Cells expressing anti-CD70 chimeric receptors and uses thereof

Abstract

The presently disclosed subject matter provides cells, compositions, and methods for enhancing immune responses to tumor antigens. These cells comprise a first antigen-recognizing receptor that targets CD70. These cells have improved activity and / or efficiency. The present invention also provides a method for reducing tumor burden in a subject with renal cell carcinoma, pancreatic cancer, or ovarian cancer, comprising administering to the subject an effective amount of cells comprising a TCR-like fusion molecule that targets CD70.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 380,482, filed October 21, 2022, the contents of which are incorporated by reference in their entirety and to which priority is claimed.

[0002] Sequence Listing A sequence listing conforming to the rules of WIPO Standard ST.26 is incorporated herein by reference. The sequence listing has been submitted via the PatentCenter as an electronic document coded as XML in UTF-8 text. The electronic document, created on October 18, 2023, is entitled "072734_1498_SL" and is 102,400 bytes in size.

[0003] Introduction The presently disclosed subject matter provides cells, compositions, and methods for enhancing immune responses to tumor antigens. These cells comprise an antigen-recognition receptor (e.g., a TCR-like fusion molecule) that targets CD70. The cells disclosed herein have improved activity against solid tumors (e.g., renal cell carcinoma). [Background technology]

[0004] Cell-based immunotherapy is a potentially curative therapy for the treatment of cancer. T cells and other immune cells can be modified to target tumor antigens through the introduction of genetic material encoding antigen-recognition receptors, such as TCR-like fusion molecules. Patient-engineered CAR T cells have shown remarkable efficacy against a variety of liquid and solid malignancies. However, treatment failure and relapse occur in a large proportion of patients. Therefore, there remains a need for improved immunotherapies. Summary of the Invention [Means for solving the problem]

[0005] The presently disclosed subject matter provides methods of reducing tumor burden and / or preventing and / or treating tumors in a subject with renal cell carcinoma, pancreatic cancer, or ovarian cancer. Additionally or alternatively, the presently disclosed subject matter provides methods of reducing tumor burden and / or preventing and / or treating tumors in a subject, wherein the tumor is renal cell carcinoma, pancreatic cancer, or ovarian cancer.

[0006] In certain embodiments, the method comprises administering to a subject an effective amount of cells comprising a TCR-like fusion molecule that targets CD70. In certain embodiments, the method reduces the number of tumor cells, reduces tumor size, and / or eradicates the tumor in the subject.

[0007] The presently disclosed subject matter further provides a method for preventing and / or treating a tumor in a subject in need thereof, the method comprising: obtaining a tumor sample from the subject having an undetectable CD70 polypeptide level; detecting a CD70 polynucleotide by FISH; and administering to the subject an effective amount of cells comprising a TCR-like fusion molecule that targets CD70 if the CD70 polynucleotide is detected.The presently disclosed subject matter further provides a method for preventing and / or treating a tumor in a subject in need thereof, the method comprising: obtaining a tumor sample from the subject having an undetectable CD70 polypeptide level; a) contacting the sample with an Ezh2 inhibitor; and b) administering to the subject an effective amount of cells comprising a TCR-like fusion molecule that targets CD70 if the CD70 polypeptide is detected in the sample.

[0008] In certain embodiments, the TCR-like fusion molecule comprises i) a first antigen-binding chain comprising an antigen-binding fragment of an antibody heavy chain variable region (VH), and ii) a second antigen-binding chain comprising an antigen-binding fragment of an antibody light chain variable region (VL), wherein the first and second antigen-binding chains a) each comprise a TRAC polypeptide or a TRBC polypeptide, and b) bind to a second antigen, and the TCR-like fusion molecule binds to the second antigen in an HLA-independent manner.

[0009] In certain embodiments, at least one of the TRAC polypeptide and the TRBC polypeptide is endogenous. In certain embodiments, the first and second antigen binding chains are about 1 x 10 -8 In certain embodiments, the first and second antigen-binding chains bind to the second antigen with a dissociation constant (KD) of about 5×10 M or less. -9 It binds to a second antigen with a dissociation constant (KD) of M or less.

[0010] In certain embodiments, the first antigen binding chain comprises an antigen-binding fragment of an antibody VH and a TRBC polypeptide, and the second antigen binding chain comprises an antigen-binding fragment of an antibody VL and a TRAC polypeptide. In certain embodiments, the first antigen binding chain comprises an antigen-binding fragment of an antibody VH and a TRAC polypeptide, and the second antigen binding chain comprises an antigen-binding fragment of an antibody VL and a TRBC polypeptide.

[0011] In certain embodiments, i) the first antigen binding chain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 34, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 35, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 36, and the second antigen binding chain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 37, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 38, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 39. In certain embodiments, the first antigen binding chain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 37, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 38, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 39, and the second antigen binding chain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 34, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 35, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 36.

[0012] In certain embodiments, the first antigen binding chain comprises CDR1, CDR2, and CDR3 of the heavy chain variable region set forth in SEQ ID NO: 40, and the second antigen binding chain comprises CDR1, CDR2, and CDR3 of the light chain variable region set forth in SEQ ID NO: 42. In certain embodiments, the first antigen binding chain comprises CDR1, CDR2, and CDR3 of the light chain variable region set forth in SEQ ID NO: 42, and the second antigen binding chain comprises CDR1, CDR2, and CDR3 of the heavy chain variable region set forth in SEQ ID NO: 40. In certain embodiments, the first antigen binding chain comprises the heavy chain variable region set forth in SEQ ID NO: 40, and the second antigen binding chain comprises CDR1, CDR2, and CDR3 of the light chain variable region set forth in SEQ ID NO: 42. In certain embodiments, the first antigen binding chain light chain variable region set forth in SEQ ID NO: 42 and the second antigen binding chain comprise the heavy chain variable region set forth in SEQ ID NO: 40.

[0013] In certain embodiments, the first and second antigen-binding chains can associate with a CD3ζ polypeptide. In certain embodiments, the first and second antigen-binding chains can activate the CD3ζ polypeptide upon binding to a second antigen. In certain embodiments, activation of the CD3ζ polypeptide can activate a cell.

[0014] In certain embodiments, the cell further comprises a gene disruption of the TRAC locus. In certain embodiments, the cell further comprises a gene disruption of the CD70 locus. In certain embodiments, the cell further comprises a gene disruption of the TRAC locus and CD70.

[0015] In certain embodiments, the tumor comprises tumor cells with low CD70 antigen density. In certain embodiments, the tumor has a low tumor cell frequency of CD70+ tumor cells. In certain embodiments, the tumor comprises a CD70 polypeptide that is undetectable by immunodiffusion, immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, Western blotting, binder-ligand assay, immunohistochemical techniques, agglutination, complement assay, high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), hyperdiffusion chromatography, or a combination thereof. In certain embodiments, the tumor and / or neoplasm comprises a CD70 polypeptide that is undetectable by immunohistochemistry (IHC). In certain embodiments, the tumor comprises a CD70 polypeptide that is undetectable by immunohistochemistry (IHC).

[0016] In certain embodiments, the cell is a lymphoid lineage cell or a myeloid lineage cell. In certain embodiments, the lymphoid lineage cell is selected from the group consisting of a T cell, a B cell, a natural killer (NK) cell, and a dendritic cell. In certain embodiments, the cell is a T cell. In certain embodiments, the T cell is derived from an induced pluripotent stem cell. In certain embodiments, the T cell is a CD8 + In certain embodiments, CD8 + The T cells are CD4-independent. In certain embodiments, the T cells are selected from the group consisting of cytotoxic T lymphocytes (CTLs), γδ T cells, tumor-infiltrating lymphocytes (TILs), regulatory T cells, and natural killer T (NKT) cells.

[0017] In certain embodiments, the cell further comprises a chimeric antigen receptor (CAR) that targets a second antigen. In certain embodiments, the CAR comprises an extracellular antigen-binding domain that binds to the first antigen and an intracellular signaling domain that can deliver an activation signal to the cell. In certain embodiments, the intracellular signaling domain of the CAR comprises a CD3ζ polypeptide. In certain embodiments, the CD3ζ polypeptide is a native CD3ζ polypeptide or a modified CD3ζ polypeptide. In certain embodiments, the modified CD3ζ polypeptide comprises a native ITAM1, an ITAM2 variant consisting of two loss-of-function mutations, and an ITAM3 variant consisting of two loss-of-function mutations. In certain embodiments, the intracellular signaling domain of the CAR further comprises at least one costimulatory signaling region. In certain embodiments, the at least one costimulatory signaling region comprises at least the intracellular domain of a costimulatory molecule or a portion thereof. In certain embodiments, the costimulatory molecule is selected from the group consisting of CD28, 4-1BB, OX40, CD27, CD40, CD154, CD97, CD11a / CD18, ICOS, DAP-10, CD2, CD150, CD226, and NKG2D. In certain embodiments, the CAR comprises a transmembrane domain.

[0018] In certain embodiments, the cell further comprises a chimeric costimulatory receptor (CCR). In certain embodiments, the CCR comprises an extracellular antigen-binding domain that binds to a third antigen and an intracellular domain that can deliver a costimulatory signal to the cell but does not alone deliver an activating signal to the cell. In certain embodiments, the intracellular domain of the CCR comprises at least the intracellular domain of a costimulatory molecule, or a portion thereof. In certain embodiments, the costimulatory molecule is selected from the group consisting of CD28, 4-1BB, OX40, CD27, CD40, CD154, CD97, CD11a / CD18, ICOS, DAP-10, CD2, CD150, CD226, and NKG2D.

[0019] In certain embodiments, the second antigen is a tumor antigen or a pathogen antigen. In certain embodiments, the tumor antigen is CD19, IL1RAP, ABCG2, AChR, ACKR6, ADAMTS13, ADGRE2, ADGRE2 (EMR2), ADORA3, ADRA1D, AGER, ALS2, an antigen of a cytomegalovirus (CMV)-infected cell (e.g., a cell surface antigen), ANO9, AQP2, ASIC3, ASPRV1, ATP6V0A4, B3GNT4, B7-H3, BCMA, BEST4, C3orf35, CADM3, CAIX, CAPN3, CCDC155, CCR1, CD10, CD117, C D123, CD133, CD135(FLT3), CD138, CD20, CD22, CD244(2B4), CD25, CD26, CD30, CD300LF, CD312, CD32, CD321, CD33, CD34, CD36, CD38, CD41, CD44, CD44V6, CD47, CD49f, CD56, CD7, CD71, CD74, CD8, CD82, CD96, CD98, CD99, CDH13, CDHR1, CEA, CEACAM6, CHST3, CLEC12A, CLEC1A, CLL1, CNIH2, COL1 5A1, COLEC12, CPM, CR1, CX3CR1, CXCR4, CYP4F11, DAGLB, DARC, DFNB31, DGKI, EGF1R, EGFR-VIII, EGP-2, EGP-40, ELOVL6, EMB, EMC10, EMR2, ENG, E pCAM, EphA2, EPHA4, ERBB, ERBB2, Erb-B3, Erb-B4, E-selectin, EXOC3L4, EXTL3, FAM186B, FBP, FCGR1A, FKBP1B, FLRT1, folate receptor-α, FOLR2, FRMD5, GABRB 2, GAS2, GD2, GD3, GDPD3, GNA14, GNAZ, GPR153, GPR56, GYPA, HEPHL1, HER-2, hERT, HILPDA, HLA-DR, HOOK1, hTERT, HTR2A, ICAM1, IGFBP3, IL10RB, IL20RB, IL23R, ILDR1, interleukin-13 receptor subunit alpha-2 (IL-13Rα2), ITFG3, ITGA4, ITGA5, ITGA8, ITGAX, ITGB5, ITGB8, JAM3, KCND1, KCNJ5, KCNK13KCNN4, KCNV2, KDR, KIF19, KIF26B, kappa light chain, L1CAM, LAX1, LEPR, Lewis Y (CD174), Lewis Y (LeY), LILRA2, LILRA6, LILRB2, LILRB3, LILRB4, LOXL4, LPAR2, LRRC37A3, LRRC8E, LRRN2, LRRTM2, LTB4R, MAGE-A1, MAGEA3, MANSC1, MART1, GP100, MBOAT1, MBOAT7, melanoma antigen family A, mesothelin (MSLN), MFAP3L, MMP25, MRP1, MT-ND1, mucin 1 (MUC1), mucin 16 (MUC16), MYADM, MYADML2, NGFR, NK CS1, NKG2D ligand, NLGN3, NPAS2, NY-ESO-1, oncofetal antigen (h5T4), OTOA, P2RY13, p53, PDE3A, PEAR1, PIEZO1, PLXNA4, PLXNC1, PNPLA3, PPFIA4, PPP2R5B, PRAME, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), proteinase 3 (PR1), PSD2, PTPRJ, RDH16, receptor tyrosine-protein kinase Erb-B2, RHBDL3, RNF173, R NF183, ROR1, RYR2, SCIN, SCN11A, SCN2A, SCNN1D, SEC31B, SEMA4A, SH3PXD2A, SIGLEC11, SIRPB1, SLC16A6, SLC19A1, SLC22A5, SLC25A36, SLC 25A41, SLC30A1, SLC34A3, SLC43A3, SLC44A1, SLC44A3, SLC45A3, SLC6A16, SLC6A6, SLC8A3, SLC9A1, SLCO2B1, SPAG17, STC1, STON2, SUN3, Saba Ivin, SUSD2, SYNC, TACSTD2, TAS1R3, TEX29, TFR2, TIM-3 (HAVCR2), TLR2, TMEFF2, TMEM145, TMEM27, TMEM40, TMEM59L, TMEM89, TMPRSS5, TNFRSF14, TNFRSF1B, TRIM55, TSPEAR, TTYH3, tumor-associated glycoprotein 72 (TAG-72), tyrosinase, vascular endothelial growth factor R2 (VEGF-R2), VLA-4, Wilms tumor protein (WT-1), WNT4, WT1,and ZDHHC11.

