Multi-part receptors and signaling complexes

Abstract

The present disclosure provides adoptive T-cell therapies having an improved architecture for targeting antigens and recruiting multimeric immune signaling complexes for treating, preventing, or ameliorating at least one symptom of cancer, infectious diseases, autoimmune diseases, inflammatory diseases, and immunodeficiency or conditions associated therewith.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 329,003, filed on April 8, 2022, under 35 U.S.C. §119(e), which is incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure relates to improved adoptive cell therapy. More specifically, the present disclosure relates to improved immune receptor signaling molecules, cells, and methods of using the same for regulating spatial and / or temporal control of immune receptor signaling initiation and downstream responses during adoptive immunotherapy. Background Art

[0004] The global cancer burden doubled between 1975 and 2000. Cancer 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 increase to 22 million over the next two decades.

[0005] While chemotherapy and subsequently biologics have been the standard of care for decades, adoptive cell therapy is emerging as a powerful paradigm for delivering complex biological signals to treat cancer. Compared to small molecules and biologic drug compositions, adoptive cell therapy has the potential to perform unique therapeutic tasks due to its extremely large number of sensory and response programs and increasingly well-defined genetic control mechanisms. To realize this therapeutic value, cells need to be equipped with mechanisms for sensing and integrating chemical and / or biological information associated with the local physiological environment.

[0006] In recent years, assembling cells with chimeric antigen receptors (CARs) or transgenic T cell receptors (TCRs) has been shown to be an effective way to target immune cells to specific antigens (e.g., tumor antigens), stimulate T cells to activate signal transduction, and ultimately attack and kill antigen-related cells (e.g., cancer cells). Despite these successes, there are still fundamental differences and limitations between the two architectures, including i) sensitivity, ii) antigen recognition, iii) antigen-independent signaling activity, and iv) lack of adjustability. Therefore, there is still a need for improved targeting and signaling mechanisms that more effectively sense and respond to target antigens and related cells. Summary of the invention

[0007] The present disclosure generally relates in part to engineered immune receptor complexes that can both 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 the same, compositions thereof, and methods of making and using the same to treat conditions or diseases (e.g., cancer or autoimmunity).

[0008] In one aspect, disclosed herein is a non-natural cell comprising a signaling component comprising (i) a first multimerization domain comprising a FRB polypeptide or a variant thereof or a FKBP polypeptide or a variant thereof, (ii) a first polypeptide linker, and (iii) a CD3ε polypeptide or a variant thereof; and a targeting component comprising (i) an anti-CLL1 scFv or a single domain antibody (sdAb), (ii) an anti-CD33 scFv or a single domain antibody (sdAb), (iii) a second polypeptide linker, (iv) a second multimerization domain comprising a FRB polypeptide or a variant thereof or a FKBP polypeptide or a variant thereof, (v) a CD4 hinge polypeptide, (vi) a CD4 transmembrane polypeptide, and (vii) a truncated CD4 intracellular polypeptide.

[0009] In some embodiments, the targeting component does not comprise a functional intracellular domain or a co-stimulatory 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 an amino acid sequence as shown in SEQ ID NO: 41.

[0010] In some embodiments, the CD3ε polypeptide comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 32. In one embodiment, the CD3ε polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 32. In some embodiments, the CD3ε polypeptide comprises both an extracellular portion and an intracellular portion.

[0011] In some embodiments, when the signaling component and the targeting component are expressed, the FRB and FKBP polypeptides are located extracellularly. In some embodiments, the first multimerization domain is different from the second multimerization domain. In some embodiments, the first multimerization domain comprises a FRB polypeptide or a variant thereof, and the second multimerization domain comprises a FKBP12 polypeptide or a variant thereof. In some embodiments, 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. In one embodiment, the FRB polypeptide is a FRB T2098L variant. In some embodiments, the FRB polypeptide comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1 or comprising a sequence as shown in SEQ ID NO: 1. In some embodiments, the FRB polypeptide comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 2 or comprising a sequence as shown in SEQ ID NO: 2. In some embodiments, the FKBP12 polypeptide comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 3, or comprises the sequence as shown in SEQ ID NO: 3. In some embodiments, the FKBP12 polypeptide comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 4, or comprises the sequence as shown in SEQ ID NO: 4.

[0012] In some embodiments, the signaling component and the multimerization domain of the targeting component are associated with a bridging factor. In some embodiments, the bridging factor is selected from the group consisting of rapamycin or a rapamycin analog 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) or a derivative thereof for FK506 binding protein (FKBP), wherein the bridging factor promotes the formation of a polypeptide complex, and the bridging factor is associated with the multimerization domain of the signaling component and the targeting component and is disposed between the multimerization domains. In some embodiments, the bridging factor is AP1903, AP20187, AP21967 (also known as C16-(S)-7-methylindorapamycin), everolimus, novolimus, pimecrolimus, ridaforolimus, sirolimus, tacrolimus, temsirolimus, umirolimus, zotarolimus, or BPC015.

[0013] In some embodiments, the first polypeptide linker is a linker of 2 to 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 of 2 to 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 of 2 to 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, 2x G4S, 3xG4s, 4xG4S, and any combination thereof. In some embodiments, the third polypeptide linker is a G4S linker.

[0014] In some embodiments, the CD4 transmembrane domain comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 45. In some embodiments, the CD4 transmembrane polypeptide comprises the amino acid sequence as shown in SEQ ID NO: 45. In some embodiments, the truncated intracellular CD4 polypeptide comprises the amino acid sequence as shown in SEQ ID NO: 48. In one embodiment, the truncated intracellular CD4 polypeptide comprises the amino acid sequence as shown in SEQ ID NO: 49.

[0015] In some embodiments, the anti-CLL1 antibody and the anti-CD33 antibody are each sdAb. In some embodiments, the sdAb is a camel VHH, a nanobody or a heavy chain antibody only (HcAb). In one embodiment, the sdAb is a camel VHH. In some embodiments, the scFv or the sdAb is human or humanized. In some embodiments, the anti-CLL1 scFv or sdAb comprises CDR1, CDR2 and CDR3 amino acid sequences as shown in SEQ ID NO: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 with SEQ ID NO:75. In some embodiments, the anti-CLL1 scFv or sdAb comprises a sequence as shown in SEQ ID NO:75. In some embodiments, the anti-CD33 scFv or sdAb comprises the CDR1, CDR2 and CDR3 amino acid sequences as shown in SEQ ID NOs: 89, 90 and 91, respectively. In some embodiments, the anti-CD33 scFv or sdAb comprises a sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 72. In some embodiments, the anti-CD33 scFv or sdAb comprises the sequence as shown in SEQ ID NO: 72.

[0016] In some embodiments, the signaling component 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 with SEQ ID NO: 96. In one embodiment, the CD8 signal sequence comprises an amino acid sequence as shown in SEQ ID NO: 96. In some embodiments, the targeting component 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 with SEQ ID NO: 95. In one embodiment, the IgK signal sequence comprises an amino acid sequence as shown in SEQ ID NO: 95. In some embodiments, the signaling component comprises a sequence having at least 90%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 111. In one embodiment, the signaling component comprises a sequence as shown in SEQ ID NO: 111. In some embodiments, the targeting component comprises a sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 122. In one embodiment, the targeting component comprises a sequence as shown in SEQ ID NO: 122.

[0017] In some embodiments, the non-natural cell comprises a fusion polypeptide comprising the targeting component and the signaling component. 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 as shown in SEQ ID NO: 151.

[0018] In some embodiments, the cell comprises a first nucleic acid molecule encoding the signaling component. In some embodiments, the cell comprises a second nucleic acid molecule encoding the targeting component. In some embodiments, the cell comprises a nucleic acid molecule encoding both the signaling component and the targeting component.

[0019] In some embodiments, the cell further expresses exogenous co-stimulatory factors, immunomodulatory factors, agonists of co-stimulatory factors, antagonists of immunosuppressive factors, immune cell engagers, flip receptors or any combination thereof. In some embodiments, the cell further expresses exogenous lymphocyte receptors or auxiliary receptors. In some embodiments, the exogenous lymphocyte receptors or auxiliary receptors are selected from the group consisting of: TCRα (TCR alpha), TCRβ (TCR beta), TCRγ (TCR gamma), TCRδ (TCR delta), CD4, CD8, pre-T cell receptor α (pTα), Fc receptor α (FcRα), Fc receptor β (FcRβ), Fc receptor γ (FcRγ), natural killer group 2 member D (NKG2D), CD79A, CD79B and any combination thereof. In some embodiments, the cell further expresses exogenous TCR.In some embodiments, the exogenous TCR binds to a target antigen selected from the group consisting of alpha-fetoprotein (AFP), B melanoma antigen (BAGE) family members, brothers of regulatory factors at imprinted sites (BORIS), cancer-testis antigen, cancer-testis antigen 83 (CT-83), carbonic anhydrase IX (CA1X), carcinoembryonic antigen (CEA), cytomegalovirus (CMV) antigen, antigen recognized by cytotoxic T cells (CTL) on melanoma (CAMEL), Epstein-Barr virus (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), antigen preferentially expressed 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 X 8 (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.

[0020] 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 + cells, CD4 + cells and / or CD8 + 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 an infected site, ascites, pleural effusion, spleen tissue or a tumor. In one embodiment, the non-natural cell is a separated non-natural cell. In one embodiment, the non-natural cell is obtained from a subject. In one embodiment, the non-natural 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 with SEQ ID NO: 137. In one embodiment, the cell further comprises an amino acid sequence as shown 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 as shown in SEQ ID NO:138.

[0021] On the one hand, disclosed herein is a fusion polypeptide comprising a signaling component, wherein the signaling component comprises (i) a first multimerization domain, wherein the first multimerization domain comprises a FRB polypeptide or a variant thereof or a FKBP polypeptide or a variant thereof, (ii) a first polypeptide linker, and (iii) a CD3ε polypeptide or a variant thereof; and a polypeptide cleavage signal; and a targeting component, wherein the targeting component comprises (i) an anti-CLL1 scFv or a single domain antibody (sdAb), (ii) an anti-CD33 scFv or a single domain antibody (sdAb), (iii) a second polypeptide linker, (iv) a second multimerization domain, wherein the second multimerization domain comprises a FRB polypeptide or a variant thereof or a FKBP polypeptide or a variant thereof, (v) a CD4 hinge polypeptide, (vi) a CD4 transmembrane polypeptide, and (vii) a truncated CD4 intracellular polypeptide.

[0022] In some embodiments, the targeting component does not comprise a functional intracellular domain or a co-stimulatory 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 an amino acid sequence as shown in SEQ ID NO: 41.

[0023] In some embodiments, the CD3ε polypeptide comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 32. In one embodiment, the CD3ε polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 32. In some embodiments, the CD3ε polypeptide comprises both an extracellular portion and an intracellular portion.

[0024] In some embodiments, when the signaling component and the targeting component are expressed, the FRB and FKBP polypeptides are located extracellularly. In some embodiments, the first multimerization domain is different from the second multimerization domain. In some embodiments, the first multimerization domain comprises a FRB polypeptide or a variant thereof, and the second multimerization domain comprises a FKBP12 polypeptide or a variant thereof. In some embodiments, 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. In one embodiment, the FRB polypeptide is a FRB T2098L variant. In some embodiments, the FRB polypeptide comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1 or comprising a sequence as shown in SEQ ID NO: 1. In some embodiments, the FRB polypeptide comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 2 or comprising a sequence as shown in SEQ ID NO: 2. In some embodiments, the FKBP12 polypeptide comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 3, or comprises the sequence as shown in SEQ ID NO: 3. In some embodiments, the FKBP12 polypeptide comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 4, or comprises the sequence as shown in SEQ ID NO: 4.

[0025] In some embodiments, the signaling component and the multimerization domain of the targeting component are associated with a bridging factor. In some embodiments, the bridging factor is selected from the group consisting of rapamycin or a rapamycin analog 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) or a derivative thereof for FK506 binding protein (FKBP), wherein the bridging factor promotes the formation of a polypeptide complex, and the bridging factor is associated with the multimerization domain of the signaling component and the targeting component and is disposed between the multimerization domains. In some embodiments, the bridging factor is AP1903, AP20187, AP21967 (also known as C16-(S)-7-methylindorapamycin), everolimus, novolimus, pimecrolimus, ridaforolimus, sirolimus, tacrolimus, temsirolimus, umirolimus, zotarolimus, or BPC015.

[0026] In some embodiments, the first polypeptide linker is a linker of 2 to 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 of 2 to 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 of 2 to 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, 2x G4S, 3xG4s, 4xG4S and any combination thereof. In one embodiment, the third polypeptide linker is a G4S linker.

[0027] In some embodiments, the CD4 transmembrane domain comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 45. In one embodiment, the CD4 transmembrane polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 45. In one embodiment, the truncated intracellular CD4 polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 48. In one embodiment, the truncated intracellular CD4 polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 49. In some embodiments, the anti-CLL1 antibody and the anti-CD33 antibody are each sdAbs. In some embodiments, the sdAb is a camel VHH, a nanobody or a heavy chain antibody (HcAb) only. In one embodiment, the sdAb is a camel VHH. In some embodiments, the scFv or the sdAb is human or humanized. In some embodiments, the anti-CLL1 scFv or sdAb comprises CDR1, CDR2 and CDR3 amino acid sequences as shown in SEQ ID NO: 92, 93 and 94, respectively. In some embodiments, the anti-CLL1 scFv or sdAb comprises a sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 75. In some embodiments, the anti-CLL1 scFv or sdAb comprises a sequence as set forth in SEQ ID NO: 75. In some embodiments, the anti-CD33 scFv or sdAb comprises a CDR1, CDR2 and CDR3 amino acid sequences as set forth in SEQ ID NOs: 89, 90 and 91, respectively. In some embodiments, the anti-CD33 scFv or sdAb comprises a sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 72. In some embodiments, the anti-CD33 scFv or sdAb comprises a sequence as set forth in SEQ ID NO: 72.

[0028] In some embodiments, the signaling component 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 with SEQ ID NO: 96. In one embodiment, the CD8 signal sequence comprises an amino acid sequence as shown in SEQ ID NO: 96. In some embodiments, the targeting component 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 with SEQ ID NO: 95. In one embodiment, the IgK signal sequence comprises an amino acid sequence as shown in SEQ ID NO: 95. In some embodiments, the signaling component comprises a sequence having at least 90%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 111. In one embodiment, the signaling component comprises a sequence as shown in SEQ ID NO: 111. In some embodiments, the targeting component comprises a sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 122. In one embodiment, the targeting component comprises a sequence as shown in SEQ ID NO: 122.

[0029] In some embodiments, the fusion polypeptide comprises a fusion polypeptide comprising the targeting component and the signaling component. 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 as shown in SEQ ID NO: 151.

[0030] In some embodiments, the polypeptide cleavage signal is a viral self-cleavage polypeptide. In some embodiments, the polypeptide cleavage signal is a viral self-cleavage 2A polypeptide.

[0031] In one aspect, the present disclosure provides a nucleic acid molecule encoding any one of the fusion polypeptides disclosed herein.

[0032] In another aspect, the present disclosure provides a cell comprising any of the fusion polypeptides disclosed herein.

[0033] In another aspect, the present disclosure provides a cell comprising any of the nucleic acid molecules disclosed herein.

[0034] In some embodiments, the cell further expresses exogenous co-stimulatory factors, immunomodulatory factors, agonists of co-stimulatory factors, antagonists of immunosuppressive factors, immune cell engagers, flip receptors or any combination thereof. In some embodiments, the cell further expresses exogenous lymphocyte receptors or auxiliary receptors. In some embodiments, the exogenous lymphocyte receptors or auxiliary receptors are selected from the group consisting of: TCRα (TCR alpha), TCRβ (TCR beta), TCRγ (TCR gamma), TCRδ (TCR delta), CD4, CD8, pre-T cell receptor α (pTα), Fc receptor α (FcRα), Fc receptor β (FcRβ), Fc receptor γ (FcRγ), natural killer group 2 member D (NKG2D), CD79A, CD79B and any combination thereof. In some embodiments, the cell further expresses exogenous TCR.In some embodiments, the exogenous TCR binds to a target antigen selected from the group consisting of alpha-fetoprotein (AFP), B melanoma antigen (BAGE) family members, brothers of regulatory factors at imprinted sites (BORIS), cancer-testis antigen, cancer-testis antigen 83 (CT-83), carbonic anhydrase IX (CA1X), carcinoembryonic antigen (CEA), cytomegalovirus (CMV) antigen, antigen recognized by cytotoxic T cells (CTL) on melanoma (CAMEL), Epstein-Barr virus (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), antigen preferentially expressed 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 X 8 (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.

[0035] 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 + cells, CD4 + cells and / or CD8 + 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 an infection site, 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.

[0036] On the one hand, disclosed herein is a polypeptide complex comprising a signaling component, wherein the signaling component comprises (i) a first multimerization domain, wherein the first multimerization domain comprises a FRB polypeptide or a variant thereof or a FKBP polypeptide or a variant thereof, (ii) a first polypeptide linker, and (iii) a CD3ε polypeptide or a variant thereof; and a targeting component, wherein the targeting component comprises (i) an anti-CLL1 scFv or a single domain antibody (sdAb), (ii) an anti-CD33 scFv or a single domain antibody (sdAb), (iii) a second polypeptide linker, (iv) a second multimerization domain, wherein the second multimerization domain comprises a FRB polypeptide or a variant thereof or a FKBP polypeptide or a variant thereof, (v) a CD4 hinge polypeptide, (vi) a CD4 transmembrane polypeptide, and (vii) a truncated CD4 intracellular polypeptide.

[0037] In some embodiments, the targeting component does not comprise a functional intracellular domain or co-stimulatory 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 some embodiments, the CD4 hinge polypeptide comprises an amino acid sequence as shown 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 shown in SEQ ID NO: 32. In some embodiments, the CD3ε polypeptide comprises both an extracellular portion and an intracellular portion.

[0038] In some embodiments, when the signaling component and the targeting component are expressed, the FRB and FKBP polypeptides are located extracellularly. In some embodiments, the first multimerization domain is different from the second multimerization domain. In some embodiments, the first multimerization domain comprises a FRB polypeptide or a variant thereof, and the second multimerization domain comprises a FKBP12 polypeptide or a variant thereof. In some embodiments, 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. In one embodiment, the FRB polypeptide is a FRB T2098L variant. In some embodiments, the FRB polypeptide comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1 or comprising a sequence as shown in SEQ ID NO: 1. In some embodiments, the FRB polypeptide comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 2 or comprising a sequence as shown in SEQ ID NO: 2. In some embodiments, the FKBP12 polypeptide comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 3, or comprises the sequence as shown in SEQ ID NO: 3. In some embodiments, the FKBP12 polypeptide comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 4, or comprises the sequence as shown in SEQ ID NO: 4.

[0039] In some embodiments, the signaling component and the multimerization domain of the targeting component are associated with a bridging factor. In some embodiments, the bridging factor is selected from the group consisting of: rapamycin or a rapamycin analog thereof, gibberellins or derivatives thereof, abscisic acid (ABA) or derivatives thereof, methotrexate or derivatives thereof, cyclosporin A or derivatives thereof, FK506 / cyclosporin A or derivatives thereof, trimethoprim (Tmp) synthetic ligand (SLF) or derivatives thereof for FK506 binding protein (FKBP), wherein the bridging factor promotes the formation of a polypeptide complex, and the bridging factor is associated with the multimerization domain of the signaling component and the targeting component and is positioned between the multimerization domains. In some embodiments, the bridging factor is AP1903, AP20187, AP21967 (also known as C16-(S)-7-methylindorapamycin), everolimus, norvolimus, pimecrolimus, rufolimus, sirolimus, tacrolimus, temsirolimus, urolimus, zotarolimus or BPC015. In some embodiments, the first polypeptide linker is a linker of 2 to 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 of 2 to 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, 2x G4S, 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 having a length of 2 to 40 amino acids. In some embodiments, the third polypeptide linker is selected from the group consisting of: GG, GS, SG, SS, GSS, SSG, GSG, SGS, SGG, G4S, 2x G4S, 3xG4s, 4xG4S, and any combination thereof. In one embodiment, the third polypeptide linker is a G4S linker.

[0040] In some embodiments, the CD4 transmembrane domain comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 45. In one embodiment, the CD4 transmembrane polypeptide comprises the amino acid sequence as shown in SEQ ID NO: 45. In one embodiment, the truncated intracellular CD4 polypeptide comprises the amino acid sequence as shown in SEQ ID NO: 48. In one embodiment, the truncated intracellular CD4 polypeptide comprises the amino acid sequence as shown in SEQ ID NO: 49.

[0041] In some embodiments, the anti-CLL1 antibody and the anti-CD33 antibody are each sdAb. In some embodiments, the sdAb is a camel VHH, a nanobody or a heavy chain antibody only (HcAb). In one embodiment, the sdAb is a camel VHH. In some embodiments, the scFv or the sdAb is human or humanized. In some embodiments, the anti-CLL1 scFv or sdAb comprises CDR1, CDR2 and CDR3 amino acid sequences as shown in SEQ ID NO: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 with SEQ ID NO:75. In some embodiments, the anti-CLL1 scFv or sdAb comprises a sequence as shown in SEQ ID NO:75. In some embodiments, the anti-CD33 scFv or sdAb comprises the CDR1, CDR2 and CDR3 amino acid sequences as shown in SEQ ID NOs: 89, 90 and 91, respectively. In some embodiments, the anti-CD33 scFv or sdAb comprises a sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 72. In some embodiments, the anti-CD33 scFv or sdAb comprises the sequence as shown in SEQ ID NO: 72.

[0042] In some embodiments, the signaling component 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 with SEQ ID NO: 96. In one embodiment, the CD8 signal sequence comprises an amino acid sequence as shown in SEQ ID NO: 96. In some embodiments, the targeting component 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 with SEQ ID NO: 95. In one embodiment, the IgK signal sequence comprises an amino acid sequence as shown in SEQ ID NO: 95. In some embodiments, the signaling component comprises a sequence having at least 90%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 111. In one embodiment, the signaling component comprises a sequence as shown in SEQ ID NO: 111. In some embodiments, the targeting component comprises a sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 122. In one embodiment, the targeting component comprises a sequence as shown in SEQ ID NO: 122. In some embodiments, the polypeptide complex comprises a fusion polypeptide comprising the targeting component and the signaling component. In some embodiments, the fusion polypeptide comprises a sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 151. In one embodiment, the fusion polypeptide comprises a sequence as shown in SEQ ID NO: 151.

[0043] In one aspect, the present disclosure provides a nucleic acid molecule encoding both the signaling component and the targeting component of any one of the polypeptide complexes disclosed herein.

[0044] In another aspect, the present disclosure provides a cell comprising any of the polypeptide complexes disclosed herein.

[0045] In another aspect, the present disclosure provides a cell comprising any of the nucleic acid molecules disclosed herein.

[0046] In some embodiments, the cell further expresses exogenous co-stimulatory factors, immunomodulatory factors, agonists of co-stimulatory factors, antagonists of immunosuppressive factors, immune cell engagers, flip receptors or any combination thereof. In some embodiments, the cell further expresses exogenous lymphocyte receptors or auxiliary receptors. In some embodiments, the exogenous lymphocyte receptors or auxiliary receptors are selected from the group consisting of: TCRα (TCR alpha), TCRβ (TCR beta), TCRγ (TCR gamma), TCRδ (TCR delta), CD4, CD8, pre-T cell receptor α (pTα), Fc receptor α (FcRα), Fc receptor β (FcRβ), Fc receptor γ (FcRγ), natural killer group 2 member D (NKG2D), CD79A, CD79B and any combination thereof. In some embodiments, the cell further expresses exogenous TCR.In some embodiments, the exogenous TCR binds to a target antigen selected from the group consisting of alpha-fetoprotein (AFP), B melanoma antigen (BAGE) family members, brothers of regulatory factors at imprinted sites (BORIS), cancer-testis antigen, cancer-testis antigen 83 (CT-83), carbonic anhydrase IX (CA1X), carcinoembryonic antigen (CEA), cytomegalovirus (CMV) antigen, antigen recognized by cytotoxic T cells (CTL) on melanoma (CAMEL), Epstein-Barr virus (EBV) antigen, G anti- 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), antigen preferentially expressed 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 X 8 (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.

[0047] 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 + cells, CD4+ cells and / or CD8 + 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 an infection site, 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.

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

[0050] In another aspect, the present disclosure provides an RNA encoding any one of the signaling components and targeting components, or any one of the fusion polypeptides, or any one of the polypeptide complexes disclosed herein.

[0051] In another aspect, the present disclosure provides a vector comprising any of the polynucleotides disclosed herein.

[0052] 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 adenoviral vector, an adeno-associated virus (AAV) vector, a herpes virus vector, a vaccinia virus vector, or a retroviral vector. In some embodiments, the retroviral vector is a lentiviral vector. In some embodiments, the lentiviral vector is selected from the group consisting of: human immunodeficiency virus 1 (HIV-1); human immunodeficiency virus 2 (HIV-2), Vesna-Medi virus (VMV) 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).

[0053] In one aspect, the present disclosure provides a cell comprising any of the fusion polypeptides, polynucleotides or vectors disclosed herein.

[0054] 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 expresses 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.

[0055] In one aspect, the present disclosure provides a composition comprising any of the cells or vectors disclosed herein.

[0056] In another aspect, the present disclosure provides a composition comprising a physiologically acceptable carrier and any of the cells or vectors disclosed herein.

[0057] In another aspect, the present disclosure provides a method of treating a subject in need thereof, the method comprising administering to the subject an effective amount of any of the compositions disclosed herein.

[0058] In another aspect, the present disclosure provides a method for treating, preventing or ameliorating at least one symptom of cancer, infectious diseases, autoimmune diseases, inflammatory diseases and immunodeficiency or conditions associated therewith, the method comprising administering to a subject an effective amount of any one of the compositions disclosed herein.

[0059] In another aspect, the present disclosure provides a method of treating solid cancer, the method comprising administering to a subject an effective amount of any one of the compositions disclosed herein.

[0060] 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, 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.

[0061] On the one hand, the present disclosure provides a method for treating a hematological malignancy, the method comprising administering to a subject an effective amount of any 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

[0062] Figures 1A-1H The architectures of representative engineered immune receptors and fusion constructs are shown. Specifically, Figures 1A-1C Expression constructs and components of several exemplary engineered immune receptors and fusion constructs of the invention are depicted. Figure 1D-1H is a sketch depicting how a representative engineered immunoreceptor comprising a signaling component and a targeting component can form a complex with an immune receptor (e.g., TCR) to activate receptor signaling when a target cell expressing a target antigen is present, and in certain embodiments when a bridging factor is present. As a non-limiting example, in Figure 1D In the construct SR022 / SR001, a signaling component including an FRB multimerization domain, a linker, and a CD3ε actuator domain is included; and a targeting component including a CD4 transmembrane domain, a CD4 hinge, a FKBP multimerization domain, a linker, and a CD33 VHH targeting domain. When a cell expressing CD33 is present, the CD33 VHH targeting domain binds to the target cell, bringing it into close contact with an immune cell (e.g., a T cell) expressing an engineered immune receptor. When a bridging factor is present, the multimerization domains then interact, allowing the signaling component to activate the immune receptor (e.g., TCR).

[0063] Figure 2 Shown are the positive percentages and median fluorescence intensity of SR024 components on T cells as measured by flow cytometry.

[0064] Figure 3 Shown are IFNγ, IL2, IL4 and TNF secretion from non-transduced cells and SR024-T cells co-cultured with high CD33 expressing cancer cell lines in the presence of rapamycin.

[0065] Figure 4 Shown is IFNγ secretion from non-transduced cells and SR024-T cells cultured alone in the presence and absence of rapamycin.

[0066] Figure 5A and 5B The cytotoxicity and proliferation of control and SR024 T cells co-cultured with high CD33 expressing cancer cell lines are shown, respectively.

[0067] Figure 6 Shown are the percent positivity and median fluorescence intensity of SR020, SR022 and SR024 components on T cells as measured by flow cytometry.

[0068] Fig. 7A Shown are IFNγ, IL2, IL4 and TNF secretion from untransduced cells, 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.

