stem cells (T SCM ), central memory T cells (T CM ), effector memory T cells (T EM ), and effector T cells (T EFF ).

[0312] As will be appreciated by those skilled in the art, other cells may also be used as immune effector cells, including one or more engineered immune receptor components as contemplated herein. In certain embodiments, immune effector cells also include NK cells, NKT cells, neutrophils, and macrophages. Immune effector cells also include precursors of effector cells, where such precursor cells may be induced to differentiate into immune effector cells in vivo or in vitro. Thus, in certain embodiments, immune effector cells include precursors of immune effector cells, such as hematopoietic stem cells (HSCs) contained within the CD34+ population of cells derived from, for example, umbilical cord blood, bone marrow, or mobilized peripheral blood, which differentiate into mature immune effector cells when administered to a subject, or may be induced to differentiate in vitro into mature immune effector cells.

[0313] The term "CD34+ cells," as used herein, refers to cells that express the CD34 protein on their cell surface. "CD34," as used herein, refers to a cell surface glycoprotein (e.g., sialomucin protein) that often acts as a cell-cell adhesion factor and is involved in T cell entry into lymph nodes. CD34+ cell populations include hematopoietic stem cells (HSCs), which upon administration to a patient differentiate and contribute to all hematopoietic lineages, including T cells, NK cells, NKT cells, neutrophils, and cells of the monocyte / macrophage lineage.

[0314] Methods for generating immune effector cells expressing one or more engineered immune receptor components contemplated herein are provided in certain embodiments. In one embodiment, the method comprises transfecting or transducing immune effector cells isolated from an individual with one or more nucleic acids and / or vectors, or a combination thereof, such that the immune effector cells include one or more engineered immune receptor components contemplated herein. In one embodiment, the method comprises transfecting or transducing immune effector cells isolated from an individual, such that the immune effector cells express one or more engineered immune receptor components contemplated herein and an exogenous lymphocyte receptor or engineered antigen receptor (e.g., TCR, CAR, CCR, zetakines, or flip receptor). In certain embodiments, immune effector cells are isolated from an individual and genetically modified without further manipulation in vitro. Such cells may then be readministered directly to the individual. In a further embodiment, the immune effector cells are first activated in vitro and stimulated to proliferate, and then genetically modified. In this regard, the immune effector cells may be cultured before and / or after being genetically modified.

[0315] In certain embodiments, the cells are human cells. In certain embodiments, a source of cells is obtained from a subject prior to in vitro manipulation or genetic modification of the immune effector cells described herein. In certain embodiments, the modified immune effector cells comprise T cells.

[0316] T cells can be obtained from many sources, including, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue at the site of infection, ascites, pleural effusion, spleen tissue, and tumors. In one embodiment, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to those of skill in the art, such as sedimentation, e.g., FICOLL™ separation, etc.

[0317] In other embodiments, isolated or purified T cell populations are used. In some embodiments, after isolation of PBMCs, both cytotoxic and helper T lymphocytes can be sorted into naive, memory and effector T cell subpopulations before or after activation, expansion and / or genetic modification.

[0318] In one embodiment, the isolated or purified population of T cells includes, but is not limited to, CD3 + , CD4 + , CD8 + , or a combination thereof.

[0319] In one embodiment, T cells are isolated from an individual and first activated and stimulated to proliferate in vitro before being modified to express one or more engineered immune receptor components.

[0320] To achieve a sufficient therapeutic dose of a T cell composition, the T cells are often subjected to one or more rounds of stimulation, activation, and / or expansion. In certain embodiments, T cells can generally be activated and expanded using methods described in, for example, U.S. Patent Nos. 6,352,694, 6,534,055, 6,905,680, 6,692,964, 5,858,358, 6,887,466, 6,905,681, 7,144,575, 7,067,318, 7,172,869, 7,232,566, 7,175,843, 5,883,223, 6,905,874, 6,797,514, and 6,867,041, each of which is incorporated by reference herein in its entirety. In certain embodiments, T cells are activated and expanded for about 6 hours, about 12 hours, about 18 hours, or about 24 hours prior to introduction of a vector or polynucleotide encoding one or more engineered immune receptor components, optionally in combination with an exogenous lymphocyte receptor or engineered antigen receptor as contemplated herein.

[0321] In one embodiment, the T cells are simultaneously activated when they are modified.

[0322] In various embodiments, a method of generating immune effector cells includes activating a population of cells comprising T cells and expanding the population of T cells. T cell activation can be achieved by providing a primary stimulatory signal via the TCR / CD3 complex of the T cells and a secondary co-stimulatory signal via an accessory molecule such as CD28.

[0323] The TCR / CD3 complex may be stimulated by contacting the T cell with a suitable CD3 binding agent, such as a CD3 ligand or an anti-CD3 monoclonal antibody. Illustrative examples of CD3 antibodies include, but are not limited to, OKT3, G19-4, BC3 and 64.1.

[0324] In addition to the primary stimulatory signal provided through the TCR / CD3 complex, induction of a T cell response requires a secondary costimulatory signal. In certain embodiments, a CD28 binding agent may be used to provide the costimulatory signal. Illustrative examples of CD28 binding agents include, but are not limited to, natural CD28 ligands, such as the natural ligands for CD28 (e.g., members of the B7 family of proteins, such as B7-1 (CD80) and B7-2 (CD86); and anti-CD28 monoclonal antibodies or fragments thereof capable of cross-linking the CD28 molecule, such as monoclonal antibodies 9.3, B-T3, XR-CD28, KOLT-2, 15E8, 248.23.2, and EX5.3D10.

[0325] In one embodiment, molecules that provide a primary stimulatory signal, eg, a molecule that provides stimulation via the TCR / CD3 complex, and a costimulatory molecule are linked to the same surface.

[0326] In certain embodiments, the binding agents that provide the stimulatory and costimulatory signals are localized on the cell surface, which can be achieved by transfecting or transducing the cells with a nucleic acid encoding the binding agent in a form suitable for expression on the cell surface, or by linking the binding agent to the cell surface.

[0327] In another embodiment, molecules that provide a primary stimulatory signal, eg, a molecule that provides stimulation via the TCR / CD3 complex, and a costimulatory molecule are presented on an antigen presenting cell.

[0328] In one embodiment, the molecule that provides the primary stimulatory signal, eg, a molecule that provides stimulation via the TCR / CD3 complex, and the costimulatory molecule are displayed on separate surfaces.

[0329] In certain embodiments, one of the binding substances providing the stimulatory and costimulatory signals is soluble (provided in solution) and the other substance is provided on one or more surfaces. In certain embodiments, the binding substances providing the stimulatory and costimulatory signals are both provided in soluble form (provided in solution). In various embodiments, the methods for generating T cells contemplated herein include activating T cells with soluble anti-CD3 and / or soluble anti-CD28 antibodies, or fragments thereof. In various embodiments, the methods for generating T cells contemplated herein include activating T cells with surface-bound anti-CD3 and / or surface-bound anti-CD28 antibodies, or fragments thereof. In various embodiments, the methods for generating T cells contemplated herein include activating T cells with bead-bound anti-CD3 and / or bead-bound anti-CD28 antibodies, or fragments thereof.

[0330] In one embodiment, expansion of T cells activated by the methods contemplated herein further comprises culturing the population of cells comprising the T cells for a period of time ranging from a few hours (about 3 hours) to about 7 days to about 28 days, or any integer number of hours therebetween. In another embodiment, the T cell composition may be cultured for 14 days. In a particular embodiment, the T cells are cultured for about 21 days. In another embodiment, the T cell composition is cultured for about 2-3 days. Several cycles of stimulation / activation / expansion may also be desired.

[0331] In certain embodiments, suitable conditions for T cell culture include a suitable medium (e.g., Minimal Essential Media or RPMI Media 1640 or X-vivo 15 (Lonza)) and one or more factors necessary for proliferation and activity, including, but not limited to, serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, IL-21, GM-CSF, IL-10, IL-12, IL-15, TGFβ, and TNF-α, or any other additives suitable for the growth of cells known to one of skill in the art.

[0332] Further illustrative examples of cell culture media include, but are not limited to, RPMI 1640, Clicks, AIM-V, DMEM, MEM, a-MEM, IMDM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and / or cytokines in sufficient amounts for the growth and proliferation of T cells.

[0333] Antibiotics, such as penicillin and streptomycin, are included only in the experimental cultures, not in the cultures of cells injected into the subject. Target cells are maintained under conditions necessary to support growth, such as an appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air plus 5% CO2).

[0334] In certain embodiments, PBMCs, or isolated T cells, are contacted with stimulatory and costimulatory agents, such as anti-CD3 and anti-CD28 antibodies, typically bound to beads or other surfaces, in culture medium containing appropriate cytokines, such as IL-2, IL-7, and / or IL-15.

[0335] In other embodiments, artificial APCs (aAPCs) are generated by engineering K562, U937, 721.221, T2 and C1R cells to induce stable expression and secretion of various costimulatory molecules and cytokines. In certain embodiments, K32 aAPCs or U32 aAPCs are used to direct the presentation of one or more antibody-based stimulatory molecules on the aAPC cell surface. A population of T cells can be expanded with aAPCs expressing various costimulatory molecules, including but not limited to CD137L (4-1BBL), CD134L (OX40L) and / or CD80 or CD86. Finally, aAPCs provide an efficient platform for expanding genetically modified T cells and maintaining CD28 expression on CD8 T cells. The aAPCs presented in WO03 / 057171 and US2003 / 0147869 are incorporated herein by reference in their entirety.

[0336] In certain embodiments, a polynucleotide encoding one or more engineered immune receptor components is introduced into a T cell population. In certain embodiments, a polynucleotide encoding one or more engineered immune receptor components is introduced into a T cell population expressing an exogenous lymphocyte receptor or an engineered antigen receptor. The polynucleotide may be introduced into the T cells by microinjection, transfection, lipofection, heat shock, electroporation, transduction, gene gun, microinjection, DEAE-dextran mediated transfer, and the like.

[0337] In a preferred embodiment, the polynucleotide is introduced into the T cell by viral transduction.

[0338] The polynucleotide is administered to immune effector cells or CD34 + Illustrative examples of viral vector systems suitable for introduction into cells include, but are not limited to, adeno-associated virus (AAV), retrovirus, herpes simplex virus, adenovirus, and vaccinia virus vectors for gene transfer.

[0339] In one embodiment, the polynucleotide is introduced into the T cell by AAV transduction. In one embodiment, the polynucleotide is introduced into the T cell by retroviral transduction. In one embodiment, the polynucleotide is introduced into the T cell by lentiviral transduction. In one embodiment, the polynucleotide is introduced into the T cell by adenoviral transduction. In one embodiment, the polynucleotide is introduced into the T cell by herpes simplex virus transduction. In one embodiment, the polynucleotide is introduced into the T cell by vaccinia virus transduction.

[0340] H. Compositions and Formulations Compositions contemplated herein may include one or more engineered immune receptor polypeptides, polynucleotides encoding engineered immune receptor polypeptides, vectors comprising same, genetically modified immune effector cells, cross-linking agents, and the like. Compositions include, but are not limited to, pharmaceutical compositions. A "pharmaceutical composition" refers to a composition formulated in a pharma- ceutical or physiologically acceptable solution for administration to a cell or animal, alone or in combination with one or more other therapeutic modalities. It will also be understood that, if desired, the composition may also be administered in combination with other agents, such as, for example, cytokines, growth factors, hormones, small molecules, chemotherapeutic agents, prodrugs, drugs, antibodies, or various other pharma- ceutical active agents. Indeed, there is also no limit to other components that may be included in the composition, provided that the additional agents do not adversely affect the ability of the composition to deliver the intended therapy.