[0020] In certain embodiments, the cells further comprise at least one exogenous costimulatory ligand. In certain embodiments, the at least one exogenous costimulatory ligand is selected from the group consisting of a tumor necrosis factor (TNF) family member, an immunoglobulin (Ig) superfamily member, and a combination thereof. In certain embodiments, the TNF family member is selected from the group consisting of 4-1BBL, OX40L, CD70, FasL, GITRL, TNF-related apoptosis-inducing ligand (TRAIL), CD30L, LIGHT (TNFSF14), and CD40L.

[0021] In certain embodiments, the Ig superfamily member is selected from the group consisting of CD80, CD86, ICOSLG, and combinations thereof. In certain embodiments, the at least one exogenous costimulatory ligand comprises CD80. In certain embodiments, the at least one exogenous costimulatory ligand comprises 4-1BBL. In certain embodiments, the cells comprise two exogenous costimulatory ligands. In certain embodiments, the at least two exogenous costimulatory ligands comprise CD80 and 4-1BBL.

[0022] In certain embodiments, the cells further comprise a fusion polypeptide comprising a) the extracellular domain and transmembrane domain of a costimulatory ligand and b) the intracellular domain of a first costimulatory molecule. In certain embodiments, the costimulatory ligand is selected from the group consisting of a tumor necrosis factor (TNF) family member, an immunoglobulin (Ig) superfamily member, and a combination thereof. In certain embodiments, the TNF family member is selected from the group consisting of 4-1BBL, OX40L, CD70, GITRL, CD40L, and a combination thereof. In certain embodiments, the Ig superfamily member is selected from the group consisting of CD80, CD86, ICOSLG, and a combination thereof. In certain embodiments, the costimulatory ligand is CD80. In certain embodiments, the first costimulatory molecule is selected from the group consisting of CD28, 4-1BB, OX40, ICOS, DAP-10, CD27, CD40, NKG2D, CD2, and a combination thereof. In certain embodiments, the first costimulatory molecule is 4-1BB. In certain embodiments, the costimulatory ligand is CD80 and the first costimulatory molecule is 4-1BB. In certain embodiments, the fusion polypeptide further comprises the intracellular domain of a second costimulatory molecule. In certain embodiments, the second costimulatory molecule is selected from the group consisting of CD28, 4-1BB, OX40, ICOS, DAP-10, CD27, CD40, NKG2D, CD2, and combinations thereof. In certain embodiments, the second costimulatory molecule is CD28. In certain embodiments, the costimulatory ligand is CD80, the first costimulatory molecule is 4-1BB, and the second costimulatory molecule is CD28.

[0023] In certain embodiments, the cells are autologous. In certain embodiments, the cells are allogeneic.

[0024] The following detailed description, which is provided by way of example and is not intended to limit the subject matter disclosed herein to the specific embodiments described, can be understood in conjunction with the accompanying drawings. [Brief explanation of the drawings]

[0025] [Figure 1] A cartoon illustrating the challenges facing CARs in solid tumors is shown. [Figure 2A] Two RCC PDX models, "K5" and "K7" (histology, prior treatment, and genetic alterations) are characterized. [Figure 2B] 1 shows CD70 expression in K5 and K7 cells in vitro. [Figure 2C] 1 shows a schematic diagram of a CD70-28z1XX CAR with a CD28 costimulatory domain and an attenuated zeta chain signaling domain, designated "1XX." [Figure 3A] 1 shows the set up of a renal cell carcinoma PDX model, and CD70 CAR-mediated killing in vitro. [Figure 3B] Figure 1 shows the setup of a renal cell carcinoma PDX model. Figure 2 shows a K7 RCC PDX lung metastasis model obtained by tail vein administration. [Figure 3C] Figure 1 shows the setup of a renal cell carcinoma PDX model. Figure 2 shows a K5 RCC PDX lung metastasis model obtained by tail vein administration. [Figure 3D] Figure 3E shows the setup of a renal cell carcinoma PDX model. K5 and K7 RCC PDX primary site models obtained by orthotopic kidney administration are shown. [Figure 3E] shows the setup of a renal cell carcinoma PDX model. [Figure 4A] Figure 1 shows the effect of anti-CD70 CAR T cells in vivo. Figure 1 shows the effect of anti-CD70 CAR T cells in a K7 RCC PDX lung metastasis model obtained by tail vein administration. [Figure 4B] Figure 1 shows the effect of anti-CD70 CAR T cells in vivo. Figure 1 shows the effect of anti-CD70 CAR T cells in the K5 RCC PDX model obtained by orthotopic kidney administration. [Figure 4C] Shows the effect of anti-CD70 CAR T cells in the K5 RCC PDX lung metastasis model obtained by tail vein administration. [Figure 4D]Shows the effect of anti-CD70 CAR T cells in a K7 RCC PDX primary site model obtained by orthotopic kidney administration. [Figure 5] The distribution of RCC tumors (labeled with firefly luciferase) and CAR T cells (labeled with Gaussia luciferase) is shown in a lung metastasis model, an orthotopic model, and in mice bearing tumors at both lung and kidney tumor sites, demonstrating that tumors were cleared from the lung but not the kidney, while CAR T cells were transported to both sites. [Figure 6-1] Figure 1 shows FACS analysis of CD70 expression in the lungs and kidneys of untreated mice. CD70 is homogeneous and high in the lungs, resulting in complete tumor eradication in the lung metastasis model by CD70 CAR T cells. CD70 expression is heterogeneous in the kidney orthotopic site (MFI negative in the CD70-negative population), explaining the incomplete tumor eradication by CD70 CAR T cells. [Figure 6-2] Same as above. [Figure 7] (20) Sorting and bulk RNA sequencing of lung, heterogeneous CD70lo, or CD70+ kidney tumors from untreated mice. Bulk RNA sequencing shows very low levels of CD70 expression by transcripts in the CD70lo kidney tumor population. Other genes not expressed in RCC tumors are listed for comparison. [Figure 8A-1] Figure 1 shows FACS analysis of in vitro cell cultures that restore CD70 expression.Culture of untreated K5 kidney tumors with in vivo kidney populations with low density CD70 (CD70 MFI negative population) restored CD70 expression in vitro. [Figure 8A-2] Same as above. [Figure 8B-1] Figure 1 shows FACS analysis of in vitro cell cultures that restore CD70 expression. Culture of untreated K7 with an in vivo kidney population with low density CD70 (CD70 MFI negative population) restored CD70 expression in vitro. [Figure 8B-2] Same as above. [Figure 9A]Epigenetic analysis of the CD70 locus in the indicated cell types. [Figure 9B] Epigenetic analysis of the CAIX locus in the indicated cell types. [Figure 9C] Epigenetic analysis shows the interaction of certain methylation regulators (Ezh2, H3K4me3, H3K27me3) with the CD70 locus. [Figure 9D] Figure 1 shows epigenetic analysis. Figure 2 shows the effect of tazemetostat (Ezh2 inhibitor) on ex vivo culture of selected heterogeneous kidney tumors and treatment with the Ezh2 inhibitor for 8 days. [Figure 10A] 1 shows the effect of CD70 overexpression on tumor clearance. mCerulean CD70-containing constructs and their expression. [Figure 10B] 1 shows the effect of CD70 overexpression on tumor clearance. 2 shows the effect of overexpression of mCerulean CD70 fusion protein on NALM6. [Figure 10C]

[0023] Figure 1 shows the effect of CD70 overexpression on tumor clearance.