[0069] Figure 7B Shown is IFNγ secretion from untransduced cells, SR020-T cells, SR022-T cells, and SR024-T cells co-cultured with high, medium, and low CD33 expressing cancer cell lines, respectively, in the presence of rapamycin.

[0070] Figure 7C Shown is IL2 secretion from untransduced cells, SR020-T cells, SR022-T cells, and SR024-T cells co-cultured with high, medium, and low CD33 expressing cancer cell lines, respectively, in the presence of rapamycin.

[0071] Figure 8 Shown is IFNγ secretion from untransduced cells alone, SR020-T cells, SR022-T cells, and SR024-T cells in the presence and absence of rapamycin.

[0072] Fig.9A and 9B The cytotoxicity and proliferation of control, SR020-T cells, SR022-T cells, and SR024-T cells, each co-cultured with a high CD33-expressing cancer cell line, are shown, respectively.

[0073] Fig.10 Shown are the positive percentage and median fluorescence intensity of SR021 components on T cells as measured by flow cytometry.

[0074] Fig.11 Shown are IFNγ, IL2, IL4 and TNF secretion from non-transduced cells and SR021-T cells co-cultured with high CD33 expressing cancer cell lines in the presence of rapamycin.

[0075] Fig.12 Shown is IFNγ secretion from untransduced cells and SR021-T cells co-cultured with individual high CD33 expressing cancer cell lines in the presence and absence of rapamycin.

[0076] Fig.13A and 13B Cytotoxicity and proliferation of control and SR021-T cells co-cultured with high CD33 expressing cancer cell lines are shown, respectively.

[0077] Fig.14 Shown are the percent positivity and median fluorescence intensity of SR022 and SR001 components on T cells as measured by flow cytometry.

[0078] Fig.15 Shown are IFNγ, IL2, and TNF secretion from untransduced cells, SR022-T cells, and SR001-T cells, each co-cultured with a high CD33 expressing cancer cell line in the presence of rapamycin.

[0079] Fig.16 Shown is IFNγ secretion from untransduced cells, SR022-T cells, and SR001-T cells each co-cultured with a high CD33 expressing cancer cell line in the presence and absence of rapamycin.

[0080] Fig.17A and 17B The cytotoxicity and proliferation of control and SR001-T cells, each co-cultured with a high CD33-expressing cancer cell line, are shown, respectively.

[0081] Fig.18 Shown are the positive percentage and median fluorescence intensity of SR028 components on T cells as measured by flow cytometry.

[0082] Fig.19 Shown are IFNγ, IL2, IL4 and TNF secretion from non-transduced cells and SR028-T cells co-cultured with high CD33 expressing cancer cell lines in the presence of rapamycin.

[0083] Fig. 20 Shown is IFNγ secretion from non-transduced cells and SR028-T cells co-cultured with high CD33 expressing cancer cell lines in the presence and absence of rapamycin.

[0084] Fig.21A and 21B The cytotoxicity and proliferation of control and SR028-T cells, each co-cultured with a high CD33-expressing cancer cell line, are shown, respectively.

[0085] Fig. 22 Shown are the percent positivity and median fluorescence intensity of SR004 and SR006 components on T cells as measured by flow cytometry.

[0086] Fig.23Shown are IFNγ, IL2, IL4 and TNF secretion from untransduced cells, SR004-T cells and SR006-T cells, each co-cultured with a high CD33 expressing cancer cell line in the presence of rapamycin.

[0087] Fig.24 Shown is IFNγ secretion from untransduced cells, SR004-T cells, and SR006-T cells each co-cultured with a high CD33 expressing cancer cell line in the presence and absence of rapamycin.

[0088] Fig.25A and 25B The cytotoxicity and proliferation of control, SR004-T cells, and SR006-T cells, each co-cultured with a high CD33-expressing cancer cell line, are shown, respectively.

[0089] Fig.26A and 26B Shown are the percent positivity and median fluorescence intensity of SR008 and SR030 components on T cells as measured by flow cytometry.

[0090] Fig.26C and 26D Shown are the phenotypes of CD4+ T cells and CD8+ T cells transduced with vectors encoding the indicated constructs.

[0091] Fig.27A and 27B Shown are IFNγ, IL2, IL4, and TNF secretion from untransduced cells, SR008-T cells, and SR030-T cells, each co-cultured with a high CD33-expressing cancer cell line or a high CLL1-expressing cancer cell line in the presence of rapamycin.

[0092] Fig.27C Shown is IFNγ secretion from non-transduced cells alone, SR008-T cells, and SR030-T cells in the presence or absence of rapamycin.

[0093] Fig.28A and 28B The cytotoxicity of non-transduced cells, SR008-T cells, and SR030-T cells each co-cultured with a high CD33-expressing cancer cell line or a high CLL1-expressing cancer cell line is shown, respectively.

[0094] Fig.29 Shown are the percent positivity and median fluorescence intensity of SR001 and SR001-28 components on T cells as measured by flow cytometry.

[0095] Fig.30Shown are IFNγ, IL2, IL4 and TNF secretion from untransduced cells, SR001-T cells and SR001-28-T cells, each co-cultured with a high CD33 expressing cancer cell line in the presence of rapamycin.

[0096] Fig.31A and 31B The cytotoxicity and proliferation of control, SR001-T cells, and SR001-28-T cells, each co-cultured with a high CD33-expressing cancer cell line, are shown, respectively.

[0097] Fig.32A and 32B In vivo tumor growth in NSG mouse xenografts and controls following administration of 10E6 SR001±41BB or CD28 co-stimulatory domains is shown. All tumor controls were regulated by rapamycin.

[0098] Fig.32C and 32D Shown are in vivo tumor growth in NSG mouse xenografts and controls following administration of 3E6 SR001±41BB or CD28 co-stimulatory domains in the presence of rapamycin.

[0099] Fig.33 A and 33B show the percent positivity and median fluorescence intensity of SR022±CD4 or CD8 coreceptor signaling domain as measured by flow cytometry.

[0100] Fig.34 Shown are IFNγ and IL2 secretion from untransduced cells, SR022-T cells ± CD4 or CD8 coreceptor signaling domains, each co-cultured with a high CD33 expressing cancer cell line in the presence or absence of rapamycin.

[0101] Figures 35A-35C Shown are vector copy number, percent positive, and geometric mean fluorescence intensity of tetramer and TCR β chain staining for untransduced TCR, transgenic TCR, SR001, or a combination of transgenic TCR and SR001 as measured by flow cytometry.

[0102] Fig.35D and 35E Shown are the percent positive and geometric mean fluorescence intensity of anti-VHH staining of untransduced TCR, transgenic TCR, SR001, or the combination of transgenic TCR and SR001 as measured by flow cytometry.

[0103] Fig.36A and 36BShown are IFNγ, IL2, and TNFα secretion from untransduced TCR, 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 presence and absence of rapamycin.

[0104] Fig.36C The cytotoxicity of non-transduced TCR cells, 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 is shown.

[0105] Fig.37A and 37B Shown is IL2 secretion from untransduced or SR001 co-cultured with very low CD33 or CLL1 expressing cancer cell lines, respectively, in the presence of rapamycin.

[0106] Fig.38 In vivo tumor growth in NSG mouse xenografts and controls following administration of 10E6 SR007 or comparator conditioned CAR T cells (targeting CD33 or CLL1) is shown. All tumor controls were conditioned by rapamycin.

[0107] Figures 39A-39C Shown are vector copy number, percentage of positive staining for FRB and VHH expression in the absence or presence of rapamycin.

[0108] Fig.40A and 40B Shown is IFNγ secretion by SR10167 alone or SR10168-T cells ± rapamycin compared to regulated CAR controls.

[0109] Fig.41A and 41B Shown is IFNγ secretion by SR10167 or SR10168-T cells ± rapamycin when cultured with CD19+ target cell lines compared to regulated CAR controls.

[0110] Figures 42A-42D Shown are IL2 and TNFα secretion by SR10167 or SR10168-T cells ± rapamycin when cultured with CD19+ target cell lines compared to conditioned CAR controls.

[0111] Fig.43A and 43BCytotoxicity of control, SR10167-T cells and regulated CAR comparator-T cells co-cultured with CD19+Jeko-1 or Daudi cell lines, respectively, is shown. All tumor controls were regulated by rapamycin.

[0112] Fig.44 Sketches of representative constitutively active (non-regulatable) engineered immune receptors and fusion constructs are shown.

[0113] Fig.45A and 45B Shown are the positive percentages and median fluorescence intensity of VHH staining for expression of SR292, SR293, SR296, SR001, and relevant comparator molecules, including non-regulated CAR, regulated CAR, and non-regulated TCR-based architecture comparators.

[0114] Fig.46A and 46B Shown are IFNγ and IL2 secretion by SR292, SR293, SR296, SR001 and related comparator molecules (including non-regulated CAR, regulated CAR and non-regulated TCR-based architecture comparators) when cultured with CD33+ target cell lines.

[0115] Fig.47 IFNγ secretion by individual T cells expressing SR292, SR293, SR296, SR001 and related comparator molecules (including non-regulated CAR, regulated CAR and non-regulated TCR-based architecture comparators) is shown.

[0116] Fig.48A and 48B Shown are cytotoxicity and T cell proliferation of control, SR292, SR293, SR296, and a non-regulated CAR after co-culture with a CD33+ target line.

[0117] Fig.49 Shown are the positive percentages and median fluorescence intensity of FRB staining for expression of SR001 ± IL7-receptor α, common γ chain, IL2-receptor β, or both common γ chain and IL2-receptor β.

[0118] Figures 50A-50D Shown are IFNγ and IL2 secretion by SR001 ± IL7-receptor α, common γ chain, IL2-receptor β, or both common γ chain and IL2-receptor β when cultured with CD33+ target cell lines in the presence and absence of rapamycin.

[0119] Fig.51Shown are 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 culture with CD33+ target lines.

[0120] Fig.52 Shown are the median fluorescence intensities of FRB or PD-1 staining of T cells expressing SR300 (affinity-enhanced PD1) or SR301 (wild-type PD1).

[0121] Figures 53A-53D Shown are the cytotoxicity of T cells expressing SR300 or SR301 when cultured with a PDL1+ target cell line or the same cell line with PDL1 and PDL2 knockout in the presence and absence of rapamycin.

[0122] Fig.54 Shown are the positive percentages and median fluorescence intensity of the expressed VHH staining of the SR354 constructs.

[0123] Fig.55A and 55B Shown is IFNγ secretion by NK cells expressing SR354 when cultured with a CD33+ target cell line or the same cell line with CD33 knockout.

[0124] Fig.55C and 55D Shown is the cytotoxicity of NK cells expressing SR354 when cultured with a CD33+ target cell line or the same cell line with CD33 knockout.

[0125] Fig.56 Shown are the positive percentages of SR303 components on T cells as measured by flow cytometry.

[0126] Fig.57A and 57B Shown is IFNγ secretion by SR303 cells when cultured with the ROR1+ target cell line in the presence and absence of rapamycin.

[0127] Fig.57C IFNγ secretion by individual T cells expressing the SR303 molecule is shown.

[0128] A brief description of sequence identifiers

[0129] SEQ ID NOs: 1-4 show the amino acid sequences of exemplary FRB and FKBP12 polypeptides.

[0130] SEQ ID NOs: 5-10 show the amino acid sequences of exemplary antibody-derived heterodimerization domains.

[0131] SEQ ID NOs: 11-31 show the amino acid sequences of exemplary linkers.

[0132] SEQ ID NOs:32-40 show the amino acid sequences of exemplary actuator domains.

[0133] SEQ ID NOs:41-44 show the amino acid sequences of exemplary hinge domains.

[0134] SEQ ID NOs:45-47 show the amino acid sequences of exemplary membrane-spanning polypeptides.

[0135] Amino acid sequences of exemplary truncated intracellular CD4 polypeptides are shown in SEQ ID NOs: 48 and 49. Amino acid sequences of exemplary targeting domains are shown in SEQ ID NOs: 50-94.

[0136] SEQ ID NOs:95-97 show the amino acid sequences of exemplary signal sequences.

[0137] SEQ ID NOs:98-106 set forth the amino acid sequences of exemplary intracellular signaling domains.

[0138] SEQ ID NOs: 107-115 show the amino acid sequences of exemplary signaling components.

[0139] SEQ ID NOs: 116-136 show the amino acid sequences of exemplary targeting components.

[0140] SEQ ID NOs: 137 and 138 show the amino acid sequences of exemplary co-signaling components.

[0141] SEQ ID NOs: 139-170 show the amino acid sequences of exemplary fusion polypeptides.

[0142] SEQ ID NOs: 171-192 show the amino acid sequences of the protease cleavage site and the self-cleaving polypeptide cleavage site.

[0143] SEQ ID NO: 193 shows the spacer amino acid sequence.

[0144] SEQ ID NO: 194 shows the furin recognition amino acid sequence.

[0145] SEQ ID NOs: 195-197 show the TEV (Tobacco Etch Virus) protease cleavage site.

[0146] SEQ ID NO: 198 shows the kozak amino acid sequence.

[0147] In the foregoing sequences, X, if present, refers to any amino acid or the absence of an amino acid. DETAILED DESCRIPTION

[0148] A. Overview

[0149] Cancer is one of the leading causes of death worldwide. Although adoptive cell therapy has been used to successfully treat some hematological malignancies, the treatment of solid tumors using chimeric antigen receptor (CAR) T cells and T cells expressing T cell receptors (TCRs) specific for tumor antigens remains largely ineffective.

[0150] In addition, although TCRs are known to be very sensitive to low levels of target antigens, they lack the ability to observe non-MHC-presented antigens. On the other hand, although 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 are prone to tonic and / or antigen-independent signaling. In addition, T cells engineered to express CARs or TCRs generally lack spatial and temporal control of T cell activity and / or exhibit insufficient activation of T cell signaling pathways.

[0151] Lack of control over the activity of engineered T cells can trigger a range of side effects, many of which start out small but rapidly worsen. A particularly serious complication is cytokine release syndrome (CRS) or "cytokine storm," in which CAR T cells induce massive and potentially lethal cytokine release. CRS can produce dangerously high fever, extreme fatigue, dyspnea, and a sharp drop in blood pressure. CRS can also produce a second wave of side effects involving the nervous system, including neurotoxicity, tremors, headaches, confusion, loss of balance, difficulty speaking, epilepsy, and hallucinations. Insufficient activation of T cell signaling pathways can result in the inability to eradicate cancer and / or lead to untreatable cancer. The compositions and methods contemplated herein provide solutions to these and other problems that plague adoptive cell therapy.

[0152] Thus, the present disclosure generally relates to improved compositions and methods for modulating the antigen recognition capacity, sensitivity and / or spatial and temporal control of adoptive cell therapy by using engineered immune receptor complexes that bind to selected target antigens and can recruit and activate native or transgenic immune receptor signaling complexes.

[0153] Without wishing to be bound by any particular theory, the engineered immune receptor compositions and methods contemplated herein provide many advantages over existing CAR T cells and TCR T cell therapies in the art, including but not limited to spatial and temporal control of immune effector cell signal transduction, binding and signal transduction activity, and activation of signal transduction pathways without the need for MHC complex recognition. In some embodiments, temporal control triggers the engineered immune receptor signal transduction mechanism by the association of the targeting component and the signal transduction component mediated by the bridging factor. In other embodiments, the mechanism is triggered by the association of the multimerization domain, without the need for a bridging factor. Spatial control participates in the signal transduction mechanism by the recognition of the target antigen by the extracellular domain or the targeting domain of the targeting component, and the signal transduction component comprises an actuator domain that forms a complex with a lymphocyte immune receptor. In this way, when there is a target cell expressing the target antigen, and in certain embodiments, when there is a bridging factor, the immune effector cell activates the receptor signal transduction.

[0154] In various embodiments, the present disclosure contemplates signaling components and targeting components that generate immune receptor-based responses to cells expressing target antigens without recognition of natural immune receptor target antigens. In various embodiments, the present disclosure contemplates signaling components and targeting components that generate immune receptor-based anti-cancer responses to cancers expressing target antigens without recognition of the target antigen and / or MHC complex of a natural or engineered target antigen by a lymphocyte immune receptor.

[0155] In certain embodiments, the engineered immune receptor complex includes a signaling component comprising a multimerization domain polypeptide or a variant thereof and an actuator domain or a variant thereof (e.g., CD3ε, CD3δ, CD3γ, FcεR1γ, Igα / CD79a, Igβ / CD79b, DAP10 or DAP12 polypeptide); and a targeting component comprising an extracellular domain, the extracellular domain comprising a targeting domain that binds to a target antigen expressed on a target cell, a multimerization domain polypeptide or a variant thereof, a transmembrane domain, and a hinge domain that is optionally disposed between the multimerization domain and the transmembrane domain. In some embodiments, in the presence of a bridging factor, the signaling and binding / targeting components associate with each other through the bridging factor to form a functionally active immune receptor. In some embodiments, a bridging factor is not required for component association.

[0156] In certain embodiments, the multimerization domains of the signaling component and the targeting component are located extracellularly. Compared to intracellular localization, extracellular localization of the multimerization domain provides many advantages, including but not limited to more effective localization of the targeting domain, higher temporal sensitivity to bridging factor regulation, and less toxicity due to the ability to use non-immunosuppressive doses of specific bridging factors.

[0157] Contemplated herein are polynucleotides encoding engineered immune receptors, targeting components, signaling components, protein / polypeptide complexes, and fusion proteins; polypeptides comprising engineered immune receptors, targeting components, signaling components, protein / polypeptide complexes, and fusion proteins; cells comprising polynucleotides encoding and / or expressing engineered immune receptors, targeting components, and signaling components; vectors encoding engineered immune receptors, targeting components, signaling components, 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).

[0158] Techniques for 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 generally can be performed as described in various general and more specific references in microbiology, molecular biology, biochemistry, molecular genetics, cell biology, virology, and immunology that are cited and discussed throughout the present specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Current Protocols in Molecular Biology (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, Vols. I and II (IRL Press, Oxford Univ. Press USA, 1985); Current Protocols in Immunology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Glover, DNA Cloning: A Practical Approach, Vols. I and II (IRL Press, Oxford Univ. Press USA, 1985); Protocols in Immunology)》(Editors: John E.Coligan,Ada M.Kruisbeek,David H.Margulies,EthanM.Shevach, Warren Strober 2001 John Wiley & Sons, New York, NY); Real-Time PCR: Current Technology and Applications, ed. 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 Hybridization: A Review of the Invention. The Hybridization (B.Hames and S.Higgins, 1985); Transcription and Translation (B.Hames and S.Higgins, 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 ed., 2010, Humana Press); Immobilized Cells and Enzymes (IRL Press, 1986); Methods in Enzymology (Academic Press, Inc., New York); Gene Transfer Vectors for Mammalian Cells (2003). Cells (JH Miller and MP Calos, eds., 1987, Cold Spring Harbor Laboratory Press); Harlow and Lane, Antibodies (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1998); Immunochemical Methods in Cell and Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook of Experimental Immunology, Vols. I-IV (DM Weir and CC Blackwell, eds., 1986); Roitt, Essential Immunology, 6th ed. (Blackwell Scientific Publications, Oxford, 1988); Current Protocols in Immunology (Current Protocols in Immunology, 1996). Immunology" (QE Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach and W. Strober, eds., 1991); Annual Review of Immunology; and monographs in journals such as Advances in Immunology.

[0159] B. Definition

[0160] Before describing the present disclosure in greater detail, it may be helpful to understand the present disclosure by providing definitions of certain terms to be used herein.

[0161] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar to or equivalent to the methods and materials described herein may be used to practice or test specific embodiments, preferred embodiments of compositions, methods and materials are described herein. For purposes of this disclosure, the following terms are defined as follows.

[0162] The articles "a," "an," and "the" are used herein to refer to one or more than one (ie, at least one, or one or more) of the grammatical object of the article. By way of example, "an element" means one element or one or more elements.

[0163] The use of alternatives (eg, "or") should be understood to mean either one, both, or any combination of the alternatives.

[0164] The term "and / or" should be understood to mean one or both of the alternatives.

[0165] 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 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% relative to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0166] In one embodiment, a range, for example, 1 to 5, about 1 to 5, or about 1 to about 5, refers to every value encompassed within the range. For example, in one non-limiting and exemplary embodiment, the range "1 to 5" is equivalent to stating 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.

[0167] As used herein, the term "substantially" refers to an amount, 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 of a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, "substantially the same" refers to an effect produced by an amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length, e.g., a physiological effect that is about the same as a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0168] Throughout this specification, unless the context requires otherwise, the term "comprise / comprises / comprising" should be understood to imply the inclusion of the stated step or element or group of steps or elements, but not the exclusion of any other step or element or any other group of steps or elements. "Consisting of" means including and limited to what follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are essential or required, and no other elements can be present. "Consisting essentially of" means including any element listed after the phrase, and is limited to other elements that do not interfere with or affect the activity or action described in the disclosure regarding the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are essential or required, and no other elements are present that substantially affect the activity or action of the listed elements.

[0169] References throughout this specification to "one embodiment," "an embodiment," "a specific embodiment," "a related embodiment," "an embodiment," "another embodiment," or "another embodiment," or combinations thereof, mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the above 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. It should also be understood that the affirmative recitation of a feature in one embodiment serves as a basis for excluding the feature in a specific embodiment.

[0170] As used herein, the term "TCR complex" refers to a complex formed by the association of CD3 with TCR. For example, the TCR complex may be composed of: a CD3γ chain, a CD3δ chain, two CD3ε chains, a homodimer of a CD3ζ chain, a TCRα chain, and a TCRβ chain. In some embodiments, the TCR complex may be composed of: a CD3γ chain, a CD3δ chain, two CD3ε chains, a homodimer of a CD3ζ chain, a TCRγ chain, and a TCRδ chain.

[0171] As used herein, "component of a TCR complex" refers to a TCR chain (i.e., TCRα, TCRβ, TCRγ or TCRδ), a CD3 chain (i.e., CD3γ, CD3δ, CD3ε or CD3ζ), or a complex formed by two or more TCR chains or CD3 chains (e.g., a complex of TCRα and TCRβ, a complex of TCRγ and TCRδ, a complex of CD3ε and CD3δ, a complex of CD3γ and CD3ε, or a sub-TCR complex of TCRα, TCRβ, CD3γ, CD3δ and two CD3ε chains).

[0172] As used herein, "actuator polypeptide", "actuator domain" or "actuator" refers to a polypeptide that directly or indirectly associates, integrates or complexes with a multimeric immune receptor complex to promote signal transduction 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. When the actuator domain contains a signaling domain or motif (such as an immunoreceptor tyrosine-based activation motif (ITAM)), it may directly contribute to the cellular response. In other embodiments, the actuator domain will indirectly promote the cellular response by associating with one or more other proteins that directly signal and thereby 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.

[0173] As used herein, "multimerization domain" or "multimerization domain polypeptide" refers to a polypeptide that preferentially interacts or associates with another different polypeptide, directly or via a bridging molecule, e.g., a chemically inducible dimer, wherein the interaction of different multimerization domains substantially facilitates or efficiently promotes multimerization (i.e., the formation of a dimer, trimer, or multipart complex, which may be a homodimer, heterodimer, homotrimer, heterotrimer, homomultimer, heteromultimer). A multimerization domain may be derived from natural, synthetic, semisynthetic, or recombinant sources.

[0174] Illustrative examples of multimerization domains suitable for use in specific embodiments contemplated herein include FK506 binding protein (FKBP) polypeptides or variants thereof, FKBP-rapamycin binding (FRB) polypeptides or variants thereof, calcineurin polypeptides or variants thereof, cyclophilin polypeptides or variants thereof, bacterial dihydrofolate reductase (DHFR) polypeptides or variants thereof, PYR1-like 1 (PYL1) polypeptides or variants thereof, abscisic acid insensitive 1 (ABI1) polypeptides or variants thereof, GIB1 polypeptides or variants thereof, or GAI polypeptides or variants thereof.

[0175] As used herein, the term "FKBP-rapamycin binding polypeptide" refers to a FRB polypeptide. In specific embodiments, the FRB polypeptide is a FKBP12-rapamycin binding polypeptide. FRB polypeptides suitable for use in specific embodiments contemplated herein typically contain at least about 85 to about 100 amino acid residues. In certain embodiments, with reference to GenBank accession number L34075.1, the FRB polypeptide comprises a 93 amino acid sequence Ile-2021 to Lys-2113 and a mutation of T2098L (T82L is the equivalent position in the 93 amino acid FRB polypeptide). As used herein, the terms "FRB star", "FRBstar" or "FRB*" refer to such FRB T2098L (T82L) mutants. The FRB polypeptide contemplated herein binds to the FKBP polypeptide via a bridging factor, thereby forming a ternary complex.

[0176] As used herein, the term "FK506 binding protein" refers to a FKBP polypeptide. In a specific embodiment, the FKBP polypeptide is a FKBP12 polypeptide or a FKBP12 polypeptide comprising an F36V mutation. In certain embodiments, the FKBP domain may also be referred to as a "rapamycin binding domain". Information on 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 polypeptide contemplated herein binds to the FRB polypeptide via a bridging factor, thereby forming a ternary complex.

[0177] "Bridging factor" refers to a molecule that associates with two or more multimerization domains and is disposed between the two or more multimerization domains. In specific embodiments, the multimerization domain substantially contributes to or efficiently promotes the formation of a polypeptide complex only in the presence of a bridging factor. In specific embodiments, the multimerization domain does not contribute to or efficiently promote the formation of a polypeptide complex in the absence of a bridging factor. Illustrative examples of bridging factors suitable for use in specific embodiments contemplated herein include, but are not limited to, AP21967, rapamycin (sirolimus) or rapamycin analogs, coumermycin or derivatives thereof, gibberellins or derivatives thereof, abscisic acid (ABA) or derivatives thereof, methotrexate or derivatives thereof, cyclosporin A or derivatives thereof, FKCsA or derivatives thereof, trimethoprim (Tmp) synthetic ligand (SLF) for FKBP or derivatives thereof, or any combination thereof.

[0178] Rapamycin analogs (rapalogs) include, but are not limited to, those disclosed in U.S. Pat. No. 6,649,595, the structure of which is incorporated herein by reference in its entirety. In certain embodiments, the bridging factor is a rapamycin analog that has a significantly lower immunosuppressive effect than rapamycin. In a preferred embodiment, the rapamycin analog is AP21967 (also known as C-16-(S)-7-methylindorapamycin, IC 50 = 10 nM, chemically modified non-immunosuppressive rapamycin analogs). Other exemplary rapamycin analogs suitable for use in the specific embodiments contemplated herein include, but are not limited to, AP1903, AP20187, everolimus, norvolimus, pimecrolimus, defostiolimus, sirolimus, tacrolimus, temsirolimus, usololimus, zotarolimus, or BPC015.

[0179] "Significantly reduced immunosuppressive effect" means at least less than 0.1 to 0.005 times the immunosuppressive effect observed or expected for the same dose measured clinically or in an appropriate in vitro (e.g., inhibition of T cell proliferation) or in vivo surrogate of human immunosuppressive activity.

[0180] A "transmembrane domain" or "TM domain" is a domain that anchors a polypeptide to the plasma membrane of a cell. A TM domain may be derived from natural, synthetic, semisynthetic or recombinant sources.

[0181] As used herein, the term "extracellular domain" refers to a domain or portion of a polypeptide located or present outside a cell. As disclosed herein, the extracellular domain of a targeting component comprises at least one targeting domain, which redirects activation of an immune receptor complex (e.g., TCR) to, for example, one or more selected target antigens on a target cell (e.g., a cancer cell) when the targeting component is associated with a signaling component (e.g., by connecting the multimerization domain of the targeting component to the multimerization domain of the signaling component via a bridging factor, or by association of the multimerization domain of the targeting component without a bridging factor to the multimerization domain of the signaling component).

[0182] 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, activation, cytokine production, proliferation, and cytotoxic activity, including the release of cytotoxic factors, or other cellular responses elicited by antigen binding to receptors expressed on immune effector cells. An "intracellular signaling domain" or "intracellular domain" refers to a portion of a protein that transduces an effector function signal and directs the cell to perform a specialized function. Although the entire intracellular signaling domain can generally be employed, 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 can be used in place of the entire domain, as long as it transduces an effector function signal. The term intracellular signaling domain means any truncated portion that is necessary or sufficient to transduce an effector function signal comprising an intracellular signaling domain.