[0341] The phrase "pharmaceutical acceptable" is used herein to refer to those compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0342] The term "pharmaceutical acceptable carrier" refers to a diluent, adjuvant, excipient, or vehicle administered with a crosslinking agent, polypeptide, polynucleotide, vector containing the same, or genetically modified immune effector cells. Illustrative examples of pharmaceutical carriers can be sterile liquids, such as cell culture media, water, and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Saline solutions and dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients in certain embodiments include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

[0343] In one embodiment, the composition comprising a pharma- ceutically acceptable carrier is suitable for administration to a subject. In certain embodiments, the composition comprising a carrier is suitable for parenteral administration, such as intravascular (intravenous or intraarterial), intraperitoneal or intramuscular administration. In certain embodiments, the composition comprising a pharma- ceutically acceptable carrier is suitable for intraventricular, intrathecal or intrathecal administration. Pharmaceutically acceptable carriers include sterile aqueous solutions, cell culture media, or dispersions. The use of such media and agents for pharma- ceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the crosslinking agent, polypeptide, polynucleotide, vector comprising them, or genetically modified immune effector cells, their use in the pharmaceutical composition is contemplated.

[0344] In certain embodiments, compositions contemplated herein comprise genetically modified T cells and a pharma- ceutically acceptable carrier. Compositions, including cell-based compositions contemplated herein, may be administered separately by enteral or parenteral administration, or may be administered in combination with other appropriate compounds to achieve a desired therapeutic goal.

[0345] In certain embodiments, compositions contemplated herein comprise a cross-linking agent and a pharma- ceutically acceptable carrier.

[0346] A pharma- ceutically acceptable carrier must be of sufficiently high purity and of sufficiently low toxicity to be suitable for administration to the human subject being treated, and must also maintain or enhance the stability of the composition. A pharma-ceutically acceptable carrier may be liquid or solid, and is selected to provide the desired volume, concentration, when mixed with the other components of the composition, having regard to the intended mode of administration. For example, the pharma- ceutically acceptable carrier may be, but is not limited to, a binder (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropylmethylcellulose, etc.), a filler (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylates, calcium hydrogen phosphate, etc.), a lubricant (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metal stearates, hydrogenated vegetable oils, corn starch, polyethylene glycol, sodium benzoate, sodium acetate, etc.), a disintegrant (e.g., starch, sodium starch glycolate, etc.), or a wetting agent (e.g., sodium lauryl sulfate, etc.). Other suitable pharma-ceutically acceptable carriers for the compositions contemplated herein include, but are not limited to, water, salt solutions, alcohol, polyethylene glycol, gelatin, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, and the like.

[0347] Such carrier solutions may also contain buffers, diluents, and other suitable additives. As used herein, the term "buffer" refers to a solution or liquid whose chemical composition neutralizes acids or bases without causing a significant change in pH. Examples of buffers contemplated herein include, but are not limited to, Dulbecco's phosphate buffered saline (PBS), Ringer's solution, 5% dextrose solution (D5W), normal / physiological saline (0.9% NaCl).

[0348] The pharma- ceutically acceptable carrier may be present in an amount sufficient to maintain the pH of the composition at about 7. Alternatively, the composition has a pH in the range of about 6.8 to about 7.4, such as 6.8, 6.9, 7.0, 7.1, 7.2, 7.3 and 7.4. In yet another embodiment, the composition has a pH of about 7.4.

[0349] The compositions contemplated herein may comprise a non-toxic pharma- ceutically acceptable medium. The compositions may be suspensions. As used herein, the term "suspension" refers to a non-adherent state in which cells do not bind to a solid support. For example, cells maintained as suspensions may be stirred or agitated and do not adhere to a support such as a culture dish.

[0350] In certain embodiments, compositions contemplated herein are formulated in a suspension, where the engineered T cells are dispersed in an acceptable liquid medium or solution, such as in an intravenous (IV) bag, such as in an acceptable liquid medium or solution, such as saline or serum-free medium. Acceptable diluents include, but are not limited to, water, PlasmaLyte, Ringer's solution, isotonic sodium chloride solution (saline), serum-free cell culture medium, and medium suitable for cryogenic storage, such as Cryostor® medium.

[0351] In certain embodiments, the pharma- ceutically acceptable carrier is substantially free of naturally occurring proteins of human or animal origin and is suitable for preserving the composition comprising the engineered T cell population. Therapeutic compositions are intended for administration to a human patient and therefore are substantially free of cell culture components such as, for example, bovine serum albumin, horse serum, and fetal bovine serum.

[0352] In some embodiments, the composition is formulated in a pharma- ceutically acceptable cell culture medium. Such a composition is suitable for administration to a human subject. In certain embodiments, the pharma-ceutically acceptable cell culture medium is a serum-free medium.

[0353] Serum-free media have several advantages over serum-containing media, including a simpler and more defined composition, reduced levels of contamination, elimination of potential sources of infectious pathogens, and lower cost. In various embodiments, serum-free media are animal-free and may optionally be protein-free. Optionally, the media may contain biopharmaceutical acceptable recombinant proteins. "Animal-free" media refers to media whose components are derived from non-animal sources. Recombinant proteins replace natural animal proteins in animal-free media, whose nutrients are derived from synthetic, plant, or microbial sources. "Protein-free" media, in contrast, are defined as being substantially protein-free.

[0354] Illustrative examples of serum-free media for use in certain compositions include, but are not limited to, QBSF-60 (Quality Biological, Inc.), StemPro-34 (Life Technologies), and X-VIVO 10.

[0355] In one embodiment, a composition comprising the modified T cells is formulated in PlasmaLyte.

[0356] In various embodiments, compositions comprising modified T cells are formulated in a cryopreservation medium. For example, a cryopreservation medium containing a cryopreservative may be used to maintain high cell activity after thawing. Illustrative examples of cryopreservation media used in certain compositions include, but are not limited to, CryoStor CS10, CryoStor CS5, and CryoStor CS2.

[0357] In one embodiment, the composition is formulated in a solution comprising 50:50 PlasmaLyte A and CryoStor CS10.

[0358] In certain embodiments, the compositions are substantially free of mycoplasma, endotoxin, and microbial contamination. "Substantially free" with respect to endotoxin means lower endotoxin per cell dose than permitted by the FDA for biologics, which is 5 EU / kg total endotoxin per day, and 350 EU per total cell dose for an average 70 kg human. In certain embodiments, compositions contemplated herein contain about 0.5 EU / mL to about 5.0 EU / mL, or about 0.5 EU / mL, 1.0 EU / mL, 1.5 EU / mL, 2.0 EU / mL, 2.5 EU / mL, 3.0 EU / mL, 3.5 EU / mL, 4.0 EU / mL, 4.5 EU / mL, or 5.0 EU / mL.

[0359] In certain embodiments, the formulation of pharma- ceutical acceptable carrier solutions is known in the art, as is the development of appropriate dosing and treatment regimens for use of the particular compositions described herein in a variety of treatment regimens, including, for example, enteral and parenteral, e.g., intravascular, intravenous, intraarterial, intraosseous, intraventricular, intracerebral, intracranial, intrathecal, intrathecal, and intramedullary administration and formulations. Those of skill in the art will appreciate that certain embodiments contemplated herein are known, for example, in the pharmaceutical arts, and can be readily understood by reference to, for example, Remington: The Science and Practice of Pharmacy, volume I and volume II.22. nd It will be understood that the compositions may include other formulations such as those described in The Pharmaceuticals and Medical Devices Association, Vol. 12, No. 1, 2012, Edition. Edited by Loyd V. Allen Jr. Philadelphia, PA: Pharmaceutical Press; 2012, which is incorporated herein by reference in its entirety.

[0360] In certain embodiments, the composition comprises a quantity of immune effector cells expressing one or more engineered immune receptor components as contemplated herein. In certain embodiments, the composition comprises a quantity of immune effector cells expressing an exogenous lymphocyte receptor or an engineered antigen receptor, and one or more engineered immune receptor components as contemplated herein. As used herein, the term "amount" refers to an "effective amount" or "effective amount" of cells comprising one or more engineered immune receptor components as contemplated herein to achieve a beneficial or desired prophylactic or therapeutic result, including a clinical result, in the presence of a crosslinking agent.

[0361] A "prophylactically effective amount" refers to an amount of cells containing one or more engineered immune receptor components, etc., as contemplated herein, that is effective to achieve a desired prophylactic result in the presence of a crosslinking agent. Typically, but not necessarily, a prophylactic dose is used prior to or in subjects at an earlier stage of disease, so the prophylactically effective amount will be less than the therapeutically effective amount.

[0362] A "therapeutically effective amount" refers to an amount of cells containing one or more engineered immune receptor components as contemplated herein, which in the presence of a cross-linking agent is effective to "treat" a subject (e.g., a patient). When a therapeutic amount is indicated, the exact amount of composition, cells, cross-linking agent, etc. to be administered can be determined by a physician, taking into account individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject).

[0363] Generally, the pharmaceutical compositions comprising the immune effector cells described herein are administered in a concentration of 10 2 ~10 10 Cells / kg body weight, preferably 10 5 ~10 6It can be said that a dosage of cells / kg body weight can be administered, including all integer values ​​within this range. The number of cells depends on the end use for which the composition is intended, as well as the type of cells contained therein. For the uses provided herein, the cells are generally in liter or less volumes, which may be 500 mL or less, or even 250 mL or 100 mL or less. Thus, the desired cell density is typically 10 6 More than 10 cells / ml, typically 10 7 More than 10 cells / ml, typically 10 8 Clinically relevant immune cell counts are 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 or 10 12 In some embodiments, the total number of cells injected may be divided into multiple injections equal to or greater than 10 cells, specifically because all cells injected are redirected to a specific target antigen. 6 / kilogram (10 per patient) 6 -10 11 ) may be administered.

[0364] If desired, treatment may also include administration of mitogens (e.g., PHA) or lymphokines, cytokines and / or chemokines (e.g., IFN-γ, IL-2, IL-12, TNF-alpha, IL-18, and TNF-beta, GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MIP1α, etc.) to enhance the induction of an immune response, as described herein.

[0365] In general, compositions comprising cells activated and expanded as described herein may be utilized in the treatment and prevention of diseases occurring in immunocompromised individuals. In particular, the compositions contemplated herein are used in the treatment of cancer. In certain embodiments, the immune effector cells may be administered as a pharmaceutical composition alone or in combination with carriers, diluents, excipients, and / or other components, such as IL-2 or other cytokines or cell populations.

[0366] In certain embodiments, a pharmaceutical composition comprises a quantity of genetically modified T cells in combination with one or more pharma- ceutical or physiologically acceptable carriers, diluents, or excipients.

[0367] In certain embodiments, a pharmaceutical composition comprises an amount of a cross-linking agent in combination with one or more pharma- ceutical or physiologically acceptable carriers, diluents, or excipients.

[0368] In certain embodiments, the composition comprises an effective amount of immune effector cells comprising one or more engineered immune receptor components as contemplated herein, alone or in combination with a crosslinking agent and / or one or more therapeutic agents, such as radiation therapy, chemotherapy, transplantation, immunotherapy, hormonal therapy, photodynamic therapy, etc. The composition may be administered in combination with an antibiotic. Such therapeutic agents may be accepted in the art as standard of care, for example, for a particular disease state as described herein, such as a particular cancer. Exemplary therapeutic agents contemplated include cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory agents, chemotherapeutic agents, radiation therapy, therapeutic antibodies, or other active and adjunctive agents.

[0369] In certain embodiments, a composition comprising an effective amount of immune effector cells comprising one or more engineered immune receptor components as contemplated herein is administered to a subject, and a composition comprising an effective amount of a cross-linking agent is administered to the subject before, during, in combination with, or subsequent to the cell composition, and optionally repeatedly administered to the subject.

[0370] In certain embodiments, compositions comprising immune effector cells comprising one or more engineered immune receptor components as contemplated herein may be administered in combination with any number of chemotherapeutic agents.

[0371] A variety of other therapeutic agents may be used in conjunction with the compositions described herein. In one embodiment, a composition comprising immune effector cells comprising one or more engineered immune receptor components as contemplated herein is administered with an anti-inflammatory agent. Anti-inflammatory agents or drugs include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone), aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF medications, cyclophosphamide, and non-steroidal anti-inflammatory agents (NSAIDs), including mycophenolate.