[0024] Figure 1 shows that overexpression of mCerulean CD70 fusion protein on NALM6 did not impair in vitro killing in CTLs for 18 hours. [Figure 10D] Figure 1 shows the effect of CD70 overexpression on tumor clearance. Figure 1 shows that overexpression of CD70 on K5 and K7 PDX orthotopic models resulted in tumor clearance in vivo. [Figure 10E] Figure 1 shows the effect of CD70 overexpression on tumor clearance. Figure 1 shows that overexpression of CD70 on K5 and K7 PDX orthotopic models resulted in tumor clearance in vivo. [Figure 11A-1] 1 shows the expression levels of CAIX. 2 shows FACS analysis of CAIX expression in vitro. [Figure 11A-2] Same as above. [Figure 11B-1]1 shows the expression level of CAIX. 2 shows CD70 / CAIX expression in K5 untreated mice. [Figure 11B-2] Same as above. [Figure 11C-1] CAIX expression levels are shown. CD70 / CAIX expression in K7 untreated mice is shown. [Figure 11C-2] Same as above. [Figure 12A] 1 shows a dual targeting approach using CD70 and CAIX. 28z1XX CAR T in vitro killing. [Figure 12B] 1 shows a dual targeting approach using CD70 and CAIX. Dual targeting of CD70 and CAIX improved tumor control for K5. [Figure 12C-1] 1 shows a dual targeting approach using CD70 and CAIX. The remaining CD70-positive population after CAIX 28z1XX CAR treatment is shown. [Figure 12C-2] Same as above. [Figure 12D-1] 1 shows a dual targeting approach using CD70 and CAIX. 2 shows that CD70 and CAIX dual targeting did not result in tumor clearance of residual low-density antigen populations. [Figure 12D-2] Same as above. [Figure 12E-1] 1 shows a dual targeting approach using CD70 and CAIX. 2 shows that CD70 and CAIX dual targeting did not result in tumor clearance of residual low-density antigen populations. [Figure 12E-2] Same as above. [Figure 12F-1] 1 shows a dual targeting approach using CD70 and CAIX. 2 shows FACS analysis illustrating a comparison of tumor profiles. [Figure 12F-2] Same as above. [Figure 12G] 1 shows a dual targeting approach using CD70 and CAIX. 2 shows the effect of anti-CD70 CAR, anti-CAIX CAR, and dual-transduced T cells on tumor models. [Figure 12H]1 shows a dual targeting approach using CD70 and CAIX. 2 shows the effect of anti-CD70 CAR, anti-CAIX CAR, and dual-transduced T cells on tumor models. [Figure 12I] 1 shows a dual targeting approach using CD70 and CAIX. 2 shows the effect of anti-CD70 CAR, anti-CAIX CAR, and dual-transduced T cells on tumor models. [Figure 13A] 1 shows an analysis of tumor cells expressing the low-density antigens of CD70 and CAIX. CD70 / CAIX expression is shown in untreated kidney tumors (four quadrants were sorted: CD70-CAIX- / CD70+CAIX- / CD70-CAIX+ / CD70+CAIX+) and in untreated tumors at the lung site (CD70+CAIX- population was sorted). [Figure 13B] Analysis of tumor cells expressing CD70 and CAIX low-density antigens. Bulk RNA sequencing of CD70 / CAIX-sorted tumor populations from kidney and lung sites of K5 RCC PDX. [Figure 13C] Analysis of tumor cells expressing CD70 and CAIX low-density antigens. Bulk RNA sequencing of CD70 / CAIX-sorted tumor populations from kidney and lung regions of K7 RCC PDX. [Figure 13D] Figure 1 shows analysis of tumor cells expressing CD70 and CAIX low density antigens. Figure 2 shows mass spectrometry analysis of CD70 / CAIX sorted tumor populations. [Figure 13E-1] Analysis of tumor cells expressing low density antigens of CD70 and CAIX. Time course of CD70 / CAIX expression in untreated kidney tumors. [Figure 13E-2] Same as above. [Figure 13F] Analysis of tumor cells expressing CD70 and CAIX low density antigens. CD70 antigen quantification in untreated kidney tumors: # mol / cell (CD70-CAIX- population is negative for CD70 by MFI and quantification). [Figure 13G]Analysis of tumor cells expressing CD70 and CAIX low density antigens. CAIX antigen quantification in untreated renal tumors: #mol / cell. [Figure 13H] Analysis of tumor cells expressing CD70 and CAIX low-density antigens. CD70 antigen quantification in ex vivo CD70-CAIX-sorted kidney tumors at day 2, with very low levels of CD70 expression recovering after 48 hours of culture. [Figure 13I] Analysis of tumor cells expressing CD70 and CAIX low density antigens. CAIX antigen quantification in ex vivo sorted kidney tumors on day 2 (48 hours in culture). [Figure 14A] Figure 1 shows the efficacy of a low antigen targeting strategy: 70-HIT expressing a costimulatory ligand (80 / 41BBL) eliminated K5 orthotopic kidney tumors. [Figure 14B] Figure 1 shows the efficacy of a low antigen targeting strategy: 70-HIT expressing a costimulatory ligand (80 / 41BBL) eliminated K5 orthotopic kidney tumors. [Figure 14C] Figure 1 shows the efficacy of a low antigen targeting strategy: 70-HIT expressing a costimulatory ligand (80 / 41BBL) eliminated K5 orthotopic kidney tumors. [Figure 14D] Figure 1 shows the efficacy of a low antigen targeting strategy: 70-HIT expressing a costimulatory ligand (80 / 41BBL) eliminated K5 orthotopic kidney tumors. [Figure 14E] Figure 1 shows the efficacy of a low antigen targeting strategy: 70-HIT expressing a costimulatory ligand (80 / 41BBL) eliminated K7 orthotopic kidney tumors. [Figure 15] Figure 1 shows the T cell phenotypic characteristics at kidney tumor sites for both K5 and K7 at d7 and d14 after CD70 HIT (expressing CD80 and 4-1BBL polypeptides) or CD7028z1XX CAR T cell therapy. The number of CAR+ or HIT+ T cells (left column), the number of tumor cells (middle column), and the expression profile of triple inhibitory receptors PD-1, TIM-3, and LAG-3 (right column) are shown. [Figure 16A]1 shows that the in vivo efficacy of T cells expressing CD70 HIT, CD80 polypeptide, and 4-1BBL polypeptide cannot be explained by bystander killing of CD70-negative tumor cells. 1 shows an in vitro cytotoxicity assay of HIT CD70 against K5 and K7 PDX lines with CD70 knockout. [Figure 16B] We show that the in vivo efficacy of T cells expressing CD70 HIT, CD80 polypeptide, and 4-1BBL polypeptide cannot be explained by bystander killing of CD70-negative tumor cells. We also show that CD70 HIT T cells expressing CD80 and 4-1BBL polypeptide are unable to eradicate RCC tumors with CD70 knockout in vivo. Furthermore, mixing 75-80% wild-type RCC PDX tumors with 20-25% CD70 knockout RCC PDX tumors did not result in bystander killing of the knockout tumors in vivo. [Figure 16C] We show that the in vivo efficacy of T cells expressing CD70 HIT, CD80 polypeptide, and 4-1BBL polypeptide cannot be explained by bystander killing of CD70-negative tumor cells. We also show that CD70 HIT T cells expressing CD80 and 4-1BBL polypeptide are unable to eradicate RCC tumors with CD70 knockout in vivo. Furthermore, mixing 75-80% wild-type RCC PDX tumors with 20-25% CD70 knockout RCC PDX tumors did not result in bystander killing of the knockout tumors in vivo. [Figure 17A] Flow cytometry characterization of CD70 heterogeneous pancreatic ductal adenocarcinoma (PDAC2) PDX lines, mechanisms of CD70 regulation, and cytotoxic effects of T cells expressing CD70 HIT, CD80 polypeptide, and 4-1BBL polypeptide against orthotopic pancreatic cancer derived from PDAC2. Characterization of PDAC2 (pancreatic ductal adenocarcinoma) PDX lines. [Figure 17B]Flow cytometry characteristics of CD70 heterogeneous pancreatic ductal adenocarcinoma (PDAC2) PDX lines, mechanisms of CD70 regulation, and cytotoxic effects of T cells expressing CD70 HIT, CD80 polypeptide, and 4-1BBL polypeptide against PDAC2-derived orthotopic pancreatic cancer. Cytotoxic effects against PDAC2 cancer in vitro. [Figure 17C] Flow cytometry characteristics of CD70 heterogeneous pancreatic ductal adenocarcinoma (PDAC2) PDX lines, mechanisms of CD70 regulation, and cytotoxic effects of T cells expressing CD70 HIT, CD80 polypeptide, and 4-1BBL polypeptide against orthotopic pancreatic cancer derived from PDAC2 are shown. The effect of Ezh2 inhibitors on PDAC2 cells is shown. [Figure 17D] Figure 1 shows flow cytometry characteristics of CD70 heterogeneous pancreatic ductal adenocarcinoma (PDAC2) PDX lines, the mechanism of CD70 regulation, and the cytotoxic effect of T cells expressing CD70 HIT, CD80 polypeptide, and 4-1BBL polypeptide against PDAC2-derived orthotopic pancreatic cancer. Immunoblotting of PDAC2 in vitro tumors treated with vehicle, 3 μM Ezh2 inhibitor, or 10 μM Ezh2 inhibitor is shown. [Figure 17E] Flow cytometry characterization of CD70 heterogeneous pancreatic ductal adenocarcinoma (PDAC2) PDX lines, mechanisms of CD70 regulation, and cytotoxic effects of T cells expressing CD70 HIT, CD80 polypeptide, and 4-1BBL polypeptide against PDAC2-derived orthotopic pancreatic cancer. In vivo efficacy of HIT CD70 T cells expressing costimulatory (CD80 / 4-1BBL) against a PDAC2 orthotopic pancreatic PDX model is shown. [Figure 18A] 1 shows the properties of PANC-1 cells as a model of CD70+ tumor cells for pancreatic cancer and the in vivo efficacy of HIT CD70 T cells against pancreatic cancer. [Figure 18B] 1 shows the properties of PANC-1 cells as a model of CD70+ tumor cells for pancreatic cancer and the in vivo efficacy of HIT CD70 T cells against pancreatic cancer. [Figure 19A]1 shows the expression profile of CD70 in the SK-OV3 ovarian cancer cell line. [Figure 19B] 1 shows tumor engraftment of the SK-OV3 cell line. [Figure 19C] Cytotoxic effects against orthotopic and intraperitoneal ovarian cancer derived from SK-OV3 are shown. [Figure 19D] Cytotoxic effects against orthotopic and intraperitoneal ovarian cancer derived from SK-OV3 are shown. [Figure 19E] Cytotoxic effects against orthotopic and intraperitoneal ovarian cancer derived from SK-OV3 are shown. [Figure 20A] Immunohistochemistry (IHC) and FISH analysis of CD70 in samples. IHC and FISH analysis of CD70 in RCC kidney tumor patient and K7 PDX samples. [Figure 20B] Immunohistochemistry (IHC) and FISH analysis of CD70 in samples. FISH analysis of CD70 in PDAC2 cell samples. DETAILED DESCRIPTION OF THE INVENTION

[0026] The presently disclosed subject matter provides compositions, e.g., modified immune cells, useful for immunotherapy (e.g., T cell immunotherapy). The compositions, e.g., modified immune cells, disclosed herein comprise a TCR-like fusion molecule that targets CD70. The presently disclosed subject matter also provides methods for producing such compositions, as well as methods of using such compositions to treat and / or prevent tumors (e.g., cancer, e.g., solid tumors, e.g., renal cell carcinoma (RCC)). The presently disclosed subject matter is based, at least in part, on the discovery that 70-HIT T cells can eradicate solid tumors (e.g., renal cell carcinoma, ovarian cancer, pancreatic cancer).

[0027] Non-limiting embodiments of the presently disclosed subject matter are illustrated by the specification and examples.

[0028] For clarity of disclosure, and not by way of limitation, this detailed description is divided into the following subsections. 1. Definition, 2.Cells, 3. Nucleic acid compositions and vectors, 4. Formulation and Administration 5. Treatment method, 6. Kits, and 7. Exemplary embodiments.

[0029] 1.Definition Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art. The following references provide those skilled in the art with general definitions of many of the terms used in the subject matter disclosed herein: Singleton et al., Dictionary of Microbiology and Molecular Biology (2002); nd ed.1994), The Cambridge Dictionary of Science and Technology(Walker ed.,1988), The Glossary of Genetics,5 th Ed., R. Rieger et al. (eds.), Springer Verlag (1991), and Hale & Marham, The Harper Collins Dictionary of Biology (1991).

[0030] As used herein, the term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 3 or more than 3 standard deviations, in accordance with practice in the art. Alternatively, "about" can mean a range of up to 20%, e.g., up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude of a value, e.g., within 5-fold or within 2-fold.

[0031] As used herein, a "costimulatory molecule" refers to a cell surface molecule other than an antigen receptor or its ligand that can provide an efficient response of a lymphocyte to an antigen. In certain embodiments, a costimulatory molecule can provide optimal lymphocyte activation.

[0032] As used herein, a "costimulatory ligand" refers to a molecule that, upon binding to its receptor (e.g., a costimulatory molecule), generates a costimulatory response, e.g., an intracellular response that results in stimulation when an antigen-recognizing receptor (e.g., a chimeric antigen receptor (CAR)) binds to its target antigen.

[0033] "Immunoresponsive cell" refers to a cell that functions in an immune response or a precursor, or progeny thereof. In certain embodiments, an immunoresponsive cell is a cell of the lymphoid lineage. Non-limiting examples of cells of the lymphoid lineage include T cells, natural killer (NK) cells, B cells, and stem cells from which lymphoid cells can differentiate. In certain embodiments, an immunoresponsive cell is a cell of the myeloid lineage.

[0034] "Activating an immunoresponsive cell" refers to the induction of intracellular signal transduction or changes in protein expression that result in the initiation of an immune response. For example, clustering of CD3 chains in response to ligand binding and immunoreceptor tyrosine-based inhibitory motifs (ITAMs) generates a signaling cascade. In certain embodiments, binding of an endogenous TCR or an exogenous CAR to an antigen leads to the formation of an immunological synapse, which involves the clustering of many molecules near the bound receptor (e.g., CD4 or CD8, CD3γ / δ / ε / ζ, etc.). This clustering of membrane-bound signaling molecules allows the ITAM motifs contained within the CD3 chains to become phosphorylated. This phosphorylation, in turn, initiates the T cell activation pathway, ultimately activating transcription factors such as NF-κB and AP-1. These transcription factors induce global gene expression in T cells, increasing IL-2 production for proliferation and expression of master regulator T cell proteins, thereby initiating a T cell-mediated immune response.

[0035] "Stimulating immunoresponsive cells" refers to signals that result in a robust and sustained immune response. In various embodiments, this occurs after activation of immune cells (e.g., T cells) or is mediated simultaneously through receptors including, but not limited to, CD28, CD137 (4-1BB), OX40, CD40, ICOS, DAP-10, CD27, NKG2D, CD2, CD150, and CD226. Receiving multiple stimulatory signals can be important for mounting a robust and long-lasting T cell-mediated immune response. T cells can quickly become inhibited and unresponsive to antigen. While the effects of these costimulatory signals can vary, they generally result in increased gene expression to generate long-lived, proliferative, anti-apoptotic T cells that strongly respond to antigen for complete and sustained eradication.

[0036] As used herein, the term "antigenic heterogeneity" refers to the differential expression of numerous antigens (e.g., tumor antigens, e.g., CD70, CD312) that result in variation in tumor cell phenotype and distribution of tumor antigen-positive cells.

[0037] As used herein, the term "low antigen density" refers to target molecules (e.g., antigens) having a cell surface density of less than about 5,000 molecules per cell. In certain embodiments, low antigen density refers to a cell surface density of less than about 4,000 molecules per cell, less than about 3,000 molecules per cell, less than about 2,000 molecules per cell, less than about 1,500 molecules per cell, less than about 1,000 molecules per cell, less than about 500 molecules per cell, less than about 200 molecules per cell, or less than about 100 molecules per cell. In certain embodiments, low antigen density refers to a cell surface density of less than about 2,000 molecules per cell. In certain embodiments, low antigen density refers to a cell surface density of less than about 1,500 molecules per cell. In certain embodiments, low antigen density refers to a cell surface density of less than about 1,000 molecules per cell. In certain embodiments, low antigen density is a cell surface density of about 4,000 molecules per cell to about 2,000 molecules per cell, about 2,000 molecules per cell to about 1,000 molecules per cell, about 1,500 molecules per cell to about 1,000 molecules per cell, about 2,000 molecules per cell to about 500 molecules per cell, about 1,000 molecules per cell to about 200 molecules per cell, or about 1,000 molecules per cell to about 100 molecules per cell.

[0038] As used herein, the term "low tumor cell frequency" refers to target cells having a target cell frequency of less than about 50% per tumor. In certain embodiments, a low tumor cell frequency is less than about 40% per tumor, less than about 30% per tumor, less than about 20% per tumor, less than about 15% per tumor, less than about 10% per tumor, less than about 5% per tumor, less than about 2% per tumor, or less than about 1% per tumor. In certain embodiments, a low tumor cell frequency is less than about 2% per tumor. In certain embodiments, a low tumor cell frequency is less than about 1.5% per tumor. In certain embodiments, a low tumor cell frequency is less than about 1% per tumor. In certain embodiments, low tumor cell frequency is between about 40% per tumor and about 20% per tumor, between about 20% per tumor and about 10% per tumor, between about 15% per tumor and about 10% per tumor, between about 20% per tumor and about 5% per tumor, between about 10% per tumor and about 2% per tumor, or between about 10% per tumor and about 1% per tumor.