[0183] It is well known that the signal generated by the TCR alone is insufficient to fully activate T cells and that secondary or co-stimulatory signals are also required. Therefore, T cell activation can be considered to be mediated by two different classes of intracellular signaling domains: primary signaling domains that initiate antigen-dependent primary activation by the TCR (e.g., the TCR / CD3 complex), and co-stimulatory signaling domains that act in an antigen-independent manner to provide secondary or co-stimulatory signals.

[0184] As used herein, the term "costimulatory signaling domain" or "costimulatory domain" refers to the intracellular signaling domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide a second signal required for efficient activation and function of T lymphocytes after binding to an antigen. Exemplary examples of such costimulatory molecules from which costimulatory domains can be separated 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), CD27 8 (ICOS), DNAX-activating protein 10 (DAP10), FYN, adaptor for activating T-cell 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 T-cell receptor zeta chain-associated protein kinase 70 (ZAP70).

[0185] "Hinge domain" refers to a polypeptide that plays a role in spatially positioning a domain away from the effector cell surface to achieve appropriate cell / cell contact, antigen binding and activation. In particular embodiments, the 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 a natural, synthetic, semisynthetic or recombinant source. The hinge domain may include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region.

[0186] "Linker" or "linker polypeptide" refers to a plurality of amino acid residues added between polypeptide domains for proper spacing and conformation of the molecule. In a specific embodiment, the linker is a variable region linker sequence. A "variable region linker sequence" is an amino acid sequence that connects the V H Domain and V LThe linker is a spacer that is compatible with the interaction of the two sub-binding domains, so that the resulting polypeptide retains specific binding affinity for the same target molecule as an antibody comprising the same light chain variable region and heavy chain variable region. In a specific embodiment, the linker separates one or more heavy chain variable domains or light chain variable domains, hinge domains, multimerization domains, transmembrane domains, costimulatory domains, and / or primary signaling domains.

[0187] Illustrative examples of linkers suitable for use in the specific 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., Proceedings of the National Academy of Sciences of the United States of America (PNAS) 5525-5530 (1997)); GGRR (SEQ ID NO: 18) (Pomerantz et al., 1995, supra); (GGGGS) n , wherein n=1, 2, 3, 4 or 5 (SEQ ID NOs: 11-15) (Kim et al., Proc. Natl. Acad. Sci. USA 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); LRQRDGERP (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 the DNA binding site and the peptide itself (Desjarlais and Berg, Proc. Natl. Acad. Sci. USA 90:2256-2260 (1993), Proc. Natl. Acad. Sci. USA 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)).

[0188] "Spacer domain" refers to a polypeptide that separates two domains. In one embodiment, the spacer domain moves or targets the domain away from the effector cell surface to enable appropriate cell / cell contact, antigen binding and activation (Patel et al., Gene Therapy, 1999; 6: 412-419). In a specific embodiment, the spacer domain separates one or more heavy chain variable domains or light chain variable domains, multimerization domains, transmembrane domains, costimulatory domains and / or primary signaling domains. The spacer domain can be derived from a natural, synthetic, semisynthetic or recombinant source. In certain embodiments, the spacer domain is a part of an immunoglobulin, including but not limited to one or more heavy chain constant regions, for example, CH2 and CH3. The spacer domain may include a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region amino acid sequence.

[0189] "Antigen (Ag)" refers to a compound, composition or substance that can stimulate antibody production or T cell response in an animal, including compositions injected or absorbed into an animal (such as compositions including cancer-specific proteins). Exemplary antigens include, but are not limited to, lipids, carbohydrates, polysaccharides, glycoproteins, peptides or nucleic acids. Antigens react with products having specific humoral or cellular immunity, including those induced by heterologous antigens such as the disclosed antigens.

[0190] "Target antigen" or "target antigen of interest" refers to a molecule expressed on the cell surface of a target cell to which a binding domain or targeting domain contemplated herein is designed to bind. In particular embodiments, the target antigen is an epitope of a polypeptide expressed on the surface of a cancer cell.

[0191] As used herein, the term "targeting domain" refers to a domain that provides a polypeptide with the ability to specifically bind to a target antigen of interest, such as a domain of a targeting component. The targeting domain can be derived from a natural, synthetic, semisynthetic or recombinant source. The targeting domain can, for example, comprise an antibody or antigen-binding fragment thereof or, for example, an extracellular domain.

[0192] As used herein, the term "specific binding affinity" or "specifically binds or specifically bound or specific binding" or "specific targeting" describes that a targeting domain binds to a target antigen with a binding affinity greater than background binding. 5 M -1 Affinity or K aThe targeting domain "specifically binds" to the target antigen if the targeting domain binds or associates with the antigen at a specific equilibrium association constant (i.e., the equilibrium association constant for a specific binding interaction in units of 1 / M). In certain embodiments, the targeting domain (or fusion protein comprising the same) binds or associates with the antigen at a specific equilibrium association constant of greater than or equal to 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 K a Binds to the target. A "high affinity" targeting domain (or a single-chain fusion protein thereof) refers to a K a For 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 higher targeting domain.

[0193] The terms "selectively binds" or "selectively bound" or "selectively binding" or "selectively targets" describe the preferential binding of a molecule to a target molecule in the presence of multiple off-target molecules (on-target binding).

[0194] "Antibody" refers to a binding agent that is a polypeptide comprising at least a light or heavy chain immunoglobulin variable region that specifically recognizes and binds an epitope of an antigen, such as a lipid, carbohydrate, polysaccharide, glycoprotein, peptide, or nucleic acid containing antigenic determinants, such as those recognized by immune cells.

[0195] "Epitope" or "antigenic determinant" refers to the region of an antigen to which a binding agent binds.

[0196] Antibodies include antigen-binding fragments thereof, such as camelid Ig, llama Ig, alpaca Ig, Ig NAR, Fab' fragment, F(ab')2 fragment, bispecific Fab dimer (Fab2), trispecific Fab trimer (Fab3), Fv, single-chain Fv protein ("scFv"), bis-scFv, (scFv)2, miniantibodies, diabodies, triabodies, tetrabodies, disulfide-stabilized Fv proteins ("dsFv") and single domain antibodies (sdAb, camelid VHH, nanobodies) as well as the portion of the full-length antibody responsible for antigen binding. The term also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (e.g., 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., WH Freeman & Co., New York, 1997.

[0197] As used herein, the term "cancer" generally refers to a class of diseases or conditions in which abnormal cells divide without control and may invade nearby tissue.

[0198] As used herein, the term "malignant" refers to a group of cancer cells that show one or more of uncontrolled growth (i.e., division beyond normal limits), invasion (i.e., invasion and destruction of adjacent tissues), and metastasis (i.e., spread to other locations in the body via the lymph or blood). As used herein, the term "metastasis" refers to the spread of cancer from one part of the body to another. Tumors formed by cells that have spread are called "metastatic tumors" or "metastasis". Metastatic tumors contain cells similar to those in the original (primary) tumor.

[0199] As used herein, the term "benign" or "non-malignant" refers to a tumor that can grow larger but does not spread to other parts of the body. Benign tumors are self-limited and usually do not invade or metastasize.

[0200] "Cancer cell" refers to a single cell of a cancerous growth or tissue. Cancer cells include solid cancers and liquid cancers. "Tumor" or "tumor cell" generally refers to a swelling or lesion formed by an abnormal growth of cells, which can be benign, pre-malignant or malignant. Most cancers form tumors, but liquid cancers such as leukemias do not necessarily form tumors. For those cancers that form tumors, the terms cancer (cell) and tumor (cell) are used interchangeably. The amount of tumor in an individual is the "tumor burden" which can be measured as the number, volume or weight of tumors.

[0201] The term "relapse" refers to the return of cancer diagnosis or signs and symptoms after a period of improvement or remission.

[0202] "Remission" is also called "clinical remission" and includes both partial remission and complete remission. In a partial remission, some but not all signs and symptoms of cancer have disappeared. In a complete remission, all signs and symptoms of cancer have disappeared, although the cancer may still be in the body.

[0203] "Refractory" means that the cancer is resistant or unresponsive to therapy with a particular therapeutic agent. A cancer can be refractory at the start of treatment (i.e., unresponsive to initial exposure to the therapeutic agent), or because it develops resistance to the therapeutic agent during the first treatment period or during subsequent treatment periods.

[0204] "Antigen negative" refers to cells that do not express an antigen or express negligible amounts of an undetectable antigen. In one embodiment, an antigen negative cell does not bind to a receptor directed to the antigen. In one embodiment, an antigen negative cell does not substantially bind to a receptor directed to the antigen.

[0205] As used herein, the terms "individual" and "subject" are often used interchangeably and refer to any animal that exhibits symptoms of cancer or other immune disorders that can be treated with the compositions and methods contemplated elsewhere herein. Suitable subjects (e.g., patients) include experimental animals (such as mice, rats, rabbits, or guinea pigs), farm animals, and domestic animals or pets (such as cats or dogs). Non-human primates and preferably human patients are included. Typical subjects include human patients who have, have been diagnosed as having, or are at risk of having cancer or another immune disorder.

[0206] As used herein, the term "patient" refers to a subject who has been diagnosed as having cancer or another immune disorder that can be treated with the compositions and methods disclosed elsewhere herein.

[0207] 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 even include a minimal reduction in one or more measurable markers of the disease or condition being treated. Optionally, treatment may involve a reduction in the disease or condition or a delay in the progression of the disease or condition, such as a delay in tumor growth. "Treatment" does not necessarily indicate complete eradication or cure of a disease or condition or its associated symptoms.

[0208] As used herein, "prevent" and similar words such as "prevented / preventing" indicate the use of preventing, inhibiting, or reducing the likelihood of the occurrence or recurrence of a disease or condition. Prevention also refers to delaying the onset or recurrence of a disease or condition or delaying the onset or recurrence of symptoms of a disease or condition. As used herein, "prevention" and similar words also include reducing the intensity, effects, symptoms, and / or burden of a disease or condition before the onset or recurrence of the disease or condition.

[0209] As used herein, the phrase "improving at least one symptom of..." refers to reducing one or more symptoms of the disease or condition being treated in the subject. In certain embodiments, the disease or condition being treated is cancer, wherein the one or more symptoms reduced include, but are not limited to, weakness, fatigue, shortness of breath, easy bruising and bleeding, frequent infections, swollen lymph nodes, abdominal swelling or pain (due to enlarged abdominal organs), bone or joint pain, bone fractures, unintentional weight loss, loss of appetite, night sweats, persistent mild fever, and decreased urination (due to impaired kidney function).

[0210] "Enhancement" or "promotion" or "increase" or "amplification" generally refers to the ability of the composition contemplated herein to produce, induce or cause a greater physiological response (i.e., downstream effect) compared to the response caused by a vehicle or control molecule / composition. A measurable physiological response may include an increase in T cell expansion, activation, persistence, cytokine secretion, and / or an increase in cancer cell killing ability, as well as other aspects apparent from the 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 times, 1.2 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times or more (e.g., 500 times, 1000 times) (including all integers and decimal points therebetween and above 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) of the response produced by a vehicle or control composition.

[0211] "Reduced" or "diminished" or "lessened" or "reduced" or "mitigated" generally refers to the ability of the compositions contemplated herein to produce, elicit or cause less physiological responses (i.e., downstream effects) than responses elicited by vehicle or control molecules / compositions. A "reduced" or "reduced" amount is generally a "statistically significant" amount and may include a decrease of 1.1-fold, 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold or more (e.g., 500-fold, 1000-fold) (including all integers and decimal points between and above 1, such as 1.5, 1.6, 1.7, 1.8, etc.) of the response produced by vehicle, control compositions, or responses in a particular cell lineage (reference response).

[0212] "Maintain" 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 comparable physiological response (i.e., downstream effect) in a cell compared to the response elicited by a vehicle, a control molecule / composition, or a response in a particular cell lineage. A comparable response is one that is not significantly different or measurably different from a reference response.

[0213] Additional definitions are set forth throughout this disclosure.

[0214] C. Engineered immune receptor architecture

[0215] In a specific embodiment, it is envisioned that the cytotoxicity of immune effector cells is redirected toward cells expressing target antigens and recruiting one or more engineered immune receptors of 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 promote the transduction of immunostimulatory signals in immune effector cells when exposed to a predefined or selected target antigen and associated / multimerized with an immune receptor complex. In certain embodiments, a multimerizing agent or bridging factor is required to promote the multimerization of non-naturally occurring polypeptides, and thereby stimulate the activity and function of immune effector cells by the activation of immune receptor complexes. In a preferred embodiment, an engineered immune receptor or system is a multi-chain chimeric receptor comprising a signaling component associated with a member of an immune receptor complex and a targeting component that redirects or supplements the antigen-specificity of a receptor / complex.

[0216] 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 supplied exogenously as a polypeptide. In a specific embodiment, the targeting component pre-loaded with a bridging factor is supplied exogenously to a cell expressing the signaling component.

[0217] 1. Signal transduction components

[0218] A "signaling component" refers to a polypeptide comprising one or more multimerization domains (e.g., a FRB or FKBP polypeptide) or variants thereof and a CD3ε domain / polypeptide or a functional fragment or variant thereof, which is capable of recruiting an immune receptor complex (e.g., a TCR complex).

[0219] In various embodiments, the CD3ε peptide or its functional fragment or variant comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 32. In some embodiments, the CD3ε peptide or its functional fragment or variant comprises an amino acid sequence having at least 95% identity with SEQ ID NO: 32. In some embodiments, the CD3ε peptide or its functional fragment or variant comprises an amino acid sequence having at least 96% identity with SEQ ID NO: 32. In some embodiments, the CD3ε peptide or its functional fragment or variant comprises an amino acid sequence having at least 97% identity with SEQ ID NO: 32. In some embodiments, the CD3ε peptide or its functional fragment or variant comprises an amino acid sequence having at least 98% identity with SEQ ID NO: 32. In some embodiments, the CD3ε peptide or its functional fragment or variant comprises an amino acid sequence having at least 99% identity with SEQ ID NO: 32. In a specific embodiment, the CD3ε peptide comprises an amino acid sequence as shown in SEQ ID NO: 32.

[0220] In various embodiments, the CD3ε polypeptide is a human CD3ε polypeptide.

[0221] In particular embodiments, the signaling component comprises a FRB or FRB T2098L multimerization domain / polypeptide, a linker polypeptide, and a CD3ε polypeptide.

[0222] In various embodiments, the signaling component comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 111. In various embodiments, the signaling component comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 111. In various embodiments, the signaling component comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 111. In various embodiments, the signaling component comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 111. In various embodiments, the signaling component comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 111. In various embodiments, the signaling component comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 111. In specific embodiments, the signaling component comprises an amino acid sequence as shown in SEQ ID NO: 111.

[0223] In various embodiments, when the signaling component is expressed, the multimerization domain (e.g., FRB or FKBP) is localized extracellularly.

[0224] In certain embodiments, the signaling component comprises a FRB polypeptide or a variant thereof. In various embodiments, the signaling component comprises a FRB polypeptide or a variant thereof comprising a T2098L (T82L) mutation. 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 as shown in SEQ ID NO: 1. In certain embodiments, the FRB polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 2. In specific embodiments, the FRB polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:2. In specific embodiments, the FRB polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:2. In specific embodiments, the FRB polypeptide comprises an amino acid sequence that is at least 96% identical to SEQ ID NO:2. In specific embodiments, the FRB polypeptide comprises an amino acid sequence that is at least 97% identical to SEQ ID NO:2. In specific embodiments, the FRB polypeptide comprises an amino acid sequence that is at least 98% identical to SEQ ID NO:2. In specific embodiments, the FRB polypeptide comprises an amino acid sequence that is at least 99% identical to SEQ ID NO:2. In specific embodiments, the FRB polypeptide comprises an amino acid sequence as shown in SEQ ID NO:2.

[0225] In certain embodiments, the signaling component comprises a FKBP12 polypeptide or a variant thereof. In various embodiments, the signaling component comprises a FKBP12 polypeptide comprising a 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 an amino acid sequence as shown in SEQ ID NO: 3. In certain embodiments, the FKBP12 polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 4. In one embodiment, the FKBP12 polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 4. In one embodiment, the FKBP12 polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 4. In one embodiment, the FKBP12 polypeptide comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 4. In one embodiment, the FKBP12 polypeptide comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 4. In one embodiment, the FKBP12 polypeptide comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 4. In one embodiment, the FKBP12 polypeptide comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 4. In a specific embodiment, the FKBP12 polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 4.

[0226] In various embodiments, the first multimerization domain (e.g., FRB or FKBP) and the CD3ε domain / polypeptide are separated by a first polypeptide linker of 2 to 40 amino acids in length. In various embodiments, preferably a short oligopeptide linker or polypeptide linker of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids in length connects one or more domains in the signaling component. Glycine-serine based linkers provide particularly suitable linkers. In some embodiments, the first polypeptide linker is selected from the group consisting of: GG, GS, SG, SS, GSS, SSG, GSG, SGS, SGG, G4S, 2xG4S, 3xG4S, 4xG4S and any combination 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 (GGGGSGGGGSGGGGS; SEQ ID NO: 13) linker. In some embodiments, the first polypeptide linker is a 4xG4S (GGGGSGGGGSGGGGSGGGGS; SEQ ID NO: 14) linker. In some embodiments, the first polypeptide linker is a 5xG4S (GGGGSGGGGSGGGGSGGGGSGGGGS; SEQ ID NO: 15) linker. In one embodiment, the linker has a sequence as set forth in SEQ ID NO: 16. In one embodiment, the linker has a sequence as set forth in SEQ ID NO: 17. In one embodiment, the linker has a sequence as set forth in SEQ ID NO: 18. In one embodiment, the linker has a sequence as set forth in SEQ ID NO: 19. In one embodiment, the linker has a sequence as set forth in SEQ ID NO: 20. In one embodiment, the linker has a sequence as set forth in SEQ ID NO: 21. In one embodiment, the linker has a sequence as set forth in SEQ ID NO: 22. In one embodiment, the linker has a sequence as shown in SEQ ID NO:23. In one embodiment, the linker has a sequence as shown in SEQ ID NO:24. In one embodiment, the linker has a sequence as shown in SEQ ID NO:25. In one embodiment, the linker has a sequence as shown in SEQ ID NO:26. In one embodiment, the linker has a sequence as shown in SEQ ID NO:27. In one embodiment, the linker has a sequence as shown in SEQ ID NO:28. In one embodiment, the linker has a sequence as shown in SEQ ID NO:29.In one embodiment, the linker has the sequence shown in SEQ ID NO: 30. In one embodiment, the linker has the sequence shown in SEQ ID NO: 31.

[0227] In various embodiments, the signaling components contemplated herein comprise a signal peptide, such as a secretory signal polypeptide or a signal sequence. Illustrative examples of signal peptides / sequences suitable for use in specific signaling components include, but are not limited to, IgG1 heavy chain signal polypeptides, Igκ light chain signal polypeptides, CD8α signal polypeptides, or human GM-CSF receptor α signal polypeptides. In various embodiments, the signaling component comprises an Igκ light chain signal polypeptide. In some embodiments, the Igκ light chain signal polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 95. In various embodiments, the signaling component comprises a CD8α signal polypeptide. In some embodiments, the CD8α light chain signal polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 96. In various embodiments, the signaling component comprises a PD1 signal polypeptide. In one embodiment, the PD1 signal polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 97.

[0228] In specific embodiments, the signaling component comprises a FRB polypeptide or a variant thereof, a 3xG4S linker and a CD3ε polypeptide or a variant thereof. In specific embodiments, the signaling component comprises a signaling sequence, a FRB polypeptide or a variant thereof, a 3xG4S linker and a CD3ε polypeptide or a variant thereof. In specific embodiments, the signaling component comprises a CD8 signaling sequence, a FRB* polypeptide or a variant thereof, a 3xG4S linker and a CD3ε polypeptide or a variant thereof. In specific embodiments, the signaling component comprises a CD8 signaling sequence (e.g., as shown in SEQ ID NO: 96), a FRB* polypeptide or a 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).

[0229] 2. Targeting components

[0230] "Targeting component" refers to a polypeptide comprising an extracellular domain, one or more multimerization domains and a transmembrane domain, the conjugation of which with a signaling component 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 and / or bind to one or more target antigens. In specific embodiments, the targeting component comprises a first targeting domain, a second multimerization domain, a hinge domain and a transmembrane domain.

[0231] In certain embodiments, the targeting component comprises an extracellular domain comprising at least one targeting domain, wherein the targeting domain is an antibody or antigen-binding fragment thereof to one or more target antigens. Antigen-binding fragments suitable for use in certain embodiments contemplated herein to one or more target antigens include antigen-binding fragments selected from the group consisting of: camel Ig, llama Ig, alpaca Ig, Ig NAR, Fab' fragment, F(ab')2 fragment, bispecific Fab dimer (Fab2), trispecific Fab trimer (Fab3), Fv, single-chain Fv protein ("scFv"), double scFv, (scFv)2, mini-antibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, disulfide-stabilized Fv proteins ("dsFv"), and single domain antibodies (e.g., sdAb, camel VHH or nanobodies).

[0232] 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 certain embodiments, the sdAb is a camel VHH, a nano antibody or a heavy chain antibody only (HcAb). In certain embodiments, the sdAb is a camel VHH. In certain embodiments, the scFv or the sdAb is human or humanized. In certain embodiments, the VHH is humanized.

[0233] The present disclosure provides a platform technology that can be applied to many types and fragments of targeting domains and should not be limited to, for example, one specific targeting domain, such as an antibody, antigen-binding fragment, extracellular domain, etc., for targeting a specific target antigen.

[0234] 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 component comprises an extracellular domain comprising a second targeting domain.

[0235] 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 certain embodiments, the sdAb is a camel VHH, a nano antibody or a heavy chain antibody only (HcAb). In certain embodiments, the sdAb is a camel VHH. In certain embodiments, the scFv or the sdAb is human or humanized. In certain embodiments, the VHH is humanized.

[0236] The extracellular domain contemplated in specific embodiments 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 peptide comprises a sequence having at least 90% identity with SEQ ID NO: 72. In one embodiment, the targeting domain peptide comprises a sequence having at least 95% identity with SEQ ID NO: 72. In one embodiment, the targeting domain peptide comprises a sequence having at least 96% identity with SEQ ID NO: 72. In one embodiment, the targeting domain peptide comprises a sequence having at least 97% identity with SEQ ID NO: 72. In one embodiment, the targeting domain peptide comprises a sequence having at least 98% identity with SEQ ID NO: 72. In one embodiment, the targeting domain peptide comprises a sequence that is at least 99% identical to SEQ ID NO: 72. In one embodiment, the targeting domain peptide comprises a sequence as set forth in SEQ ID NO:72.

[0237] 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 peptide comprises a sequence having at least 90% identity with SEQ ID NO:75. In one embodiment, the targeting domain peptide comprises a sequence having at least 95% identity with SEQ ID NO:75. In one embodiment, the targeting domain peptide comprises a sequence having at least 96% identity with SEQ ID NO:75. In one embodiment, the targeting domain peptide comprises a sequence having at least 97% identity with SEQ ID NO:75. In one embodiment, the targeting domain peptide comprises a sequence having at least 98% identity with SEQ ID NO:75. In one embodiment, the targeting domain peptide comprises a sequence having at least 99% identity with SEQ ID NO:75. In one embodiment, the targeting domain peptide comprises a sequence as shown in SEQ ID NO:75.

[0238] In one embodiment, the targeting component 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.

[0239] 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

[0240] The VHH comprises a CDR1 comprising SEQ ID NO:92, a CDR2 comprising SEQ ID NO:93, and a CDR3 comprising SEQ ID NO:94.

[0241] 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

[0242] The VHH comprises a CDR1 comprising SEQ ID NO:92, a CDR2 comprising SEQ ID NO:93, and a CDR3 comprising SEQ ID NO:94.

[0243] In various embodiments, the first targeting domain and the second targeting domain are separated by a third polypeptide linker having a length of 2 to 40 amino acids. 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.

[0244] In certain embodiments, the targeting component comprises one or more multimerization domains.

[0245] Illustrative examples of multimerization domains suitable for use in the specific targeting components contemplated herein include, but are not limited to, FK506 binding protein (FKBP) polypeptides or variants thereof, FKBP-rapamycin binding (FRB) polypeptides or variants thereof, calcineurin polypeptides or variants thereof, cyclophilin polypeptides or variants thereof, bacterial dihydrofolate reductase (DHFR) polypeptides or variants thereof, PYR1-like 1 (PYL1) polypeptides or variants thereof, and abscisic acid insensitive 1 (ABI1) polypeptides or variants thereof.

[0246] In various embodiments, the targeting component comprises a FRB polypeptide. In various embodiments, the targeting component comprises a FRB polypeptide or a variant thereof comprising a T2098L (T82L) mutation. In specific embodiments, the FRB polypeptide comprises the amino acid sequence as shown in SEQ ID NO: 1. In specific embodiments, the FRB polypeptide comprises the amino acid sequence as shown in SEQ ID NO: 2.

[0247] In various embodiments, the targeting component comprises a FKBP12 polypeptide or a variant thereof. In various embodiments, the targeting component comprises a FKBP12 polypeptide comprising a F36V mutation. In specific embodiments, the FKBP12 polypeptide comprises the amino acid sequence as shown in SEQ ID NO:3. In specific embodiments, the FKBP12 polypeptide comprises the amino acid sequence as shown in SEQ ID NO:4.

[0248] Other illustrative examples of multimerization domains suitable for use in the specific targeting components contemplated herein include antibody-derived heterodimerization domains. Such antibody heterodimerization domains include, but are not limited to, lock and key, DEKK, SEEDbody, DuoBody, dual variable domain immunoglobulin (DVD-Ig) and tandem Fab immunoglobulin (FIT-Ig) multimerization domains (see, e.g., Ma et al., Frontiers in Immunology (Front Immunol) . May 5, 2021; 12: 626616).

[0249] In various embodiments, the extracellular domain or the first targeting domain and the second multimerization domain are separated by a second polypeptide linker of 2 to 40 amino acids in length. In various embodiments, preferably a short oligopeptide linker or polypeptide linker of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids in length connects one or more domains in the targeting component. A linker based on glycine-serine provides a particularly suitable linker. In certain 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 certain embodiments, the second polypeptide linker is a G4S (GGGGS; SEQ ID NO: 11) linker. In some embodiments, the second polypeptide linker is a 2xG4S (GGGGSGGGGS; SEQ ID NO: 12) linker. In some embodiments, the second polypeptide linker is a 3xG4S (GGGGSGGGGSGGGGS; SEQ ID NO: 13) linker. In some embodiments, the second polypeptide linker is a 4xG4S (GGGGSGGGGSGGGGSGGGGS; SEQ ID NO: 14) linker. In some embodiments, the second polypeptide linker is a 5xG4S (GGGGSGGGGSGGGGSGGGGSGGGGS; SEQ ID NO: 15) linker. In some embodiments, the second polypeptide linker comprises an amino acid sequence as shown in any one or more of SEQ ID NOs: 16-31.

[0250] 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 that is at least 90% identical to SEQ ID NO: 41. In some embodiments, the CD4 hinge comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 41. In some embodiments, the CD4 hinge comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 41. In some embodiments, the CD4 hinge comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 41. In some embodiments, the CD4 hinge comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 41. In some embodiments, the CD4 hinge comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 41. In some embodiments, the CD4 hinge comprises an amino acid sequence as shown in SEQ ID NO: 41.

[0251] In some embodiments, the targeting component comprises a linker comprising a GGR amino acid sequence. In some embodiments, the targeting component comprises a linker comprising an amino acid sequence as shown in SEQ ID NO: 13 or SEQ ID NO: 14.

[0252] In certain embodiments, the targeting component comprises a transmembrane domain.

[0253] Illustrative examples of transmembrane domains suitable for use in the specific targeting components contemplated herein include, but are not limited to, the transmembrane regions of the α chain, β chain, γ chain, or δ chain 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, amniotic membrane protein (AMN), and programmed cell death 1 (PDCD1). In various embodiments, the targeting component comprises a CD4 transmembrane domain, a CD28 transmembrane domain, or a CD8α transmembrane domain.