[0372] Illustrative examples of therapeutic antibodies suitable for combination therapy with modified T cells comprising one or more engineered immune receptor components contemplated herein include, but are not limited to, atezolizumab, avelumab, bavituximab, bevacizumab (Avastin), bivatuzumab, blinatumomab, conatumumab, daratumumab, durigotumab, dacetuzumab, darotuzumab, and darotuzumab. , durvalumab, elotuzumab (HuLuc63), gemtuzumab, ibritumomab, indatuximab, inotuzumab, ipilimumab, lorvotuzumab, lucatumumab, milatuzumab, moxetumomab, nivolumab, ocaratuzumab, ofatumumab, pembrolizumab, rituximab, siltuximab, teprotumumab, and ublituximab.

[0373] In some embodiments, the compositions described herein are administered in conjunction with a cytokine. As used herein, "cytokine" refers to a general term for proteins released by one cell group and acting as intercellular mediators on another cell. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones.

[0374] I. Treatment method Immune effector cells modified to express one or more engineered immune receptor components and / or exogenous lymphocyte receptors or engineered antigen receptors contemplated herein provide improved adoptive immunotherapy methods for use in, or for preventing, treating, or ameliorating at least one symptom associated with an immune-related disease or disorder, such as cancer, an autoimmune disease, an immune deficiency, an inflammatory disease, GVHD, or an infectious disease.

[0375] Engineered immune receptor systems, e.g., immune effector cells comprising targeting and signaling components, provide improved adoptive immunotherapy methods for use in, or for preventing, treating, or ameliorating at least one symptom associated with an immune-related disease or disorder, e.g., cancer, an autoimmune disease, an immunodeficiency, an inflammatory disease, GVHD, or an infectious disease.

[0376] In certain embodiments, immune effector cells modified to express one or more engineered immune receptor components provide an improved method of adoptive immunotherapy to fine-tune the safety and efficacy of cytotoxic responses against target cells expressing the target antigen, e.g., tumor cells, while reducing the risk of on-target antigen, off-target cellular cytotoxicity (recognizing the target antigen on normal non-target cells).

[0377] In certain embodiments, a method for preventing, treating, or ameliorating at least one symptom of an immune-related disease or disorder (e.g., cancer, an autoimmune disease, an immune deficiency, an inflammatory disease, GVHD, or an infectious disease) comprises administering to a subject an effective amount of modified immune effector cells or T cells (comprising one or more components of an engineered immune receptor system and an engineered / exogenous TCR, CAR, or other therapeutic transgene) to redirect the cells to target cells. Genetically modified cells are more effective and safer cellular immunotherapy by delivering chemically tunable immunostimulatory signals.

[0378] In certain embodiments, one or more immune effector cells, e.g., T cells, are modified to express both a targeting moiety and a signaling moiety. In this case, the modified cells are administered to a subject in need thereof and directed to the target cells via interaction of the signaling moiety expressed on the immune effector cells with the target antigen expressed on the target cells. A cross-linking agent is administered to the subject before, at about the same time as, or after the modified cells are administered to the subject. In the presence of the cross-linking agent, a complex forms between the targeting moiety bound to the target antigen, the cross-linking agent, and the signaling moiety bound to the immune receptor complex. Upon complex formation, the signaling moiety and the targeting moiety transmit an immune stimulatory signal to the immune effector cells, which synergize with the immune receptor signal to then elicit a cytotoxic response from the immune effector cells against the target cells.

[0379] In various embodiments, immune effector cells comprising one or more engineered immune receptor components and / or exogenous lymphocyte receptors or engineered antigen receptors fine-tune the safety and efficacy of the cytotoxic response against target cells using a dual targeting strategy in which one or more target cells express one or more target antigens recognized by the exogenous lymphocyte receptors or engineered antigen receptors and the engineered immune receptors.

[0380] In certain embodiments, one or more immune effector cells, e.g., T cells, are modified to express both a targeting moiety and a signaling moiety as well as an exogenous lymphocyte receptor or an engineered antigen receptor, e.g., a TCR, a CAR, a CCR, or a flip receptor. In this case, the modified cells are administered to a subject in need thereof and home to the target cells via the interaction of a targeting moiety that binds to a first target antigen and an antigen receptor that binds to a second target antigen, where one or both target antigens are expressed on the target cell or population of target cells. The interaction of an antigen receptor (e.g., a TCR) with a target antigen on the target cell can elicit a cytotoxic response from the immune effector cells against the target cell. In some embodiments, a cross-linking agent is administered to the subject prior to the modified cells, at about the same time as the modified cells, or after the modified cells are administered to the subject. In the presence of the cross-linking agent, a complex forms between the targeting moiety that binds to the first target antigen, the cross-linking agent, and the signaling moiety that binds to the immune receptor complex. Upon formation of the complex, the signaling component and the targeting component transmit an immunostimulatory signal to the immune effector cell which, in turn, synergizes with the immune receptor signal to induce or augment a cytotoxic response from the immune effector cell against the target cell.

[0381] In certain embodiments, one or more immune effector cells, e.g., T cells, are modified to express both a targeting moiety and a signaling moiety. In this case, the modified cells are administered to a subject in need thereof and directed to the target cells via interaction of the signaling moiety expressed on the immune effector cells with the target antigen expressed on the target cells. In some embodiments, a cross-linking agent is administered to the subject prior to the modified cells, at about the same time as the modified cells, or after the modified cells are administered to the subject. In the presence of the cross-linking agent, a complex forms between the targeting moiety bound to the target antigen, the cross-linking agent, and the signaling moiety comprising a multimerization domain, optionally a linker polypeptide, and an actuator domain (e.g., Fcγ, CD3ε, CD3δ, or CD3γ or a fragment thereof). Upon formation of the complex, the signaling moiety and the targeting moiety transmit an immune stimulatory signal to the immune effector cells, which synergize with an immune receptor signal to then elicit a cytotoxic response from the immune effector cells against the target cells.

[0382] In certain embodiments, one or more immune effector cells, e.g., T cells, are modified to express both the targeting moiety and the signaling moiety, as well as an exogenous lymphocyte receptor or an engineered antigen receptor, e.g., an engineered / exogenous TCR. In this case, the modified cells are administered to a subject in need thereof and home to the target cells via the interaction of a targeting moiety that binds to a first target antigen and an immune receptor that binds to a second target antigen, where one or both target antigens are expressed on the target cell or population of target cells. The interaction of the TCR with the target antigen on the target cell can elicit a cytotoxic response from the immune effector cells against the target cell. A cross-linking agent is administered to the subject before, at about the same time as, or after the modified cells are administered to the subject. In the presence of the cross-linking agent, a complex forms between the targeting moiety that binds to the first target antigen, the cross-linking agent, and the signaling moiety that includes a multimerization domain, optionally a linker polypeptide, and Fcγ, CD3ε, CD3δ, or CD3γ, or a fragment thereof. Upon formation of the complex, the signaling component and the targeting component transmit an immunostimulatory signal to the immune effector cell which, in turn, synergizes with the immune receptor signal to induce or augment a cytotoxic response from the immune effector cell against the target cell.

[0383] In certain embodiments, the engineered immune effector cells contemplated herein are used in the treatment of solid tumors or cancer.

[0384] In certain embodiments, the engineered immune effector cells contemplated herein are selected from the group consisting of, but not limited to, adrenal carcinoma, adrenocortical carcinoma, anal carcinoma, appendix carcinoma, astrocytoma, atypical teratoid tumor / atypical rhabdomyoid tumor, basal cell carcinoma, bile duct carcinoma, bladder carcinoma, bone cancer, brain / CNS cancer, breast cancer, bronchial tumor, cardiac tumor, cervical cancer, bile duct carcinoma, chondrosarcoma, chordoma, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma in situ (DCIS), endometrial cancer, epithelioma, esophageal cancer, nasal neuroblastoma, Ewing's sarcoma, extracranial embryonal cell carcin ... alveolar tumor, extragonadal germ cell tumor, eye cancer, fallopian tube cancer, fibrous tissue sarcoma, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, glioma, glioblastoma, head and neck cancer, hemangioblastoma, hepatocellular carcinoma, hypopharyngeal cancer, intraocular melanoma, Kaposi's sarcoma, renal cancer, laryngeal cancer, leiomyosarcoma, tongue cancer, liposarcoma, hepatoma, lung cancer, non-small cell lung cancer, embryonal carcinoid tumor, malignant mesothelioma, medullary carcinoma, medulloblastoma, meningioma, melanoma, Merkel cell carcinoma, midline carcinoma, mouth cancer, myxosarcoma, myelodysplastic syndrome, myeloproliferative neoplasm, cancer of the nasal cavity and paranasal sinuses, nasopharyngeal carcinoma, neuroblastoma, oligodendroglioma, oral cancer, oral cavity in the treatment of solid tumors or cancers, including oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, islet cell tumors, papillary carcinoma, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pinealoma, pituitary tumor, pleuropulmonary blastoma, primary pleurioma, prostate cancer, rectal cancer, retinoblastoma, renal cell carcinoma, renal pelvis and ureter cancer, rhabdomyosarcoma, salivary gland cancer, sebaceous gland cancer, skin cancer, soft tissue sarcoma, squamous cell carcinoma, small cell lung cancer, small intestine cancer, gastric cancer, sweat gland carcinoma, synovial tumor, testicular cancer, pharyngeal cancer, thymic cancer, thyroid cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vascular cancer, vulvar cancer, and Wilms' tumor

[0385] In certain embodiments, the engineered immune effector cells contemplated herein are used in the treatment of solid tumors or cancers, including, but not limited to, 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, and oligodendroglioma.

[0386] In certain embodiments, the engineered immune effector cells contemplated herein are used in the treatment of solid tumors or cancers, including, but not limited to, non-small cell lung cancer, metastatic colorectal cancer, glioblastoma, head and neck cancer, pancreatic cancer, and breast cancer.

[0387] In certain embodiments, the engineered immune effector cells contemplated herein are used in the treatment of glioblastoma.

[0388] In certain embodiments, the engineered immune effector cells contemplated herein are used in the treatment of liquid or hematological cancers.

[0389] In certain embodiments, the engineered immune effector cells contemplated herein are used in the treatment of B-cell malignancies, including, but not limited to, leukemia, lymphoma, and multiple myeloma.

[0390] In certain embodiments, the engineered immune effector cells contemplated herein are directed to, but are not limited to, leukemias, lymphomas, and multiple myelomas: acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, erythroleukemia, hairy cell leukemia (HCL), chronic lymphocytic leukemia (CLL), and chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia (CMML), and polycythemia vera, Hodgkin's lymphoma, nodular lymphocyte-predominant Hodgkin's lymphoma, Burkholderia myeloma, and multiple myeloma. It is used in the treatment of liquid cancers including idiopathic myeloma, small lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, mantle cell lymphoma, marginal zone lymphoma, mycosis fungoides, anaplastic large cell lymphoma, Sezary syndrome, precursor T-lymphoblastic lymphoma, multiple myeloma, overt multiple myeloma, smoldering multiple myeloma, plasma cell leukemia, nonsecretory myeloma, IgD myeloma, osteosclerotic myeloma, solitary bone plasmacytoma, and extramedullary plasmacytoma.

[0391] In certain embodiments, the engineered immune effector cells contemplated herein are used in the treatment of acute myeloid leukemia (AML).

[0392] Preferred cells for use in the methods contemplated herein include autologous / autogeneic ("self") cells, preferably hematopoietic cells, more preferably T cells, and more preferably immune effector cells.

[0393] In certain embodiments, the method comprises administering to a patient in need thereof a therapeutically effective amount of modified immune effector cells expressing one or more engineered immune receptor components and, optionally, an exogenous lymphocyte receptor or an engineered antigen receptor or another targeting or signaling component, or a composition comprising the same, and, optionally, also administering a cross-linking agent to the subject. In certain embodiments, the cells are used in the treatment of a patient at risk of developing an immune disorder. Thus, certain embodiments include the treatment, prevention, or amelioration of at least one symptom of an immune-related disease or disorder, e.g., cancer or an autoimmune disease, comprising administering to a subject in need thereof a therapeutically effective amount of modified immune effector cells as contemplated herein, and, optionally, a cross-linking agent.

[0394] The amount and frequency of administration of the engineered immune effector cells, targeting moieties, and / or cross-linking agents will depend on factors such as the condition of the patient, the type and severity of the patient's disease, and appropriate dosage amounts and dosing schedules may be determined through clinical trials.