[0039] As used herein, the term "antigen-recognizing receptor" refers to a receptor that can activate an immune cell or immunoresponsive cell (e.g., a T cell) in response to its binding to an antigen.

[0040] As used herein, the term "antibody" refers not only to intact antibody molecules but also to fragments of antibody molecules that retain immunogen-binding ability. Such fragments are also well known in the art and are commonly used both in vitro and in vivo. Thus, as used herein, the term "antibody" refers not only to intact immunoglobulin molecules but also to the well-known active fragments F(ab')2 and Fab. F(ab')2 and Fab fragments, which lack the Fe fragment of intact antibodies, are cleared from the circulation more rapidly and may exhibit less nonspecific tissue binding than intact antibodies (Wahl et al., J. Nucl. Med. 24:316-325 (1983). As used herein, antibodies include natural whole antibodies, bispecific antibodies, chimeric antibodies, Fab, Fab', single-chain variable fragments (scFv), fusion polypeptides, and non-traditional antibodies. In certain embodiments, antibodies are glycoproteins comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (referred to herein as V H ) and heavy chain constant (C H The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. Each light chain contains a light chain variable region (referred to herein as V L ) and light chain constant C L The light chain constant region consists of one domain, C L It consists of V H and V L The regions can be further subdivided into regions of hypervariability, called complementarity-determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). H and V Lis composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0041] As used herein, "CDR" is defined as the complementarity determining region amino acid sequences of an antibody, which are the hypervariable regions of immunoglobulin heavy and light chains. See, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 4 th See USDapartment of Health and Human Services, National Institutes of Health (1987). Generally, antibodies contain three heavy chain and three light chain CDRs or CDR regions in the variable region. CDRs provide the majority of contact residues for antibody binding to an antigen or epitope. In certain embodiments, CDR regions are delineated using the Kabat system (Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242). In certain embodiments, CDR regions are delineated using the PyIgClassify system (Adolf-Bryfogle et al., Nucleic acids research 43.D1 (2015):D432-D438).

[0042] As used herein, the term "linker" is intended to mean a functional group (e.g., chemical or polypeptide) that covalently bonds two or more polypeptides or nucleic acids so as to connect them to one another. As used herein, a "peptide linker" is a peptide linker that couples two proteins together (e.g., V H and V L In certain embodiments, the linker is a G4S linker. In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1, as provided below: GGGGSGGGGSGGGGS [SEQ ID NO: 1]

[0043] In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO:2, provided below: GGGGSGGGGSGGGSGGGGS [SEQ ID NO: 2]

[0044] In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO:3, provided below: GGGGSGGGGSGGGGSGGGSGGGGS [SEQ ID NO: 3]

[0045] In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO:4, provided below: GGGGSGGGGSGGGGSGGGGSGGGSGGGGS [SEQ ID NO: 4]

[0046] In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 5, provided below: GGGGS [SEQ ID NO: 5]

[0047] In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO:6, provided below: GGGGSGGGGS [SEQ ID NO: 6]

[0048] As used herein, the term "single-chain variable fragment" or "scFv" refers to a V H ::V L Covalently linked immunoglobulin heavy chains (V) to form heterodimers H ) and light chain (V L ) is a fusion protein of the variable region of V H and V L are either directly joined or joined by a peptide-encoding linker (e.g., 10, 15, 20, 25 amino acids), and V H N-terminus of V L or V H The C-terminus of V L The linker is usually rich in glycine for flexibility and rich in serine or threonine for solubility. Despite the removal of the constant region and the introduction of the linker, the scFv protein retains the specificity of the original immunoglobulin. Single-chain Fv polypeptide antibodies are composed of V, V, VH ... H and V LThe polypeptide can be expressed from a nucleic acid containing a sequence encoding the polypeptide. See also U.S. Patent Nos. 5,091,513, 5,132,405, and 4,956,778, and U.S. Patent Publication Nos. 2005 / 0196754 and 2005 / 0196754. Antagonist scFvs with inhibitory activity have been described (e.g., Zhao et al., Hyrbidoma (Larchmt) 2008 27(6):455-51; Peter et al., J Cachexia Sarcopenia Muscle 2012 August 12; Shieh et al., J Imunol 2009 183(4):2277-85; Giomarelli et al., Thromb Haemost 2007 97(6):955-63; Fife et al., J Clin Invst 2006 116(8):2252-61; Brocks et al., Immunotechnology 1997 3(3):173-84; Moosmayer et al., Ther Immunol 1995 2 (10:31-40). Agonist scFvs with stimulatory activity have been described (see, e.g., Peter et al., J Bioi Chern 2003 25278(38):36740-7; Xie et al., Nat Biotech 1997 15(8):768-71; Ledbetter et al., Crit Rev Immunol 1997 17(5-6):427-55; Ho et al., BioChim Biophys Acta 2003 1638(3):257-66).

[0049] As used herein, the term "affinity" refers to a measure of binding strength. Affinity may depend on the closeness of the stereochemical compatibility between the antibody binding site and the antigenic determinant, the size of the contact area between them, and / or the distribution of charged and hydrophobic groups. As used herein, the term "affinity" also includes "avidity," which refers to the strength of antigen-antibody binding after the formation of a reversible complex. Methods for calculating the affinity of an antibody for an antigen are known in the art, and include, but are not limited to, various antigen binding experiments, such as functional assays (e.g., flow cytometry assays).

[0050] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a molecule comprising an extracellular antigen-binding domain fused to an intracellular signaling domain capable of activating or stimulating an immune cell or immunoresponsive cell, and a transmembrane domain. In certain embodiments, the extracellular antigen-binding domain of a CAR comprises an scFv. An scFv can be derived by fusing the variable heavy and variable light regions of an antibody. Alternatively or additionally, an scFv can be derived from a Fab (e.g., obtained from a Fab library instead of from an antibody). In certain embodiments, an scFv is fused to a transmembrane domain and then to an intracellular signaling domain. In certain embodiments, a CAR is selected to have high binding affinity or avidity for an antigen.

[0051] As used herein, the terms "substantially identical" or "substantially homologous" refer to a polypeptide or nucleic acid molecule that exhibits at least about 50% identity or homology to a reference amino acid sequence (e.g., any of the amino acid sequences described herein) or a reference nucleic acid sequence (e.g., any of the nucleic acid sequences described herein). In certain embodiments, such a sequence is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% identical or homologous to the amino acid or nucleic acid sequence used for comparison.

[0052] Sequence identity can be measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, the BLAST program can be used, with a probability score of e-3 to e-100 indicating closely related sequences.

[0053] The percent homology between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) as incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Additionally, the percent homology between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) as incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6. Additionally or alternatively, the amino acid sequences of the subject matter disclosed herein can be further used as a "query sequence" to perform searches against public databases, for example, to identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. To obtain amino acid sequences homologous to particular sequences disclosed herein (e.g., heavy and light chain variable region sequences), BLAST protein searches can be performed with the XBLAST program, score=50, word length=3. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0054] As used herein, the term "conservative sequence modification" refers to an amino acid modification that does not significantly affect or change the binding characteristics of the antigen-recognizing receptor (e.g., the extracellular antigen-binding domain of a CAR) disclosed herein, including the amino acid sequence. Conservative modifications can include amino acid substitutions, additions, and deletions. Modifications can be introduced into the extracellular antigen-binding domain of a CAR of the present disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Amino acids can be classified into groups according to physicochemical properties, such as charge and polarity. A conservative amino acid substitution is one in which an amino acid residue is replaced with an amino acid from the same group. For example, amino acids can be classified by charge, with positively charged amino acids including lysine, arginine, and histidine, negatively charged amino acids including aspartic acid and glutamic acid, and neutrally charged amino acids including alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Additionally, amino acids can be classified by polarity, with polar amino acids including arginine (basic polar), asparagine, aspartic acid (acidic polar), glutamic acid (acidic polar), glutamine, histidine (basic polar), lysine (basic polar), serine, threonine, and tyrosine, and nonpolar amino acids including alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. Thus, one or more amino acid residues in a CDR region can be replaced with other amino acid residues from the same group, and the altered antibody can be tested for retained function (i.e., the functions set forth in (c)-(l) above) using the functional assays described herein. In certain embodiments, no more than one, no more than two, no more than three, no more than four, or no more than five residues in a designated sequence or CDR region are altered.

[0055] By "disease" is meant any condition, disease, or disorder that damages or interferes with the normal function of a cell, tissue, or organ, for example, neoplasms and pathogenic infection of cells.

[0056] "Effective amount" means an amount sufficient to have a therapeutic effect. In certain embodiments, an "effective amount" is an amount sufficient to prevent, ameliorate, or inhibit the continued proliferation, growth, or metastasis (e.g., invasion or migration) of a neoplasm.

[0057] "Endogenous" means a nucleic acid molecule or polypeptide that is normally expressed in a cell or tissue.

[0058] "Exogenous" refers to a nucleic acid molecule or polypeptide that is not endogenously present in a cell. Thus, the term "exogenous" will encompass foreign, heterologous, and any recombinant nucleic acid molecule or polypeptide expressed in a cell, such as overexpressed nucleic acid molecules and polypeptides. An "exogenous" nucleic acid refers to a nucleic acid that is not present in a native, wild-type cell; for example, an exogenous nucleic acid may differ from its endogenous counterpart by sequence, position / location, or both. For clarity, an exogenous nucleic acid may have the same or a different sequence compared to its native, endogenous counterpart, may be introduced into the cell itself or its precursor by genetic engineering, and may optionally be linked to alternative regulatory sequences, such as a non-native promoter or secretory sequence.

[0059] By "increase" is meant a positive change of at least about 5%. The change may be about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, about 100%, or more.

[0060] By "decreasing" is meant a negative change of at least about 5%. The change may be about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, or even about 100%.

[0061] The terms "isolated," "purified," or "biologically pure" refer to material that is free to varying degrees from components that normally accompany it as found in its native state. "Isolated" refers to a degree of separation from the original source or surroundings. "Purified" refers to a degree of separation greater than isolation. A "purified" or "biologically pure" protein is sufficiently free from other substances so that any impurities do not substantially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. The term "purified" can indicate that a nucleic acid or protein yields essentially one band in an electrophoretic gel. For proteins that can be subject to modifications, such as phosphorylation or glycosylation, different modifications can yield different isolated proteins that can be purified separately.

[0062] The term "isolated cell" means a cell that is separated from molecules and / or cellular components that naturally accompany the cell.

[0063] As used herein, the term "antigen-binding domain" refers to a domain that is capable of specifically binding to a particular antigenic determinant or set of antigenic determinants present on a cell.

[0064] "Neoplasm" or "malignancy" refers to a disease characterized by the pathological proliferation of cells or tissues and their subsequent migration or invasion into other tissues or organs. Neoplastic growth is typically uncontrolled and progressive, occurring under conditions that do not induce or cause the cessation of normal cell proliferation. Neoplasms can affect various cell types, tissues, or organs, including, but not limited to, the bladder, bone, brain, breast, cartilage, glia, esophagus, fallopian tubes, gallbladder, heart, intestine, kidney, liver, lung, lymph nodes, nervous tissue, ovaries, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid, trachea, urinary tract, ureter, urethra, uterus, and vagina, or organs selected from these tissues or cell types. Neoplasms include cancers, such as sarcomas, carcinomas, or plasmacytomas (malignant tumors of plasma cells). In certain embodiments, the neoplasm is cancer.

[0065] By "specifically binds" is meant a polypeptide or fragment thereof that recognizes and binds to a biological molecule (e.g., a polypeptide) of interest, but does not substantially recognize and bind to other molecules in a sample, e.g., a biological sample, which naturally includes the polypeptides disclosed herein.

[0066] As used herein, the term "tumor antigen" refers to an antigen (e.g., a polypeptide) that is uniquely or differentially expressed on tumor cells compared to normal or non-neoplastic cells. In certain embodiments, a tumor antigen includes any polypeptide expressed by a tumor that can activate or induce an immune response via an antigen-recognizing receptor, or that can suppress an immune response via receptor-ligand binding.

[0067] The terms "comprises" and "comprising" are intended to have the broad meaning ascribed to them in U.S. patent law and may mean "includes," "including," etc.

[0068] As used herein, the term "treatment" refers to clinical intervention in an attempt to alter the disease course of the individual or cell being treated, and can be performed either for prophylaxis or during the course of clinical pathology. The therapeutic effect of treatment includes, but is not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and remission or improved prognosis. By preventing the progression of a disease or disorder, treatment can not only prevent deterioration due to the disorder in a subject affected or diagnosed with, or suspected of having, the disorder, but also prevent the onset of the disorder or symptoms of the disorder in a subject at risk of, or suspected of having, the disorder.