[0254] In a specific embodiment, the targeting component comprises a CD4 transmembrane domain. In some embodiments, the CD4 transmembrane domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 45. In some embodiments, the CD4 transmembrane domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 45. In some embodiments, the CD4 transmembrane domain comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 45. In some embodiments, the CD4 transmembrane domain comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 45. In some embodiments, the CD4 transmembrane domain comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 45. In some embodiments, the CD4 transmembrane domain comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 45. In some embodiments, the CD4 transmembrane domain comprises an amino acid sequence as shown in SEQ ID NO: 45.

[0255] In certain embodiments, the targeting component 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 an amino acid sequence as 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 that is at least 95% identical to SEQ ID NO: 49. In one embodiment, the truncated CD4ic domain comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 49. In one embodiment, the truncated CD4ic domain comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 49. In one embodiment, the truncated CD4ic domain comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 49. In one embodiment, the truncated CD4ic domain comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 49. In various embodiments, the truncated CD4ic domain comprises an amino acid sequence as shown in SEQ ID NO: 49.

[0256] In certain embodiments, the targeting component does not comprise a primary signaling domain (e.g., CD3z). In various embodiments, the targeting component does not comprise an intracellular signaling domain or a co-stimulatory domain.

[0257] In various embodiments, the targeting component further comprises a truncated intracellular CD4 polypeptide. In some embodiments, the truncated intracellular CD4 polypeptide comprises the amino acid sequence as shown in SEQ ID NO: 48. In some embodiments, the truncated intracellular CD4 polypeptide comprises the amino acid sequence as shown in SEQ ID NO: 49.

[0258] In a preferred embodiment, the targeting component comprises an extracellular domain, the extracellular domain comprising a first targeting domain comprising a VHH or scFv that binds to CLL1, a linker polypeptide, a second targeting domain comprising a VHH or scFv that binds to CD33, a linker polypeptide, a FRB or FKBP12 multimerization domain, a CD4 hinge, a CD4 transmembrane domain, and a truncated CD4ic domain. In a preferred embodiment, the targeting component comprises an extracellular domain, the extracellular domain comprising a first targeting domain comprising a VHH or scFv that binds to CD33, a linker polypeptide, a second targeting domain comprising a VHH or scFv that binds to CLL1, a linker polypeptide, a FRB or FKBP12 multimerization domain, a CD4 hinge, a CD4 transmembrane domain, and a truncated CD4ic domain.

[0259] In one embodiment, the targeting component comprises a sequence that is at least 90% identical to SEQ ID NO: 122. In one embodiment, the targeting component comprises a sequence that is at least 95% identical to SEQ ID NO: 122. In one embodiment, the targeting component comprises a sequence that is at least 96% identical to SEQ ID NO: 122. In one embodiment, the targeting component comprises a sequence that is at least 97% identical to SEQ ID NO: 122. In one embodiment, the targeting component comprises a sequence that is at least 98% identical to SEQ ID NO: 122. In one embodiment, the targeting component comprises a sequence that is at least 99% identical to SEQ ID NO: 122. In one embodiment, the targeting component comprises a sequence as set forth in SEQ ID NO: 122.

[0260] In various embodiments, the targeting components contemplated herein comprise a signal peptide, such as a secretory signal polypeptide or sequence. Illustrative examples of signal polypeptides / sequences suitable for use in a particular targeting 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 α signal polypeptide.

[0261] In various preferred embodiments, the targeting component comprises an Igκ light chain signal polypeptide. In some embodiments, the Igκ light chain signal polypeptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 95. In some embodiments, the Igκ light chain signal polypeptide comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 95. In some embodiments, the Igκ light chain signal polypeptide comprises an amino acid sequence having at least 96% identity to SEQ ID NO: 95. In some embodiments, the Igκ light chain signal polypeptide comprises an amino acid sequence having at least 97% identity to SEQ ID NO: 95. In some embodiments, the Igκ light chain signal polypeptide comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 95. In some embodiments, the Igκ light chain signal polypeptide comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 95. In some embodiments, the Igκ light chain signal polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 95.

[0262] In certain embodiments, the targeting component comprises a CLL1 targeting domain, a 3xG4S linker, a CD33 targeting domain, a 3xG4S linker, a FKBP polypeptide or a variant thereof, a CD4 hinge domain, a CD4 transmembrane domain, and a truncated intracellular CD4 polypeptide. In certain embodiments, the targeting component comprises a signaling sequence, a CLL1 targeting domain, a 3xG4S linker, a CD33 targeting domain, a 3xG4S linker, a FKBP polypeptide or a variant thereof, a CD4 hinge domain, a CD4 transmembrane domain, and a truncated intracellular CD4 polypeptide. In certain embodiments, the targeting component comprises an IgK signaling sequence, a CLL1 VHH targeting domain, a 3xG4S linker, a CD33 targeting domain, a 3xG4S linker, a FKBP polypeptide or a variant thereof, a CD4 hinge domain, a CD4 transmembrane domain, and a truncated intracellular CD4 polypeptide. In certain embodiments, the targeting component comprises an IgK signaling sequence (e.g., as set forth in SEQ ID NO:95), a CLL1 VHH targeting domain (e.g., comprising CDR1 as set forth in SEQ ID NO:92, CDR2 as set forth in SEQ ID NO:93, and SEQ ID NO:94; and / or a VHH as set forth in SEQ ID NO:75), a 3xG4S linker (e.g., as set forth in SEQ ID NO:13), a CD33 VHH targeting domain (e.g., comprising CDR1 as set forth in SEQ ID NO:89, CDR2 as set forth in SEQ ID NO:90, CDR3 as set forth in SEQ ID NO:91; and / or a VHH comprising SEQ ID NO:72), a 3xG4S linker (e.g., as set forth in SEQ ID NO:13), a FKBP polypeptide or variant thereof (e.g., as set forth in SEQ ID NO:3), a CD4 hinge domain (e.g., as set forth in SEQ ID NO:41), a CD4 transmembrane domain (e.g., as set forth in SEQ ID NO:43), a CD4 polypeptide (e.g., as set forth in SEQ ID NO:44), a CD4 polypeptide (e.g., as set forth in SEQ ID NO:45), a CD4 polypeptide (e.g., as set forth in SEQ ID NO:46), a CD4 polypeptide (e.g., as set forth in SEQ ID NO:47), a CD4 polypeptide (e.g., as set forth in SEQ ID NO:48), a CD4 polypeptide (e.g., as set forth in SEQ ID NO:49), a CD4 polypeptide (e.g., as set forth in SEQ ID NO:50), a CD4 polypeptide (e.g., as set forth in SEQ ID NO:51), a CD4 polypeptide (e.g., as set forth in SEQ ID NO:52), NO:45) and a truncated intracellular CD4 polypeptide (e.g., as shown in SEQ ID NO:48 or SEQ ID NO:49).

[0263] 3. Bridging Factor

[0264] The bridging factors envisioned in the specific embodiments herein mediate or promote the association of one or more signaling components with one or more targeting components through the multimerization domains in the corresponding components. The bridging factor associates with the multimerization domain and is placed between the multimerization domains to promote the association of the signaling component with the targeting component. In the presence of the bridging factor, when the targeting component binds to the target antigen on the target cell, the targeting component and the signaling component bind to the immune receptor complex and initiate the immune effector cell activity against the target cell. In the absence of the bridging factor, the targeting component does not associate with the signaling component, does not recruit the immune receptor complex, and the engineered receptor has no activity against the target antigen of the targeting component.

[0265] In particular embodiments, the signaling component and the targeting component comprise a homologous multimerization domain pair 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).

[0266] In certain embodiments, the multimerization domains of the signaling component and the targeting component are associated with a bridging factor selected from the group consisting of rapamycin or a rapamycin analog thereof, kumarins or a derivative thereof, gibberellins or a derivative thereof, abscisic acid (ABA) or a derivative thereof, methotrexate or a derivative thereof, cyclosporine A or a derivative thereof, FK506 / cyclosporine A (FKCsA) or a derivative thereof, and a trimethoprim (Tmp) synthetic ligand (SLF) for FK506 binding protein (FKBP) or a derivative thereof.

[0267] In certain embodiments, the signaling component and the targeting component comprise one or more FRB and / or FKBP multimerization domains or variants thereof. In certain embodiments, the signaling component comprises a FKBP12 multimerization domain or a variant thereof, and the targeting component comprises a FRB multimerization domain or a variant thereof. In certain embodiments, the signaling component comprises a FRB multimerization domain or a variant thereof, and the targeting component comprises a FKBP12 multimerization domain or a variant thereof. In certain preferred embodiments, the signaling component comprises a FKBP12 or FKBP12 F36V multimerization domain or a variant thereof, and the targeting component comprises a FRBT2098L multimerization domain or a variant thereof. In certain preferred embodiments, the CD3 signaling component comprises a FRB T2098L (T82L) multimerization domain or a variant thereof, and the targeting component comprises a FKBP12 or FKBP12 F36V multimerization domain or a variant thereof.

[0268] Illustrative examples of bridging factors suitable for use in the specific embodiments contemplated herein include, but are not limited to, AP1903, AP20187, AP21967, (also known as C-16-(S)-7-methylindorapamycin), everolimus, norvolimus, pimecrolimus, dafoslimus, sirolimus, tacrolimus, temsirolimus, usololimus, zotarolimus, or BPC015. In certain preferred embodiments, the bridging factor is AP21967. In certain preferred embodiments, the bridging factor is a non-immunosuppressive dose of sirolimus (rapamycin).

[0269] D. Antigen receptors and binding molecules

[0270] In certain embodiments, the immune effector cells contemplated herein further express exogenous lymphocyte receptors or engineered antigen receptors and one or more components of engineered immune receptor complexes or systems as described herein. In some embodiments, exogenous lymphocyte receptors or engineered antigen receptors are chimeric antigen receptors (CAR), chimeric costimulatory receptors (CCR), T cell receptors (TCR), αβT cell receptors (αβ-TCR), γδT cell receptors (γδ-TCR), zetakine or flip receptors. In some embodiments, the immune effector cells contemplated herein further express exogenous costimulators, immunomodulators, agonists of costimulatory factors, antagonists of immunosuppressive factors, immune cell engagers or any combination thereof. In some embodiments, the immune effector cells contemplated herein further express BiTE.

[0271] In various embodiments, the exogenous lymphocyte receptor or engineered antigen receptor is an engineered αβ or γδT cell receptor (TCR), a chimeric antigen receptor (CAR) or a chimeric costimulatory receptor (CCR). In specific embodiments, the exogenous lymphocyte receptor is an engineered αβ or γδT cell receptor. In specific embodiments, the engineered antigen receptor is a chimeric antigen receptor (CAR). In specific embodiments, the engineered antigen receptor is a chimeric costimulatory receptor (CCR). In various embodiments, the immune effector cell contemplated herein comprises an engineered / exogenous TCR and an engineered immune receptor component as described herein. Without wishing to be bound by any particular theory, the signaling component of the engineered immune receptor components / systems contemplated herein comprises a multimerization domain fused to an actuator domain (e.g., CD3ε, CD3δ, CD3γ or Fcγ subunit) that associates with multimeric immune receptor complexes and thereby, in the presence of a bridging factor, recruits the immune receptor complexes by multimerization with a targeting component comprising a second multimerization domain.

[0272] In one embodiment, T cell engineering is performed 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 cell engineering is performed by introducing a polynucleotide or vector encoding an engineered antigen receptor and a polynucleotide or vector encoding one or more components of an immune receptor system. In one embodiment, T cells engineered to express engineered immune receptors are further engineered by introducing a polynucleotide or vector encoding one or more components of an immune receptor system.

[0273] Naturally occurring T cell receptors contain two subunits, an alpha chain and a beta chain subunit (αβTCR) or a gamma chain and a delta chain subunit (γδTCR), each of which is a unique protein produced by a recombination event in the genome of each T cell. TCR libraries can be screened for their selectivity for specific target antigens. In this way, natural TCRs with high affinity and responsiveness to the target antigen can be selected, cloned and subsequently introduced into a T cell population for adoptive immunotherapy. In one embodiment, TCR is an αβTCR. In one embodiment, TCR is a γδTCR.

[0274] In one embodiment, T cells are modified by introducing TCR subunits with the ability to form TCRs, and the TCRs confer specificity to T cells for cells expressing target antigens. In a specific embodiment, compared to naturally occurring subunits, the subunits have one or more amino acid substitutions, deletions, insertions or modifications, as long as the subunits can form TCRs and enable transfected T cells to home target cells and participate in immune-related cytokine signaling. Preferably, the engineered TCR also binds to target cells displaying related peptides (e.g., tumor-associated peptides) with high affinity, and optionally mediates efficient killing of target cells presenting related peptides in vivo.

[0275] The nucleic acid encoding the engineered TCR is preferably separated from its natural environment in the (naturally occurring) chromosome of T cells, and can be incorporated into a suitable vector (as described elsewhere herein). In a specific embodiment, the nucleic acid and the vector comprising the nucleic acid can be transferred to cells, preferably T cells. Then, the modified T cells can express one or more chains of the TCR encoded by one or more transduced nucleic acids. In a preferred embodiment, TCR is an exogenous TCR because it is introduced into T cells that do not express specific TCR under normal circumstances. In a specific embodiment, the basic aspect of TCR is that it has high affinity for antigens presented by major histocompatibility complex (MHC) or similar immune components. Compared with TCR, CAR is engineered to bind to the target antigen in an MHC-independent manner.

[0276] The TCR can be expressed with an additional polypeptide linked to the amino-terminal portion or carboxyl-terminal portion of the α chain or β chain of the TCR or the γ chain or δ chain of the TCR, as long as the additional polypeptide does not interfere with the ability of the α chain or β chain to form a functional T cell receptor and MHC-dependent antigen recognition.

[0277] Antigens recognized by the TCRs contemplated in specific embodiments include, but are not limited to, cancer antigens, including antigens on both hematological cancers and solid tumors. Exemplary antigens include, but are not limited to, alpha-fetoprotein (AFP), members of the B melanoma antigen (BAGE) family, brothers of regulatory factors at imprinted sites (BORIS), cancer-testis antigen, cancer-testis antigen 83 (CT-83), carbonic anhydrase IX (CA1X), carcinoembryonic antigen (CEA), cytomegalovirus (CMV) antigen, antigen recognized by cytotoxic T cells (CTL) on 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-RasG12V, 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), antigen preferentially expressed in melanoma (PRAME), survivin, synovial sarcoma X1 (SSX1), synovial sarcoma X 2 (SSX2), synovial sarcoma X 3 (SSX3), synovial sarcoma X 4 (SSX4), synovial sarcoma X 5 (SSX5), synovial sarcoma X 8 (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).

[0278] In other embodiments, the immune effector cells contemplated herein include CAR and engineered immune receptor components as described herein.Chimeric antigen receptor (CAR) is a molecule that combines the specificity of an antibody for a target antigen (eg, a tumor antigen) with a T cell receptor activating intracellular domain to produce a chimeric protein that exhibits specific anti-tumor cell immune activity.As used herein, the term "chimeric" describes being composed of different proteins or DNA portions from different sources.

[0279] In other embodiments, the immune effector cells contemplated herein include CCR and engineered immune receptor components as described herein. Unlike CAR, chimeric co-stimulatory receptors (CCRs) are molecules that combine antibody-based specificity for a desired antigen with T cell receptor co-stimulatory domains but lack a primary signaling domain (see, e.g., WO2020 / 252110 and WO2021 / 211948).

[0280] In other embodiments, the immune effector cells contemplated herein express bispecific T cell engagers (BiTEs) and engineered immune receptor components as described herein. BiTEs are molecules comprising two different antibody fragments (e.g., single-chain variable fragments (scFv)) connected by a small linker peptide, wherein one antibody fragment binds to a component (e.g., CD3 chain) of a T cell receptor (TCR) complex and the other antibody fragment binds to another antigen (e.g., a tumor-associated antigen).

[0281] In other embodiments, the immune effector cells contemplated herein comprise flipped receptors and engineered immune receptor components as described herein. Flipped receptors comprise the extracellular portion of a receptor, a transmembrane domain, and the intracellular signaling portion of a different receptor. Thus, flipped receptors "convert" the binding properties of one receptor type into an intracellular signaling event of another receptor type. See, for example, WO2018 / 094244, WO2016 / 122738, WO2014 / 172584, and WO2012 / 138858.

[0282] In certain embodiments, 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), alpha 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 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), EGFR family including ErbB2 (HER2), IGF2BP3 / A3, IL-10Rα, IL-13Rα2, κ, cancer / testis antigen 2 (LAGE-1A), K-Ras, K-Ras G12C, K-RasG12D, K-Ras G12V, λ, 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-associated protein A (MICA), MHCClass I chain-associated 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, breakpoint X 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).

[0283] In specific 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.

[0284] In various embodiments, the immune effector cells contemplated herein comprise one or more chains of a zetakine receptor and an engineered immune receptor component as described herein. Zetakine is a chimeric transmembrane immune receptor comprising an extracellular domain, a transmembrane region, and an intracellular signaling domain, wherein the extracellular domain comprises a soluble receptor ligand connected to a carrier region capable of tethering the extracellular domain to the cell surface. When expressed on the surface of T lymphocytes, zetakine directs T cell activity to those cells expressing receptors for which the soluble receptor ligand is specific. Zetakine chimeric immune receptors redirect the antigen specificity of T cells, especially in the treatment of various cancers via the autocrine / paracrine cytokine system utilized by human malignancies.

[0285] In other embodiments, immune effector cells are modified by introducing Fc receptor (FcR) subunits with the ability to bind to the fragment crystallizable (Fc) portion / region of an antibody and engineered immune receptor components as described herein. In some embodiments, the FcR subunits may be from Fc-γ receptors, Fc-α receptors, or Fc-ε receptors. See, e.g., Mkaddem et al., Frontiers in Immunology. 2019 Apr 12; 10:811.

[0286] In other embodiments, immune effector cells are modified by introducing NKG2D receptors and engineered immune receptor components as described herein.

[0287] E. Peptide

[0288] Various polypeptides are contemplated herein, including but not limited to engineered immune receptor systems, targeting components, signaling components, immune receptors and their associated subunits or adapter molecules, exogenous lymphocyte receptors, engineered antigen receptors, TCR, FcR, CAR, CCR, BiTE, zetakine, flip receptors, exogenous co-stimulatory factors, immunomodulatory factors, agonists of co-stimulatory factors, antagonists of immunosuppressive factors, immune cell engagers, or any combination thereof. Fusion proteins comprising the aforementioned polypeptides and fragments thereof are also contemplated herein. In a preferred embodiment, the polypeptide comprises an amino acid sequence shown in any one of SEQ ID NO: 107-115, 116-138, or 139-170.

[0289] Unless otherwise indicated, "polypeptide", "peptide" and "protein" are used interchangeably and are defined according to the conventional meaning, i.e., as an amino acid sequence. In one embodiment, "polypeptide" includes fusion polypeptides and other variants. The polypeptides can be prepared using any of a variety of well-known recombinant and / or synthetic techniques. The polypeptide is not limited to a specific length, for example, it can include a full-length protein sequence, a fragment of a full-length protein, or a fusion protein, and can include post-translational modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, etc., as well as other modifications known in the art that occur naturally and non-naturally. In certain preferred embodiments, fusion polypeptides, polypeptides, fragments thereof and other variants are prepared, obtained or isolated from one or more human polypeptides.

[0290] As used herein, "isolated peptide" or "isolated polypeptide" and the like refer to a peptide or polypeptide molecule that is isolated and / or purified in vitro from the cellular environment and from association with other components of the cell, i.e., the peptide or polypeptide molecule is not significantly associated with in vivo substances. In specific embodiments, the isolated polypeptide is a synthetic polypeptide, a semisynthetic polypeptide, or a polypeptide obtained or derived from a recombinant source.

[0291] 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 naturally occurring or may be generated synthetically, e.g., 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 a polypeptide by introducing one or more substitutions, deletions, additions and / or insertions into the polypeptide. In specific embodiments, polypeptides include polypeptides 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 to any of the reference sequences contemplated herein, typically wherein the variant maintains at least one biological activity of the reference sequence. In specific embodiments, the biological activity is binding affinity. In specific embodiments, the biological activity is enzymatic activity.

[0292] In certain embodiments, the engineered immune receptor components as described herein recruit multimeric immune receptor complexes, the multimeric immune receptor complexes 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 specific embodiments, the multimerization domains are identical; in certain embodiments, the first multimerization domain is different from the second multimerization domain. The first multimerization domain and the second multimerization domain significantly contribute to or efficiently promote the formation of a polypeptide complex (e.g., in the presence of a bridging factor). In the absence of the first multimerization domain, the second multimerization domain, or the bridging factor, if the association between the first fusion polypeptide and the second fusion polypeptide is statistically significantly reduced, then one or more interactions between the first multimerization domain and the second multimerization domain substantially contribute to or efficiently promote the multimerization of the first fusion polypeptide and the second fusion polypeptide. In certain embodiments, when the first fusion polypeptide and the second fusion polypeptide are co-expressed in the presence of a bridging factor, at least about 60%, for example, 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 single-chain polypeptide and the second single-chain polypeptide form multimers with each other.

[0293] Polypeptide variants include biologically active "polypeptide fragments". Illustrative examples of biologically active polypeptide fragments include binding domains, intracellular signaling domains, etc. 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 activity of a naturally occurring polypeptide. In certain embodiments, a polypeptide fragment may include an amino acid chain of at least 5 to about 1700 amino acids in length. It should be understood that in certain embodiments, the fragment length is 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, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700 or more amino acids.

[0294] In certain embodiments, the polypeptides described herein may include one or more amino acids denoted as "X". "X" if 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 includes a sequence of consecutive X residues that cumulatively represent any amino acid sequence.

[0295] As described above, polypeptides can be altered in a variety of ways, including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are well known in the art. For example, amino acid sequence variants of reference polypeptides can be prepared by mutations of DNA. Methods for mutagenesis and nucleotide sequence changes are well 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. Pat. No. 4,873,192; Watson, JD et al., Molecular Biology of the Gene, 4th ed., Benjamin / Cummings, Menlo Park, Calif., 1987) and references cited herein. Guidance on appropriate amino acid substitutions that do not affect the biological activity of a protein of interest can be found in Dayhoff et al., (1978), Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC).

[0296] In certain embodiments, polypeptide variants include one or more conservative substitutions." conservative substitutions "are substitutions in which amino acids are replaced by another amino acid with similar properties so that the secondary structure and hydrophilicity of the polypeptide are substantially unchanged. Modifications can be made to the structure of the polynucleotides and polypeptides envisioned in a particular embodiment and still obtain functional molecules encoding variants or derivative polypeptides with desired properties. When it is desired to change the amino acid sequence of a polypeptide to produce an equivalent or even improved variant polypeptide, those skilled in the art can, for example, change one or more of the codons of the coding DNA sequence according to Table 1.

[0297] Table 1 - Amino acid codons

[0298]

[0299]

[0300] Computer programs such as DNASTAR, DNAStrider, G, Mac Vector or Vector NTI software known in the art can be used to find guidance on determining which amino acid residues can be substituted, inserted or deleted without eliminating biological activity. Preferably, the amino acid changes of the protein variants disclosed herein are conservative amino acid changes, that is, the replacement of similarly charged or uncharged amino acids. Conservative amino acid changes involve the replacement of an amino acid in a family of amino acids related in terms of 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), non-polar amino acids (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) and polar uncharged amino acids (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan and tyrosine are sometimes collectively classified as aromatic amino acids. In peptides or proteins, suitable conservative amino acid substitutions are known to those skilled in the art and can generally be carried out without changing the biological activity of the resulting molecule. Those skilled in the art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide will not substantially alter the biological activity (see, e.g., Watson et al., Molecular Biology of the Gene, 4th ed., 1987, The Benjamin / Cummings Pub. Co., p. 224).

[0301] In one embodiment, when expression of two or more polypeptides is desired, the polynucleotide sequences encoding the two or more polypeptides may be separated by an IRES sequence.

[0302] The polypeptides contemplated in particular embodiments include fusion polypeptides. In particular embodiments, fusion polypeptides and polynucleotides encoding fusion polypeptides are provided. Fusion polypeptides and fusion proteins refer to polypeptides having at least two, three, four, five, six, seven, eight, nine or ten or more polypeptide fragments. 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.

[0303] In certain embodiments, two or more engineered immune receptor components and / or other polypeptides can be expressed as a fusion protein comprising one or more self-cleaving peptide sequences between the polypeptides as disclosed elsewhere herein.

[0304] In certain embodiments, the fusion polypeptide comprises a targeting component and one or more signaling components.

[0305] A fusion polypeptide may comprise: one or more polypeptide domains or fragments, including but not limited to a signal peptide, a cell permeating peptide domain (CPP), a binding domain, a signaling domain, an epitope tag (e.g., maltose binding protein ("MBP"), glutathione S transferase (GST), HIS6, MYC, FLAG, V5, VSV-G, and HA); a polypeptide linker; and a polypeptide cleavage signal. A fusion polypeptide is typically C-terminally linked to an N-terminus, but it may also be C-terminally linked to a C-terminus, N-terminally linked to an N-terminus, or N-terminally linked to a C-terminus. In a particular embodiment, the polypeptides of the fusion protein may be in any order. A fusion polypeptide or fusion protein may also comprise conservatively modified variants, polymorphic variants, alleles, mutants, subsequences, and interspecies homologs, as long as the desired activity of the fusion polypeptide is maintained. A fusion polypeptide may be produced by chemical synthesis or by chemical linkage between two parts or may generally be prepared using other standard techniques. The linked DNA sequence comprising the fusion polypeptide is operably linked to a suitable transcriptional or translational control element as disclosed elsewhere herein.

[0306] Fusion polypeptides may optionally include one or more joints that can be used to connect one or more polypeptides or domains within a polypeptide. Peptide joint sequences can be used to separate any two or more polypeptide components by a sufficient distance to ensure that each polypeptide folds into its appropriate secondary structure and tertiary structure so that the polypeptide domains can perform their desired functions. Such peptide joint sequences are incorporated into fusion polypeptides using standard techniques in the art. Suitable peptide joint sequences can be selected based on the following factors: (1) they can adopt a flexible extended conformation; (2) they cannot adopt a secondary structure that can interact with functional epitopes on signal transduction components and targeting components; and (3) lack hydrophobic residues or charged residues that can react with functional epitopes of polypeptides. In a particular embodiment, preferred peptide joint sequences contain Gly, Asn, and Ser residues. Other near-neutral amino acids, such as Thr and Ala, can also be used in the joint sequence. Amino acid sequences that can be effectively used 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. When a particular fusion polypeptide fragment comprises a non-essential N-terminal amino acid region that can be used to separate the functional domains and prevent spatial interference, a linker sequence is not required. In a particular embodiment, a preferred linker is typically a flexible amino acid subsequence that is synthesized as part of a recombinant fusion protein. The linker polypeptide can be between 1 and 200 amino acids in length, between 1 and 100 amino acids in length, or between 1 and 50 amino acids in length, including all integer values ​​therebetween.

[0307] 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).

[0308] Suitable protease cleavage sites and self-cleaving peptides are known to the skilled person (see for example 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, the following cleavage sites: potato virus Y NIa protease (e.g., tobacco etch virus protease), potato virus Y HC protease, potato virus Y P1 (P35) protease, byo virus NIa protease, byo virus RNA-2 encoded protease, foot-and-mouth disease virus L protease, enterovirus 2A protease, rhinovirus 2A protease, small RNA 3C protease, cowpea mosaic virus 24K protease, nematode-transmitted polyhedron virus 24K protease, RTSV (rice tungro spherical virus) 3C-like protease, PYVF (European parsnip yellow spot virus) 3C-like protease, heparin, thrombin, factor Xa and enterokinase. Due to its high cleavage stringency, in one embodiment TEV (Tobacco Etch Virus) protease cleavage sites are preferred, such as EXXYXQ (G / S) (SEQ ID NO: 195), such as 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).

[0309] In particular embodiments, the polypeptide cleavage signal is a viral self-cleavage peptide or a ribosomal skipping sequence.