[0395] In one exemplary embodiment, the effective amount of engineered immune effector cells provided to a subject is at least 2×10 6 Cells / kg, at least 3x10 6 Cells / kg, at least 4x10 6 Cells / kg, at least 5x10 6 Cells / kg, at least 6x10 6 Cells / kg, at least 7x10 6Cells / kg, at least 8x10 6 Cells / kg, at least 9x10 6 cells / kg or at least 10x10 6 cells / kg or more, including all doses of cells in between.

[0396] In another exemplary embodiment, the effective amount of engineered immune effector cells provided to a subject is about 2×10 6 cells / kg, approximately 3x10 6 cells / kg, approximately 4x10 6 cells / kg, approximately 5x10 6 cells / kg, approximately 6x10 6 cells / kg, approximately 7x10 6 cells / kg, approximately 8x10 6 cells / kg, approximately 9x10 6 cells / kg, or approximately 10x10 6 cells / kg or more, including all doses of cells in between.

[0397] In another exemplary embodiment, the effective amount of engineered immune effector cells provided to a subject is about 2×10 6 cells / kg ~ approx. 10x10 6 cells / kg, approximately 3x10 6 cells / kg ~ approx. 10x10 6 cells / kg, approximately 4x10 6 cells / kg ~ approx. 10x10 6 cells / kg, approximately 5x10 6 cells / kg ~ approx. 10x10 6 cells / kg, 2x10 6 cells / kg ~ approx. 6x10 6 cells / kg, 2x10 6 cells / kg ~ approx. 7x10 6 cells / kg, 2x10 6 cells / kg ~ approx. 8x10 6 cells / kg, 3x10 6 cells / kg ~ approx. 6x10 6 cells / kg, 3x10 6 cells / kg ~ approx. 7x10 6 cells / kg, 3x10 6 cells / kg ~ approx. 8x10 6cells / kg, 4x10 6 cells / kg ~ approx. 6x10 6 cells / kg, 4x10 6 cells / kg ~ approx. 7x10 6 cells / kg, 4x10 6 cells / kg ~ approx. 8x10 6 cells / kg, 5x10 6 cells / kg ~ approx. 6x10 6 cells / kg, 5x10 6 cells / kg ~ approx. 7x10 6 cells / kg, 5x10 6 cells / kg ~ approx. 8x10 6 cells / kg or 6x10 6 cells / kg ~ approx. 8x10 6 cells / kg and includes all cell doses in between.

[0398] Those skilled in the art will recognize that multiple administrations of the compositions contemplated in certain embodiments may be required to affect the desired treatment. For example, the compositions may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times over a period of 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 5 years, 10 years, or more. The modified immune effector cells, engineered immune receptor components, and / or cross-linking agents may be administered in the same or different compositions; in one or more compositions at the same time; or in more than one composition at different times. The modified immune effector cells, engineered immune receptor components (e.g., targeting moieties), and / or cross-linking agents may be administered via the same route of administration or different routes.

[0399] In some embodiments, it may be desirable to administer activated T cells to a subject, then draw blood (or perform apheresis) from the subject, activate the T cells derived therefrom, and reinfuse these activated and expanded T cells back into the patient. This process may be performed multiple times, every few weeks. In some embodiments, the T cells may be activated from a blood draw of 10cc to 400cc. In some embodiments, the T cells are activated from a blood draw of 20cc, 30cc, 40cc, 50cc, 60cc, 70cc, 80cc, 90cc, 100cc, 150cc, 200cc, 250cc, 300cc, 350cc, or 400cc or more. Without being bound by theory, the use of a multiple blood draw / multiple reinfusion protocol may aid in the selection of certain populations of T cells.

[0400] In one embodiment, a method of treating a subject diagnosed with an immune-related disease or disorder (e.g., cancer or an autoimmune disease) comprises removing immune effector cells from the subject, modifying the immune effector cells by introducing into the cells one or more vectors encoding one or more engineered immune receptor components, producing a population of modified immune effector cells, and administering the population of modified immune effector cells to the same subject. In a preferred embodiment, the immune effector cells comprise T cells.

[0401] In one embodiment, a method of treating a subject diagnosed with cancer or an autoimmune disease comprises administering to the subject one or more vectors encoding one or more engineered immune receptor components. In a preferred embodiment, the immune effector cells comprise T cells.

[0402] In one embodiment, a method of treating a subject diagnosed with cancer comprises removing immune effector cells from the subject, modifying the immune effector cells by introducing into the cells one or more vectors encoding one or more engineered immune receptor components and, optionally, an exogenous lymphocyte receptor or an engineered antigen receptor or another targeting or signaling component, producing a population of modified immune effector cells, and administering the population of modified immune effector cells to the same subject. In a preferred embodiment, the immune effector cells comprise T cells.

[0403] In one embodiment, a method of treating a subject diagnosed with cancer comprises administering to the subject one or more vectors encoding one or more engineered immune receptor components. In a preferred embodiment, the immune effector cells comprise T cells.

[0404] Methods of administering the cell compositions contemplated in certain embodiments include any method effective to effect the reintroduction of engineered immune effector cells ex vivo, or any method effective to effect the reintroduction of engineered precursors of immune effector cells that differentiate into mature immune effector cells upon introduction into a subject. One method involves modifying peripheral blood T cells ex vivo by introducing into the cells one or more vectors encoding one or more engineered immune receptor components and, optionally, an exogenous lymphocyte receptor or an engineered antigen receptor or another targeting or signaling component, and returning the transduced cells to the subject.

[0405] Methods of administering the cell compositions contemplated in certain embodiments include any method effective to effect the reintroduction of engineered immune effector cells ex vivo, or any method effective to effect the reintroduction of engineered precursors of immune effector cells that differentiate into mature immune effector cells upon introduction into a subject. One method involves modifying peripheral blood T cells ex vivo by introducing into the cells one or more vectors encoding one or more engineered immune receptor components and, optionally, an exogenous lymphocyte receptor or an engineered antigen receptor or another targeting or signaling component, and returning the transduced cells to the subject.

[0406] Methods for administering the vector compositions contemplated in certain embodiments include any method that is effective to result in engineered immune effector cells in vivo.

[0407] [Table 12-1] [Table 12-2] [Table 12-3] [Table 12-4] [Table 12-5] [Table 12-6] [Table 12-7] [Table 12-8] [Table 12-9] [Table 12-10] [Table 12-11] [Table 12-12] [Table 12-13] [Table 12-14] [Table 12-15] [Table 12-16] [Table 12-17] [Table 12-18] [Table 12-19] [Table 12-20]

[0408] All publications, patent applications, and issued patents cited in this specification are herein incorporated by reference to the same extent as if each individual publication, patent application, or issued patent was specifically and individually indicated to be incorporated by reference.

[0409] Although the foregoing embodiments have been described in detail in the figures and examples for purposes of clarity and understanding, it will be readily apparent to those skilled in the art in light of the teachings contemplated herein that certain changes and modifications may be made without departing from the spirit or scope of the appended claims. The following examples are provided for illustration only and not for limitation. Those skilled in the art will readily recognize a variety of non-critical parameters that can be changed or modified to produce essentially similar results. EXAMPLES

[0410] Working Example 1 Rapamycin-induced TCR (T cell receptor) assembly. Lentiviral vectors containing constructs containing at least a multimerization domain (eg, a rapamycin-inducible dimerization domain), a CD3 subunit, and an extracellular antigen targeting domain were designed, cloned, and sequence verified. The constructs contain various combinations of the following units: a signal sequence (e.g., a signal sequence derived from CD8α or IgK), one or more multimerization domains (e.g., FK506 binding protein (FKBP12 or FKBP) and FKBP-rapamycin binding protein (FRB or FRB*)), a CD3 subunit, one or more viral self-cleaving peptides (e.g., P2A or T2A self-cleaving peptides), one or more extracellular antigen targeting domains (e.g., an antibody-derived targeting domain or a natural ligand-derived targeting domain), various hinge and transmembrane domains (e.g., derived from CD4 or CD28), one or more intracellular domains (e.g., a costimulatory domain), and one or more co-receptor or cytokine receptor domains (see Figures 1A-1H). Transgenic TCRs have also been expressed along with the above components in some constructs.

[0411] Example 2 Evaluation of rapamycin-inducible TCR T cells without the hinge domain that anchors FKBP12. T cells expressing rapamycin-inducible T cell receptor (SR-T cells) were generated using a 10-day transduction and expansion process and then assessed for expression and biological activity against specific target antigens (see Leung et al., JCI Insight. 2019 Apr 30;5(11)). Briefly, peripheral blood mononuclear cells (PBMCs) were cultured in IL-2-containing medium and activated with antibodies against human CD3 and human CD28. PBMCs were transduced one day after initiation of culture using a lentiviral vector encoding a rapamycin-inducible TCR without a hinge domain between the FKBP12 multimerization domain and the CD4 transmembrane domain (CD4tm) of the targeting moiety (SR024), and the cells were then transferred 48 hours later into a 24-well G-REX® culture system (Wilson Wolf). After a total of 10 days in culture, SR-T cells were assessed for rapamycin-dependent, antigen-dependent activity, as well as rapamycin-independent, antigen-independent background activity.

[0412] SR-T cells (SR024-T cells) transduced with the lentiviral vector described above were analyzed for extracellular expression of key components of the rapamycin-inducible system, including the targeting domain (e.g., anti-CD33 VHH), FRB multimerization domain, and FKBP12 multimerization domain, by flow cytometry using recombinant CD33-Fc fusion, anti-FRB antibody, and anti-FKBP12 antibody, respectively. As shown in Figure 2, the FRB and CD33 components were highly expressed compared to untransduced control cells.

[0413] The rapamycin-dependent antigen-dependent activity of SR024-T cells was assessed in co-culture with target antigen-positive tumor cells in the presence of 1 nM rapamycin. Specifically, IFNγ, IL2, IL4, and TNFα production was quantified using MSD from the supernatants of cultures with SR024-T cells and cell lines engineered to express high levels of CD33 (Figure 3). SR024-T cells released each of these four cytokines at levels highly distinguishable from baseline (untransduced control; UTD). In addition, T cells were cultured alone, without the presence of antigen, both in the presence and without 1 nM rapamycin (Figure 4). In both cases, SR024-T cells produced 2 orders of magnitude less IFNγ than when they were cultured in the presence of antigen.

[0414] The cytotoxicity of SR024-T cells was assessed by incubating them with cells expressing the target antigen and nuclear red fluorescent molecules. Both cells expressing nuclear fluorescence (antigen-positive target line) and cells not expressing nuclear fluorescence (SR-T cells) were tracked for approximately 200 hours using an Incucyte® live cell imaging system (Sartorius). SR024-T cells did not effectively kill CD33+ target cells (Figure 5A), but expanded significantly in these cultures compared to T cell controls (Figure 5B).

[0415] Example 3 Evaluation of rapamycin-inducible TCR T cells with FKBP12 anchored to the CD4 or CD28 hinge domain. T cells expressing rapamycin-inducible T cell receptor (SR-T cells) were generated using a 10-day transduction and expansion process, and then evaluated for expression and biological activity against specific target antigens. Briefly, PBMCs were cultured in IL-2-containing medium and activated with antibodies against human CD3 and human CD28. PBMCs were transduced on the first day after initiation of culture using lentiviral vectors (SR022, SR020, and SR024) encoding rapamycin-inducible TCR with FKBP12 containing targeting moieties anchored to CD4 or CD28 hinge domains (CD4h or CD28h) or without hinge domains, and the cells were then transferred to 24-well G-REX® culture systems 48 hours later. After a total of 10 days in culture, SR-T cells were evaluated for rapamycin-dependent, antigen-dependent activity; as well as rapamycin-independent, antigen-independent background activity.

[0416] SR-T cells transduced with the lentiviral vectors described above (SR022-, SR020-, and SR024-T cells) were analyzed for extracellular expression of key components of the rapamycin-inducible system, including the targeting domain (e.g., anti-CD33 VHH), FRB multimerization domain, and FKBP12 multimerization domain, by flow cytometry using recombinant CD33-Fc fusion, anti-FRB antibody, and anti-FKBP12 antibody, respectively. The FRB and CD33 components were highly expressed compared to non-transduced control cells (Figure 6).