[0069] As used herein, an "individual" or "subject" refers to a vertebrate, e.g., a human or a non-human animal, e.g., a mammal. Mammals include, but are not limited to, humans, primates, farm animals, sport animals, rodents, and pets. Non-limiting examples of non-human animal subjects include rodents, such as mice, rats, and hamsters, and non-human primates, such as guinea pigs, rabbits, dogs, cats, sheep, pigs, goats, cows, horses, and apes and monkeys. As used herein, the term "immunocompromised" refers to a subject with an immunodeficiency. The subject is highly vulnerable to opportunistic infections, infections caused by organisms that do not normally cause disease in humans with healthy immune systems but may affect people with dysfunctional or suppressed immune systems.

[0070] As used herein, a "functional fragment" of a molecule or polypeptide includes a fragment of a molecule or polypeptide that retains at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% of the primary function of the molecule or polypeptide.

[0071] Other aspects of the presently disclosed subject matter are described in the disclosure that follows and are within the scope of the presently disclosed subject matter.

[0072] 2.Cells The presently disclosed subject matter provides cells comprising an antigen-recognizing receptor that targets CD70. In certain embodiments, the antigen-recognizing receptor is a TCR-like fusion molecule.

[0073] In certain embodiments, the cell is selected from the group consisting of a cell of the lymphoid lineage and a cell of the myeloid lineage. In certain embodiments, the cell is an immunoresponsive cell. In certain embodiments, the immunoresponsive cell is a cell of the lymphoid lineage.

[0074] In certain embodiments, the cells are lymphoid lineage cells. Lymphoid lineage cells can provide antibody production, regulation of the cellular immune system, detection of foreign substances in the blood, detection of cells foreign to the host, etc. Non-limiting examples of lymphoid lineage cells include T cells, natural killer (NK) cells, B cells, dendritic cells, and stem cells from which lymphoid cells can be differentiated. In certain embodiments, the stem cells are pluripotent stem cells (e.g., embryonic stem cells).

[0075] In certain embodiments, the cell is a T cell. T cells can be lymphocytes that mature in the thymus and are primarily involved in cell-mediated immunity. T cells participate in the adaptive immune system. T cells of the presently disclosed subject matter include helper T cells, cytotoxic T cells, memory T cells (central memory T cells, stem cell-like memory T cells (or stem-like memory T cells)), as well as two types of effector memory T cells: e.g., T EM Cells and T EMRAThe T cells can be any type of T cell, including, but not limited to, CD4 T cells, regulatory T cells (also known as suppressor T cells), tumor-infiltrating lymphocytes (TIL), natural killer T cells, mucosal-associated invariant T cells, and γδ T cells. Cytotoxic T cells (CTL or killer T cells) are a subset of T lymphocytes that can induce the death of infected somatic cells or tumor cells. The patient's own T cells may be genetically modified to target specific antigens through the introduction of antigen-recognition receptors, such as CAR or TCR. T cells can be CD4 + T cells or CD8 + In certain embodiments, the T cells are CD4 + In certain embodiments, the T cells are CD8 + In certain embodiments, CD8 + The T cells are CD4-independent. In certain embodiments, the T cells are derived from induced pluripotent stem cells (iPSCs). In certain embodiments, the T cells are CD8 + T cells, CD8 + T cells are derived from iPSCs.

[0076] In certain embodiments, the cells are NK cells. Natural killer (NK) cells are lymphocytes that are part of cell-mediated immunity and act during innate immune responses. NK cells do not require prior activation to exert cytotoxic effects on target cells.

[0077] Types of human lymphocytes of the presently disclosed subject matter include, but are not limited to, peripheral donor lymphocytes. For example, Sadelain, M., et al. 2003 Nat Rev Cancer 3:35-45 (disclosing peripheral donor lymphocytes genetically modified to express CAR), Morgan, RA, et al. 2006 Science 314:126-129 (disclosing peripheral donor lymphocytes genetically modified to express a full-length tumor antigen-recognizing T cell receptor complex containing α and β heterodimers), Panelli, MC, et al. 2000 J Immunol 164:495-504, Panelli, MC, et al. 2000 J Immunol 164:4382-4392 (disclosing lymphocyte cultures derived from tumor-infiltrating lymphocytes (TILs) in tumor biopsies), and Dupont, J., et al. 2005 Cancer Res 65:5417-5427, Papanicolaou, GA, et al. 2003 Blood 102:2498-2505 (disclosing selectively in vitro expanded antigen-specific peripheral blood leukocytes using artificial antigen presenting cells (AAPC) or pulsed dendritic cells).

[0078] In certain embodiments, the cells (e.g., T cells) are autologous. In certain embodiments, the cells (e.g., T cells) are non-autologous. In certain embodiments, the cells (e.g., T cells) are allogeneic. In certain embodiments, the cells (e.g., T cells) are derived from in vitro engineered progenitor or stem cells.

[0079] In certain embodiments, the cells are cells of the myeloid lineage. Non-limiting examples of cells of the myeloid lineage include monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes, and stem cells from which myeloid cells can differentiate.

[0080] In certain embodiments, the stem cells are pluripotent stem cells (eg, embryonic stem cells or induced pluripotent stem cells).

[0081] 2.1. Antigen Recognition Receptors The antigen-recognizing receptor (e.g., a first antigen-recognizing receptor) targets an antigen (e.g., a first antigen). The antigen (e.g., a first antigen) can be a tumor antigen or a pathogen antigen. In certain embodiments, the antigen-recognizing receptor (e.g., a first antigen-recognizing receptor) is a chimeric receptor. In certain embodiments, the chimeric receptor is a TCR-like fusion molecule.

[0082] 2.1.1. Antigen In certain embodiments, the antigen is a tumor antigen. In certain embodiments, the tumor antigen is an antigen with low antigen density. In certain embodiments, the tumor antigen is expressed on cells with low tumor cell frequency.

[0083] Any tumor antigen (antigenic peptide) can be used in the tumor-related embodiments described herein. Sources of antigens include, but are not limited to, cancer proteins. The antigen (e.g., first antigen) can be expressed as a peptide or as an intact protein or portion thereof. The intact protein or portion thereof can be naturally occurring or mutagenized. Non-limiting examples of tumor antigens include CD19, CD70, IL1RAP, ABCG2, AChR, ACKR6, ADAMTS13, ADGRE2, ADGRE2 (EMR2), ADORA3, ADRA1D, AGER, ALS2, antigens of cytomegalovirus (CMV)-infected cells (e.g., cell surface antigens), ANO9, AQP2, ASIC3, ASPRV1, ATP6V0A4, B3GNT4, B7-H3, BCMA, BEST4, C3orf35, CAD M3, CAIX, CAPN3, CCDC155, CCR1, CD10, CD117, CD123, CD133, CD135(FLT3), CD138, CD20, CD22, CD244(2B4), CD25, CD26, CD2 76, CD30, CD300LF, CD312, CD32, CD321, CD33, CD34, CD36, CD38, CD41, CD44, CD44V6, CD47, CD49f, CD56, CD7, CD71, CD74, CD8 , CD82, CD96, CD98, CD99, CDH13, CDHR1, CEA, CEACAM6, CHST3, CLEC12A, CLEC1A, CLL1, CNIH2, COL15A1, COLEC12, CPM, CR1, C X3CR1, CXCR4, CYP4F11, DAGLB, DARC, DFNB31, DGKI, EGF1R, EGFR-VIII, EGP-2, EGP-40, ELOVL6, EMB, EMC10, EMR2, ENG, EpCAM , EphA2, EPHA4, ERBB, ERBB2, Erb-B3, Erb-B4, E-selectin, EXOC3L4, EXTL3, FAM186B, FBP, FCGR1A, FKBP1B, FLRT1, folate receptor-a, FO LR2, FRMD5, GABRB2, GAS2, GD2, GD3, GDPD3, GNA14, GNAZ, GPR153, GPR56, GYPA, HEPHL1, HER-2, hERT, HILPDA, HLA-DR, HOOK1,hTERT, HTR2A, ICAM1, IGFBP3, IL10RB, IL20RB, IL23R, ILDR1, interleukin-13 receptor subunit alpha-2 (IL-13Rα2), ITFG3, ITGA4, ITGA5, ITGA8, ITGAX, ITGB5, ITGB8, JAM3, KCND1, KCNJ5, KCNK13, KCNN4, KCNV2, KDR, KIF19, KIF26B, κ-light chain, L1CAM, LAX1, LEPR, Lewis Y (CD174), Lewis Y (LeY), LILRA2, LILRA6, LILRB2, LILRB3, LILRB4, LOXL4, LPAR2, LRRC37A3, LRRC8E, LRRN2, LRRTM2, LTB4R, MAGE-A1, MAGEA3, MANSC1, MART1, GP100, MBOAT1, MBOAT7, melanoma antigen family A, mesothelin (MSLN), MFAP3L, MMP25, MRP1, MT-ND1, mucin 1 (M UC1), mucin 16 (MUC16), MYADM, MYADML2, NGFR, NKCS1, NKG2D ligand, NLGN3, NPAS2, NY-ESO-1, oncofetal antigen (h5T4), OTOA, P2RY13, p53, PDE3A, PEAR1, PIEZO1, PLXNA4, PLXNC1, PNPLA3, PPFIA4, PPP2R5B, PRAME, prostate stem cell antigen (PSCA), prostate-specific membrane antigen Protease 3 (PR1), PSD2, PTPRJ, RDH16, receptor tyrosine-protein kinase Erb-B2, RHBDL3, RNF173, RNF183, ROR1, RYR2, SCIN, SCN11A, SCN2A, SCNN1D, SEC31B, SEMA4A, SH3PXD2A, SIGLEC11, SIRPB1, SLC16A6, SLC19A1, SLC22A5, SLC25A36, S LC25A41, SLC30A1, SLC34A3, SLC43A3, SLC44A1, SLC44A3, SLC45A3, SLC6A16, SLC6A6, SLC8A3, SLC9A1, SLCO2B1, SPA G17, STC1, STON2, SUN3, Survivin, SUSD2, SYNC, TACSTD2, TAS1R3, TEX29, TFR2, TIM-3(HAVCR2), TLR2, TMEFF2, TMEM145,Examples of antigens include TMEM27, TMEM40, TMEM59L, TMEM89, TMPRSS5, TNFRSF14, TNFRSFlB, TRIM55, TSPEAR, TTYH3, tumor-associated glycoprotein 72 (TAG-72), tyrosinase, vascular endothelial growth factor R2 (VEGF-R2), VLA-4, Wilms tumor protein (WT-1), WNT4, WT1, and ZDHHC11. In certain embodiments, the antigen is CD70.

[0084] In certain embodiments, the antigen (eg, the first antigen) is a pathogen antigen. Non-limiting examples of viruses include Retroviridae (e.g., human immunodeficiency viruses, such as HIV-1 (also referred to as HDLV-III, LAVE, or HTLV-III / LAV, or other isolates such as HIV-III and HIV-LP), Picornaviridae (e.g., poliovirus, hepatitis A virus, enterovirus, human coxsackievirus, rhinovirus, echovirus), Calciviridae (e.g., strains that cause gastroenteritis), Togaviridae (e.g., equine encephalitis virus, rubella virus), Flaviviridae (e.g., dengue virus, encephalitis virus, yellow fever virus), Coronoviridae (e.g., coronavirus), Rhabdoviridae (e.g., vesicular stomatitis virus, rabies virus), Filoviridae (e.g., Ebola virus), Paramyxoviridae (e.g., parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus), Orthomyxoviridae (e.g., influenza virus), Bungaviridae (e.g., Hantavirus, viruses, Bungaviruses, Phleboviruses, and Nairaviruses), Arenaviridae (hemorrhagic fever viruses), Reoviridae (e.g., reoviruses, orbivulus, and rotaviruses), Birnaviridae, Hepadnaviridae (hepatitis B virus), Parvoviridae (parvoviruses), Papovaviridae (papillomaviruses, polyomaviruses), Adenoviridae (most adenoviruses), Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella-zoster virus, saproviridae, varicella-zoster virus ... These include cytomegalovirus (CMV), herpesviruses, poxviridae (variola virus, vaccinia virus, poxvirus), and iridoviridae (e.g., African swine fever virus), as well as unclassified viruses such as the causative agent of hepatitis delta (thought to be a defective satellite of hepatitis B virus), non-A, non-B hepatitis agents (Class 1 = internally transmitted, Class 2 = parenterally transmitted (i.e., hepatitis C)), Norwalk and related viruses, and astroviruses.