[0310] Illustrative examples of ribosomal skipping sequences include, but are not limited to, 2A or 2A-like sites, sequences or domains (Donnelly et al., 2001. Journal of General Virology 82: 1027-1041). In a specific embodiment, the viral 2A peptide is a foot-and-mouth disease virus 2A peptide, a potato virus Y 2A peptide or a cardiovirus 2A peptide.

[0311] In one embodiment, the viral 2A peptide is selected from the group consisting of: foot-and-mouth disease virus (FMDV) 2A peptide, equine rhinitis virus type A (ERAV) 2A peptide, bright veined moth virus (TaV) 2A peptide, porcine teschovirus-1 (PTV-1) 2A peptide, Theiler virus 2A peptide and encephalomyocarditis virus 2A peptide.

[0312] Illustrative examples of 2A sites are provided in Table 2.

[0313] Table 2:

[0314] SEQ ID NO:171 GSGATNFSLLKQAGDVEENPGP SEQ ID NO:172 ATNFSLLKQAGDVEENPGP SEQ ID NO:173 LLKQAGDVEENPGP SEQ ID NO:174 GSGEGRGSLLTCGDVEENPGP SEQ ID NO:175 EGRGSLLTCGDVEENPGP SEQ ID NO:176 LLTCGDVEENPGP SEQ ID NO:177 GSGQCTNYALLKLAGDVESNPGP SEQ ID NO:178 QCTNYALLKLAGDVESNPGP SEQ ID NO:179 LLKLAGDVESNPGP SEQ ID NO:180 GSGVKQTLNFDLLKLAGDVESNPGP SEQ ID NO:181 VKQTLNFDLLKLAGDVESNPGP SEQ ID NO:182 LLKLAGDVESNPGP SEQ ID NO:183 LLNFDLLKLAGDVESNPGP SEQ ID NO:184 TLNFDLLKLAGDVESNPGP SEQ ID NO:185 LLKLAGDVESNPGP SEQ ID NO:186 NFDLLKLAGDVESNPGP SEQ ID NO:187 QLLNFDLLKLAGDVESNPGP SEQ ID NO:188 APVKQTLNFDLLKLAGDVESNPGP SEQ ID NO:189 VTELLYRMKRAETYCPRPLLAIHPTEARHKQKIVAPVKQT SEQ ID NO:190 LNFDLLKLAGDVESNPGP SEQ ID NO:191 LLAIHPTEARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP SEQ ID NO:192 EARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP

[0315] In certain embodiments, the self-cleaving polypeptide (e.g., 2A peptide) comprises a SGSG (SEQ ID NO: 193) spacer sequence. In certain embodiments, the self-cleaving polypeptide (e.g., 2A peptide) comprises a furin recognition site, such as a RAKR (SEQ ID NO: 194) spacer sequence.

[0316] In preferred embodiments, the polypeptide or fusion polypeptide comprises one or more engineered immune receptors or components. In preferred embodiments, the fusion polypeptide comprises one or more engineered immune receptors or components separated by one or more self-cleaving polypeptides.

[0317] In specific embodiments, the fusion polypeptide comprises a signaling component comprising a first multimerization domain and an actuator domain (e.g., a CD3ε, CD3δ, or CD3γ domain) that forms a complex with and recruits an immune receptor complex; a viral self-cleaving 2A polypeptide; and a targeting component comprising 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, and a transmembrane domain.

[0318] In specific embodiments, the fusion polypeptide comprises a signaling component comprising a first multimerization domain and an actuator domain (e.g., a CD3ε, CD3δ, or CD3γ domain) that forms a complex with and recruits an immune receptor complex; a viral self-cleaving 2A polypeptide; and a targeting component comprising 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 domain, and a CD4 transmembrane domain.

[0319] In specific embodiments, the fusion polypeptide comprises a signaling component comprising a first multimerization domain and an actuator domain (e.g., a CD3ε, CD3δ, or CD3γ domain) that forms a complex with an immune receptor complex and recruits the immune receptor complex; a viral self-cleaving 2A polypeptide; and a targeting component comprising 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 domain, a CD4 transmembrane domain, and a truncated CD4ic domain.

[0320] In a specific embodiment, the fusion polypeptide comprises a signaling component comprising a FRB or FKBP12 multimerization domain and an actuator domain (e.g., a CD3ε, CD3δ, or CD3γ domain) that forms a complex with an immune receptor complex and recruits the immune receptor complex; a viral self-cleaving 2A polypeptide; and a targeting component comprising an extracellular domain (comprising a VHH or scFv that binds to a target antigen), a FRB or FKBP12 multimerization domain, a CD4 hinge, a CD4 transmembrane domain, and a truncated CD4ic domain.

[0321] In a specific embodiment, the fusion polypeptide comprises a signaling component comprising an antibody-derived heterodimerization domain, a multimerization domain and an actuator domain (e.g., a CD3ε, CD3δ, or CD3γ domain) that forms a complex with an immune receptor complex and recruits the immune receptor complex; a viral self-cleaving 2A polypeptide; and a targeting component comprising a VHH or scFv that binds to a target antigen, an antibody-derived heterodimerization domain, a CD4 hinge, a CD4 transmembrane domain, a truncated CD4ic domain, and optionally a co-stimulatory or cytokine receptor intracellular signaling domain.

[0322] In specific embodiments, the fusion polypeptide comprises the amino acid sequence as set forth in any one of SEQ ID NOs: 139-170.

[0323] In particular embodiments, the fusion polypeptide comprises a signaling component comprising a first multimerization domain and an actuator domain (e.g., an Fcγ domain / polypeptide) that forms a complex with and recruits an Fc receptor complex; a viral self-cleaving 2A polypeptide; and a targeting component comprising 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.

[0324] In particular embodiments, the fusion polypeptide comprises a signaling component comprising a first multimerization domain and an actuator domain (e.g., an Fcγ domain / polypeptide) that forms a complex with and recruits an Fc receptor complex; a viral self-cleaving 2A polypeptide; and a targeting component comprising 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.

[0325] In specific embodiments, the fusion polypeptide comprises a signaling component comprising a first multimerization domain and an actuator domain (e.g., an Fcγ domain / polypeptide) that forms a complex with and recruits an Fc receptor complex; a viral self-cleaving 2A polypeptide; and a targeting component comprising 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 truncated CD4ic domain.

[0326] In a specific embodiment, the fusion polypeptide comprises a signaling component comprising a FRB or FKBP12 multimerization domain and an actuator domain (e.g., an Fcγ domain / polypeptide) that forms a complex with an Fc receptor complex and recruits the Fc receptor complex; a viral self-cleaving 2A polypeptide; and a targeting component comprising an extracellular domain (comprising a VHH or scFv that binds to a target antigen), a FRB or FKBP12 multimerization domain, a CD4 hinge, a CD4 transmembrane domain, and a truncated CD4ic domain.

[0327] In a specific embodiment, the fusion polypeptide comprises a signaling component comprising an antibody-derived heterodimerization domain and an actuator domain (e.g., an Fcγ domain / polypeptide) that forms a complex with an Fc receptor complex and recruits the Fc receptor complex; a viral self-cleaving 2A polypeptide; and a targeting component 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 truncated CD4ic domain, and optionally a co-stimulatory or cytokine receptor intracellular signaling domain.

[0328] In specific embodiments, the fusion polypeptide comprises the amino acid sequence as set forth in any one of SEQ ID NOs: 139-170.

[0329] In one embodiment, the fusion polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 151. In one embodiment, the fusion polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 151. In one embodiment, the fusion polypeptide comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 151. In one embodiment, the fusion polypeptide comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 151. In one embodiment, the fusion polypeptide comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 151. In one embodiment, the fusion polypeptide comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 151. In one embodiment, the fusion polypeptide comprises the amino acid sequence as set forth in SEQ ID NO: 151.

[0330] In one embodiment, the fusion polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 137. In one embodiment, the fusion polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 137. In one embodiment, the fusion polypeptide comprises a sequence that is at least 96% identical to SEQ ID NO: 137. In one embodiment, the fusion polypeptide comprises a sequence that is at least 97% identical to SEQ ID NO: 137. In one embodiment, the fusion polypeptide comprises a sequence that is at least 98% identical to SEQ ID NO: 137. In one embodiment, the fusion polypeptide comprises a sequence that is at least 99% identical to SEQ ID NO: 137. In one embodiment, the fusion polypeptide comprises a sequence as set forth in SEQ ID NO: 137.

[0331] In one embodiment, the fusion polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 138. In one embodiment, the fusion polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 138. In one embodiment, the fusion polypeptide comprises a sequence that is at least 96% identical to SEQ ID NO: 138. In one embodiment, the fusion polypeptide comprises a sequence that is at least 97% identical to SEQ ID NO: 138. In one embodiment, the fusion polypeptide comprises a sequence that is at least 98% identical to SEQ ID NO: 138. In one embodiment, the fusion polypeptide comprises a sequence that is at least 99% identical to SEQ ID NO: 138. In one embodiment, the fusion polypeptide comprises a sequence as set forth in SEQ ID NO: 138.

[0332] F. Polynucleotides

[0333] In specific embodiments, polynucleotides encoding an engineered immune receptor system, one or more engineered immune receptor components, signaling components, targeting components, exogenous lymphocyte receptors, engineered TCRs, CARs, CCRs, BiTEs, zetakine, flip receptors, exogenous co-stimulatory factors, immunomodulatory factors, agonists of co-stimulatory factors, antagonists of immunosuppressive factors, immune cell engagers, fusion proteins comprising the aforementioned polypeptides, and fragments thereof.

[0334] As used herein, the term "polynucleotide" or "nucleic acid" refers to deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and DNA / RNA hybrids. Polynucleotides can be single-stranded or double-stranded and are recombinant, synthetic or isolated. Polynucleotides include, but are not limited to: pre-mRNA, messenger RNA (mRNA), RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, 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. Polynucleotides refer to polymeric forms 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, and all intermediate lengths, either ribonucleotides or deoxyribonucleotides or modified forms of either type of nucleotide. It will be readily appreciated that in this context, "intermediate lengths" means any length between the recited values, such as 6, 7, 8, 9, etc.; 101, 102, 103, etc.; 151, 152, 153, etc.; 201, 202, 203, etc. In specific embodiments, the 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.

[0335] As used herein, "isolated polynucleotides" refers to polynucleotides that have been purified from sequences on both sides that are in a naturally occurring state, for example, a DNA fragment that has been removed from sequences that are usually adjacent to the fragment. In specific embodiments, "isolated polynucleotides" refers to complementary DNA (cDNA), recombinant DNA, or other polynucleotides that do not exist in nature but have been made by human hands. In specific embodiments, isolated polynucleotides are synthetic polynucleotides, semisynthetic polynucleotides, or polynucleotides obtained or derived from recombinant sources.

[0336] In various embodiments, the polynucleotide comprises an mRNA encoding a polypeptide contemplated herein. In certain embodiments, the mRNA comprises a cap, one or more nucleotides, and a poly(A) tail.

[0337] In certain embodiments, the polynucleotides described herein, including polynucleotides encoding one or more engineered immune receptor components, can be codon optimized. As used herein, the term "codon optimized" refers to replacing codons in a polynucleotide encoding a polypeptide to increase the expression, stability and / or activity of the polypeptide. Factors affecting codon optimization include, but are not limited to, one or more of the following: (i) changes in codon bias or synthetically constructed bias tables between two or more organisms or genes; (ii) changes in codon bias within an organism, gene or genome; (iii) systematic changes in codons including background; (iv) changes in codons according to their decoding tRNA; (v) changes in codons according to GC% in general or at one position in a triplet; (vi) changes in similarity to a reference sequence, such as a naturally occurring sequence; (vii) changes in codon frequency cutoffs; (viii) structural properties of mRNA transcribed from a DNA sequence; (ix) prior knowledge of the function of the DNA sequence on which the codon substitution set is designed; (x) systematic changes in the codon set for each amino acid; and / or (xi) isolated removal of spurious translation start sites.

[0338] As used herein, the term "nucleotide" refers to a heterocyclic nitrogenous base that is N-glycosidically linked to a phosphorylated sugar. Nucleotides are understood to include natural bases and a variety of modified bases recognized in the art. Such bases are generally located at the 1' position of the nucleotide sugar portion. Nucleotides generally include 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., a sugar lacking the hydroxyl group present in ribose.

[0339] Illustrative examples of polynucleotides include, but are not limited to, polynucleotides encoding the polypeptides shown in SEQ ID NOs: 107-170.

[0340] In various exemplary embodiments, the 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, and expression vectors, viral vectors, and transfer plasmids comprising the polynucleotides contemplated herein.

[0341] As used herein, the terms "polynucleotide variant" and "variant" and the like refer to polynucleotides that exhibit substantial sequence identity to a reference polynucleotide sequence or a polynucleotide that hybridizes to a reference sequence under stringent conditions as defined below. 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" encompass polynucleotides in which one or more nucleotides have been added or deleted, or modified or replaced with a different nucleotide. In this regard, it is well understood in the art that certain changes, including mutations, additions, deletions, and substitutions, may be made to a reference polynucleotide, whereby the altered polynucleotide retains the biological function or activity of the reference polynucleotide.

[0342] As used herein, the statement "sequence identity" or, for example, including "a sequence that is 50% identical to ... " refers to the degree to which the sequences are identical on a nucleotide-by-nucleotide basis or on an amino acid-by-amino acid basis within a comparison window. Therefore, the "percentage of sequence identity" can be calculated in the following manner: comparing two optimally aligned sequences within the comparison window, determining the number of positions where consistent nucleic acid bases (e.g., A, T, C, G, I) or consistent 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) appear in the two sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., window size) and multiplying the result by 100 to obtain the percentage of sequence identity. %, 93%, 94%, 95%, 86%, 97%, 98%, or 99% sequence identity to any of the reference sequences described herein.

[0343] As used herein, the term "nucleic acid cassette" or "expression cassette" refers to a gene sequence in a vector that can express RNA and subsequently express a polypeptide. In one embodiment, the nucleic acid cassette contains a gene of interest, e.g., a polynucleotide of interest. In another embodiment, the nucleic acid cassette contains 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. The vector may include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleic acid cassettes. The nucleic acid cassette is positioned and sequentially oriented within the vector so that the nucleic acid in the cassette can be transcribed into RNA and translated into a protein or polypeptide when necessary, undergoes appropriate post-translational modifications required for activity in a transformed cell, and is translocated to a suitable compartment by targeting a suitable intracellular compartment to an extracellular compartment or secreting it to an extracellular compartment to have biological activity. Preferably, the cassette has its 3' and 5' ends adapted to be ready for insertion into a vector, e.g., it has a restriction endonuclease site at each end. The cassette can be removed and inserted into a plasmid or viral vector as a single unit.

[0344] The polynucleotides include one or more polynucleotides of interest. As used herein, the term "polynucleotide of interest" refers to a polynucleotide that encodes a polypeptide or fusion polypeptide or a polynucleotide that serves as a template for a transcriptional inhibitory polynucleotide, as contemplated herein.

[0345] As disclosed elsewhere herein or as known in the art, regardless of the length of the coding sequence itself, the polynucleotides contemplated herein can 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 sites, FRT sites, and Att sites), stop codons, transcription termination signals, and polynucleotides encoding self-cleaving polypeptides, epitope tags, such that the overall length of the polynucleotides can vary significantly. Thus, it is contemplated that polynucleotide fragments of virtually any length can be employed, with the overall length preferably being limited by ease of preparation and use in the intended recombinant DNA protocol.

[0346] Any of various mature techniques known and available in the art can be used to prepare, manipulate, express and / or deliver polynucleotides. In order to express the desired polypeptide, the nucleotide sequence encoding the polypeptide can be inserted into an appropriate vector.

[0347] Illustrative examples of vectors include, but are not limited to, plasmids, autonomously replicating sequences, and transposable elements, such as Sleeping Beauty, PiggyBac.

[0348] 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.

[0349] Illustrative examples of viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40).

[0350] Illustrative examples of expression vectors include, but are not limited to, pClneo vector (Promega) for expression in mammalian cells; pLenti4 / V5-DEST for lentivirus-mediated gene transfer and expression in mammalian cells; TM 、pLenti6 / V5-DEST TM and pLenti6.2 / V5-GW / lacZ (Invitrogen). In certain embodiments, the coding sequence of the polypeptide disclosed herein can be ligated into such expression vectors for expressing the polypeptide in mammalian cells.

[0351] In a specific embodiment, the vector is an episomal vector or a vector maintained outside the chromosome. As used herein, the term "episomal" refers to a vector that can replicate without being integrated into the chromosomal DNA of the host and will not be gradually lost from the host cell of division, which also means that the vector replicates outside the chromosome or in a free manner.

[0352] "Expression control sequences," "control elements," or "regulatory sequences" present in an expression vector are those non-translated regions of the vector, including origin of replication, selection cassette, promoter, enhancer, translation initiation signal (Shine Dalgarno sequence or Kozak sequence), introns, polyadenylation sequence, 5' and 3' non-translated regions, which interact with host cell proteins for transcription and translation. The strength and specificity of such elements may vary. Depending on the vector system and host utilized, any number of suitable transcription and translation elements may be used, including ubiquitous promoters and inducible promoters.

[0353] In a particular embodiment, the polynucleotide includes a vector, and the vector includes but is not limited to an expression vector and a viral vector. The vector may include one or more exogenous, endogenous or heterologous control sequences, such as promoters and / or enhancers. An "endogenous control sequence" is a sequence naturally connected to a given gene in a genome. An "exogenous" control sequence is a control sequence that is placed in juxtaposition with a gene by genetic manipulation (i.e., molecular biotechnology) so that the transcription of the gene is guided by the connected enhancer / promoter. A "heterologous control sequence" is an exogenous sequence from a different species from a cell manipulated by the gene. A "synthetic" control sequence may include one or more endogenous and / or exogenous sequences and / or elements of a sequence that provides the best promoter and / or enhancer activity for a particular therapy as determined in vitro or in a computer.

[0354] As used herein, the term "promoter" refers to a recognition site for a polynucleotide (DNA or RNA) to which RNA polymerase binds. RNA polymerase initiates and transcribes a polynucleotide operably linked to a promoter. In a specific embodiment, a promoter operable in a mammalian cell includes an AT-rich region located approximately 25 to 30 bases upstream of the transcription start site and / or another sequence found 70 to 80 bases upstream of the transcription start site: a CNCAAT region, wherein N can be any nucleotide.

[0355] The term "enhancer" refers to a DNA segment containing a sequence that can provide enhanced transcription and in some cases can function independently of its orientation relative to another control sequence. An enhancer can function synergistically or additively with a promoter and / or another enhancer element. The term "promoter / enhancer" refers to a DNA segment containing a sequence that can provide both promoter and enhancer functions.

[0356] The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship that permits the components to function in their intended manner. In one embodiment, the term refers to a functional connection between a nucleic acid expression control sequence (e.g., a promoter and / or enhancer) and a second polynucleotide sequence, e.g., a polynucleotide of interest, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.

[0357] As used herein, the term "constitutive expression control sequence" refers to a promoter, enhancer, or promoter / enhancer that continuously or continually permits transcription of an operably linked sequence. A constitutive expression control sequence can be a "ubiquitous" promoter, enhancer, or promoter / enhancer that permits expression in a wide variety of cell and tissue types or a "cell-specific," "cell type-specific," "cell lineage-specific," or "tissue-specific" promoter, enhancer, or promoter / enhancer that permits expression in a restricted variety of cell and tissue types, respectively.

[0358] Exemplary ubiquitous expression control sequences suitable for use in specific embodiments include, but are not limited to, cytomegalovirus (CMV) immediate early promoter, 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, H5, P7.5 promoter and P11 promoter from vaccinia virus, elongation factor 1-alpha (EF1a) promoter, early growth response 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 ROSA26 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 substituted (MND) U3 promoter (Haas et al., Journal of Virology. 2003; 77(17): 9439-9450).

[0359] In one embodiment, the vector includes the MNDU3 promoter.

[0360] In one embodiment, the vector comprises an EF1a promoter comprising the first intron of the human EF1a gene.

[0361] In one embodiment, the vector includes the EF1a promoter lacking the first intron of the human EF1a gene.

[0362] In a particular embodiment, it may be desirable to use cell-, cell type-, cell lineage-, or tissue-specific expression control sequences to achieve cell type-specific, lineage-specific, or tissue-specific expression of a desired polynucleotide sequence (e.g., to express a particular nucleic acid encoding a polypeptide in only a subset of cell types, cell lineages, or tissues, or during a specific developmental stage).

[0363] In a particular embodiment, it may be desirable to express the polynucleotide under a T cell-specific promoter.

[0364] 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 having a specific physiological, biological or disease state, etc. This definition is not intended to exclude cell type or tissue specific expression. Certain embodiments provide conditional expression of a polynucleotide of interest, for example, controlling expression by subjecting a cell, tissue, organism, etc., to a treatment or condition that results in expression of the polynucleotide or results in increased or decreased expression of a polynucleotide encoded by the polynucleotide of interest.

[0365] Illustrative examples of inducible promoters / systems include, but are not limited to, steroid-inducible promoters, such as promoters of genes encoding glucocorticoid or estrogen receptors (inducible by treatment with the corresponding hormones), thiol metal binding protein promoters (inducible by treatment with various heavy metals), MX-1 promoters (inducible by interferons), the "GeneSwitch" mifepristone regulatable system (Sirin et al., 2003, Gene, 323:67), cumate-inducible gene switch (WO 2002 / 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.

[0366] As used herein, "internal ribosome entry site" or "IRES" refers to an element that promotes direct internal ribosome entry into a cistron (protein coding region) such as an initiation codon such as ATG, thereby resulting in cap-independent translation of the gene. See, for example, 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 skilled in the art include the IRES described in U.S. Pat. No. 6,692,736. Other examples of "IRES" known in the art include, but are not limited to, IRES obtainable from picorna viruses (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, Ephelomencycarditis virus (EMCV) commercially available from Novagen (Duke et al., 1992. J. Virol. 66(3):1602-9) and VEGF IRES (Huez et al., 1998. Mol. Cell. Biol. 18(11):6178-90). IRES have been reported in viral genomes of species from the families Picornaviridae, Dicistroviridae, and Flaviviridae, as well as in HCV, Friend murine leukemia virus (FrMLV), and Moloney murine leukemia virus (MoMLV).

[0367] In one embodiment, the IRES used in the polynucleotides contemplated herein is the EMCV IRES.

[0368] In a specific embodiment, the polynucleotide is a consensus Kozak sequence. As used herein, the term "Kozak sequence" refers to a short nucleotide sequence that greatly promotes the initial binding of mRNA to the small subunit of the ribosome 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).

[0369] The elements guiding the efficient termination and polyadenylation of heterologous nucleic acid transcripts will increase heterologous gene expression. The transcription termination signal is usually present in the downstream of the polyadenylation signal. In a specific embodiment, the vector includes a polyadenylation sequence at the 3' place of the polynucleotide encoding the polypeptide to be expressed. As used herein, the term "polyA site" or "polyA sequence" represents a DNA sequence that guides the termination and polyadenylation of nascent RNA transcripts by RNA polymerase II. The polyadenylation sequence can promote mRNA stability by adding a polyA tail to the 3' end of the coding sequence, and therefore helps to improve translation efficiency. Cutting and polyadenylation are guided by the poly (A) sequence in RNA. The core poly (A) sequence of mammalian pre-mRNA has two recognition elements flanking the cutting-polyadenylation site. Generally, the almost unchanged AAUAAA hexamer is located at the upstream 20-50 nucleotides of the more variable element rich in U or GU residues. The cutting of the nascent transcript occurs between these two elements and is coupled to up to 250 adenosines added to the 5' cleavage product. In certain embodiments, the core poly(A) sequence is an ideal polyA sequence (e.g., AATAAA, ATTAAA, AGTAAA). In certain embodiments, the poly(A) sequence is an SV40 polyA sequence, a bovine growth hormone polyA sequence (BGHpA), a rabbit β-globin polyA sequence (rβgpA), a variant thereof, or another suitable heterologous or endogenous polyA sequence known in the art. In certain embodiments, the poly(A) sequence is synthetic.

[0370] In certain embodiments, the polynucleotides encoding one or more polypeptides or fusion polypeptides can be introduced into immune effector cells, such as T cells, by non-viral and viral methods. In certain embodiments, the delivery of one or more polynucleotides can be provided by the same method or by different methods and / or by the same vector or by different vectors.

[0371] 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 typically connected to, (e.g., inserted into) a carrier nucleic acid molecule. The vector may contain sequences that direct autonomous replication in a cell, or may contain sequences sufficient to allow integration into host cell DNA. In a specific embodiment, a non-viral vector is used to deliver one or more polynucleotides contemplated herein to a T cell.

[0372] 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.

[0373] Exemplary methods of non-viral delivery of polynucleotides contemplated in particular embodiments include, but are not limited to, electroporation, sonoporation, lipofection, microinjection, biolistic methods, virosomes, liposomes, immunoliposomes, nanoparticles, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, DEAE-dextran-mediated transfer, gene guns, and heat shock.

[0374] Illustrative examples of polynucleotide delivery systems suitable for use in particular embodiments contemplated in particular embodiments include, but are not limited to, systems provided by Amaxa Biosystems, Maxcyte, Inc., BTX Molecular Delivery Systems, and Copernicus Therapeutics Inc. Lipid transfection reagents are commercially available (e.g., Transfectam TM Lipofectin TM ). Cationic lipids and neutral lipids suitable for efficient receptor recognition lipid transfection 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, bacteria-derived, inanimate nanocell-based delivery is also contemplated in certain embodiments.

[0375] As is apparent to those skilled in the art, the term "viral vector" is widely used to refer to nucleic acid molecules (e.g., transfer plasmids) comprising viral-derived nucleic acid elements that generally promote the transfer of nucleic acid molecules or integration into the genome of a cell, or to viral particles that mediate nucleic acid transfer. Viral particles will typically contain various viral components and sometimes also host cell components in addition to nucleic acid. The term "viral vector" or "slow virus vector" may refer to a virus or viral particle that can transfer nucleic acid into a cell or to the nucleic acid itself that is transferred. Viral vectors and transfer plasmids contain structural and / or functional genetic elements that are primarily derived from viruses.

[0376] As described below, viral vectors including polynucleotides contemplated in specific embodiments can be delivered in vivo by administration to individual patients, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous or intracranial infusion) or topical application. Alternatively, the vector can be delivered ex vivo to cells, such as cells transplanted from individual patients (e.g., mobilized peripheral blood, lymphocytes, bone marrow aspirates, tissue biopsies, etc.) or universal donor hematopoietic stem cells, which are then reimplanted in the patient.

[0377] In one embodiment, a viral vector comprising the polynucleotides envisioned herein is directly used to transduce cells in vivo to an organism or a subject. Alternatively, naked DNA can be used. Any approach that is usually used to introduce a molecule and finally contact blood or tissue cells is used, including but not limited to injection, infusion, topical application and electroporation. Suitable methods for using this nucleic acid are available to those skilled in the art and are well known, and although more than one approach can be used to use a particular composition, a particular approach can usually provide a more direct and more effective reaction than another approach.

[0378] Illustrative examples of viral vector systems suitable for use in particular embodiments contemplated in particular embodiments include, but are not limited to, adeno-associated virus (AAV), retrovirus, herpes simplex virus, adenovirus, and vaccinia virus vectors.

[0379] 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, such as T cells, by transducing the cells with a recombinant adeno-associated virus (rAAV) comprising the one or more polynucleotides.

[0380] AAV is a small (about 26nm) replication-deficient, mainly free-type non-enveloped virus. AAV can infect dividing and non-dividing cells, and its genome can be incorporated into the genome of the host cell. Recombinant AAV (rAAV) is usually composed of at least a transgene and its regulatory sequences and 5' and 3' AAV inverted terminal repeats (ITRs). The length of the ITR sequence is about 145bp. In a specific embodiment, rAAV includes ITR and capsid sequences separated from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or AAV10.

[0381] In some embodiments, chimeric rAAV is used to isolate ITR sequences from one AAV serotype and to isolate capsid sequences from different AAV serotypes. For example, rAAV having ITR sequences derived from AAV2 and capsid sequences derived from AAV6 is referred to as AAV2 / AAV6. In specific embodiments, the rAAV vector may 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, rAAV includes ITR sequences derived from AAV2 and capsid sequences derived from AAV6. In a preferred embodiment, rAAV includes ITR sequences derived from AAV2 and capsid sequences derived from AAV2.