[0417] The rapamycin-dependent, antigen-dependent activity of SR022-, SR020-, and SR024-T cells was assessed in co-culture with target antigen-positive tumor cells in the presence of 1 nM rapamycin. Specifically, IFNγ, IL2, IL4, and TNFα production was quantified using MSD from the supernatants of cultures with SR022-, SR020-, or SR024-T cells; and cell lines engineered to express high levels of CD33 (Figure 7A). Both SR022- and SR020-T cells released each of these four cytokines at levels highly distinguishable from baseline (untransduced control; UTD). SR020 showed reduced IFNγ, IL2, and TNFα compared to SR022, but IL4 was comparable. SR024-T cells had reduced cytokine release across all four cytokines compared to SR022- and SR020-T cells.

[0418] T cells were also co-cultured with CD33+ cell lines with high, medium, and low antigen densities. SR022- and SR020-T cells produce more IFNγ than SR024-T cells at all antigen densities, especially at low antigen density (Figure 7B). In addition, SR022- and SR020-T cells produce IL2 in cultures at all antigen densities, whereas SR024-T produced little or no IL2 (Figure 7C). SR022-T cells secrete more IFNγ and IL2 across high, medium, and low CD33+ antigen density target lines. T cells were also cultured alone without the presence of antigen, in the presence and without 1 nM rapamycin (Figure 8). In the absence of antigen, SR024, SR022, and SR020-T cells produced 2 orders of magnitude less IFNγ than when antigen was present.

[0419] The cytotoxicity of SR024-, SR022-, and SR020-T cells was assessed by incubating them with cells expressing the target antigen and nuclear red stain. Both cells expressing the nuclear stain (antigen-positive target line) and cells not expressing the nuclear stain (SR-T cells) were tracked for approximately 200 hours using an Incucyte® live cell imaging system (Sartorius). While SR024-T cells exhibited minimal killing of CD33+ target cells (Figure 9A), they expanded significantly in these cultures compared to T cell controls (Figure 9B). SR022-T cells were able to effectively kill (Figure 9A) and expand (Figure 9B) CD33+ target cells compared to T cell controls. While SR020-T cells expanded well compared to controls (Figure 9B), they showed intermediate performance in the cytotoxicity assay (Figure 9A). Overall, SR022-T cells performed significantly better in the cytotoxicity assays compared to SR020- or SR024-T cells.

[0420] Figures 7A-7C, 8, and 9A-9B demonstrate that SR020 and SR022, which include a hinge domain, exhibit improved cytokine release profiles and increased killing of CD33+ target cells compared to SR024, which lacks a hinge domain (see Figures 1A and 1D). Without wishing to be bound by any particular theory, it is contemplated that the addition of a hinge domain to a signaling moiety improves the function of the structure by allowing the first multimerization domain of the signaling moiety to better align spatially with the multimerization domain of the targeting moiety, thereby surprisingly increasing the efficacy of the targeting moiety that includes the hinge domain compared to a targeting moiety lacking the hinge domain.

[0421] Example 4 Evaluation of rapamycin-induced TCR T cells with FRB attached to CD3γ. T cells expressing rapamycin-inducible T cell receptor (SR-T cells) were generated using a 10-day transduction and expansion process and then assessed for expression and biological activity against specific target antigens. Briefly, peripheral blood mononuclear cells (PBMCs) were cultured in IL-2-containing medium and activated with antibodies against human CD3 and human CD28. PBMCs were transduced on the first day after initiation of culture using a lentiviral vector encoding rapamycin-inducible TCR with FRB attached to CD3γ instead of CD3ε (SR021), and cells were then transferred to a 24-well G-REX® culture system 48 hours later. After a total of 10 days in culture, SR-T cells were assessed for rapamycin-dependent, antigen-dependent activity, as well as rapamycin-independent, antigen-independent background activity.

[0422] SR-T cells transduced with the lentiviral vector described above (SR021-T cells) were analyzed for extracellular expression of key components of the rapamycin-inducible system, including the targeting domain (e.g., anti-CD33 VHH), FRB dimerization domain, and FKBP12 dimerization domain, by flow cytometry using recombinant CD33-Fc fusion, anti-FRB antibody, and anti-FKBP12 antibody, respectively. FRB and CD33 components were highly expressed compared to non-transduced control cells (Figure 10).

[0423] The rapamycin-dependent antigen-dependent activity of SR021-T cells was evaluated in co-culture with target antigen-positive tumor cells in the presence of 1 nM rapamycin. Specifically, IFNγ, IL2, IL4, and TNFα production were quantified using MSD from the supernatants of cultures with SR021-T cells and cell lines engineered to express high levels of CD33 (Figure 11). SR021-T cells released each of these four cytokines at levels highly distinguishable from baseline (untransduced control; UTD). In addition, T cells were cultured alone, both without and in the presence of 1 nM rapamycin, without the presence of antigen (Figure 12). In the absence of antigen, SR021-T cells produced 2 orders of magnitude less IFNγ than in the presence of antigen.

[0424] Cytotoxicity of SR021-T cells against cells expressing the target antigen was assessed using a target line expressing a nuclear red stain and tracked over time using an Incucyte® live cell imaging system (Sartorius). Both cells expressing the nuclear stain (antigen positive target line) and cells not expressing the nuclear stain (SR-T cells) were tracked for approximately 200 hours. SR021-T cells exhibited a tendency to kill CD33+ cells (Figure 13A) and proliferated significantly compared to T cell controls (Figure 13B).

[0425] Example 5 Evaluation of rapamycin-inducible TCR T cells with FRB*(T2098L). T cells expressing rapamycin-inducible T cell receptor (SR-T cells) were generated using a 10-day transduction and expansion process and then evaluated for expression and biological activity against specific target antigens. Briefly, peripheral blood mononuclear cells (PBMCs) were cultured in IL-2-containing medium and activated with antibodies against human CD3 and human CD28. PBMCs were transduced on day 1 after initiation of culture using lentiviral vectors (SR022 and SR001, respectively) encoding rapamycin-inducible TCRs with either FRB or mutated FRB-FRB (T2098L) that allow binding of a specific rapamycin analog (denoted as FRB*), and the cells were then transferred to a 24-well G-REX® culture system 48 hours later. After a total of 10 days in culture, SR-T cells were evaluated for rapamycin-dependent, antigen-dependent activity, as well as rapamycin-independent, antigen-independent background activity.

[0426] SR-T cells transduced with the lentiviral vectors described above (SR022-T cells and SR001-T cells for FRB and FRB*, respectively) were analyzed for extracellular expression of key components of the rapamycin-inducible system, including the targeting domain (e.g., anti-CD33 VHH) and the FRB multimerization domain, by flow cytometry using recombinant CD33-Fc fusions and anti-FRB antibodies, respectively. FRB and CD33 components were highly expressed compared to non-transduced control cells (Figure 14).

[0427] The rapamycin-dependent antigen-dependent activity of SR022-T cells and SR001 T cells was evaluated in co-culture with target antigen-positive tumor cells in the presence of 1 nM rapamycin. Specifically, IFNγ, IL2, and TNFα production was quantified using MSD from the supernatants of cultures with SR022- or SR001-T cells and cell lines engineered to express high levels of CD33 (Figure 15). Both SR022- and SR001-T cells released each of these three cytokines at levels highly distinguishable from baseline (untransduced control; UTD). In addition, T cells were cultured alone, both without and in the presence of 1 nM rapamycin, and without the presence of antigen (Figure 16). Without the presence of antigen, SR022- and SR001-T cells produced 3 orders of magnitude less IFNγ than when antigen was present.

[0428] Cytotoxicity of SR001-T cells against cells expressing the target antigen was assessed using a target line expressing a nuclear red stain and tracked over time using an Incucyte® live cell imaging system (Sartorius). Both cells expressing the nuclear stain (antigen positive target line) and cells not expressing the nuclear stain (SR-T cells) were tracked for approximately 160 hours. SR001-T cells effectively killed CD33+ target cells (Figure 17A) and proliferated significantly compared to T cell controls (Figure 17B).

[0429] Example 6 Evaluation of rapamycin-inducible TCR T cells with FKBP12 distal to the antigen-targeting domain. In another experiment, T cells expressing rapamycin-inducible T cell receptor (SR-T cells) were generated using a 10-day transduction and expansion process and then evaluated for expression and biological activity against specific target antigens. Briefly, peripheral blood mononuclear cells (PBMCs) were cultured in IL-2-containing medium and activated with antibodies against human CD3 and human CD28. PBMCs were transduced on the first day after initiation of culture using a lentiviral vector encoding the rapamycin-inducible TCR with CD4 hinge (CD4h), transmembrane (CD4tm), and minimal intracellular (CD4ic) domains, anchoring the antigen targeting domain, followed by the FKBP12 multimerization domain (SR028), and then the cells were transferred to a 24-well G-REX® culture system 48 hours later. After a total of 10 days in culture, the SR-T cells were evaluated for rapamycin-dependent, antigen-dependent activity, as well as rapamycin-independent, antigen-independent background activity.

[0430] SR-T cells (SR028-T cells) transduced with the lentiviral vector described above were analyzed for extracellular expression of key components of the rapamycin-inducible system, including the targeting domain (e.g., anti-CD33 VHH), FRB multimerization domain, and FKBP12 multimerization domain, by flow cytometry using recombinant CD33-Fc fusion, anti-FRB antibody, and anti-FKBP12 antibody, respectively. The FRB component, FKBP12 component, and CD33 component were highly expressed compared to non-transduced control cells (Figure 18).

[0431] The rapamycin-dependent antigen-dependent activity of SR028-T cells was evaluated in co-culture with target antigen-positive tumor cells in the presence of 1 nM rapamycin. Specifically, IFNγ, IL2, IL4, and TNFα production was quantified using MSD from the supernatant of cultures with SR028-T cells and a cell line engineered to express high levels of CD33 (Figure 19). SR028-T cells released each of these four cytokines at levels highly distinguishable from baseline (untransduced control; UTD). In addition, T cells were cultured alone, both without and in the presence of 1 nM rapamycin, and without the presence of antigen. SR028-T cells did not produce IFNγ in the absence of rapamycin, while when 1 nM rapamycin was added to SR028-T cells without the presence of antigen, IFNγ was released at levels comparable to those in the presence of antigen (Figure 20). SR028-T cells produced significant amounts of IFNγ in the absence of antigen, comparable to that produced in the presence of antigen.

[0432] Cytotoxicity of SR028-T cells against cells expressing the target antigen was assessed using a target line expressing a nuclear red stain and tracked over time using an Incucyte® live cell imaging system (Sartorius). Both cells expressing the nuclear stain (antigen positive target line) and cells not expressing the nuclear stain (SR-T cells) were tracked for approximately 200 hours. SR028-T cells effectively killed CD33+ target cells (Figure 21A) and proliferated significantly compared to T cell controls (Figure 21B).

[0433] Example 7 Evaluation of rapamycin-inducible TCR T cells with multi-antigen targeting. In another experiment, T cells expressing rapamycin-inducible T cell receptor (SR-T cells) were generated using a 10-day transduction and expansion process, and then evaluated for expression and biological activity against specific target antigens. Briefly, peripheral blood mononuclear cells (PBMCs) were cultured in IL-2-containing medium and activated with antibodies against human CD3 and human CD28. PBMCs were transduced one day after initiation of culture using a lentiviral vector encoding a rapamycin-inducible TCR that affixes an FKBP12 or FRB multimerization domain and two antigen targeting domains (SR004 and SR006) with a CD4 hinge, transmembrane domain, and intracellular domain, and then the cells were transferred to a 24-well G-REX® culture system (Wilson Wolf) 48 hours later. After a total of 10 days in culture, SR-T cells were evaluated for rapamycin-dependent, antigen-dependent activity, as well as rapamycin-independent, antigen-independent background activity.

[0434] SR-T cells transduced with the lentiviral vectors described above (SR004-T cells and SR006-T cells) were analyzed for extracellular expression of key components of the rapamycin-inducible system, including targeting domains (e.g., anti-CD33 and anti-CLL1 VHHs) and FRB multimerization domains, by flow cytometry using recombinant CD33-Fc fusions, recombinant CLL1-Fc fusions, and anti-FRB antibodies, respectively. The FRB, anti-CD33, and anti-CLL1 components were highly expressed compared to untransduced control cells (Figure 22).