[0085] Non-limiting examples of bacteria include Pasteurella, Staphylococci, Streptococcus, Escherichia coli, Pseudomonas species, and Salmonella species.The main ingredients of the fermentation product are Helicobacter pylori, Borelia burgdorferi, Legionella, Legionella pneumophilia, and Mycobacteria sps(from M. tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae, M. leprae) Staphylococcus aureus, Staphylococcus epidermidis, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (A Streptococcus), Streptococcus agalactiae (B, Streptococcus) faecalis anthracis, corynebacterium diphtheriae, corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridium spp multocida、Bacteroides sp.、Fusobacterium nucleatum、Streptobacillus moniliformis、Treponema pallidium、Treponema pertenua、Leptospira、Rickettsia、Actinomyces israelli is a very good friend.Mycoplasma, Pseudomonas aeruginosa, Pseudomonas fluorescens, Corynobacteria diphtheriae, Bartonella henselae, Bartonella quintana, Coxiella burnetii, chlamydia, shigella, Yersinia enterocolitica, Yersinia pseudotuberculosis, Listeria monocytogenes, Mycoplasma spp., Vibrio cholerae, Borrelia, Francisella, Brucella melitensis, Proteus mirabilis, and Proteus.

[0086] In certain embodiments, the pathogen antigen is a viral antigen present in cytomegalovirus (CMV), a viral antigen present in Epstein-Barr virus (EBV), a viral antigen present in human immunodeficiency virus (HIV), or a viral antigen present in influenza virus.

[0087] 2.1.2.TCR-like fusion molecules In certain embodiments, the antigen-recognizing receptor is a TCR-like fusion molecule. Non-limiting examples of TCR fusion molecules include HLA-independent TCR-based chimeric antigen receptors (also known as "HITs" and disclosed, for example, in International Patent Application No. PCT / US19 / 017525, the entire contents of which are incorporated by reference), and T cell receptor fusion constructs (TRuCs) (for example, those disclosed in Baeuerle et al., "Synthetic TRuC receptors engaging the complete T cell receptor for potent anti-tumor response," Nature Communications volume 10, Article number: 2087 (2019), the entire contents of which are incorporated by reference).

[0088] In certain embodiments, the TCR-like fusion molecule is a recombinant T cell receptor (TCR). In certain embodiments, the recombinant TCR comprises at least one antigen-binding chain. In certain embodiments, the antigen-binding domain of the recombinant TCR comprises a ligand for a cell surface receptor, a receptor for a cell surface ligand, an antigen-binding portion of an antibody or fragment thereof, or an antigen-binding portion of a TCR. In certain embodiments, the recombinant TCR comprises two antigen-binding chains, i.e., a first antigen-binding chain and a second antigen-binding chain. In certain embodiments, the first and second antigen-binding chains each comprise a constant domain. In certain embodiments, the recombinant TCR binds to an antigen (e.g., a first antigen or a second antigen) in an HLA-independent manner. Thus, in certain embodiments, the recombinant TCR is an HLA-independent (or non-HLA-restricted) TCR (referred to as "HIT").

[0089] In certain embodiments, the first antigen-binding chain comprises a heavy chain variable region (V H In certain embodiments, the second antigen-binding chain comprises an antigen-binding fragment of the light chain variable region (V L In certain embodiments, the first antigen-binding chain comprises an antigen-binding fragment of the V H and the second antigen-binding chain comprises an antigen-binding fragment of the antibody V L and antigen-binding fragments of the

[0090] In certain embodiments, the constant domain comprises a TCR constant region selected from the group consisting of a native or modified TRAC polypeptide, a native or modified TRBC polypeptide, a native or modified TRDC polypeptide, a native or modified TRGC polypeptide, and any variant or functional fragment thereof. In certain embodiments, the constant domain comprises a native or modified TRAC polypeptide. In certain embodiments, the constant domain comprises a native or modified TRBC polypeptide. In certain embodiments, the first antigen binding chain comprises a TRAC polypeptide and the second antigen binding chain comprises a TRBC polypeptide. In certain embodiments, the first antigen binding chain comprises a TRBC polypeptide and the second antigen binding chain comprises a TRAC polypeptide.

[0091] In certain embodiments, the first antigen-binding chain is H and a TRAC polypeptide, and the second antigen-binding chain comprises the V L and TRBC polypeptides.

[0092] In certain embodiments, the first antigen-binding chain is H and a TRBC polypeptide, wherein the second antigen-binding chain comprises the V L and TRAC polypeptides.

[0093] In certain embodiments, at least one of the TRAC polypeptide and the TRBC polypeptide is endogenous. In certain embodiments, the TRAC polypeptide is endogenous. In certain embodiments, the TRBC polypeptide is endogenous. In certain embodiments, both the TRAC polypeptide and the TRBC polypeptide are endogenous.

[0094] In certain embodiments, the antigen binding chain can associate with a CD3ζ polypeptide. In certain embodiments, when the antigen binding chain binds to an antigen, it can activate the CD3ζ polypeptide associated with the antigen binding chain. In certain embodiments, activation of the CD3ζ polypeptide can activate immunoresponsive cells. In certain embodiments, the TCR-like fusion molecule can incorporate into the CD3 complex and provide HLA-independent antigen recognition. In certain embodiments, the TCR-like fusion molecule replaces the endogenous TCR in the CD3 / TCR complex.

[0095] In certain embodiments, the first and second antigen binding chains are about 2 x 10 -7 The dissociation constant (K D ) binds to the antigen. In certain embodiments, the first and second antigen binding chains bind to the antigen with high binding affinity. In certain embodiments, the K D is about 2 x 10 -7 M or less, approximately 1×10 -7 M or less, approximately 9 x 10 -8 M or less, approximately 1×10 -8 M or less, approximately 9 x 10 -9 M or less, about 5 x 10 -9 M or less, approximately 4 x 10 -9 M or less, about 3 x 10 -9 Below, approximately 2×10 -9 M or less, or about 1 x 10 -9 In certain embodiments, K D is approximately 1 x 10 -8 In certain embodiments, K D is about 3 x 10 -9 In certain embodiments, K D is about 5 x 10 -9 In certain embodiments, K D is approximately 1 x 10 -9 M ~ approx. 1×10 -8 M. In certain embodiments, K D is approximately 1.5 x 10 -9 M ~ approx. 1×10 -8 M. In certain embodiments, K Dis about 5 x 10 -9 M ~ approx. 1×10 -8 It's M.

[0096] In certain embodiments, the constant domain comprises a TCR constant region, e.g., a T cell receptor alpha constant region (TRAC), a T cell receptor beta constant region (TRBC, e.g., TRBC1 or TRBC2), a T cell receptor gamma constant region (TRGC, e.g., TRGC1 or TRGC2), a T cell receptor delta constant region (TRDC), or any variant or functional fragment thereof.

[0097] In certain embodiments, the first antigen-binding chain or the second antigen-binding chain comprises a constant domain comprising a native or modified TRAC polypeptide. In certain embodiments, the TRAC polypeptide comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 7 or a fragment thereof, and / or may optionally contain up to one, up to two, or up to three conservative amino acid substitutions. In certain embodiments, the TRAC polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 7. SEQ ID NO: 7 is provided below. IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS [SEQ ID NO: 7]

[0098] An exemplary nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:7 is set forth in SEQ ID NO:8, provided below. ATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCA TGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC [SEQ ID NO: 8]

[0099] In certain embodiments, a TRAC polypeptide comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 9, or a fragment thereof, and / or may optionally contain up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, a TRAC polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 9. SEQ ID NO: 9 is provided below. IPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS [SEQ ID NO: 9]

[0100] An exemplary nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:9 is set forth in SEQ ID NO:10, provided below. ATTCCCAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTT TGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC [SEQ ID NO: 10]

[0101] In certain embodiments, a TRAC polypeptide comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to an amino acid sequence encoded by a transcript expressed by the gene of NCBI Genbank ID: 28755, NG_001332.3, range 925603-930229 (SEQ ID NO: 11), or a fragment thereof, and / or may optionally contain up to one, up to two, or up to three conservative amino acid substitutions. In certain embodiments, a TRAC polypeptide comprises or consists of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 11. SEQ ID NO: 11 is provided below.

[0102] In certain embodiments, the first antigen-binding chain or the second antigen-binding chain comprises a constant domain comprising a native or modified TRBC polypeptide. In certain embodiments, the TRBC polypeptide is a TRBC2 polypeptide. In certain embodiments, the TRBC2 polypeptide comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 12 or a fragment thereof, and / or may optionally contain up to one, up to two, or up to three conservative amino acid substitutions. In certain embodiments, the TRBC2 polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 12. SEQ ID NO: 12 is provided below. DLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG [SEQ ID NO: 12]

[0103] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO:12 is set forth in SEQ ID NO:13, provided below. [SEQ ID NO: 13]

[0104] In certain embodiments, the TRBC2 polypeptide is a TRBC2 polypeptide. In certain embodiments, the TRBC2 polypeptide comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 14 or a fragment thereof, and / or may optionally contain up to one, up to two, or up to three conservative amino acid substitutions. In certain embodiments, the TRBC2 polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 14. SEQ ID NO: 14 is provided below. LEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG [SEQ ID NO: 14]

[0105] An exemplary nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:14 is set forth in SEQ ID NO:15, provided below. [SEQ ID NO: 15]

[0106] In certain embodiments, the TRBC polypeptide is a TRBC1 polypeptide. In certain embodiments, the TRBC1 polypeptide comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 16 or a fragment thereof, and / or may optionally contain up to one, up to two, or up to three conservative amino acid substitutions. In certain embodiments, the TRBC1 polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 16. SEQ ID NO: 16 is provided below. LNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF [SEQ ID NO: 16]

[0107] In certain embodiments, the TRBC1 polypeptide comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 17 or a fragment thereof, and / or may optionally contain up to one, up to two, or up to three conservative amino acid substitutions. In certain embodiments, the TRBC1 polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 17. SEQ ID NO: 17 is provided below. DLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF [SEQ ID NO: 17]

[0108] An exemplary nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:17 is set forth in SEQ ID NO:18, provided below. [SEQ ID NO: 18]

[0109] In certain embodiments, the TRBC polypeptide comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence or fragment thereof encoded by a transcript expressed by the gene of NCBI Genbank ID: 28639, NG_001333.2, range 645749-647196 (TRBC1, SEQ ID NO: 19), NCBI Genbank ID: 28638, NG_001333.2, range 655095-656583 (TRBC2, SEQ ID NO: 20), and / or may optionally contain up to one, up to two, or up to three conservative amino acid substitutions. In certain embodiments, the TRBC polypeptide comprises or consists of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 19. In certain embodiments, the TRBC polypeptide comprises or consists of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 20. SEQ ID NOs: 19 and 20 are provided below.

[0110] In certain embodiments, the first antigen-binding chain or the second antigen-binding chain comprises a constant domain comprising a native or modified TRGC polypeptide. In certain embodiments, the TRGC polypeptide is a native or modified TRGC1 polypeptide. In certain embodiments, the TRGC1 polypeptide comprises or consists of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:21, provided below. In certain embodiments, the TRGC1 polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO:21. DKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNTILGSQEGNTMKTNDTYMKFSWLTVPEKSLDKEHRCIVRHENNKNGVDQEIIFPPIKTDVITMDPKDNCSKDANDTLLLQLTNTSAYYMYLLLLLKSVVYFAIITCCLLRRTAFCCNGEKS [SEQ ID NO: 21]

[0111] In certain embodiments, the TRGC polypeptide is a native or modified TRGC2 polypeptide. In certain embodiments, the TRGC2 polypeptide comprises or consists of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 22, provided below. In certain embodiments, the TRGC2 polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22. DKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDIIKIHWQEKKSNTILGSQEGNTMKTNDTYMKFSWLTVPEESLDKEHRCIVRHENNKNGIDQEIIFPPIKTDVTTVDPKYNYSKDANDVITMDPKDNWSKDANDTLLLQLTNTSAYYTYLLLLLKSVVYFAIITCCLLRRTAFCCNGEKS [SEQ ID NO: 22]

[0112] In certain embodiments, the TRGC polypeptide comprises or consists of an amino acid sequence that is at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homologous or identical to an amino acid sequence or fragment thereof encoded by a transcript expressed by a gene of NCBI Genbank ID: 6966, NG_001336.2, range 108270-113860 (TRGC1, SEQ ID NO: 23), NCBI Genbank ID: 6967, NG_001336.2, range 124376-133924 (TRGC2, SEQ ID NO: 24), and / or may optionally contain up to one, up to two, or up to three conservative amino acid substitutions. In certain embodiments, the TRGC polypeptide comprises or consists of an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 5236. In certain embodiments, the TRGC polypeptide comprises or consists of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 24. SEQ ID NOs: 23 and 24 are provided below. [SEQ ID NO: 24]

[0113] In certain embodiments, the first antigen-binding chain or the second antigen-binding chain comprises a constant domain comprising a native or modified TRDC polypeptide. In certain embodiments, the TRDC polypeptide comprises or consists of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 25, provided below. In certain embodiments, the TRDC polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 25. SQPHTKPSVFVMKNGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAVKLGKYEDSNSVTCSVQHDNKTVHSTDFEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTEKVNMMSLTVLGLRMLFAKTVAVNFLLTAKLFFL [SEQ ID NO: 25]