[0382] In some embodiments, engineering and selection methods can be applied to AAV capsids to make them more likely to transduce cells of interest.

[0383] The construction, preparation, and purification of rAAV vectors have been disclosed, for example, in U.S. Pat. Nos. 9,169,494; 9,169,492; 9,012,224; 8,889,641; 8,809,058; and 8,784,799, each of which is incorporated herein by reference in its entirety.

[0384] 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, such as T cells, by transducing the cells with a retrovirus (e.g., a lentivirus) comprising the one or more polynucleotides.

[0385] 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 subsequently covalently integrates its genomic DNA into the host genome. Exemplary retroviruses suitable for use in specific embodiments include, but are not limited to, Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), Gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), foamy virus, Friedreich murine leukemia virus, murine stem cell virus (MSCV) and Rous sarcoma virus (RSV) and lentivirus.

[0386] As used herein, the term "lentivirus" refers to a group (or genus) of complex retroviruses. Exemplary lentiviruses include, but are not limited to: HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); visna-maedi virus (VMV) virus; 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.

[0387] In various embodiments, the 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, export element, poly(A) sequence, and may optionally include WPRE or HPRE, insulator elements, selectable markers, and cellular suicide genes, as discussed elsewhere herein.

[0388] In certain embodiments, the lentiviral vectors contemplated herein may be integrative or non-integrative or integration-defective lentiviruses. As used herein, the term "integration-defective lentivirus" or "IDLV" refers to a lentivirus having an integrase that lacks the ability to integrate the viral genome into the genome of a host cell. Integration-incompetent viral vectors have been described in patent application WO 2006 / 010834, which is incorporated herein by reference in its entirety.

[0389] Exemplary mutations in the HIV-1 pol gene suitable for reducing integrase activity include, but are not limited to: H12N, H12C, H16C, H16V, S81 R, D41A, K42A, H51A, Q53C, D55V, D64E, D64V, E69A, K71A, E85A, E87A, D116 N, D1161, D116A, N120G, N1201, N120E, E152G, E152A, D35E, K156E, K156A, E 157A, K159E, K159A, K160A, R166A, D167A, E170A, H171A, K173A, K186Q, K1 86T, K188T, E198A, R199c, R199T, R199A, D202A, K211A, Q214L, Q216L, Q221 L, W235F, W235E, K236S, K236A, K246A, G247W, D253A, R262A, R263A and K264H.

[0390] The term "long terminal repeat (LTR)" refers to a domain of base pairs located at the end of retroviral DNA that is a direct repeat in its native sequence context and contains the U3, R and U5 regions.

[0391] As used herein, the term "FLAP element" or "cPPT / FLAP" refers to a nucleic acid whose sequence includes the central polypurine tract and central termination sequence (cPPT and CTS) of a retrovirus, e.g., HIV-1 or HIV-2. Suitable FLAP elements are described in U.S. Pat. No. 6,682,907 and Zennou et al., 2000, Cell, 101:173.

[0392] As used herein, the term "packaging signal" or "packaging sequence" refers to a psi[Ψ] sequence located within the retroviral genome that is required for inserting the viral RNA into the viral capsid or particle, see, e.g., Clever et al., 1995. J. of Virology, Vol. 69, No. 4; pp. 2101-2109.

[0393] The term "export element" refers to a cis-acting post-transcriptional regulatory element that regulates the transport of RNA transcripts from the nucleus to the cytoplasm of a cell. Examples of RNA export elements include, but are not limited to, the human immunodeficiency virus (HIV) rev response element (RRE) (see, e.g., Culle et al., 1991. Journal of Virology. 65:1053; and Cullen et al., 1991. Cell 58:423) and the hepatitis B virus post-transcriptional regulatory element (HPRE).

[0394] In a specific embodiment, the expression of heterologous sequences in viral vectors is increased by incorporating post-transcriptional regulatory elements, efficient polyadenylation sites, and optionally transcription termination signals into the vector. A variety of post-transcriptional regulatory elements can increase the expression of heterologous nucleic acids on proteins, such as woodchuck hepatitis virus post-transcriptional regulatory elements (WPRE; Zufferey et al., 1999, Journal of Virology, 73: 2886); post-transcriptional regulatory elements present in hepatitis B virus (HPRE) (Huang et al., Mol. Cell. Biol., 5: 3864); etc. (Liu et al., 1995, Genes Dev., 9: 1766).

[0395] Since LTR is modified, the lentiviral vector preferably contains several safety enhancements. "Self-inactivation" (SIN) vector refers to a replication-defective vector, for example, a retrovirus or lentiviral vector, in which the right (3') LTR enhancer-promoter region referred to as the U3 region has been modified (for example, by deletion or substitution) to prevent viral transcription outside the first round of viral replication. Self-inactivation is preferably achieved by introducing a deletion in the U3 region of the 3'LTR of the vector DNA (that is, the DNA for producing the vector RNA). Therefore, during reverse transcription, this deletion is transferred to the 5'LTR of the proviral DNA. In a specific embodiment, it is desirable to eliminate enough U3 sequences to greatly reduce or completely eliminate the transcriptional activity of LTR, thereby greatly reducing or eliminating the production of full-length vector RNA in transduced cells. In the case of HIV-based lentiviral vectors, it has been found that this vector tolerates significant U3 deletions, including removal of the LTRTATA box (for example, from -418 to -18 deletions), without significantly reducing vector titer.

[0396] Additional safety enhancement is provided by replacing the U3 region of the 5'LTR with a heterologous promoter to drive transcription of the viral genome during production of viral particles. Examples of heterologous promoters that can be used include, for example, the viral 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) promoters.

[0397] As used herein, the term "pseudotype" or "pseudotyping" refers to a virus whose viral envelope protein has been replaced with a viral envelope protein of another virus that has preferred properties. For example, HIV can be pseudotyped with the vesicular stomatitis virus G protein (VSV-G) envelope protein, which allows HIV to infect a wider range of cells because the HIV envelope protein (encoded by the env gene) normally targets the virus to the CD4 + Presenting cells.

[0398] In certain embodiments, the lentiviral vector is produced according to known methods. See, for example, 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.

[0399] According to certain specific 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 retroviral and / or lentiviral sequences from a variety of different sources can be used, or that certain lentiviral sequences can accommodate a large number of substitutions and combinations of changes without compromising the ability of the transfer vector to perform the functions described herein. In addition, 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. Pat. Nos. 6,013,516 and 5,994,136, many of which may be suitable for producing viral vectors or transfer plasmids contemplated herein.

[0400] 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 comprising the one or more polynucleotides.

[0401] Adenovirus-based vectors are capable of extremely high transduction efficiencies in many cell types and do not require cell division. Using this vector, high titers and high expression levels have been achieved. This vector can be prepared in large quantities in a relatively simple system. Most adenovirus vectors are engineered so that transgenes replace the Ad E1a, E1b and / or E3 genes; subsequently, replication-defective vectors are propagated in human 293 cells that provide the missing gene function in trans. Ad vectors can transduce many types of tissues in vivo, including non-dividing differentiated cells such as those found in the liver, kidney, and muscle. Conventional Ad vectors have a large carrying capacity.

[0402] The generation and propagation of replication-deficient current adenoviral vectors can utilize a unique helper cell line named 293, which is transformed from human embryonic kidney cells by Ad5 DNA fragments and constitutively expresses the E1 protein (Graham et al., 1977). Since the E3 region can be allocated from the adenoviral genome (Jones and Shenk, 1978), current adenoviral vectors carry foreign DNA in the E1, D3 region, or both regions with the help of 293 cells (Graham and Prevec, 1991). Adenoviral vectors have been used for eukaryotic gene expression (Levrero et al., 1991; Gomez-Foix et al., 1992) and vaccine development (Grunhaus and Horwitz, 1992; Graham and Prevec, 1992). Studies on administration of recombinant adenoviruses to different tissues include tracheal instillation (Rosenfeld et al., 1991; Rosenfeld et al., 1992), intramuscular injection (Ragot et al., 1993), peripheral intravenous injection (Herz and Gerard, 1993), and stereotaxic intracerebral inoculation (LeGal La Salle et al., 1993). An example of the use of Ad vectors in clinical trials involves polynucleotide therapy for anti-tumor immunity with intramuscular injection (Sterman et al., Gene Ther. 7:1083-9 (1998)).

[0403] 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) comprising the one or more polynucleotides.

[0404] Mature HSV virions are composed of enveloped icosahedral capsids, wherein the viral genome is composed of a 152kb linear double-stranded DNA molecule. In one embodiment, the viral vector based on HSV lacks one or more essential or non-essential HSV genes. In one embodiment, the viral vector based on HSV is replication-deficient. Most replication-deficient HSV vectors contain deletions to remove one or more early, early or late HSV genes to prevent replication. For example, the HSV vector may lack the immediate early genes selected from the group consisting of: ICP4, ICP22, ICP27, ICP47 and combinations thereof. The advantage of the HSV vector is its ability to enter a latent period that can lead to long-term DNA expression, and its large viral DNA genome that can accommodate exogenous DNA inserts up to 25kb. 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 WO 91 / 02788, WO 96 / 04394, WO 98 / 15637, and WO 99 / 06583, each of which is incorporated herein by reference in its entirety.

[0405] G. Genetically modified cells

[0406] In various embodiments, cells are modified to express engineered immune receptor systems, one or more engineered immune receptor components, signaling components, targeting components, engineered TCR, CAR, CCR, BiTE, zetakine, flip receptors, exogenous co-stimulatory factors, immunomodulatory factors, agonists of co-stimulatory factors, antagonists of immunosuppressive factors, immune cell engagers and / or fusion proteins contemplated herein. In a specific embodiment, cells are used to treat cancer. Cells can be non-genetically modified to express one or more polypeptides in the polypeptides contemplated herein, or in a specific preferred embodiment, cells can be genetically modified to express one or more polypeptides in the polypeptides contemplated herein. As used herein, the term "genetically engineered" or "genetically modified" refers to adding additional genetic material to the total genetic material in the cell in the form of DNA or RNA. In a specific embodiment, the terms "genetically modified cells", "modified cells" and "redirected cells" are used interchangeably.

[0407] In a specific embodiment, one or more engineered immune receptor components of the raised immune receptor complexes contemplated herein are introduced into immune effector cells and expressed therein, to improve the efficacy of immune effector cells. In a specific embodiment, one or more engineered immune receptor components of the raised immune receptor complexes are introduced into immune effector cells and expressed therein, and the immune effector cells are redirected to target cells by co-expressing exogenous lymphocyte receptors or engineered antigen receptors (e.g., engineered TCR or CAR) in cells.

[0408] In certain embodiments, dual-targeted immune effector cells are contemplated, wherein 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).

[0409] In particular embodiments, dual-targeted immune effector cells are contemplated wherein the target cells express CD33, CD123, CLL1, B7-H3, BCMA, CD19, CD20, CD22, CD79A, CD79B, EGFR, EGFRvIII, or a NKG2D ligand recognized by an engineered immune receptor and a target antigen recognized by an exogenous lymphocyte receptor or an engineered antigen receptor (e.g., an engineered / exogenous TCR or CAR).

[0410] 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). Exemplary immune effector cells contemplated herein are T lymphocytes, including but not limited to cytotoxic T cells (CTLs; CD8 + T cells), TIL and helper T cells (HTL; CD4 + In one embodiment, the immune effector cells include natural killer (NK) cells. In one embodiment, the immune effector cells include natural killer T (NKT) cells.

[0411] Immune effector cells can be autologous / autogeneic ("one's own") or non-autologous ("non-one's own", e.g., allogeneic, isogenic, or xenogeneic). As used herein, "autologous" refers to cells 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, "isogenic" 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 than the compared cells. In a preferred embodiment, the cells are human autologous immune effector cells.

[0412] Exemplary immune effector cells suitable for introduction of one or more engineered immunoreceptor 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. The T cell can be a T helper (Th) cell, such as a T helper 1 (Th1) cell or a T helper 2 (Th2) cell. The T cell can be a helper T cell (HTL; CD4 + T cells)CD4 + T cells, cytotoxic T cells (CTL, CD8 + T cells), CD4 + CD8 + T cells, CD4 - CD8 - T cells or any other T cell subsets. Other exemplary T cell populations suitable for use in specific embodiments include primary T cells and memory T cells. For example, T cell populations suitable for use in specific embodiments include 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 ).

[0413] As will be appreciated by those skilled in the art, other cells may also be used as immune effector cells comprising one or more engineered immune receptor components contemplated herein. In a particular embodiment, immune effector cells also include NK cells, NKT cells, neutrophils and macrophages. Immune effector cells also include the progenitor cells of effector cells, wherein this progenitor cell can be induced to differentiate into immune effector cells in vivo or in vitro. Therefore, in a particular embodiment, immune effector cells include the progenitor cells of immune effector cells, such as hematopoietic stem cells (HSC) contained in CD34+ cell groups derived from cord blood, bone marrow or mobilized peripheral blood, the HSC is differentiated into mature immune effector cells after being used in a subject, or the HSC can be induced in vitro to differentiate into mature immune effector cells.

[0414] As used herein, the term "CD34+ cells" refers to cells expressing CD34 protein on their cell surface. As used herein, "CD34" refers to cell surface glycoproteins (e.g., sialomucin) that generally act as cell-cell adhesion factors and participate in the entry of T cells into lymph nodes. The CD34+ cell population contains hematopoietic stem cells (HSCs), which differentiate and contribute to all hematopoietic lineages when administered to patients, including cells of T cell, NK cell, NKT cell, neutrophil and monocyte / macrophage lineages.

[0415] In a specific embodiment, there is provided a method for preparing an immune effector cell expressing one or more engineered immune receptor components envisioned herein. In one embodiment, the method includes transfection or transduction of immune effector cells separated from an individual, so that the immune effector cells with one or more nucleic acids and / or carriers or a combination thereof include one or more engineered immune receptor components envisioned herein. In one embodiment, the method includes transfection or transduction of immune effector cells separated from an individual, so that the immune effector cells express one or more engineered immune receptor components and exogenous lymphocyte receptors or engineered antigen receptors (e.g., TCR, CAR, CCR, zetakine or flip receptors) envisioned herein. In certain embodiments, immune effector cells are separated from an individual and genetically modified without further manipulation in vitro. Then, such cells can be directly applied to an individual again. In another embodiment, immune effector cells are first activated and stimulated to proliferate in vitro before genetic modification. In this regard, immune effector cells can be cultured before and / or after genetic modification.

[0416] In certain embodiments, the cells are human cells. In certain embodiments, the source of cells is obtained from a subject prior to the in vitro manipulation or genetic modification of the immune effector cells described herein. In certain embodiments, the modified immune effector cells include T cells.

[0417] T cells can be obtained from a variety of sources, including but not limited to peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, any number of techniques known to the skilled artisan such as sedimentation (e.g., FICOLL TM T cells are obtained by isolating (isolation) from a unit of blood collected from a subject.

[0418] In other embodiments, isolated or purified T cell populations are used. In some embodiments, following isolation of PBMCs, cytotoxic and helper T lymphocytes may be sorted into naive, memory, and effector T cell subsets before or after activation, expansion, and / or genetic modification.

[0419] In one embodiment, the isolated or purified T cell population expresses one or more of the following markers, including but not limited to: CD3 + 、CD4 + 、CD8 + or a combination thereof.

[0420] In certain embodiments, T cells are isolated from an individual and first activated and stimulated in vitro to proliferate before being modified to express one or more engineered immune receptor components.

[0421] To achieve a sufficient therapeutic dose of a T cell composition, the T cells are typically subjected to one or more rounds of stimulation, activation and / or expansion. In certain embodiments, T cells may generally be activated and expanded using methods as described, for example, in U.S. Pat. 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 herein by reference in its entirety. In specific embodiments, T cells are activated and expanded for about 6 hours, about 12 hours, about 18 hours, or about 24 hours prior to the 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.

[0422] In one embodiment, the T cells are activated while being modified.

[0423] In various embodiments, a method for producing immune effector cells comprises activating a cell population comprising T cells and expanding the T cell population. T cell activation can be achieved by providing a primary stimulation signal through a T cell TCR / CD3 complex and providing a secondary co-stimulatory signal through an auxiliary molecule (e.g., CD28).

[0424] The TCR / CD3 complex can be stimulated by contacting the T cells with an appropriate CD3 binding agent (e.g., 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.

[0425] In addition to the primary stimulation signal provided by the TCR / CD3 complex, the induction of T cell responses also requires a second co-stimulatory signal. In a particular embodiment, a CD28 binding agent can be used to provide a co-stimulatory signal. Illustrative examples of CD28 binding agents include, but are not limited to, natural CD28 ligands, such as natural ligands of CD28 (e.g., members of the B7 protein family, such as B7-1 (CD80) and B7-2 (CD86)); and anti-CD28 monoclonal antibodies or fragments thereof capable of cross-linking CD28 molecules, for example, monoclonal antibodies 9.3, B-T3, XR-CD28, KOLT-2, 15E8, 248.23.2, and EX5.3D10.

[0426] In one embodiment, the molecule that provides the primary stimulation signal, such as the molecule that provides stimulation via the TCR / CD3 complex, and the co-stimulatory molecule are coupled to the same surface.

[0427] In certain embodiments, the binding agent that provides the stimulatory and co-stimulatory signals is localized on the surface of the cell. This can be achieved by transfecting or transducing the cell with a nucleic acid encoding the binding agent in a form suitable for expression of the binding agent on the cell surface, or alternatively coupling the binding agent to the cell surface.

[0428] In another embodiment, molecules that provide a primary stimulation signal, such as molecules that provide stimulation via the TCR / CD3 complex, and co-stimulatory molecules are displayed on antigen presenting cells.

[0429] In one embodiment, the molecule that provides the primary stimulation signal, such as the molecule that provides stimulation via the TCR / CD3 complex, and the co-stimulatory molecule are provided on separate surfaces.

[0430] In a certain embodiment, one of the binding agents providing the stimulation signal and the costimulatory signal is soluble (provided in solution), and another or other binding agents are provided on one or more surfaces. In a specific embodiment, both the binding agents providing the stimulation signal and the costimulatory signal are provided in a soluble form (provided in solution). In various embodiments, the method for preparing T cells contemplated herein comprises activating T cells with soluble anti-CD3 and / or soluble anti-CD28 antibodies or fragments thereof. In various embodiments, the method for preparing T cells contemplated herein comprises activating T cells with surface-bound anti-CD3 and / or surface-bound anti-CD28 antibodies or fragments thereof. In various embodiments, the method for preparing T cells contemplated herein comprises activating T cells with bead-bound anti-CD3 and / or bead-bound anti-CD28 antibodies or fragments thereof.

[0431] In one embodiment, expanding the T cells activated by the methods contemplated herein further comprises culturing a cell population comprising T cells for several hours (about 3 hours) to about 7 days to about 28 days or any hourly integer value therebetween. In another embodiment, the T cell composition can be cultured for 14 days. In a specific embodiment, the T cells are cultured for about 21 days. In another embodiment, the T cell composition is cultured for about 2 to 3 days. It may also be desirable to perform several cycles of stimulation / activation / expansion.

[0432] In a specific embodiment, conditions suitable for T cell culture comprise an appropriate culture medium (e.g., Minimal Essential Medium or RPMI medium 1640 or X-vivo 15 (Lonza)) and one or more factors necessary for proliferation and viability, 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 cell growth known to those of skill in the art.

[0433] Additional 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, supplemented with 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 amounts sufficient to allow T cells to grow and expand.

[0434] Antibiotics (e.g., penicillin and streptomycin) are included only for experimental cultures and not for cells to be injected into a 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).

[0435] In specific embodiments, PBMCs or isolated T cells are contacted with stimulatory and co-stimulatory agents such as anti-CD3 and anti-CD28 antibodies, typically attached to beads or other surfaces, in culture medium with appropriate cytokines such as IL-2, IL-7 and / or IL-15.

[0436] In other embodiments, artificial APC (aAPC) is prepared by engineered K562, U937, 721.221, T2 and C1R cells to guide the stable expression and secretion of various co-stimulatory molecules and cytokines. In a specific embodiment, K32 or U32 aAPC is used to guide one or more antibody-based stimulatory molecules to be displayed on the surface of AAPC cells. T cell populations can be expanded by aAPC expressing various co-stimulatory molecules, including but not limited to CD137L (4-1BBL), CD134L (OX40L) and / or CD80 or CD86. Finally, aAPC provides an efficient platform for amplifying genetically modified T cells and maintaining CD28 expression on CD8 T cells. The aAPC provided in WO 03 / 057171 and US2003 / 0147869 are incorporated herein by reference in their entirety.

[0437] In a specific embodiment, polynucleotides encoding one or more engineered immune receptor components are introduced into a T cell population. In a specific embodiment, polynucleotides encoding one or more engineered immune receptor components are introduced into a T cell population expressing an exogenous lymphocyte receptor or an engineered antigen receptor. The polynucleotides can be introduced into T cells by microinjection, transfection, lipofection, heat shock, electroporation, transduction, gene gun, microinjection, DEAE-dextran mediated transfer, etc.

[0438] In a preferred embodiment, the polynucleotide is introduced into T cells by viral transduction.

[0439] Suitable for introducing polynucleotides into immune effector cells or CD34 + Illustrative examples of viral vector systems in cells include, but are not limited to, adeno-associated virus (AAV), retrovirus, herpes simplex virus, adenovirus, vaccinia virus vectors for gene transfer.

[0440] 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.

[0441] H. Compositions and Formulations

[0442] The compositions contemplated herein may include one or more engineered immune receptor polypeptides, polynucleotides encoding engineered immune receptor polypeptides, vectors comprising the polypeptides, genetically modified immune effector cells, bridging factors, and the like. Compositions include, but are not limited to, pharmaceutical compositions. "Pharmaceutical composition" refers to a composition formulated in a pharmaceutically acceptable or physiologically acceptable solution for administration to cells or animals alone or in combination with one or more other treatment modalities. It should also be understood that, if desired, the composition may also be administered in combination with other agents, such as cytokines, growth factors, hormones, small molecules, chemotherapeutic agents, prodrugs, drugs, antibodies, or other various pharmaceutically active agents. There is no practical limitation on other components that may also be included in the composition, provided that the additional agents do not adversely affect the ability of the composition to deliver the intended therapy.

[0443] The phrase "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0444] The term "pharmaceutically acceptable carrier" refers to a diluent, adjuvant, excipient or vehicle for administering a bridging factor, polypeptide, polynucleotide, a carrier including the same, or a genetically modified immune effector cell. Illustrative examples of pharmaceutical carriers can be sterile liquids, such as cell culture media, water and oils, including those of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Saline solutions and aqueous glucose and glycerol solutions can also be used as liquid carriers, especially liquid carriers for injectable solutions. In a particular embodiment, suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc. In addition to any conventional culture medium or medicament being incompatible with the active ingredient, its use in a pharmaceutical composition is contemplated. Supplementary active ingredients can also be incorporated into the composition.

[0445] In one embodiment, the composition including a pharmaceutically acceptable carrier is suitable for administration to a subject. In a specific embodiment, the composition including a carrier is suitable for parenteral administration, such as intravascular (intravenous or intraarterial), intraperitoneal or intramuscular administration. In a specific embodiment, the composition including a pharmaceutically acceptable carrier is suitable for intraventricular, intraspinal or intrathecal administration. A pharmaceutically acceptable carrier comprises a sterile aqueous solution, a cell culture medium or a dispersion. The use of these media and medicaments for pharmaceutically active substances is well known in the art. Except for any conventional culture medium or medicament incompatible with bridging factors, polypeptides, polynucleotides, carriers including them or genetically modified immune effector cells, its use in pharmaceutical compositions is envisioned.

[0446] In certain embodiments, the compositions contemplated herein include genetically modified T cells and a pharmaceutically acceptable carrier. Compositions contemplated herein, including cell-based compositions, can be administered alone or in combination with other suitable compounds via enteral or parenteral administration methods to achieve the desired therapeutic purpose.

[0447] In certain embodiments, the compositions contemplated herein include a bridging factor and a pharmaceutically acceptable carrier.

[0448] Pharmaceutically acceptable carriers must have sufficiently high purity and sufficiently low toxicity to make them suitable for application to human subjects being treated. Pharmaceutically acceptable carriers should maintain or increase the stability of the composition. Pharmaceutically acceptable carriers can be liquid or solid and are selected to provide the desired volume, consistency, etc. when combined with other components of the composition in consideration of the planned mode of administration. For example, pharmaceutically acceptable carriers can be, but are not limited to, binders (e.g., pregelatinized corn starch, polyvinyl pyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylate, calcium hydrogen phosphate, etc.); lubricants (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.); disintegrants (e.g., starch, sodium starch glycolate, etc.); or wetting agents (e.g., sodium lauryl sulfate, etc.). Other suitable pharmaceutically acceptable carriers for the compositions contemplated herein include, but are not limited to, water, saline solutions, alcohol, polyethylene glycol, gelatin, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinyl pyrrolidone, and the like.

[0449] 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 an acid or base without significantly changing the pH. Examples of buffers contemplated herein include, but are not limited to, Dulbecco's phosphate buffered saline (PBS), Ringer's solution, 5% dextrose in water (D5W), normal / physiologic saline (0.9% NaCl).

[0450] The pharmaceutically acceptable carrier may be present in an amount sufficient to maintain the pH of the composition at about 7. Alternatively, the pH of the composition ranges from 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 pH of the composition is about 7.4.

[0451] The compositions contemplated herein may include non-toxic pharmaceutically acceptable culture media. The compositions may be suspensions. As used herein, the term "suspension" refers to a non-adhesive condition in which cells are not attached to a solid support. For example, cells maintained as a suspension may be stirred or agitated and the cells are not adhered to a support, such as a culture dish.

[0452] In certain embodiments, the compositions contemplated herein are formulated in suspension, wherein the modified T cells are dispersed in an acceptable liquid medium or solution, such as saline or serum-free medium, in an intravenous (IV) bag, etc. Acceptable diluents include, but are not limited to, water, PlasmaLyte, Ringer's solution, isotonic sodium chloride (saline) solution, serum-free cell culture medium, and medium suitable for cryogenic storage, such as Culture medium.

[0453] In certain embodiments, the pharmaceutically acceptable carrier is substantially free of natural proteins of human or animal origin and is suitable for storing compositions comprising modified T cell populations. The pharmaceutical composition is intended for administration to human patients and is therefore substantially free of cell culture components such as bovine serum albumin, horse serum and fetal bovine serum.

[0454] In some embodiments, the composition is formulated in a pharmaceutically acceptable cell culture medium. Such compositions are suitable for administration to human subjects. In specific embodiments, the pharmaceutically acceptable cell culture medium is a serum-free medium.

[0455] Serum-free medium has several advantages over serum-containing medium, including that the composition is simplified and better defined, the degree of contaminants is reduced, possible sources of infectious agents are eliminated, and the cost is reduced. In various embodiments, serum-free medium is animal-free and can optionally be protein-free. Optionally, the medium can contain biopharmaceutically acceptable recombinant proteins. "Animal-free" medium refers to a medium in which the components are derived from non-animal sources. Recombinant proteins replace natural animal proteins in animal-free medium, and nutrients are obtained from synthetic, plant or microbial sources. In contrast, "protein-free" medium is defined as being substantially free of protein.

[0456] Illustrative examples of serum-free media for use in particular compositions include, but are not limited to, QBSF-60 (Quality Biological, Inc.), StemPro-34 (Life Technologies), and X-VIVO 10.

[0457] In one embodiment, the composition comprising modified T cells is formulated in PlasmaLyte.

[0458] In various embodiments, the composition comprising the modified T cells is formulated in a cryopreservation medium. For example, a cryopreservation medium with a cryopreservation agent can be used to maintain high cell viability results after thawing. Illustrative examples of cryopreservation medium used in a particular composition include, but are not limited to, CryoStor CS10, CryoStor CS5, and CryoStor CS2.

[0459] In one embodiment, the composition is formulated in a solution comprising 50:50 CryoStor A:CryoStor CS10.