[0435] The rapamycin-dependent antigen-dependent activity of SR0004 and SR0006-T cells was assessed in co-culture with target antigen-positive tumor cells in the presence of 1 nM rapamycin. Specifically, IFNγ, IL2, IL4, and TNFα production was quantified using MSD from the supernatants of cultures with SR004- and SR006-T cells and cell lines engineered to express high levels of CD33 and CLL1. SR004- and SR006-T cells released each of these four cytokines at levels that were distinguishable from baseline (untransduced control; UTD). SR004-T cells tended to show increased IFNγ and IL2 release over SR006-T cells, but exhibited significantly more TNFα release (FIG. 23). In addition, T cells were cultured alone without the presence of antigen, both without and in the presence of 1 nM rapamycin. SR004-T cells and SR006-T cells did not produce IFNγ in the absence of rapamycin, while when 1 ...

Claims

1. A non-naturally occurring cell, (a) a signaling component comprising (i) a first multimerization domain comprising an FRB polypeptide or a variant thereof or an FKBP polypeptide or a variant thereof, (ii) a first polypeptide linker, and (iii) a CD3ε polypeptide or a variant thereof; and (b) a non-native cell comprising a targeting moiety comprising: (i) an anti-CLL1 scFv or single domain antibody (sdAb); (ii) an anti-CD33 scFv or single domain antibody (sdAb); (iii) a second polypeptide linker; (iv) a second multimerization domain comprising an FRB polypeptide or a variant thereof or an FKBP polypeptide or a variant thereof; (v) a CD4 hinge polypeptide; (vi) a CD4 transmembrane polypeptide; and (vii) a truncated CD4 intracellular polypeptide.

2. The non-naturally occurring cell of claim 1 , wherein the targeting moiety does not contain a functional intracellular domain with signaling capability or a costimulatory domain.

3. 3. The non-naturally occurring cell of any one of claims 1 to 2, wherein the CD4 hinge polypeptide comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

41.

4. The non-naturally occurring cell of any one of claims 1 to 3, wherein the CD4 hinge polypeptide comprises the amino acid sequence shown in SEQ ID NO:

41.

5. 5. The non-naturally occurring cell of any one of claims 1 to 4, wherein the CD3ε polypeptide comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

32.

6. The non-naturally occurring cell of any one of claims 1 to 5, wherein the CD3ε polypeptide comprises the amino acid sequence shown in SEQ ID NO:

32.

7. The non-naturally occurring cell of any one of claims 1 to 6, wherein the CD3ε polypeptide comprises both an extracellular portion and an intracellular portion.

8. 8. The non-naturally occurring cell of any one of claims 1 to 7, wherein the FRB and FKBP polypeptides are localized extracellularly when the signaling and targeting components are expressed.

9. The non-naturally occurring cell of any one of claims 1 to 8, wherein the first and second multimerization domains are different.

10. 10. The non-naturally occurring cell of any one of claims 1 to 9, wherein the first multimerization domain comprises an FRB polypeptide or a variant thereof, and the second multimerization domain comprises an FKBP12 polypeptide or a variant thereof.

11. 11. The non-naturally occurring cell of any one of claims 1 to 10, wherein the first multimerization domain comprises a FKBP12 polypeptide or a variant thereof, and the second multimerization domain comprises a FRB polypeptide or a variant thereof.

12. The non-naturally occurring cell of any one of claims 1 to 11, wherein the FRB polypeptide is an FRB T2098L variant.

13. 13. The non-naturally occurring cell of any one of claims 1 to 12, wherein the FRB polypeptide comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to, or including, the sequence set forth as SEQ ID NO:

1.

14. 14. The non-naturally occurring cell of any one of claims 1 to 13, wherein the FRB polypeptide comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to, or including, the sequence set forth as SEQ ID NO:

2.

15. 15. The non-naturally occurring cell of any one of claims 1 to 14, wherein the FKBP12 polypeptide comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to, or including, the sequence set forth as SEQ ID NO:

3.

16. 16. The non-naturally occurring cell of any one of claims 1 to 15, wherein the FKBP12 polypeptide comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to, or including, the sequence set forth as SEQ ID NO:

4.

17. 17. The non-natural cell of any one of claims 1 to 16, wherein the multimerization domains of the signaling moiety and the targeting moiety are associated with a cross-linking agent.

18. 18. The non-naturally occurring cell of claim 17, wherein the cross-linking agent is 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, and 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.

19. 19. The non-naturally occurring cell of claim 17 or claim 18, 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.

20. The non-naturally occurring cell of any one of claims 1 to 19, wherein the first polypeptide linker is a linker between 2 and 40 amino acids in length.

21. 21. The non-naturally occurring cell of claim 20, 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.

22. The non-naturally occurring cell of claim 21 , wherein the first polypeptide linker is a 3×G4S linker.

23. The non-naturally occurring cell of any one of claims 1 to 22, wherein the second polypeptide linker is a linker between 2 and 40 amino acids in length.

24. 24. The non-naturally occurring cell of claim 23, 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.

25. The non-naturally occurring cell of claim 24, wherein the second polypeptide linker is a G4S linker.

26. The non-naturally occurring cell of any one of claims 1 to 25, wherein the anti-CLL1 scFv or sdAb and the anti-CD33 scFv or sdAb are separated by a third polypeptide linker.

27. The non-naturally occurring cell of claim 26, wherein the third polypeptide linker is a linker between 2 and 40 amino acids in length.

28. 28. The non-naturally occurring cell of claim 27, 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, and any combination thereof.

29. The non-naturally occurring cell of claim 28 , wherein the third polypeptide linker is a G4S linker.

30. 30. The non-naturally occurring cell of any of claims 1-29, 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.

31. 31. The non-naturally occurring cell of any of claims 1-30, wherein the CD4 transmembrane polypeptide comprises the amino acid sequence shown in SEQ ID NO:

45.

32. 32. The non-naturally occurring cell of any of claims 1-31, wherein the truncated intracellular CD4 polypeptide comprises the amino acid sequence shown in SEQ ID NO:

48.

33. 32. The non-naturally occurring cell of any one of claims 1 to 31, wherein the truncated intracellular CD4 polypeptide comprises the amino acid sequence shown in SEQ ID NO:

49.

34. The non-natural cell of any one of claims 1 to 33, wherein the anti-CLL1 antibody and the anti-CD33 antibody are each an sdAb.

35. 35. The non-naturally occurring cell of claim 34, wherein the sdAb is a Camelid VHH, a nanobody, or a heavy chain-only antibody (HcAb).

36. 36. The non-naturally occurring cell of any one of claims 1 to 35, wherein the sdAb is a Camelidae VHH.

37. 37. The non-naturally occurring cell of any one of claims 1 to 36, wherein the scFv or sdAb is human or humanized.

38. 38. The non-naturally occurring cell of any one of claims 1 to 37, wherein the anti-CLL1 scFv or sdAb comprises the CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 92, 93, and 94, respectively.

39. 39. The non-naturally occurring cell of any one of claims 1 to 38, wherein the anti-CLL1 scFv or sdAb comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

75.

40. The non-naturally occurring cell of any one of claims 1 to 39, wherein the anti-CLL1 scFv or sdAb comprises the sequence shown in SEQ ID NO:

75.

41. 41. The non-naturally occurring cell of any one of claims 1 to 40, wherein the anti-CD33 scFv or sdAb comprises the CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 89, 90, and 91, respectively.

42. 42. The non-naturally occurring cell of any one of claims 1 to 41, wherein the anti-CD33 scFv or sdAb comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

72.

43. The non-naturally occurring cell of any one of claims 1 to 42, wherein the anti-CD33 scFv or sdAb comprises the sequence shown in SEQ ID NO:

72.

44. 44. The non-naturally occurring cell of any one of claims 1 to 43, wherein the signaling component further comprises a signal sequence.

45. 45. The non-naturally occurring cell of claim 44, wherein the signal sequence is a CD8 signal sequence.

46. 46. ​​The non-naturally occurring cell of claim 45, wherein 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.

47. The non-naturally occurring cell of claim 46, wherein the CD8 signal sequence comprises the amino acid sequence shown as SEQ ID NO:

96.

48. 48. The non-naturally occurring cell of any one of claims 1 to 47, wherein the targeting moiety further comprises a signal sequence.

49. 49. The non-naturally occurring cell of claim 48, wherein the signal sequence is an IgK signal sequence.

50. 50. The non-naturally occurring cell of claim 49, wherein 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.

51. 51. The non-naturally occurring cell of claim 50, wherein the IgK signal sequence comprises the amino acid sequence shown as SEQ ID NO:

95.

52. 52. The non-naturally occurring cell of any one of claims 1-51, wherein the signaling component comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

111.

53. 53. The non-naturally occurring cell of claim 52, wherein the signaling component comprises the sequence shown as SEQ ID NO:

111.

54. 54. The non-naturally occurring cell of any one of claims 1 to 53, 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:

122.

55. 55. The non-naturally occurring cell of claim 54, wherein the targeting moiety comprises the sequence shown as SEQ ID NO:

122.

56. 56. The non-naturally occurring cell of any one of claims 1 to 55, comprising a fusion polypeptide comprising said targeting moiety and said signaling moiety.

57. 57. The non-naturally occurring cell of claim 56, wherein the fusion polypeptide comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

151.

58. 58. The non-naturally occurring cell of claim 57, wherein the fusion polypeptide comprises the sequence shown as SEQ ID NO:

151.

59. 59. The non-naturally occurring cell of any one of claims 1 to 58, wherein the cell comprises a first nucleic acid molecule encoding said signaling component.

60. 60. The non-naturally occurring cell of any one of claims 1 to 59, wherein the cell comprises a second nucleic acid molecule encoding the targeting moiety.

61. 61. The non-naturally occurring cell of any one of claims 1 to 60, wherein the cell comprises a nucleic acid molecule encoding both the signaling component and the targeting component.

62. 62. The non-naturally occurring cell of any one of claims 1-61, 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.

63. 63. The non-naturally occurring cell of any one of claims 1 to 62, wherein the cell further expresses an exogenous lymphocyte receptor or co-receptor.

64. 64. The non-natural cell of claim 63, 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.

65. 65. The non-naturally occurring cell of any one of claims 1 to 64, wherein the cell further expresses an exogenous TCR.

66. The exogenous TCR is capable of detecting 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), preferentially expressed in melanoma 66. The non-naturally occurring cell of claim 65, wherein the cell binds to a target antigen selected from the group consisting of antigen expressing thyroiditis (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).

67. 67. The non-naturally occurring cell of claim 65 or claim 66, wherein the exogenous TCR is an αβ-TCR or a γδ-TCR.

68. 68. The non-naturally occurring cell of any one of claims 1 to 67, wherein the cell further expresses a CAR, a CCR, or a flip receptor.

69. 69. The non-naturally occurring cell of any one of claims 1-68, wherein the cell further expresses a zetakine, an immune cell engager, or a BiTE.

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

71. 71. The non-naturally occurring cell of any one of claims 1 to 70, wherein the cell is a T cell, an αβ-T cell, or a γδ-T cell.

72. The cells are ＋ , CD4 ＋ and / or CD8 ＋ 72. The non-naturally occurring cell of any one of claims 1 to 71, which is a cell.

73. The non-naturally occurring cell of any one of claims 1 to 72, wherein the cell is an immune effector cell.

74. 74. The non-naturally occurring cell of any one of claims 1 to 73, wherein the cell is a cytotoxic T lymphocyte (CTL), a tumor infiltrating lymphocyte (TIL), or a helper T cell.

75. The non-naturally occurring cell of any one of claims 1 to 74, wherein the cell is a natural killer (NK) cell or a natural killer T (NKT) cell.

76. 76. The non-naturally occurring cell of any one of claims 1-75, 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.

77. 77. The non-naturally occurring cell of any one of claims 1 to 76, wherein the non-naturally occurring cell is an isolated non-naturally occurring cell.

78. The non-naturally occurring cell of any one of claims 1 to 77, wherein the non-naturally occurring cell is obtained from a subject.

79. The non-naturally occurring cell of any one of claims 1 to 78, wherein the non-naturally occurring cell is a human cell.