[0114] In certain embodiments, the TCR-like fusion molecule comprises a hinge / spacer region linking the first antigen-binding chain to the constant domain. In certain embodiments, the TCR-like fusion molecule comprises a hinge / spacer region linking the second antigen-binding chain to the constant domain. The hinge / spacer region may be sufficiently flexible to allow the antigen-binding chain to orient in different directions to facilitate antigen recognition. In certain embodiments, the hinge / spacer region may be a hinge region from IgG1, an immunoglobulin CH2CH3 region, a portion of CD3, a portion of a TCRα polypeptide, a portion of a TCRβ polypeptide, a portion of a CD28 polypeptide, a portion of a CD8 polypeptide, or a synthetic spacer sequence. In certain embodiments, the hinge / spacer region comprises a portion of a TCRα polypeptide. In certain embodiments, the hinge / spacer region comprises a portion of a variable region (TRAV), a portion of a diversity region (TRAD), a portion of a joining region (TRAJ), a portion of a constant region (TRAC), or a combination thereof. In certain embodiments, the hinge / spacer region comprises a portion of the TRAJ region and a portion of the TRAC region of a TCR alpha polypeptide. In certain embodiments, the hinge / spacer region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 26. In certain embodiments, the hinge / spacer region comprises or consists of amino acids 1-3 of the sequence set forth in SEQ ID NO: 26. An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 26 is set forth in SEQ ID NO: 27. SEQ ID NOs: 26 and 27 are provided below. IPNIQNPDPA [SEQ ID NO: 26] ATTCCCAATATCCAGAACCCTGACCCTGCC [SEQ ID NO: 27]

[0115] In certain embodiments, the hinge / spacer region comprises a portion of a TCR β polypeptide. In certain embodiments, the hinge / spacer region comprises a portion of the variable region (TRBV), a portion of the diversity region (TRBD), a portion of the joining region (TRBJ), a portion of the constant region (TRBC), or a combination thereof. In certain embodiments, the hinge / spacer region comprises a portion of the TRBJ region and a portion of the TRAC region (C) of a TCR β polypeptide. In certain embodiments, the hinge / spacer region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 28. In certain embodiments, the hinge / spacer region comprises or consists of amino acids 1-2 of the sequence set forth in SEQ ID NO: 28. An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 28 is set forth in SEQ ID NO: 29. SEQ ID NOs: 28 and 29 are provided below. LEDLKNVFPPE [SEQ ID NO: 28] CTGGAGGATCTGAAAAACGTGTTCCCTCCTGAA [SEQ ID NO: 29]

[0116] In certain embodiments, the antigen binding chain does not comprise an intracellular domain. In certain embodiments, the antigen binding chain can associate with a CD3ζ polypeptide. In certain embodiments, the antigen binding chain is associated with a CD3ζ polypeptide via a constant domain. In certain embodiments, the CD3ζ polypeptide is endogenous. In certain embodiments, the CD3ζ polypeptide is exogenous. In certain embodiments, binding of the antigen binding chain to a target antigen can activate the CD3ζ polypeptide associated with the antigen binding chain. In certain embodiments, the exogenous CD3ζ polypeptide is fused to or incorporated into a costimulatory molecule disclosed herein.

[0117] In certain embodiments, the TCR-like fusion molecule comprises an antigen-binding chain comprising an intracellular domain. In certain embodiments, the intracellular domain comprises a CD3ζ polypeptide. In certain embodiments, binding of the antigen-binding chain to an antigen can activate the CD3ζ polypeptide of the antigen-binding chain.

[0118] In certain embodiments, CD3ζ polypeptides comprise or consist of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous to the amino acid sequence set forth in SEQ ID NO: 53 or a fragment thereof, and / or may optionally contain up to one, up to two, or up to three conservative amino acid substitutions. In certain embodiments, CD3ζ polypeptides comprise or consist of an amino acid sequence that is a contiguous portion of SEQ ID NO: 53 that is at least about 20, or at least about 30, or at least about 40, or at least about 50, and up to about 164 amino acids in length. In certain embodiments, CD3ζ comprises or consists of the amino acid sequence of amino acids 1-164, 1-50, 50-100, 52-164, 100-150, or 150-164 of SEQ ID NO: 53. In certain embodiments, the CD3ζ polypeptide comprises or consists of amino acids 52 to 164 of SEQ ID NO:53.

[0119] In certain embodiments, a CD3ζ polypeptide comprises or consists of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to SEQ ID NO:54 or a fragment thereof, and / or may optionally contain up to one, up to two, or up to three conservative amino acid substitutions. In certain embodiments, a CD3ζ polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO:54.

[0120] In certain embodiments, the TCR-like fusion molecule comprises an antigen-binding chain comprising an intracellular domain, wherein the intracellular domain comprises a costimulatory signaling region. In certain embodiments, the intracellular domain comprises a costimulatory signaling region and a CD3ζ polypeptide. In certain embodiments, the intracellular domain comprises a costimulatory signaling region and does not comprise a CD3ζ polypeptide. In certain embodiments, the costimulatory signaling region comprises at least the intracellular domain of a costimulatory molecule disclosed herein.

[0121] In certain embodiments, the TCR-like fusion molecule can associate with the CD3 complex (also known as the "T cell co-receptor"). In certain embodiments, the TCR-like fusion molecule and the CD3 complex form an antigen-recognition receptor complex similar to a natural TCR / CD3 complex. In certain embodiments, the CD3 complex is endogenous. In certain embodiments, the CD3 complex is exogenous. In certain embodiments, the TCR-like fusion molecule replaces a natural and / or endogenous TCR in a CD3 / TCR complex. In certain embodiments, the CD3 complex comprises a CD3γ chain, a CD3δ chain, and two CD3ε chains.

[0122] In certain embodiments, the CD3 gamma chain comprises or consists of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to the amino acid sequence having NCBI Reference Number: NP_000064.1 (SEQ ID NO: 30), or a fragment thereof, and / or may optionally contain up to one, or up to two, or up to three conservative amino acid substitutions. SEQ ID NO: 30 is provided below. MEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRN [SEQ ID NO: 30]

[0123] In certain embodiments, the CD3 delta chain comprises or consists of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to the amino acid sequence having NCBI Reference Number: NP_000723.1 (SEQ ID NO: 31), or a fragment thereof, or the amino acid sequence having NCBI Reference Number: NP_001035741.1 (SEQ ID NO: 32), or a fragment thereof, and / or may optionally contain up to one, up to two, or up to three conservative amino acid substitutions. SEQ ID NOs: 31 and 32 are provided below. MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALGVFCFAGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNK [SEQ ID NO: 31] MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRTADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNK [SEQ ID NO: 32]

[0124] In certain embodiments, the CD3 epsilon chain comprises or consists of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to the amino acid sequence having NCBI Reference Number: NP_000724.1 (SEQ ID NO: 33), or a fragment thereof, and / or may optionally contain up to one, or up to two, or up to three conservative amino acid substitutions. SEQ ID NO: 33 is provided below. MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI [SEQ ID NO: 33]

[0125] In certain embodiments, TCR-like fusion molecules exhibit higher antigen sensitivity than CARs targeting the same antigen. In certain embodiments, TCR-like fusion molecules can induce an immune response when bound to an antigen with low antigen density on the surface of tumor cells. In certain embodiments, cells containing TCR-like fusion molecules can be used to treat subjects with tumor cells that have low levels of surface antigen expression, for example, to prevent disease recurrence, and the subject has undergone treatment that results in residual tumor cells. In certain embodiments, tumor cells have a low antigen density of target molecules on the surface of the tumor cells. In certain embodiments, target molecules with low antigen density on the cell surface have a density of less than about 5,000 molecules per cell, less than about 4,000 molecules per cell, less than about 3,000 molecules per cell, less than about 2,000 molecules per cell, less than about 1,500 molecules per cell, less than about 1,000 molecules per cell, less than about 500 molecules per cell, less than about 200 molecules per cell, or less than about 100 molecules per cell. In certain embodiments, target molecules having a low antigen density on the cell surface have a density of less than about 2,000 molecules per cell. In certain embodiments, target molecules having a low antigen density on the cell surface have a density of less than about 1,500 molecules per cell. In certain embodiments, target molecules having a low antigen density on the cell surface have a density of less than about 1,000 molecules per cell. In certain embodiments, target molecules having a low antigen density on the cell surface have a density of about 4,000 molecules per cell to about 2,000 molecules per cell, about 2,000 molecules per cell to about 1,000 molecules per cell, about 1,500 molecules per cell to about 1,000 molecules per cell, about 2,000 molecules per cell to about 500 molecules per cell, about 1,000 molecules per cell to about 200 molecules per cell, or about 1,000 molecules per cell to about 100 molecules per cell.

[0126] In certain embodiments, the antigen-recognizing receptor is a V H and a first antigen-binding chain comprising a constant domain comprising a TRBC polypeptide; and a V Land a second antigen binding chain comprising a constant domain comprising a TRAC polypeptide. In certain embodiments, the first antigen binding chain is a TCR-like fusion molecule comprising a "V H In certain embodiments, the second antigen-binding chain is designated "V-TRBC chain." L In certain embodiments, the first antigen-binding chain is designated as a V-TRAC chain. H and the TRBC polypeptide. In certain embodiments, the hinge region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 26 or SEQ ID NO: 28. In certain embodiments, the second antigen-binding chain comprises a V L and the TRAC polypeptide. In certain embodiments, the hinge region comprises or consists of the amino acid sequence set forth in SEQ ID NO:26 or SEQ ID NO:28.

[0127] In certain embodiments, the antigen-recognizing receptor is a V H and a first antigen-binding chain comprising a constant domain comprising a TRAC polypeptide; and a V L and a second antigen binding chain comprising a constant domain comprising a TRBC polypeptide. In certain embodiments, the first antigen binding chain is a TCR-like fusion molecule comprising a "V H In certain embodiments, the second antigen-binding chain is designated as "V-TRAC chain." L In certain embodiments, the first antigen-binding chain is designated as a V-TRBC chain. H and the TRAC polypeptide. In certain embodiments, the second antigen-binding chain comprises a hinge region between V L and the TRBC polypeptide. In certain embodiments, the first antigen-binding chain and the second antigen-binding chain bind to an antigen (e.g., human CD70).

[0128] In certain embodiments, the antigen-recognizing receptor is a V H and a first antigen-binding chain comprising a constant domain comprising a TRBC polypeptide; and a V Land a second antigen binding chain comprising a constant domain comprising a TRAC polypeptide. In certain embodiments, the first antigen binding chain is a TCR-like fusion molecule comprising a "V H In certain embodiments, the second antigen-binding chain is designated "V-TRBC chain." L In certain embodiments, the first antigen-binding chain is designated as a V-TRAC chain. H and the TRAC polypeptide. In certain embodiments, the second antigen-binding chain comprises a hinge region between V L and the TRBC polypeptide. In certain embodiments, the first antigen-binding chain and the second antigen-binding chain bind to an antigen (e.g., human CD70).

[0129] In certain embodiments, the antigen-recognizing receptor is a TCR-like fusion molecule that binds to CD70 (e.g., human CD70) and comprises two antigen-binding chains, e.g., V, that can dimerize and bind to CD70. H and a first antigen-binding chain ("V") comprising a TRBC polypeptide. H -TRBC chain") and V L and a second antigen-binding chain ("V") comprising a TRBC polypeptide. L In certain embodiments, V H comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 34, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 35, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 36. H comprises the amino acid sequence set forth in SEQ ID NO: 40. In certain embodiments, V L comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 37, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 38, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 39. Lcomprises the amino acid sequence set forth in SEQ ID NO: 42. In certain embodiments, the TRAC polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 9. In certain embodiments, the TRBC polypeptide is a TRBC2 polypeptide. In certain embodiments, the TRBC2 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 14. In certain embodiments, the TCR-like fusion molecule is designated "70-HIT" or "70H". SEQ ID NOs: 34-43 are provided in Table 1 below.

[0130] In certain embodiments, the CDR regions / sequences disclosed herein are delineated using the Kabat system (Swindells et al., J Mol Biol. 2017 Feb 3;429(3):356-364). [Table 1]

[0131] In certain embodiments, the antigen-recognizing receptor is a TCR-like fusion molecule that binds to CD70 (e.g., human CD70) and comprises two antigen-binding chains, e.g., V, that can dimerize and bind to CD70. H and a first antigen-binding chain ("V") comprising a TRBC polypeptide. H -TRBC chain") and V L and a second antigen-binding chain ("V") comprising a TRBC polypeptide. L In certain embodiments, V H is a V of the anti-CD70 antibody disclosed in International Patent Publication No. WO 2007 / 038637, which is incorporated by reference in its entirety. H In certain embodiments, the V H is the V of the anti-CD70 antibody 2H5 disclosed in International Patent Publication No. 2007 / 038637. H In certain embodiments, the V L is the V of the anti-CD70 antibody disclosed in International Patent Publication No. 2007 / 038637. HIn certain embodiments, the V L is the V of the anti-CD70 antibody 2H5 disclosed in International Patent Publication No. 2007 / 038637. H It includes the sequences CDR1, CDR2, and CDR3.