[0460] In certain embodiments, the compositions are substantially free of mycoplasma, endotoxin, and microbial contamination. With respect to endotoxin, "substantially free" means that the endotoxin content per dose of cells is lower than the content allowed by the FDA for biologics, which is 5 EU / kg body weight per day of total endotoxin, and 350 EU per total dose of cells for an average 70 kg human. In certain embodiments, the 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.

[0461] In certain embodiments, the formulation of pharmaceutically acceptable carrier solutions is well known to those skilled in the art, as is the development of suitable dosing and treatment regimens for the use of the specific compositions described herein in a variety of treatment regimens, including, for example, enteral and parenteral, such as intravascular, intravenous, intraarterial, intraosseous, intraventricular, intracerebral, intracranial, intraspinal, intrathecal, and intramedullary administration and formulation. It will be appreciated by those skilled in the art that the specific embodiments contemplated herein may include other formulations, such as those well known in the pharmaceutical arts and described, for example, in Remington: The Science and Practice of Pharmacy, Vol. I and Vol. II, 22nd ed. Edited by Loyd V. Allen Jr. Philadelphia, PA: Pharmaceutical Press; 2012, each of the above-mentioned U.S. patents is incorporated herein by reference in its entirety.

[0462] In a particular embodiment, the composition comprises an amount of immune effector cells expressing one or more engineered immune receptor components contemplated herein. In a particular embodiment, the composition comprises an amount of immune effector cells expressing exogenous lymphocyte receptors or engineered antigen receptors and one or more engineered immune receptor components contemplated herein. As used herein, the term "amount" refers to an "effective amount (an amount effective / an effective amount)" of cells comprising one or more engineered immune receptor components contemplated herein, etc., in the presence of a bridging factor for achieving a beneficial or desired preventive or therapeutic outcome (including a clinical outcome).

[0463] "Prophylactically effective amount" refers to the amount of cells that are effective for achieving the desired prevention and treatment results in the presence of bridging factors, including one or more engineered immune receptor components contemplated herein. Typically, but not necessarily, the preventive effective amount is less than the therapeutic effective amount because the preventive dose is used for subjects before or at an early stage of the disease.

[0464] "Therapeutically effective amount" refers to the amount of cells comprising one or more engineered immune receptor components contemplated herein that is effective for "treating" a subject (e.g., a patient) in the presence of a bridging factor. When a therapeutic amount is indicated, the exact amount of the composition, cells, bridging factor, etc. to be administered can be determined by a physician taking into account individual differences in age, weight, tumor size, degree of infection or metastasis, and the condition of the patient (subject).

[0465] Generally speaking, the pharmaceutical composition comprising the immune effector cells described herein can be taken as 10 2 Up to 10 10 cells / kg body weight, preferably 10 5 Up to 10 6 The number of cells will depend on the desired end use of the composition, as will the type of cells contained therein. For the purposes provided herein, the volume of cells is typically one liter or less, and may be 500 mL or less, or even 250 mL or 100 mL or less. Thus, the desired cell density is typically greater than 10 6 cells / ml and usually greater than 10 7 cells / ml, usually 10 8 Clinically relevant numbers of immune cells can be assigned to cells that accumulate to or exceed 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 or 10 12 In some embodiments, particularly since all infused cells will be redirected to a specific target antigen, 10 6 / kg(10 6 -10 11 / patient) range.

[0466] If desired, treatment may also comprise administration of mitogens (e.g., PHA) or lymphokines, cytokines and / or chemokines (e.g., IFN-γ, IL-2, IL-12, TNF-α, IL-18 and TNF-β, GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MIP1α, etc.) as described herein to enhance the induction of an immune response.

[0467] In general, compositions comprising activated and expanded cells as described herein can be used to treat and prevent diseases that occur in immunocompromised individuals. In particular, the compositions contemplated herein are used to treat cancer. In particular embodiments, immune effector cells can be administered alone or in combination with a carrier, diluent, excipient, and / or with other components such as IL-2 or other cytokines or cell populations as a pharmaceutical composition.

[0468] In certain embodiments, the pharmaceutical composition includes an amount of genetically modified T cells in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.

[0469] In certain embodiments, the pharmaceutical composition includes an amount of a bridging factor in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.

[0470] In a particular embodiment, the composition comprises an effective amount of immune effector cells comprising one or more engineered immune receptor components contemplated herein alone or in combination with a bridging factor and / or one or more therapeutic agents (such as radiotherapy, chemotherapy, transplantation, immunotherapy, hormone therapy, photodynamic therapy, etc.). The composition can also be administered in combination with an antibiotic. Such therapeutic agents are acceptable in the art as standard treatments for specific disease states such as specific cancers as described herein. Exemplary therapeutic agents contemplated include cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory agents, chemotherapeutic agents, radiotherapeutic agents, therapeutic antibodies or other active agents and adjuvants.

[0471] In a specific embodiment, a composition comprising an effective amount of immune effector cells comprising one or more engineered immune receptor components contemplated herein is administered to a subject, and a composition comprising an effective amount of a bridging factor is administered to the subject before, during, in combination with, or subsequently to the cell composition, and optionally repeatedly administered to the subject.

[0472] In certain embodiments, compositions comprising immune effector cells comprising one or more engineered immune receptor components contemplated herein may be administered in conjunction with any number of chemotherapeutic agents.

[0473] A variety of other therapeutic agents can 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 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); non-steroidal anti-inflammatory drugs (NSAIDS), including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF agents, cyclophosphamide, and mycophenolate.

[0474] 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, duligotumab, dacetuzumab, dalotuzumab, durvalumab, elotuzumab (HuL uc63), gemtuzumab, ibritumomab, indatuximab, inotuzumab, ipilimumab, lorvotuzumab, lucatumumab, milatuzumab, moxetumomab, nivolumab, ocaratuzumab, ofatumumab, pembrolizumab, rituximab, siltuximab, teprotumumab, and ublituximab.

[0475] In certain embodiments, the compositions described herein are administered in combination with cytokines. As used herein, "cytokine" is a general term for a protein released by a cell population that acts as an intercellular mediator on another cell. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones.

[0476] I. Treatment Methods

[0477] The 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 methods for improved adoptive immunotherapy for preventing, treating and ameliorating immune-related diseases or disorders (e.g., cancer, autoimmune diseases, immunodeficiency, inflammatory diseases, GVHD or infectious diseases) or for preventing, treating or ameliorating at least one symptom associated therewith.

[0478] Immune effector cells comprising an engineered immune receptor system (e.g., targeting components and signaling components) provide a method for improved adoptive immunotherapy for preventing, treating, and ameliorating immune-related diseases or disorders (e.g., cancer, autoimmune diseases, immunodeficiency, inflammatory diseases, GVHD, or infectious diseases) or for preventing, treating, and ameliorating at least one symptom associated therewith.

[0479] In particular embodiments, immune effector cells modified to express one or more engineered immune receptor components provide methods for fine-tuning the safety and efficacy of improved adoptive immunotherapy against target cells (e.g., tumor cells) expressing the target antigen while reducing the risk of on-target antigen, off-target cytotoxicity (recognition of the target antigen on normal non-target cells).

[0480] In a specific embodiment, the method for preventing, treating or improving at least one symptom of an immune-related disease or disorder (e.g., cancer, autoimmune disease, immunodeficiency, inflammatory disease, GVHD or infectious disease) comprises administering to a subject an effective amount of modified immune effector cells or T cells, wherein the modified immune effector cells or T cells comprise an engineered immune receptor system and one or more components of engineered / exogenous TCR, CAR or other therapeutic transgenes to redirect cells to target cells. By transducing chemically regulated immunostimulatory signals, genetically modified cells are more effective and safe cell immunotherapy.

[0481] In a specific embodiment, one or more immune effector cells (e.g., T cells) are modified to express both targeting components and signaling components. In this case, the modified cells are applied to a subject in need and homed to the target cells by the interaction of the signaling components expressed on the immune effector cells and the target antigens expressed on the target cells. Before the modified cells have been applied to the subject, at almost the same time as the modified cells have been applied to the subject, or after the modified cells have been applied to the subject, a bridging factor is applied to the subject. In the presence of a bridging factor, a complex is formed between the targeting components bound to the target antigen, the bridging factor, and the signaling components bound to the immune receptor complex. After the complex is formed, the signaling components and the targeting components transduce the immunostimulatory signal to the immune effector cells, which synergize with the immune receptor signals, and then trigger the cytotoxic response for the target cells from the immune effector cells.

[0482] In various embodiments, immune effector cells comprising one or more engineered immune receptor components and / or exogenous lymphocyte receptors or engineered antigen receptors use a dual targeting strategy to fine-tune the safety and efficacy of cytotoxic responses against target cells, wherein one or more target cells express one or more target antigens recognized by both the exogenous lymphocyte receptor or engineered antigen receptor and the engineered immune receptor.

[0483] In certain embodiments, one or more immune effector cells (e.g., T cells) are modified to express both targeting components and signaling components and exogenous lymphocyte receptors or engineered antigen receptors, e.g., TCR, CAR, CCR or flip receptors. In this case, the modified cells are applied to subjects in need, and the target cells are homed to by the interaction of the targeting components bound to the first target antigen and the antigen receptor bound to the second target antigen, wherein one or both target antigens are expressed on target cells or target cell colonies. The interaction of antigen receptors (e.g., TCR) with target antigens on target cells can trigger cytotoxic responses against target cells from immune effector cells. In certain embodiments, before modified cells have been applied to subjects, at almost the same time as modified cells have been applied to subjects, or after modified cells have been applied to subjects, bridging factors are applied to subjects. In the presence of bridging factors, complexes are formed between the targeting components bound to the first target antigen, bridging factors, and signaling components bound to immune receptor complexes. After complex formation, the signaling component and the targeting component transduce the immunostimulatory signal to the immune effector cells, which cooperate with the immune receptor signal and in turn initiate or enhance the cytotoxic response from the immune effector cells against the target cells.

[0484] In a specific embodiment, one or more immune effector cells (e.g., T cells) are modified to express both targeting components and signaling components. In this case, the modified cells are applied to a subject in need and homed to the target cells by the interaction of the signaling components expressed on the immune effector cells and the target antigens expressed on the target cells. In certain embodiments, before the modified cells have been applied to the subject, at almost the same time as the modified cells have been applied to the subject, or after the modified cells have been applied to the subject, a bridging factor is applied to the subject. In the presence of a bridging factor, a complex is formed between the targeting components, bridging factors, and signaling components bound to the target antigen, the signaling components comprising a multimerization domain, optionally a linker polypeptide, and an actuator domain (e.g., Fcγ, CD3ε, CD3δ, or CD3γ or its fragment). After the complex is formed, the signaling components and the targeting components transduce the immunostimulatory signal to the immune effector cells, which synergize with the immune receptor signals, and then induce a cytotoxic response to the target cells from the immune effector cells.

[0485] In a specific embodiment, one or more immune effector cells (e.g., T cells) are modified to express both targeting components and signaling components and exogenous lymphocyte receptors or engineered antigen receptors, for example, engineered / exogenous TCRs. In this case, the modified cells are applied to a subject in need, and are homed to target cells through the interaction of the targeting components bound to the first target antigen and the immune receptor bound to the second target antigen, wherein one or both target antigens are expressed on the target cell or target cell population. The interaction of TCR with the target antigen on the target cell can trigger a cytotoxic response from immune effector cells to the target cell. Before the modified cells have been applied to the subject, at almost the same time as the modified cells have been applied to the subject, or after the modified cells have been applied to the subject, a bridging factor is applied to the subject. In the presence of a bridging factor, a complex is formed between the targeting component bound to the first target antigen, the bridging factor, and the signaling component, and the signaling component comprises a multimerization domain, optionally a linker polypeptide and Fcγ, CD3ε, CD3δ or CD3γ or a fragment thereof. After complex formation, the signaling component and the targeting component transduce the immunostimulatory signal to the immune effector cells, which cooperate with the immune receptor signal and in turn initiate or enhance the cytotoxic response from the immune effector cells against the target cells.

[0486] In specific embodiments, the modified immune effector cells contemplated herein are used to treat solid tumors or cancers.

[0487] In specific embodiments, the modified immune effector cells contemplated herein are used to treat solid tumors or cancers, including, but not limited to, adrenal cancer, adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, atypical teratoid / rhabdoid tumor, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain / CNS cancer, breast cancer, bronchial tumors, cardiac tumors, cervical cancer, bile duct cancer, chondrosarcoma, chordoma, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma in situ (DCIS) endometrial cancer, ependymoma, esophageal cancer, nasal glioma, Ewing's sarcoma, Extracranial germ cell tumor, extragonadal germ cell tumor, eye cancer, fallopian tube cancer, fibrosarcoma, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid, gastrointestinal stromal tumor (GIST), germ cell tumor, glioma, glioblastoma, head and neck cancer, hemangioblastoma, hepatocellular carcinoma, hypopharyngeal cancer, intraocular melanoma, Kaposi's sarcoma, kidney cancer, laryngeal cancer, leiomyosarcoma, lip cancer, liposarcoma, liver cancer, lung cancer, non-small cell lung cancer, lung carcinoid, malignant mesothelioma, medullary carcinoma, medulloblastoma, meningioma, melanoma, Merkel cell carcinoma cell carcinoma), midline cancer, oral cancer, mucosal sarcoma, myelodysplastic syndrome, myeloproliferative neoplasms, nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oligodendroglioma, oral cancer, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, islet cell tumors, papillary carcinoma, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal tumor, pituitary tumor, pleuropulmonary blastoma, primary peritoneal cancer, 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, stomach cancer, sweat gland cancer, synovialoma, testicular cancer, pharyngeal cancer, thymus cancer, thyroid cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vascular cancer, vulvar cancer, and Wilms Tumor.

[0488] In particular embodiments, the modified immune effector cells contemplated herein are used to treat solid tumors or cancers, including but not limited to 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 and oligodendroglioma.

[0489] In specific embodiments, the modified immune effector cells contemplated herein are used to treat 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.

[0490] In specific embodiments, the modified immune effector cells contemplated herein are used to treat glioblastoma.

[0491] In specific embodiments, the modified immune effector cells contemplated herein are used to treat liquid or hematological cancers.

[0492] In specific embodiments, the modified immune effector cells contemplated herein are used to treat B-cell malignancies, including but not limited to: leukemias, lymphomas, and multiple myeloma.

[0493] In specific embodiments, the immune effector cells contemplated herein are used to treat liquid cancers, including but not limited to leukemias, lymphomas, and multiple myeloma: 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 myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML) and polycythemia vera, Hodgkin lymphoma, nodular lymphocyte-predominant Hodgkin lymphoma, Burkitt lymphoma (Burkitt lymphoma), lymphoma), 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, Sézary syndrome, precursor T-lymphoblastic lymphoma, multiple myeloma, overt multiple myeloma, smoldering multiple myeloma, plasma cell leukemia, nonsecretory myeloma, IgD myeloma, osteosclerotic myeloma, solitary plasmacytoma of bone, and extramedullary plasmacytoma.

[0494] In specific embodiments, the modified immune effector cells contemplated herein are used to treat acute myeloid leukemia (AML).

[0495] Preferred cells for use in the methods contemplated herein comprise autologous / self ("self") cells, preferably hematopoietic cells, more preferably T cells and more preferably immune effector cells.

[0496] In a particular embodiment, the method comprises administering to a patient in need a therapeutically effective amount of a modified immune effector cell or a composition comprising the same, the modified immune effector cell expressing one or more engineered immune receptor components, and optionally an exogenous lymphocyte receptor or an engineered antigen receptor or another targeting component or signaling component, and optionally also administering a bridging factor to the subject. In certain embodiments, the cells are used to treat patients at risk of developing an immune disorder. Thus, a particular embodiment comprises the treatment or prevention or improvement 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 a therapeutically effective amount of a modified immune effector cell contemplated herein and optionally a bridging factor.

[0497] The amount and frequency of administration of modified immune effector cells, targeting components and / or bridging factors will be determined by factors such as the patient's condition and the type and severity of the patient's disease, although appropriate doses and dosing schedules can be determined through clinical trials.

[0498] In an exemplary embodiment, the effective amount of modified immune effector cells provided to a subject is at least 2×10 6 cells / kg, at least 3×10 6 cells / kg, at least 4×10 6 cells / kg, at least 5×10 6 cells / kg, at least 6×10 6 cells / kg, at least 7×10 6 cells / kg, at least 8×10 6 cells / kg, at least 9×10 6 cells / kg, or at least 10×10 6 cells / kg or more, including all intermediate doses of cells.

[0499] In another exemplary embodiment, the effective amount of modified immune effector cells provided to a subject is about 2×10 6 cells / kg, about 3×10 6 cells / kg, about 4×10 6 cells / kg, about 5×10 6 cells / kg, about 6×10 6 cells / kg, about 7×10 6 cells / kg, about 8×10 6 cells / kg, about 9×10 6 cells / kg, or about 10×10 6 cells / kg or more, including all intermediate doses of cells.

[0500] In another exemplary embodiment, the effective amount of modified immune effector cells provided to a subject is about 2×10 6 cells / kg to about 10×10 6 cells / kg, about 3×10 6 cells / kg to about 10×10 6 cells / kg, about 4×10 6 cells / kg to about 10×10 6 cells / kg, about 5×10 6 cells / kg to about 10×10 6 cells / kg, 2×10 6 cells / kg to about 6×10 6 cells / kg, 2×10 6 cells / kg to about 7×10 6 cells / kg, 2×10 6 cells / kg to about 8×10 6 cells / kg, 3×10 6 cells / kg to about 6×10 6 cells / kg, 3×10 6 cells / kg to about 7×10 6 cells / kg, 3×10 6 cells / kg to about 8×10 6 cells / kg, 4×10 6 cells / kg to about 6×10 6 cells / kg, 4×10 6 cells / kg to about 7×10 6 cells / kg, 4×10 6 cells / kg to about 8×10 6 cells / kg, 5×10 6 cells / kg to about 6×10 6 cells / kg, 5×10 6 cells / kg to about 7×10 6 cells / kg, 5×10 6 cells / kg to about 8×10 6 cells / kg or 6×10 6 cells / kg to about 8×10 6 cells / kg, including all intermediate doses.

[0501] Those of ordinary skill in the art will recognize that it may be necessary to administer the compositions envisioned in a particular embodiment multiple times to affect the desired therapy. For example, the composition may be administered 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or 10 times or more over a span 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 longer. Modified immune effector cells, engineered immune receptor components, and / or bridging factors may be used in the same or different compositions; administered simultaneously in one or more compositions; or administered at different times in more than one composition. Modified immune effector cells, engineered immune receptor components (e.g., targeting components), and / or bridging factors may be administered by the same route of administration or by different routes.

[0502] In certain embodiments, it may be desirable to administer activated T cells to a subject, and then redraw blood (or perform apheresis), activate T cells therefrom, and re-infuse these activated and amplified T cells to the patient. This process may be performed multiple times every few weeks. In certain embodiments, 10cc to 400cc of blood may be drawn to activate T cells. In certain embodiments, 20cc, 30cc, 40cc, 50cc, 60cc, 70cc, 80cc, 90cc, 100cc, 150cc, 200cc, 250cc, 300cc, 350cc, or 400cc or more of blood may be drawn to activate T cells. Without being bound by theory, the use of this multiple blood draw / multiple re-infusion scheme may be used to select certain T cell populations.

[0503] In one embodiment, the method for treating a subject diagnosed with an immune-related disease or condition (e.g., cancer or autoimmune disease) comprises removing immune effector cells from the subject, modifying the immune effector cells and producing a modified immune effector cell colony by introducing one or more vectors encoding one or more engineered immune receptor components into cells, and applying the modified immune effector cell colony to the same subject. In a preferred embodiment, the immune effector cells comprise T cells.

[0504] 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.

[0505] In one embodiment, the method for treating a subject diagnosed with cancer comprises removing immune effector cells from the subject, modifying the immune effector cells by introducing one or more vectors encoding one or more engineered immune receptor components and optionally exogenous lymphocyte receptors or engineered antigen receptors or another targeting component or signaling component into the cells and producing a modified immune effector cell population, and administering the modified immune effector cell population to the same subject. In a preferred embodiment, the immune effector cells comprise T cells.

[0506] 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.

[0507] Methods for administering the cell compositions contemplated in specific embodiments include any method effective to result in reintroduction of modified immune effector cells ex vivo or modified progenitor cells reintroduced into immune effector cells that differentiate into mature immune effector cells when introduced into a subject. One method includes ex vivo modification of peripheral blood T cells by introducing into cells one or more vectors encoding one or more engineered immune receptor components and optionally exogenous lymphocyte receptors or engineered antigen receptors or another targeting component or signaling component, and returning the transduced cells to the subject.

[0508] Methods for administering the cell compositions contemplated in specific embodiments include any method effective to result in reintroduction of modified immune effector cells ex vivo or modified progenitor cells reintroduced into immune effector cells that differentiate into mature immune effector cells when introduced into a subject. One method includes ex vivo modification of peripheral blood T cells by introducing into cells one or more vectors encoding one or more engineered immune receptor components and optionally exogenous lymphocyte receptors or engineered antigen receptors or another targeting component or signaling component, and returning the transduced cells to the subject.

[0509] Methods for administering the vector compositions contemplated in particular embodiments include any method effective for generating modified immune effector cells in vivo.

[0510] J. Sequence Listing

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[0521]

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[0538] 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.

[0539] Although the foregoing embodiments have been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art, based on the teachings contemplated herein, that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. The following examples are provided for illustration only and are not limiting. One of ordinary skill in the art will readily recognize various non-critical parameters that may be changed or modified to produce substantially similar results.

[0540] Examples

[0541] Example 1

[0542] Construction of rapamycin-inducible TCR (T cell receptor)

[0543] Lentiviral vectors containing constructs including at least a multimerization domain (e.g., a rapamycin-induced dimerization domain), CD3 subunits, 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 CD8α or IgK-derived signal sequence), 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., antibody-derived targeting domains or natural ligand-derived targeting domains, various hinge and transmembrane domains, (e.g., hinge and transmembrane domains derived from CD4 or CD28), one or more intracellular domains (e.g., co-stimulatory domains), and one or more co-receptor or cytokine receptor domains (see Figures 1A-1H ). In some constructs, a transgenic TCR is also expressed along with the above components.

[0544] Example 2

[0545] Evaluation of rapamycin-induced TCR T cells without hinge-domain-anchored FKBP12

[0546] T cells expressing rapamycin-induced T cell receptors (SR-T cells) were generated using a 10-day transduction and expansion process, and then the expression and biological activity against specific target antigens were evaluated (see Leung et al., Journal of Clinical Investigation Insight (JCI Insight). 2019 April 30; 5 (11)). Briefly, peripheral blood mononuclear cells (PBMCs) were cultured in a medium containing IL-2 and activated with antibodies against human CD3 and human CD28. One day after the start of culture, PBMCs were transduced with a lentiviral vector encoding a rapamycin-induced TCR without a hinge domain between the FKBP12 multimerization domain of the targeting component (SR024) and the CD4 transmembrane domain (CD4tm), and then the cells were transferred to a 24-well plate 48 hours later. Culture system (Wilson Wolf). After a total of 10 days in culture, SR-T cells were evaluated for rapamycin-dependent, antigen-dependent activity, and rapamycin-independent, antigen-independent background activity.

[0547] SR-T cells (SR024-T cells) transduced with the above lentiviral vectors were analyzed by flow cytometry for the extracellular expression of key components of the rapamycin-induced system, including the targeting domain (e.g., anti-CD33 VHH), the FRB multimerization domain, and the FKBP12 multimerization domain (using recombinant CD33-Fc fusion, anti-FRB antibody, and anti-FKBP12 antibody, respectively). Figure 2 As shown, FRB and CD33 components were highly expressed compared to untransduced control cells.

[0548] 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, MSD of supernatants from cultures with SR024-T cells and cell lines engineered to express high levels of CD33 was used to quantify IFNγ, IL2, IL4, and TNFα production ( Figure 3 SR024-T cells released each of these 4 cytokines at levels that were highly distinguishable relative to baseline (untransduced control; UTD). In addition, T cells cultured alone in the absence of antigen ( Figure 4 ). In both cases, SR024-T cells produced 2 orders of magnitude less IFNγ than when cultured in the presence of antigen.

[0549] The cytotoxicity of SR024-T cells was assessed by incubating them with cells expressing the target antigen and a nuclear red fluorescent molecule. The live cell imaging system (Sartorius) tracked both cells expressing nuclear fluorescence (antigen-positive target lines) and cells not expressing nuclear fluorescence (SR-T cells) for approximately 200 hours. Although SR024-T cells did not effectively kill CD33+ target cells ( Figure 5A ), but they expanded significantly in these cultures compared with T cell controls ( Figure 5B ).

[0550] Example 3

[0551] Evaluation of rapamycin-inducible TCR with FKBP12 anchored to the CD4 or CD28 hinge domain

[0552] T cells

[0553] T cells expressing rapamycin-induced T cell receptors (SR-T cells) were generated using a 10-day transduction and expansion process, and then the expression and biological activity against specific target antigens were evaluated. In brief, PBMCs were cultured in medium containing IL-2 and activated with antibodies against human CD3 and human CD28. One day after the start of culture, PBMCs were transduced with a lentiviral vector encoding a rapamycin-induced TCR with a targeting component containing FKBP12 anchored to the CD4 or CD28 hinge domain (CD4h or CD28h) or without a hinge domain (SR022, SR020, and SR024), and then the cells were transferred to 24-well cells 48 hours later. Culture system. After a total of 10 days of culture, the rapamycin-dependent and antigen-dependent activities of SR-T cells, as well as the rapamycin-independent and antigen-independent background activities were evaluated.

[0554] SR-T cells (SR022-T cells, SR020-T cells, and SR024-T cells) transduced with the above lentiviral vectors were analyzed by flow cytometry for the extracellular expression of key components of the rapamycin-induced system, including targeting domains (e.g., anti-CD33 VHH), FRB multimerization domain, and FKBP12 multimerization domain (using recombinant CD33-Fc fusion, anti-FRB antibody, and anti-FKBP12 antibody, respectively). FRB and CD33 components were highly expressed ( Figure 6 ).

[0555] Rapamycin-dependent, antigen-dependent activity of SR022-T cells, SR020-T cells, and SR024-T cells was assessed in co-culture with target antigen-positive tumor cells in the presence of 1 nM rapamycin. Specifically, MSD of supernatants from cultures with SR022-T cells, SR020-T cells, or SR024-T cells and cell lines engineered to express high levels of CD33 was used to quantify IFNγ, IL2, IL4, and TNFα production ( Fig. 7A ). Both SR022-T cells and SR020-T cells released each of these 4 cytokines at levels that were highly distinguishable relative to baseline (untransduced control; UTD). SR020 showed reduced IFNγ, IL2, and TNFα, but comparable IL4 compared to SR022. SR024-T cells had reduced cytokine release in all 4 cytokines compared to SR022-T cells and SR020-T cells.

[0556] T cells were also co-cultured with CD33+ cell lines at high, medium, and low antigen densities. At all antigen densities, especially at low antigen density, SR022-T cells and SR020-T cells produced more IFNγ than SR024-T cells ( Figure 7B In addition, SR022-T cells and SR020-T cells produced IL2 in cultures at all antigen densities, whereas SR024-T cells produced almost no IL2 ( Figure 7C SR022-T cells secreted more IFNγ and IL2 in high, medium, and low CD33+ antigen density target lines. T cells were cultured alone in the absence of antigen and in the presence and absence of 1 nM rapamycin ( Figure 8 In the absence of antigen, SR024, SR022, and SR020-T cells produced IFNγ that was 2 orders of magnitude lower than in the presence of antigen.

[0557] Cytotoxicity was assessed by incubating SR024-T cells, SR022-T cells, and SR020-T cells with cells expressing target antigens and nuclear red staining. Both cells expressing nuclear stain (antigen-positive target lines) and cells not expressing nuclear stain (SR-T cells) were tracked for approximately 200 hours using a live cell imaging system (Sartorius). Although SR024-T cells showed minimal killing of CD33+ target cells ( Fig.9A ), but they expanded significantly in these cultures compared with T cell controls ( Fig. 9B ). Compared with T cell control, SR022-T cells were able to effectively kill CD33+ target cells ( Fig.9A) and amplify ( Fig. 9B Although SR020-T cells expanded well compared to the control ( Fig. 9B ), but they performed moderately in cytotoxicity assays ( Fig.9A ). Overall, SR022-T cells performed significantly better in the cytotoxicity assay compared to SR020-T cells or SR024-T cells.