80. 1. A fusion polypeptide comprising: (a) (i) a first multimerization domain comprising an FRB polypeptide or a variant thereof, or an FKBP polypeptide or a variant thereof; (ii) a first (iii) a signaling component comprising a CD3ε polypeptide or a variant thereof; and (b) a polypeptide cleavage signal; and (c) a fusion polypeptide comprising: (i) an anti-CLL1 scFv or single domain antibody (sdAb); (ii) an anti-CD33 scFv or single domain antibody (sdAb); (iii) a second polypeptide linker; (iv) a second multimerization domain comprising an FRB polypeptide or a variant thereof or an FKBP polypeptide or a variant thereof; (v) a CD4 hinge polypeptide; (vi) a CD4 transmembrane polypeptide; and (vii) a targeting moiety comprising a truncated CD4 intracellular polypeptide.

81. The fusion polypeptide of claim 80, wherein the targeting moiety does not contain a functional intracellular domain or a costimulatory domain having signaling capability.

82. The fusion polypeptide of any one of claims 80 to 81, wherein the CD4 hinge polypeptide comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

41.

83. The fusion polypeptide of any one of claims 80 to 82, wherein the CD4 hinge polypeptide comprises the amino acid sequence shown in SEQ ID NO:

41.

84. The fusion polypeptide of any one of claims 80 to 83, wherein the CD3ε polypeptide comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

32.

85. The fusion polypeptide of any one of claims 80 to 84, wherein the CD3ε polypeptide comprises the amino acid sequence shown in SEQ ID NO:

32.

86. The fusion polypeptide of any one of claims 80 to 85, wherein the CD3ε polypeptide comprises both an extracellular portion and an intracellular portion.

87. The fusion polypeptide of any one of claims 80 to 86, wherein the FRB and FKBP polypeptides are localized extracellularly when the signaling and targeting components are expressed.

88. A fusion polypeptide according to any one of claims 80 to 87, wherein the first and second multimerization domains are different.

89. The fusion polypeptide of any one of claims 80 to 88, wherein the first multimerization domain comprises an FRB polypeptide or a variant thereof, and the second multimerization domain comprises an FKBP12 polypeptide or a variant thereof.

90. The fusion polypeptide of any one of claims 80 to 89, wherein the first multimerization domain comprises an FKBP12 polypeptide or a variant thereof, and the second multimerization domain comprises an FRB polypeptide or a variant thereof.

91. The fusion polypeptide of any one of claims 80 to 90, wherein the FRB polypeptide is an FRB T2098L variant.

92. The fusion polypeptide of any one of claims 80 to 91, wherein the FRB polypeptide comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to or including the sequence set forth as SEQ ID NO:

1.

93. The fusion polypeptide of any one of claims 80 to 92, wherein the FRB polypeptide comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to or including the sequence set forth as SEQ ID NO:

2.

94. The fusion polypeptide of any one of claims 80 to 93, wherein the FKBP12 polypeptide comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to or including the sequence shown as SEQ ID NO:

3.

95. The fusion polypeptide of any one of claims 80 to 94, wherein the FKBP12 polypeptide comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to or including the sequence shown as SEQ ID NO:

4.

96. The fusion polypeptide of any one of claims 80 to 95, wherein the multimerization domains of the signaling moiety and the targeting moiety are associated with a cross-linking agent.

97. 97. The fusion polypeptide of claim 96, wherein the cross-linking agent is 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, and the cross-linking agent promotes the formation of a polypeptide complex, and the cross-linking agent is associated with and positioned between the multimerization domains of the signaling component and the targeting component.

98. The fusion polypeptide of claim 96 or claim 97, 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.

99. The fusion polypeptide of any one of claims 80 to 98, wherein the first polypeptide linker is a linker of 2 to 40 amino acids in length.

100. 100. The fusion polypeptide of claim 99, 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.

101. The fusion polypeptide of claim 100, wherein the first polypeptide linker is a 3xG4S linker.

102. The fusion polypeptide of any one of claims 80 to 101, wherein the second polypeptide linker is a linker of 2 to 40 amino acids in length.

103. 103. The fusion polypeptide of claim 102, 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.

104. The fusion polypeptide of claim 103, wherein the second polypeptide linker is a G4S linker.

105. The fusion polypeptide of any one of claims 80 to 104, wherein the anti-CLL1 scFv or sdAb and the anti-CD33 scFv or sdAb are separated by a third polypeptide linker.

106. The fusion polypeptide of claim 105, wherein the third polypeptide linker is a linker of 2 to 40 amino acids in length.

107. The fusion polypeptide of claim 106, 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, and any combination thereof.

108. The fusion polypeptide of claim 107, wherein the third polypeptide linker is a G4S linker.

109. The fusion polypeptide of any one of claims 80 to 108, 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.

110. The fusion polypeptide of any one of claims 80 to 109, wherein the CD4 transmembrane polypeptide comprises the amino acid sequence shown in SEQ ID NO:

45.

111. The fusion polypeptide of any one of claims 80 to 110, wherein the truncated intracellular CD4 polypeptide comprises the amino acid sequence shown in SEQ ID NO:

48.

112. The fusion polypeptide of any one of claims 80 to 111, wherein the truncated intracellular CD4 polypeptide comprises the amino acid sequence shown in SEQ ID NO:

49.

113. The fusion polypeptide of any one of claims 80 to 112, wherein the anti-CLL1 antibody and the anti-CD33 antibody are each an sdAb.

114. The fusion polypeptide of claim 113, wherein the sdAb is a camelid VHH, a nanobody, or a heavy chain only antibody (HcAb).

115. The fusion polypeptide of any one of claims 80 to 114, wherein the sdAb is a Camelidae VHH.

116. The fusion polypeptide of any one of claims 80 to 115, wherein the scFv or sdAb is human or humanized.

117. The fusion polypeptide of any one of claims 80 to 116, wherein the anti-CLL1 scFv or sdAb comprises the CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 92, 93, and 94, respectively.

118. The fusion polypeptide of any one of claims 80 to 117, wherein the anti-CLL1 scFv or sdAb comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

75.

119. The fusion polypeptide of any one of claims 80 to 118, wherein the anti-CLL1 scFv or sdAb comprises the sequence shown in SEQ ID NO:

75.

120. The fusion polypeptide of any one of claims 80 to 119, wherein the anti-CD33 scFv or sdAb comprises the CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 89, 90, and 91, respectively.

121. The fusion polypeptide of any one of claims 80 to 120, wherein the anti-CD33 scFv or sdAb comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

72.

122. The fusion polypeptide of any one of claims 80 to 121, wherein the anti-CD33 scFv or sdAb comprises the sequence shown in SEQ ID NO:

72.

123. The fusion polypeptide of any one of claims 80 to 122, wherein the signaling component further comprises a signal sequence.

124. The fusion polypeptide of claim 123, wherein the signal sequence is a CD8 signal sequence.

125. The fusion polypeptide of claim 124, wherein 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.

126. The fusion polypeptide of claim 125, wherein the CD8 signal sequence comprises the amino acid sequence shown as SEQ ID NO:

96.

127. The fusion polypeptide of any one of claims 80 to 126, wherein the targeting moiety further comprises a signal sequence.

128. The fusion polypeptide of claim 127, wherein the signal sequence is an IgK signal sequence.

129. The fusion polypeptide of claim 128, wherein 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.

130. The fusion polypeptide of claim 129, wherein the IgK signal sequence comprises the amino acid sequence set forth as SEQ ID NO:

95.

131. The fusion polypeptide of any one of claims 80 to 130, wherein the signaling component comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

111.

132. The fusion polypeptide of claim 131, wherein the signal transduction component comprises the sequence shown as SEQ ID NO:

111.

133. The fusion polypeptide of any one of claims 80 to 132, 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:

122.

134. The fusion polypeptide of claim 133, wherein the targeting moiety comprises the sequence shown as SEQ ID NO:

122.

135. A fusion polypeptide according to any one of claims 80 to 134, comprising a fusion polypeptide comprising said targeting moiety and said signalling moiety.

136. The fusion polypeptide of claim 135, wherein the fusion polypeptide comprises an sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

151.

137. The fusion polypeptide of claim 136, wherein the fusion polypeptide comprises the sequence shown as SEQ ID NO:

151.

138. The fusion polypeptide of any one of claims 80 to 137, wherein the polypeptide cleavage signal is a viral autocleaving polypeptide.

139. The fusion polypeptide of any one of claims 80 to 138, wherein the polypeptide cleavage signal is a viral auto-cleaving 2A polypeptide.

140. A nucleic acid molecule encoding a fusion polypeptide according to any one of claims 80 to 139.

141. A cell comprising a fusion polypeptide according to any one of claims 80 to 139.

142. A cell comprising the nucleic acid molecule of claim 140.

143. The cell of claim 140 or claim 141, 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.

144. The cell of any one of claims 141 to 143, wherein the cell further expresses an exogenous lymphocyte receptor or co-receptor.

145. The cell of claim 144, 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.

146. The cell of any one of claims 141 to 145, wherein the cell further expresses an exogenous TCR.

147. The exogenous TCR is capable of detecting 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), preferentially in melanoma 147. The cell of claim 146, 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).

148. The cell of claim 146 or claim 147, wherein the exogenous TCR is an αβ-TCR or a γδ-TCR.

149. The cell of any one of claims 141 to 148, wherein the cell further expresses a CAR, CCR, or flip receptor.

150. The cell of any one of claims 141 to 149, wherein the cell further expresses a zetakine, an immune cell engager, or a BiTE.

151. The cell of any one of claims 141 to 150, wherein the cell is a hematopoietic cell.

152. The cell of any one of claims 141 to 151, wherein the cell is a T cell, an αβ-T cell, or a γδ-T cell.

153. The cells are ＋ , CD4 ＋ and / or CD8 ＋ A cell according to any one of claims 141 to 152, which is a cell.

154. The cell of any one of claims 141 to 153, wherein the cell is an immune effector cell.

155. The cell of any one of claims 141 to 154, wherein the cell is a cytotoxic T lymphocyte (CTL), a tumor infiltrating lymphocyte (TIL), or a helper T cell.

156. The cell of any one of claims 141 to 155, wherein the cell is a natural killer (NK) cell or a natural killer T (NKT) cell.

157. 157. The cell of any one of claims 141-156, 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.

158. The cell of any one of claims 141 to 157, wherein the cell is an isolated cell.

159. The cell of any one of claims 141 to 158, wherein the cell is obtained from a subject.

160. The cell of any one of claims 141 to 159, wherein the cell is a human cell.

161. 1. A polypeptide complex comprising: (a) a signaling component comprising (i) a first multimerization domain comprising an FRB polypeptide or a variant thereof or an FKBP polypeptide or a variant thereof, (ii) a first polypeptide linker, and (iii) a CD3ε polypeptide or a variant thereof; and (b) a polypeptide complex comprising: (i) an anti-CLL1 scFv or single domain antibody (sdAb); (ii) an anti-CD33 scFv or single domain antibody (sdAb); (iii) a second polypeptide linker; (iv) a second multimerization domain comprising an FRB polypeptide or a variant thereof or an FKBP polypeptide or a variant thereof; (v) a CD4 hinge polypeptide; (vi) a CD4 transmembrane polypeptide; and (vii) a targeting moiety comprising a truncated CD4 intracellular polypeptide.

162. The polypeptide complex of claim 161, wherein the targeting moiety does not contain a functional intracellular domain or a costimulatory domain having signaling capability.

163. 163. The polypeptide complex of any one of claims 161 to 162, wherein the CD4 hinge polypeptide comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

41.

164. 164. The polypeptide complex of any one of claims 161 to 163, wherein the CD4 hinge polypeptide comprises the amino acid sequence shown in SEQ ID NO:

41.

165. 165. The polypeptide complex of any one of claims 161 to 164, wherein the CD3ε polypeptide comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

32.

166. 166. The polypeptide complex of any one of claims 161 to 165, wherein the CD3ε polypeptide comprises the amino acid sequence shown in SEQ ID NO:

32.

167. 167. The polypeptide complex of any one of claims 161 to 166, wherein the CD3ε polypeptide comprises both an extracellular portion and an intracellular portion.