[0558] Figures 7A-7C , 8 and 9A-9B show that SR020 and SR022 containing the hinge domain exhibited improved cytokine release profiles and increased CD33+ target cell killing compared to SR024 lacking the hinge domain (see Figure 1A and 1D Without wishing to be bound by any particular theory, it is contemplated that the addition of a hinge domain to the signaling component improves the functionality of the architecture by allowing the first multimerization domain of the signaling component to better align spatially with the multimerization domain of the targeting component, surprisingly increasing the efficacy of the targeting component comprising the hinge domain as compared to a targeting component lacking the hinge domain.

[0559] Example 4

[0560] Evaluation of rapamycin-induced TCR T cells with FRB attached to CD3γ

[0561] T cells expressing rapamycin-induced T cell receptors (SR-T cells) were generated using a 10-day transduction and expansion process, and then the expression and biological activity against specific target antigens were evaluated. Briefly, peripheral blood mononuclear cells (PBMCs) were cultured in medium containing IL-2 and activated with antibodies against human CD3 and human CD28. One day after the start of culture, PBMCs were transduced with a lentiviral vector (SR021) encoding a rapamycin-induced TCR with an FRB attached to CD3γ instead of CD3ε, and then the cells were transferred to 24-well plates 48 hours later. Culture system. After a total of 10 days of culture, the rapamycin-dependent and antigen-dependent activities of SR-T cells, as well as the rapamycin-independent and antigen-independent background activities were evaluated.

[0562] SR-T cells (SR021-T cells) transduced with the above lentiviral vectors were analyzed by flow cytometry for the extracellular expression of key components of the rapamycin-induced system, including targeting domains (e.g., anti-CD33 VHH), FRB dimerization domain, and FKBP12 dimerization domain (using recombinant CD33-Fc fusion, anti-FRB antibody, and anti-FKBP12 antibody, respectively). FRB and CD33 components were highly expressed ( Fig.10 ).

[0563] Rapamycin-dependent, antigen-dependent activity of SR021-T cells was assessed in co-culture with target antigen-positive tumor cells in the presence of 1 nM rapamycin. Specifically, MSD of supernatants from cultures with SR021-T cells and cell lines engineered to express high levels of CD33 was used to quantify IFNγ, IL2, IL4, and TNFα production ( Fig.11 SR021-T cells released each of these 4 cytokines at levels that were highly distinguishable relative to baseline (untransduced control; UTD). In addition, T cells cultured alone in the absence of antigen ( Fig.12 ). In the absence of antigen, SR021-T cells produced IFNγ 2 orders of magnitude lower than in the presence of antigen.

[0564] The cytotoxicity of SR021-T cells relative to cells expressing the target antigen was assessed using a target line expressing nuclear red staining and using The live cell imaging system (Sartorius) was used for tracking over time. Both cells expressing nuclear staining (antigen-positive target lines) and cells not expressing nuclear staining (SR-T cells) were tracked for approximately 200 hours. Compared with T cell controls, SR021-T cells showed a trend of killing CD33+ cells ( Fig.13A ) and significantly expanded ( Fig. 13B ).

[0565] Example 5

[0566] Evaluation of rapamycin-induced TCR T cells with FRB* (T2098L)

[0567] T cells expressing rapamycin-induced T cell receptors (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 medium containing IL-2 and activated with antibodies against human CD3 and human CD28. One day after the start of culture, PBMCs were transduced with a lentiviral vector encoding a rapamycin-induced TCR with FRB or mutant FRB-FRB (T2098L) (which allows binding to certain rapamycin analogs (denoted as FRB*)) (SR022 and SR001, respectively), and then the cells were transferred to 24-well plates 48 hours later. Culture system. After a total of 10 days of culture, the rapamycin-dependent and antigen-dependent activities of SR-T cells, as well as the rapamycin-independent and antigen-independent background activities were evaluated.

[0568] SR-T cells transduced with the above lentiviral vectors (SR022-T cells and SR001-T cells against FRB and FRB*, respectively) were analyzed by flow cytometry for the extracellular expression of key components of the rapamycin-induced system, including targeting domains (e.g., anti-CD33 VHH) and FRB multimerization domains (using recombinant CD33-Fc fusion and anti-FRB antibodies, respectively). FRB and CD33 components were highly expressed ( Fig.14 ).

[0569] Rapamycin-dependent, antigen-dependent activity of SR022-T cells, SR001 T cells was assessed in co-culture with target antigen-positive tumor cells in the presence of 1 nM rapamycin. Specifically, IFNγ, IL2, and TNFα production were quantified using MSD of supernatants from cultures with SR022-T cells or SR001-T cells and cell lines engineered to express high levels of CD33 ( Fig.15 ). Both SR022-T cells and SR001-T cells released each of these 3 cytokines at levels that were highly distinguishable relative to baseline (untransduced control; UTD). In addition, in the presence of 1 nM rapamycin, T cells cultured alone in the absence of antigen ( Fig.16 ). In the absence of antigen, IFNγ produced by SR022-T cells and SR001-T cells was 3 orders of magnitude lower than that in the presence of antigen.

[0570] The cytotoxicity of SR001-T cells relative to cells expressing the target antigen was assessed using a target line expressing nuclear red staining and using The live cell imaging system (Sartorius) was used for tracking over time. Both cells expressing nuclear staining (antigen-positive target lines) and cells not expressing nuclear staining (SR-T cells) were tracked for approximately 160 hours. Compared with T cell controls, SR001-T cells effectively killed CD33+ target cells ( Fig.17A ) and significantly expanded ( Fig. 17B ).

[0571] Example 6

[0572] Evaluation of rapamycin-inducible TCR T cells with FKBP12 located distal to the antigen targeting domain

[0573] In another experiment, T cells expressing rapamycin-induced T cell receptors (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 medium containing IL-2 and activated with antibodies against human CD3 and human CD28. One day after the start of culture, PBMCs were transduced with a lentiviral vector encoding a rapamycin-induced TCR with a CD4 hinge (CD4h), a transmembrane (CD4tm), and a minimal intracellular (CD4ic) domain anchored to an antigen targeting domain followed by a FKBP12 multimerization domain (SR028), and then the cells were transferred to a 24-well plate 48 hours later. Culture system. After a total of 10 days of culture, the rapamycin-dependent and antigen-dependent activities of SR-T cells, as well as the rapamycin-independent and antigen-independent background activities were evaluated.

[0574] SR-T cells (SR028-T cells) transduced with the above lentiviral vectors were analyzed by flow cytometry for the extracellular expression of key components of the rapamycin-induced system, including targeting domains (e.g., anti-CD33 VHH), FRB multimerization domain, and FKBP12 multimerization domain (using recombinant CD33-Fc fusion, anti-FRB antibody, and anti-FKBP12 antibody, respectively). FRB, FKBP12, and CD33 components were highly expressed ( Fig.18 ).

[0575] Rapamycin-dependent, antigen-dependent activity of SR028-T cells was assessed in co-culture with target antigen-positive tumor cells in the presence of 1 nM rapamycin. Specifically, MSD of supernatants from cultures with SR028-T cells and cell lines engineered to express high levels of CD33 was used to quantify IFNγ, IL2, IL4, and TNFα production ( Fig.19 ). SR028-T cells released each of these 4 cytokines at levels that were highly distinguishable relative to baseline (untransduced control; UTD). Additionally, T cells were cultured alone in the presence of 1 nM rapamycin in the absence of antigen. Although SR028-T cells did not produce IFNγ in the absence of rapamycin, when 1 nM rapamycin was added to SR028-T cells and antigen was not present, IFNγ was released at levels comparable to those in the presence of antigen ( Fig. 20 ). SR028-T cells produced a large amount of IFNγ in the absence of antigen compared to the amount of IFNγ produced in the presence of antigen.

[0576] The cytotoxicity of SR028-T cells relative to cells expressing the target antigen was assessed using a target line expressing nuclear red staining and using The live cell imaging system (Sartorius) was used for tracking over time. Both cells expressing nuclear staining (antigen-positive target lines) and cells not expressing nuclear staining (SR-T cells) were tracked for approximately 200 hours. Compared with T cell controls, SR028-T cells effectively killed CD33+ target cells ( Fig.21A ) and significantly expanded ( Fig.21B ).

[0577] Example 7

[0578] Evaluation of rapamycin-induced TCR T cells w...

Claims

1. A non-natural cell comprising: (a) a signaling component comprising (i) a first multimerization domain comprising a FRB polypeptide or a variant thereof or a FKBP polypeptide or a variant thereof, (ii) a first polypeptide linker, and (iii) a CD3ε polypeptide or a variant thereof; and (b) a targeting component comprising (i) an anti-CLL1 scFv or a single domain antibody (sdAb), (ii) an anti-CD33 scFv or a single domain antibody (sdAb), (iii) a second polypeptide linker, (iv) a second multimerization domain comprising a FRB polypeptide or a variant thereof or a 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-natural cell of claim 1, wherein the targeting component does not comprise a functional intracellular domain or a co-stimulatory domain having signaling capability.

3. The non-natural cell of any of the preceding claims, wherein the CD4 hinge polypeptide comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:

41.

4. The non-natural cell of any one of the preceding claims, wherein the CD4 hinge polypeptide comprises the amino acid sequence as shown in SEQ ID NO:

41.

5. The non-natural cell of any one of the preceding claims, wherein the CD3ε polypeptide comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:

32.

6. The non-natural cell of any one of the preceding claims, wherein the CD3ε polypeptide comprises the amino acid sequence as shown in SEQ ID NO:

32.

7. The non-natural cell of any one of the preceding claims, wherein the CD3ε polypeptide comprises both an extracellular portion and an intracellular portion.

8. The non-natural cell of any of the preceding claims, wherein when the signaling component and the targeting component are expressed, the FRB polypeptide and the FKBP polypeptide are localized extracellularly.

9. The non-natural cell of any of the preceding claims, wherein the first multimerization domain and the second multimerization domain are different.

10. The non-natural cell of any of the preceding claims, wherein the first multimerization domain comprises a FRB polypeptide or a variant thereof, and the second multimerization domain comprises a FKBP12 polypeptide or a variant thereof.

11. The non-natural cell of any of the preceding claims, 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-natural cell of any preceding claim, wherein the FRB polypeptide is the FRB T2098L variant.

13. The non-natural cell of any of the preceding claims, wherein the FRB polypeptide comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to, or comprises the sequence as set forth in, SEQ ID NO:

1.

14. The non-natural cell of any of the preceding claims, wherein the FRB polypeptide comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to, or comprises the sequence as set forth in, SEQ ID NO:

2.

15. The non-natural cell of any of the preceding claims, wherein the FKBP12 polypeptide comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to or comprises the sequence as set forth in SEQ ID NO:

3.

16. The non-natural cell of any of the preceding claims, wherein the FKBP12 polypeptide comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to or comprises the sequence as set forth in SEQ ID NO:

4.

17. The non-natural cell of any of the preceding claims, wherein the multimerization domains of the signaling component and the targeting component are associated with a bridging factor.

18. The non-natural cell of claim 17, wherein the bridging factor is selected from the group consisting of: rapamycin or a rapamycin analog thereof, gibberellin or a derivative thereof, abscisic acid (ABA) or a derivative thereof, methotrexate or a derivative thereof, cyclosporine A or a derivative thereof, FK506 / cyclosporine A or a derivative thereof, trimethoprim (Tmp) synthetic ligand (SLF) for FK506 binding protein (FKBP) or a derivative thereof, wherein the bridging factor promotes the formation of a polypeptide complex, and the bridging factor associates with the multimerization domains of the signaling component and the targeting component and is positioned between the multimerization domains.

19. The non-natural cell of claim 17 or claim 18, wherein the bridging factor is AP1903, AP20187, AP21967 (also known as C16-(S)-7-methylindorapamycin), everolimus, novolimus, pimecrolimus, ridaforolimus, sirolimus, tacrolimus, temsirolimus, umirolimus, zotarolimus, or BPC015.

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

21. The non-natural 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-natural cell of claim 21, wherein the first polypeptide linker is a 3xG4S linker.

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

24. The non-natural 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, 2x G4S, 3xG4s, 4xG4S, and any combination thereof.

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

26. The non-natural cell of any one of the preceding claims, wherein the anti-CLL1 scFv or sdAb and the anti-CD33 scFv or sdAb are separated by a third polypeptide linker.

27. The non-natural cell of claim 26, wherein the third polypeptide linker is a linker of 2 to 40 amino acids in length.

28. The non-natural 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, 2x G4S, 3xG4s, 4xG4S, and any combination thereof.

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

30. The non-natural cell of any of the preceding claims, wherein the CD4 transmembrane domain comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:

45.

31. The non-natural cell of any one of the preceding claims, wherein the CD4 transmembrane polypeptide comprises the amino acid sequence as shown in SEQ ID NO:

45.

32. The non-natural cell of any one of the preceding claims, wherein the truncated intracellular CD4 polypeptide comprises the amino acid sequence shown in SEQ ID NO:

48.

33. The non-natural 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 the preceding claims, wherein the anti-CLL1 antibody and the anti-CD33 antibody are each sdAbs.

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

36. The non-naturally occurring cell of any one of the preceding claims, wherein the sdAb is a camelid VHH.

37. The non-natural cell of any one of the preceding claims, wherein the scFv or the sdAb is human or humanized.

38. The non-natural cell of any of the preceding claims, 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. The non-natural cell of any of the preceding claims, wherein the anti-CLL1 scFv or sdAb comprises a sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:

75.

40. The non-natural cell of any one of the preceding claims, wherein the anti-CLL1 scFv or sdAb comprises the sequence shown in SEQ ID NO:

75.

41. The non-natural cell of any of the preceding claims, 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. The non-natural cell of any of the preceding claims, wherein the anti-CD33 scFv or sdAb comprises a sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:

72.

43. The non-natural cell of any one of the preceding claims, wherein the anti-CD33 scFv or sdAb comprises the sequence shown as SEQ ID NO:

72.

44. The non-natural cell of any of the preceding claims, wherein the signaling component further comprises a signal sequence.

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

46. ​​The non-natural cell of claim 45, wherein the CD8 signal sequence comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:

96.

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

96.

48. The non-natural cell of any of the preceding claims, wherein the targeting component further comprises a signal sequence.

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

50. The non-natural cell of claim 49, wherein the IgK signal sequence comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:

95.

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

95.

52. The non-natural cell of any of the preceding claims, wherein the signaling component comprises a sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:

111.

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

111.

54. The non-natural cell of any of the preceding claims, wherein the targeting component comprises a sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to the sequence shown in SEQ ID NO:

122.

55. The non-natural cell of claim 54, wherein the targeting component comprises the sequence shown in SEQ ID NO:

122.

56. The non-natural cell of any of the preceding claims, comprising a fusion polypeptide comprising the targeting component and the signaling component.

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

151.

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

151.

59. The non-natural cell of any of the preceding claims, wherein the cell comprises a first nucleic acid molecule encoding the signaling component.

60. The non-natural cell of any preceding claim, wherein the cell comprises a second nucleic acid molecule encoding the targeting component.

61. The non-natural cell of any of the preceding claims, wherein the cell comprises a nucleic acid molecule encoding both the signaling component and the targeting component.

62. The non-natural cell of any of the preceding claims, wherein the cell further expresses an exogenous co-stimulatory factor, an immunomodulatory factor, an agonist of a co-stimulatory factor, an antagonist of an immunosuppressive factor, an immune cell engager, a flip receptor, or any combination thereof.

63. The non-natural cell of any preceding claim, wherein the cell further expresses an exogenous lymphocyte receptor or co-receptor.

64. The non-natural cell of claim 63, wherein the exogenous lymphocyte receptor or co-receptor is selected from the group consisting of TCRα (TCR alpha), TCRβ (TCR beta), TCRγ (TCR gamma), TCRδ (TCRdelta), 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. The non-natural cell of any of the preceding claims, wherein the cell further expresses an exogenous TCR.

66. The non-natural cell of claim 65, wherein the exogenous TCR binds to a target antigen selected from the group consisting of alpha-fetoprotein (AFP), members of the B melanoma antigen (BAGE) family, brothers of regulatory factors at imprinted sites (BORIS), cancer-testis antigen, cancer-testis antigen 83 (CT-83), carbonic anhydrase IX (CA1X), carcinoembryonic antigen (CEA), cytomegalovirus (CMV) antigen, antigen recognized by cytotoxic T cells (CTL) on melanoma (CAMEL), Epstein-Barr virus (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), antigen preferentially expressed 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 X 8 (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. The non-natural cell of claim 65 or claim 66, wherein the exogenous TCR is an αβ-TCR or a γδ-TCR.

68. The non-natural cell of any of the preceding claims, wherein the cell further expresses a CAR, a CCR, or a flip receptor.

69. The non-natural cell of any of the preceding claims, wherein the cell further expresses a zetakine, an immune cell engager, or a BiTE.

70. The non-natural cell of any of the preceding claims, wherein the cell is a hematopoietic cell.

71. The non-natural cell of any of the preceding claims, wherein the cell is a T cell, an αβ-T cell, or a γδ-T cell.

72. The non-natural cell of any of the preceding claims, wherein the cell is a CD3 + cells, CD4 + cells and / or CD8 + cell.

73. The non-natural cell of any of the preceding claims, wherein the cell is an immune effector cell.

74. The non-natural cell of any of the preceding claims, wherein the cell is a cytotoxic T lymphocyte (CTL), a tumor infiltrating lymphocyte (TIL), or a helper T cell.

75. The non-natural cell of any one of the preceding claims, wherein the cell is a Natural Killer (NK) cell or a Natural Killer T (NKT) cell.

76. The non-natural cell of any of the preceding claims, 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. The non-naturally occurring cell of any one of the preceding claims, wherein the non-naturally occurring cell is an isolated non-naturally occurring cell.

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

79. The non-natural cell of any of the preceding claims, wherein the non-natural cell is a human cell.

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

81. The fusion polypeptide of claim 80, wherein the targeting component does not comprise a functional intracellular domain or a co-stimulatory 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 that is at least 90%, 95%, 96%, 97%, 98% or 99% identical 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 that is at least 90%, 95%, 96%, 97%, 98% or 99% identical 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 as 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 when the signaling component and the targeting component are expressed, the FRB polypeptide and the FKBP polypeptide are localized extracellularly.

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

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

90. The fusion polypeptide of any one of claims 80 to 89, 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.

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

92. The fusion polypeptide of any one of claims 80 to 91, wherein the FRB polypeptide comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1 or comprises the sequence as shown in 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 that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:2 or comprises the sequence as shown in 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 that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:3 or comprises the sequence as shown in 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 that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:4 or comprises the sequence as shown in SEQ ID NO:

4.

96. The fusion polypeptide of any one of claims 80 to 95, wherein the multimerization domains of the signaling component and the targeting component are associated with a bridging factor.

97. The fusion polypeptide of claim 96, wherein the bridging factor is selected from the group consisting of: rapamycin or a rapamycin analog thereof, gibberellins or derivatives thereof, abscisic acid (ABA) or derivatives thereof, methotrexate or derivatives thereof, cyclosporine A or derivatives thereof, FK506 / cyclosporine A or derivatives thereof, trimethoprim (Tmp) synthetic ligand (SLF) for FK506 binding protein (FKBP) or derivatives thereof, wherein the bridging factor promotes the formation of a polypeptide complex, and the bridging factor associates with the multimerization domains of the signaling component and the targeting component and is positioned between the multimerization domains.

98. The fusion polypeptide of claim 96 or claim 97, wherein the bridging factor is AP1903, AP20187, AP21967 (also known as C16-(S)-7-methylindorapamycin), everolimus, norvolimus, pimecrolimus, dafolimus, sirolimus, tacrolimus, temsirolimus, urolimus, 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. 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. 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, 2x G4S, 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, 2x G4S, 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 that is at least 90%, 95%, 96%, 97%, 98% or 99% identical 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 as 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 as 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 sdAbs.

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 camelid VHH.

116. The fusion polypeptide of any one of claims 80 to 115, wherein the scFv or the 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 at least 90%, 95%, 96%, 97%, 98% or 99% identical 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 that is at least 90%, 95%, 96%, 97%, 98% or 99% identical 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 that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:

96.

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

96.

127. The fusion polypeptide of any one of claims 80 to 126, wherein the targeting component 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 that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:

95.

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

95.

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

111.

132. The fusion polypeptide of claim 131, wherein the signaling component comprises the sequence shown in SEQ ID NO:

111.

133. A fusion polypeptide according to any one of claims 80 to 132, wherein the targeting component comprises a sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to the sequence shown in SEQ ID NO:

122.

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

122.

135. The fusion polypeptide of any one of claims 80 to 134, comprising a fusion polypeptide comprising the targeting component and the signaling component.

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

151.

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

151.

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

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

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

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

142. A cell comprising the nucleic acid molecule according to claim 140.

143. The cell of claim 140 or claim 141, further expressing an exogenous co-stimulatory factor, an immunomodulatory factor, an agonist of a co-stimulatory factor, an antagonist of 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 TCRα (TCR alpha), TCRβ (TCR beta), TCRγ (TCR gamma), TCRδ (TCR delta), 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 cell of claim 146, wherein the exogenous TCR binds to a target antigen selected from the group consisting of alpha-fetoprotein (AFP), members of the B melanoma antigen (BAGE) family, brothers of regulatory factors at imprinted sites (BORIS), cancer-testis antigen, cancer-testis antigen 83 (CT-83), carbonic anhydrase IX (CA1X), carcinoembryonic antigen (CEA), cytomegalovirus (CMV) antigen, antigen recognized by cytotoxic T cells (CTL) on 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), antigen preferentially expressed 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 X 8 (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, a CCR or a 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 cell according to any one of claims 141 to 152, wherein the cell is a CD3 + cells, CD4 + cells and / or CD8 + 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. The cell of any one of claims 141 to 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. A polypeptide complex comprising: (a) a signaling component comprising (i) a first multimerization domain comprising a FRB polypeptide or a variant thereof or a FKBP polypeptide or a variant thereof, (ii) a first polypeptide linker, and (iii) a CD3ε polypeptide or a variant thereof; and (b) a targeting component comprising (i) an anti-CLL1 scFv or a single domain antibody (sdAb), (ii) an anti-CD33 scFv or a single domain antibody (sdAb), (iii) a second polypeptide linker, (iv) a second multimerization domain comprising a FRB polypeptide or a variant thereof or a FKBP polypeptide or a variant thereof, (v) a CD4 hinge polypeptide, (vi) a CD4 transmembrane polypeptide, and (vii) a truncated CD4 intracellular polypeptide.

162. The polypeptide complex of claim 161, wherein the targeting component does not comprise a functional intracellular domain or co-stimulatory domain having signaling capability.

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

41.

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

41.

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

32.

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

32.

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

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

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

171. The polypeptide complex of any one of claims 161 to 170, 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.

172. The polypeptide complex of any one of claims 161 to 171, wherein the FRB polypeptide is a FRBT2098L variant.

173. The polypeptide complex of any one of claims 161 to 172, wherein the FRB polypeptide comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1 or comprises the sequence as shown in 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 that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:2 or comprises the sequence as shown in SEQ ID NO:

2.

175. The polypeptide complex of any one of claims 161 to 174, wherein the FKBP12 polypeptide comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:3 or comprises the sequence as shown in SEQ ID NO:

3.

176. The polypeptide complex of any one of claims 161 to 175, wherein the FKBP12 polypeptide comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:4 or comprises the sequence as shown in SEQ ID NO:

4.

177. The polypeptide complex of any one of claims 161 to 176, wherein the multimerization domains of the signaling component and the targeting component are associated with a bridging factor.

178. The polypeptide complex of claim 177, wherein the bridging factor is selected from the group consisting of: rapamycin or a rapamycin analog thereof, gibberellins or derivatives thereof, abscisic acid (ABA) or derivatives thereof, methotrexate or derivatives thereof, cyclosporine A or derivatives thereof, FK506 / cyclosporine A or derivatives thereof, trimethoprim (Tmp) synthetic ligand (SLF) for FK506 binding protein (FKBP) or derivatives thereof, wherein the bridging factor promotes the formation of the polypeptide complex, and the bridging factor is associated with the multimerization domains of the signaling component and the targeting component and is positioned between the multimerization domains.

179. The polypeptide complex of claim 177 or claim 178, wherein the bridging factor is AP1903, AP20187, AP21967 (also known as C16-(S)-7-methylindorapamycin), everolimus, norvolimus, pimecrolimus, dafolimus, sirolimus, tacrolimus, temsirolimus, urolimus, zotarolimus or BPC015.

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. 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, 2x G4S, 3xG4s, 4xG4S, and any combination thereof.

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

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 of 2 to 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, 2x G4S, 3xG4s, 4xG4S, and any combination thereof.

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

190. The polypeptide complex of any one of claims 161 to 189, wherein the CD4 transmembrane domain comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:

45.

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

45.

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

48.

193. The polypeptide complex of any one of claims 161 to 191, wherein the truncated intracellular CD4 polypeptide comprises the amino acid sequence as 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 sdAbs.

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

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

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

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

75.

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

72.

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 to 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 that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:

96.

207. The polypeptide complex according to claim 206, wherein the CD8 signal sequence comprises the amino acid sequence shown in SEQ ID NO:

96.

208. The polypeptide complex of any one of claims 161 to 207, wherein the targeting component 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 that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:

95.

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

95.

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

111.

213. The polypeptide complex of claim 212, wherein the signaling component comprises the sequence shown in SEQ ID NO:

111.

214. The polypeptide complex of any one of claims 161 to 213, wherein the targeting component comprises a sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to the sequence shown in SEQ ID NO:

122.

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

122.

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

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

151.

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

151.

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

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

221. A cell comprising the nucleic acid molecule according to claim 220.

222. The cell of any one of claims 220 or 221, wherein the cell further expresses an exogenous co-stimulatory factor, an immunomodulatory factor, an agonist of a co-stimulatory factor, an antagonist of an immunosuppressive factor, 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 TCRα (TCR alpha), TCRβ (TCR beta), TCRγ (TCR gamma), TCRδ (TCR delta), CD4, CD8, pre-T cell receptor α (pTα), Fc receptor α (FcRα), Fc receptor β (FcRβ), Fc receptor γ (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 cell of claim 225, wherein the exogenous TCR binds to a target antigen selected from the group consisting of: alpha-fetoprotein (AFP), members of the B melanoma antigen (BAGE) family, brothers of regulatory factors at imprinted sites (BORIS), cancer-testis antigen, cancer-testis antigen 83 (CT-83), carbonic anhydrase IX (CA1X), carcinoembryonic antigen (CEA), cytomegalovirus (CMV) antigen, antigen recognized by cytotoxic T cells (CTL) on 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), antigen preferentially expressed 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 X 8 (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 to 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 cell according to any one of claims 220 to 231, wherein the cell is a CD3 + cells, CD4 + cells and / or CD8 + 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. The cell of any one of claims 220 to 235, wherein the source of the cell is peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, 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 component and the targeting component of the fusion polypeptide of any one of claims 80 to 139 or the polypeptide complex of any one of claims 161 to 219.

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

242. An RNA encoding the signaling component and the targeting component of 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 according to 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 adenoviral vector, an adeno-associated virus (AAV) vector, a herpes virus vector, a vaccinia virus vector, or a retroviral 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) virus; 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 the fusion polypeptide of any one of claims 80 to 139, the polynucleotide of claim 240, or the vector of 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 cell according to any one of claims 251 to 244, wherein the cell expresses CD3 + 、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. 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. A composition comprising a physiologically acceptable carrier and 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.

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

261. A method for treating, preventing or ameliorating at least one symptom of cancer, infectious diseases, autoimmune diseases, inflammatory diseases and immunodeficiency or conditions associated therewith, the method comprising administering to a subject an effective amount of a composition according to claim 258 or claim 259.

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

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, 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.

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

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

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

268. The non-natural cell of any one of claims 1 to 79, wherein the cell further comprises a polypeptide that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:

137.

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

137.

270. The non-natural cell of any one of claims 1 to 79, wherein the cell further comprises a polypeptide that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:

138.

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

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