168. 168. The polypeptide complex of any one of claims 161 to 167, wherein the FRB and FKBP polypeptides are localized extracellularly when the signaling and targeting components are expressed.

169. 169. The polypeptide complex of any one of claims 161 to 168, wherein the first and second multimerization domains are different.

170. The polypeptide complex of any one of claims 161 to 169, wherein the first multimerization domain comprises an FRB polypeptide or a variant thereof, and the second multimerization domain comprises an FKBP12 polypeptide or a variant thereof.

171. The polypeptide complex of any one of claims 161 to 170, wherein the first multimerization domain comprises an FKBP12 polypeptide or a variant thereof, and the second multimerization domain comprises an FRB polypeptide or a variant thereof.

172. 172. The polypeptide complex of any one of claims 161-171, wherein the FRB polypeptide is an FRB T2098L variant.

173. 173. The polypeptide complex of any one of claims 161 to 172, wherein the FRB polypeptide comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to or including the sequence set forth as SEQ ID NO:

1.

174. The polypeptide complex of any one of claims 161 to 173, wherein the FRB polypeptide comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to or including the sequence set forth as SEQ ID NO:

2.

175. 175. The polypeptide complex of any one of claims 161 to 174, wherein the FKBP12 polypeptide comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to or including the sequence set forth as SEQ ID NO:

3.

176. 176. The polypeptide complex of any one of claims 161 to 175, wherein the FKBP12 polypeptide comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to or including the sequence set forth as SEQ ID NO:

4.

177. 177. The polypeptide complex of any one of claims 161-176, wherein the multimerization domains of the signaling component and the targeting component are associated with a cross-linking agent.

178. The polypeptide complex of claim 177, wherein the cross-linking agent is 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, and the cross-linking agent promotes the formation of a polypeptide complex, and the cross-linking agent is associated with and positioned between the multimerization domains of the signaling component and the targeting component.

179. The polypeptide complex of claim 177 or claim 178, 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.

180. 180. The polypeptide complex of any one of claims 161 to 179, wherein the first polypeptide linker is a linker of 2 to 40 amino acids in length.

181. The polypeptide complex of claim 180, 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.

182. The polypeptide complex of claim 181, wherein the first polypeptide linker is a 3xG4S linker.

183. 183. The polypeptide complex of any one of claims 161 to 182, wherein the second polypeptide linker is a linker of 2 to 40 amino acids in length.

184. The polypeptide complex of claim 183, 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.

185. The polypeptide complex of claim 184, wherein the second polypeptide linker is a G4S linker.

186. 186. The polypeptide complex of any one of claims 161 to 185, wherein the anti-CLL1 scFv or sdAb and the anti-CD33 scFv or sdAb are separated by a third polypeptide linker.

187. The polypeptide complex of claim 186, wherein the third polypeptide linker is a linker between 2 and 40 amino acids in length.

188. The polypeptide complex of claim 187, 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, and any combination thereof.

189. The polypeptide complex of claim 188, wherein the third polypeptide linker is a G4S linker.

190. 190. The polypeptide complex of any one of claims 161-189, 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.

191. 191. The polypeptide complex of any one of claims 161 to 190, wherein the CD4 transmembrane polypeptide comprises the amino acid sequence shown in SEQ ID NO:

45.

192. 192. The polypeptide complex of any one of claims 161-191, wherein the cleaved intracellular CD4 polypeptide comprises the amino acid sequence shown in SEQ ID NO:

48.

193. 192. The polypeptide complex of any one of claims 161-191, wherein the cleaved intracellular CD4 polypeptide comprises the amino acid sequence shown in SEQ ID NO:

49.

194. The polypeptide complex of any one of claims 161 to 193, wherein the anti-CLL1 antibody and the anti-CD33 antibody are each an sdAb.

195. 195. The polypeptide complex of claim 194, wherein the sdAb is a camelid VHH, a nanobody, or a heavy chain only antibody (HcAb).

196. 196. The polypeptide complex of any one of claims 161 to 195, wherein the sdAb is a Camelidae VHH.

197. 197. The polypeptide complex of any one of claims 161 to 196, wherein the scFv or sdAb is human or humanized.

198. 200. The polypeptide complex of any one of claims 161 to 197, wherein the anti-CLL1 scFv or sdAb comprises the CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 92, 93, and 94, respectively.

199. 200. The polypeptide complex of any one of claims 161 to 198, wherein the anti-CLL1 scFv or sdAb comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

75.

200. 200. The polypeptide complex of any one of claims 161 to 199, wherein the anti-CLL1 scFv or sdAb comprises the sequence shown in SEQ ID NO:

75.

201. 201. The polypeptide complex of any one of claims 161 to 200, wherein the anti-CD33 scFv or sdAb comprises the CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 89, 90, and 91, respectively.

202. 202. The polypeptide complex of any one of claims 161-201, wherein the anti-CD33 scFv or sdAb comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

72.

203. 203. The polypeptide complex of any one of claims 161 to 202, wherein the anti-CD33 scFv or sdAb comprises the sequence shown in SEQ ID NO:

72.

204. The polypeptide complex of any one of claims 161-203, wherein the signaling component further comprises a signal sequence.

205. The polypeptide complex of claim 204, wherein the signal sequence is a CD8 signal sequence.

206. The polypeptide complex of claim 205, wherein 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.

207. The polypeptide complex of claim 206, wherein the CD8 signal sequence comprises the amino acid sequence set forth as SEQ ID NO:

96.

208. The polypeptide complex of any one of claims 161-207, wherein the targeting moiety further comprises a signal sequence.

209. The polypeptide complex of claim 208, wherein the signal sequence is an IgK signal sequence.

210. The polypeptide complex of claim 209, wherein 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.

211. The polypeptide complex of claim 210, wherein the IgK signal sequence comprises the amino acid sequence shown as SEQ ID NO:

95.

212. 212. The polypeptide complex of any one of claims 161-211, wherein the signaling component comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

111.

213. The polypeptide complex of claim 212, wherein the signal transduction component comprises the sequence shown as SEQ ID NO:

111.

214. 214. The polypeptide complex of any one of claims 161 to 213, 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:

122.

215. The polypeptide complex of claim 214, wherein the targeting moiety comprises the sequence shown as SEQ ID NO:

122.

216. 216. The polypeptide complex of any one of claims 161 to 215, comprising a fusion polypeptide comprising said targeting moiety and said signaling moiety.

217. The polypeptide complex of claim 216, wherein the fusion polypeptide comprises an sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

151.

218. The polypeptide complex of claim 217, wherein the fusion polypeptide comprises the sequence shown as SEQ ID NO:

151.

219. A nucleic acid molecule encoding both the signaling component and the targeting component of a polypeptide complex of any one of claims 161-218.

220. A cell comprising a polypeptide complex according to any one of claims 161 to 219.

221. A cell comprising the nucleic acid molecule of claim 220.

222. The cell of any one of claims 220 or 221, wherein the cell further expresses an exogenous costimulatory factor, an immune modulator, an agonist for a costimulatory factor, an antagonist for an immune suppressor, an immune cell engager, a flip receptor, or any combination thereof.

223. The cell of any one of claims 220 to 222, wherein the cell further expresses an exogenous lymphocyte receptor or co-receptor.

224. The cell of claim 223, 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.

225. The cell of any one of claims 220 to 224, wherein the cell further expresses an exogenous TCR.

226. The exogenous TCR is capable of detecting 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), preferentially in melanoma 226. The cell of claim 225, 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).

227. The cell of claim 225 or claim 226, wherein the exogenous TCR is an αβ-TCR or a γδ-TCR.

228. The cell of any one of claims 220 to 227, wherein the cell further expresses a CAR, a CCR, or a flip receptor.

229. The cell of any one of claims 220-228, wherein the cell further expresses a zetakine, an immune cell engager, or a BiTE.

230. The cell of any one of claims 220 to 229, wherein the cell is a hematopoietic cell.

231. The cell of any one of claims 220 to 230, wherein the cell is a T cell, an αβ-T cell, or a γδ-T cell.

232. The cells are ＋ , CD4 ＋ and / or CD8 ＋ A cell according to any one of claims 220 to 231, which is a cell.

233. The cell of any one of claims 220 to 232, wherein the cell is an immune effector cell.

234. The cell of any one of claims 220 to 233, wherein the cell is a cytotoxic T lymphocyte (CTL), a tumor infiltrating lymphocyte (TIL), or a helper T cell.

235. The cell of any one of claims 220 to 234, wherein the cell is a natural killer (NK) cell or a natural killer T (NKT) cell.

236. 236. The cell of any one of claims 220-235, 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.

237. The cell of any one of claims 220 to 236, wherein the cell is an isolated cell.

238. The cell of any one of claims 220 to 237, wherein the cell is obtained from a subject.

239. The cell of any one of claims 220 to 238, wherein the cell is a human cell.

240. A polynucleotide encoding the signaling and targeting component of the fusion polypeptide of any one of claims 80-139 or the polypeptide complex of any one of claims 161-219.

241. A cDNA encoding the signaling and targeting components of a fusion polypeptide according to any one of claims 80-139 or a polypeptide complex according to any one of claims 161-219.

242. 2. An RNA encoding a signalling component and a targeting component according to any one of claims 1 to 79, 141 to 160 or 220 to 239, or a fusion polypeptide according to any one of claims 80 to 139, or a polypeptide complex according to any one of claims 161 to 218.

243. A vector comprising the polynucleotide of claim 240.

244. The vector of claim 243, wherein the vector is an expression vector.

245. The vector of claim 243, wherein the vector is a transposon.

246. The vector of claim 243, wherein the vector is a piggyBAC transposon or a Sleeping Beauty transposon.

247. The vector of claim 243, wherein the vector is a viral vector.

248. The vector of claim 247, 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.

249. The vector of claim 248, wherein the retroviral vector is a lentiviral vector.

250. The vector of claim 249, 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).

251. A cell comprising a fusion polypeptide according to any one of claims 80 to 139, a polynucleotide according to claim 240, or a vector according to any one of claims 243 to 250.

252. The cell of claim 251 , wherein the cell is a hematopoietic cell.

253. The cell of claim 251 or claim 252, wherein the cell is an immune effector cell.

254. The cell of any one of claims 251 to 253, wherein the cell is a T cell, an αβ T cell, or a γδ T cell.

255. The cells are ＋ , CD4 ＋ , CD8 ＋ , or a combination thereof.

256. The cell of any one of claims 251 to 255, wherein the cell is a cytotoxic T lymphocyte (CTL), a tumor infiltrating lymphocyte (TIL), or a helper T cell.

257. The cell of any one of claims 251 to 266, wherein the cell is a natural killer (NK) cell or a natural killer T (NKT) cell.

258. 2. A composition comprising a cell according to any one of claims 1 to 79, 141 to 160, 220 to 239 or 251 to 257, or a vector according to any one of claims 243 to 250.

259. 251. A composition comprising a physiologically acceptable carrier and a cell according to any one of claims 1-79, 141-160, 220-239 or 251-257, or a vector according to any one of claims 243-250.

260. A method of treating a subject in need thereof comprising administering to the subject an effective amount of the composition of claim 258 or claim 259.

261. A method for 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 the subject an effective amount of the composition of claim 258 or claim 259.

262. A method of treating a solid cancer comprising administering to the subject an effective amount of the composition of claim 258 or claim 259.

263. 263. The method of claim 262, 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.

264. The method of claim 262 or claim 263, 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.

265. A method of treating a hematological malignancy comprising administering to the subject an effective amount of the composition of claim 258 or claim 259.

266. 266. The method of claim 265, wherein the hematological malignancy is leukemia, lymphoma, or multiple myeloma.

267. 266. The method of claim 265, wherein the hematological malignancy is acute myeloid leukemia (AML).

268. 80. The non-naturally occurring cell of any one of claims 1-79, wherein the cell further comprises a polypeptide having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

137.

269. 80. The non-naturally occurring cell of any one of claims 1 to 79, wherein the cell further comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:

137.

270. 80. The non-naturally occurring cell of any one of claims 1-79, wherein the cell further comprises a polypeptide having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

138.

271. 80. The non-naturally occurring cell of any one of claims 1 to 79, wherein the cell further comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:138.

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