[0132] Various TCR-like fusion molecules are disclosed in International Patent Application Publication No. WO 2019 / 133969, which is incorporated by reference in its entirety.

[0133] 2.1.3. Delivery of antigen-recognizing receptors In certain embodiments, the antigen-recognizing receptor is delivered to the cell by a viral method. In certain embodiments, the viral method includes a viral vector. In certain embodiments, the viral vector is a retroviral vector (e.g., a gamma retroviral vector or a lentiviral vector). Other viral vectors include adenoviral vectors, adeno-associated viral vectors, vaccinia virus, bovine papillomavirus, and herpesviruses (e.g., Epstein-Barr virus).

[0134] In certain embodiments, the antigen-recognizing receptor is delivered to cells by non-viral methods. Any targeted genome editing method can also be used to deliver the first antigen-recognizing receptor to cells. In certain embodiments, the antigen-recognizing receptor is delivered to cells by methods including homologous recombination, zinc finger nuclease, meganuclease, transcription activator-like effector nuclease (TALEN), clustered regularly interspaced short palindromic repeats (CRISPR) system, or a combination thereof. In certain embodiments, the CRISPR system is used to deliver the first antigen-recognizing receptor to cells.

[0135] In certain embodiments, the cell is a T cell, and the first antigen recognition receptor is integrated into a locus within the genome of the T cell. Non-limiting examples of loci include the TRAC locus, the TRBC locus, the TRDC locus, and the TRGC locus. In certain embodiments, the locus is the TRAC locus or the TRBC locus. In certain embodiments, the cell is a T cell, and the first antigen recognition receptor is integrated into the TRAC locus. Methods of targeting a CAR to a site within the genome of a T cell are disclosed in WO2017 / 180989 and Eyquem et al., Nature. (2017 Mar 2); 543(7643): 113-117, both of which are incorporated by reference in their entireties. In certain embodiments, the cell is a T cell, and the first antigen recognition receptor is a CAR, and the first antigen recognition receptor is integrated into the TRAC locus. In certain embodiments, the cell further comprises a genetic disruption of the TRBC locus. In certain embodiments, the gene disruption of the TRBC locus results in a knockout of the TRBC locus.

[0136] 2.1.4. Secondary antigen-recognition receptor In certain embodiments, a cell disclosed herein comprising an antigen-recognizing receptor (e.g., a first antigen-recognizing receptor) further comprises a second antigen-recognizing receptor that targets a second antigen. In certain embodiments, the second antigen-recognizing receptor is a chimeric receptor. In certain embodiments, the chimeric receptor is a chimeric antigen receptor (CAR). In certain embodiments, the chimeric receptor is a chimeric ligand receptor. In certain embodiments, the chimeric receptor is a CCR. In certain embodiments, the chimeric receptor is a T cell receptor (TCR).

[0137] 2.1.4.1. Second Antigen In certain embodiments, the second antigen is a tumor antigen, such as those disclosed in Section 2.1.1. In certain embodiments, the tumor antigen is an antigen with low antigen density. In certain embodiments, the tumor antigen is expressed on cells with low tumor cell frequency.

[0138] In certain embodiments, the second antigen is selected from the group consisting of CD19, CD70, IL1RAP, ABcG2, AChR, ACKR6, ADAMTS13, ADGRE2, ADGRE2(EMR2), ADORA3, ADRA1D, AGER, ALS2, an antigen of a cytomegalovirus (CMV)-infected cell (e.g., a cell surface antigen), ANO9, AQP2, ASIC3, ASPRV1, ATP6V0A4, B3GNT4, B7-H3, BCMA, BEST4, C3orf35, CADM3, CAIX, CAPN3, CCDC155, CCR1, CD10, CD117, CD1 23, CD133, CD135(FLT3), CD138, CD20, CD22, CD244(2B4), CD25, CD26, CD276, CD30, CD300LF, CD312, CD32, CD321, CD33, CD34, CD36, CD38, CD41, CD44, C D44V6, CD47, CD49f, CD56, CD7, CD71, CD74, CD8, CD82, CD96, CD98, CD99, CDH13, CDHR1, CEA, CEACAM6, CHST3, CLEC12A, CLEC1A, CLL1, CNIH2, COL15A1, C OLEC12, CPM, CR1, CX3CR1, CXCR4, CYP4F11, DAGLB, DARC, DFNB31, DGKI, EGF1R, EGFR-VIII, EGP-2, EGP-40, ELOVL6, EMB, EMC10, EMR2, ENG, EpCAM, EphA2 , EPHA4, ERBB, ERBB2, Erb-B3, Erb-B4, E-selectin, EXOC3L4, EXTL3, FAM186B, FBP, FCGR1A, FKBP1B, FLRT1, folate receptor-a, FOLR2, FRMD5, GABRB2, GAS2, GD2, GD3 , GDPD3, GNA14, GNAZ, GPR153, GPR56, GYPA, HEPHL1, HER-2, hERT, HILPDA, HLA-DR, HOOK1, hTERT, HTR2A, ICAM1, IGFBP3, IL10RB, IL20RB, IL23R, ILDR1, interleukin-13 receptor subunit alpha-2 (IL-13Rα2), ITFG3, ITGA4, ITGA5, ITGA8, ITGAX, ITGB5, ITGB8, JAM3, KCND1, KCNJ5, KCNK13, KCNN4, KCNV2, KDR, KIF19,KIF26B, kappa light chain, L1CAM, LAX1, LEPR, Lewis Y (CD174), Lewis Y (LeY), LILRA2, LILRA6, LILRB2, LILRB3, LILRB4, LOXL4, LPAR2, LRRC37A3, LRRC8E, LRRN2, LRRTM2, LTB4R, MAGE-A1, MAGEA3, MANSC1, MART1, GP100, MBOAT1, MBOAT7, melanoma antigen family A, mesothelin (MSLN), MFAP3L, MMP25, MRP1, MT-ND1, mucin 1 (MUC1), mucin 16 (MUC16), MYADM, MYADML2, NGFR, NKCS1, NK G2D ligand, NLGN3, NPAS2, NY-ESO-1, oncofetal antigen (h5T4), OTOA, P2RY13, p53, PDE3A, PEAR1, PIEZO1, PLXNA4, PLXNC1, PNPLA3, PPFIA4, PPP2R5B, PRAME, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), proteinase 3 (PR1), PSD2, PTPRJ, RDH16, receptor tyrosine-protein kinase Erb-B2, RHBDL3, RNF173, RNF183, ROR1, RYR2, SCIN, SCN11A, SCN2A, SCNN1D, SEC31B, SEMA4A, SH3PXD2A, SIGLEC11, SIRPB1, SLC16A6, SLC19A1, SLC22A5, SLC25A36, SLC25A41, SLC30A1 , SLC34A3, SLC43A3, SLC44A1, SLC44A3, SLC45A3, SLC6A16, SLC6A6, SLC8A3, SLC9A1, SLCO2B1, SPAG17, STC1, STON2, SUN3, Survivin, SUSD2, SYNC, T ACSTD2, TAS1R3, TEX29, TFR2, TIM-3 (HAVCR2), TLR2, TMEFF2, TMEM145, TMEM27, TMEM40, TMEM59L, TMEM89, TMPRSS5, TNFRSF14, TNFRSF1B, TRIM55, TSPEAR, TTYH3, tumor-associated glycoprotein 72 (TAG-72), tyrosinase, vascular endothelial growth factor R2 (VEGF-R2), VLA-4, Wilms tumor protein (WT-1), WNT4, WT1, and ZDHHC11.

[0139] In certain embodiments, the second antigen is a pathogen antigen, such as those disclosed in Section 2.1.1.

[0140] Chimeric Antigen Receptors (CARs) CARs are engineered receptors that transfer or confer a desired specificity onto immune effector cells. CARs can be used to transfer the specificity of monoclonal antibodies into T cells, and the transfer of their coding sequences is facilitated by retroviral vectors.

[0141] There are three generations of CARs. "First generation" CARs are typically composed of an extracellular antigen-binding domain (e.g., scFv) that binds to the target antigen and an intracellular signaling domain. In certain embodiments, the CAR further comprises a transmembrane domain. "First generation" CARs provide novel antigen recognition and signal transduction via the CD3 zeta chain signaling domain in a single fusion molecule, independent of HLA-mediated antigen presentation. + and CD8 + "Second generation" CARs include those that provide both costimulatory (e.g., CD28 or 4-1BB) and activation (CD3ζ) signals. "Third generation" CARs include those that provide multiple costimulatory (e.g., CD28 and 4-1BB) and activation (CD3ζ) signals.

[0142] In certain embodiments, the second antigen-recognizing receptor is a CAR comprising an extracellular antigen-binding domain that binds to the second antigen and an intracellular signaling domain. In certain embodiments, the CAR further comprises a transmembrane domain. In certain embodiments, the CAR further comprises a hinge / spacer region.

[0143] In certain embodiments, the extracellular antigen binding domain of the CAR (e.g., scFv) is about 5×10 -7 M or less, approximately 1×10 -7 M or less, about 5 x 10 -8 M or less, approximately 1×10 -8 M or less, about 5 x 10 -9 M or less, or about 1 x 10 -9 M or less, or about 1 x 10 -10 The dissociation constant (K D In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) of the CAR binds to the first antigen at about 1 x 10 -8 K below M D binds to the first antigen.

[0144] The binding of the extracellular antigen-binding domain (e.g., in scFv) can be confirmed, for example, by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), or Western blot assay. Each of these assays generally detects the presence of a particular protein-antibody complex of interest by using a labeled reagent (e.g., antibody or scFv) specific to the complex of interest. For example, scFv can be radioactively labeled and used in a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, incorporated herein by reference). Radioisotopes can be detected by means such as the use of a gamma counter or scintillation counter, or autoradioactivity testing. In certain embodiments, the extracellular antigen-binding domain of the CAR is labeled with a fluorescent marker. Non-limiting examples of fluorescent markers include green fluorescent protein (GFP), blue fluorescent protein (e.g., EBFP, EBFP2, Azurite, and mKalama1), cyan fluorescent protein (e.g., ECFP, Cerulean, and CyPet), and yellow fluorescent protein (e.g., YFP, Citrine, Venus, and YPet).

[0145] The extracellular antigen-binding domain may comprise or be an scFv, Fab (optionally cross-linked), or F(ab)2. In certain embodiments, any of the above molecules may be included in a fusion protein with a heterologous sequence to form the extracellular antigen-binding domain. In certain embodiments, the extracellular antigen-binding domain comprises or is an scFv. In certain embodiments, the scFv is a human scFv. In certain embodiments, the scFv is a humanized scFv. In certain embodiments, the scFv is a mouse scFv.

[0146] In addition, the extracellular antigen-binding domain of the CAR may contain a leader or signal peptide that directs the nascent protein into the endoplasmic reticulum. A signal peptide or leader may be essential if the CAR is glycosylated and anchored in the cell membrane. The signal sequence or leader may be a peptide sequence (about 5, about 10, about 15, about 20, about 25, or about 30 amino acids in length) present at the N-terminus of a newly synthesized protein that directs entry into the secretory pathway. In certain embodiments, the signal peptide is covalently linked to the 5' end (N-terminus) of the extracellular antigen-binding domain of the CAR. Exemplary leader sequences include, but are not limited to, a human IL-2 signal sequence (e.g., a human IL-2 signal sequence comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 44), a mouse IL-2 signal sequence (e.g., a mouse IL-2 signal sequence comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 45), a human kappa leader sequence (e.g., a human kappa leader sequence comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 46), a mouse kappa leader sequence (e.g., a mouse kappa leader sequence comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 47), a human CD8 leader sequence (e.g., a human CD8 leader sequence comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 48), a truncated human CD8 signal peptide (e.g., a truncated human CD8 signal peptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 49), a human albumin signal sequence (e.g., a human albumin signal sequence comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 50), and a human prolactin signal sequence (e.g., a human prolactin signal sequence comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 51). SEQ ID NOs: 44-51 are provided below. MYRMQLLSCIALSLALVTNS [SEQ ID NO: 44] MYSMQLASCVTLTLVLLVNS [SEQ ID NO: 45] METPAQLLFLLLLWLPDTTG [SEQ ID NO: 46] METDTLLLWVLLLWVPGSTG [SEQ ID NO: 47] MALPVTALLLPLALLLHAARP [SEQ ID NO: 48] MALPVTALLLPLALLLHA [SEQ ID NO: 49] MKWVTFISLLFSSAYS [SEQ ID NO: 50] MDSKGSSQKGSRLLLLLVVSNLLLCQGVVS [SEQ ID NO: 51]

[0147] In certain embodiments, the signal peptide comprises a CD8 polypeptide, e.g., the CAR comprises a truncated CD8 signal peptide. In certain embodiments, the signal peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO:49.

[0148] In certain embodiments, the second antigen-recognizing receptor is a CAR that includes a transmembrane domain. Different transmembrane domains result in different receptor stability. After antigen recognition, the receptors cluster and transmit signals to cells. According to the subject matter disclosed herein, the transmembrane domain of the first antigen-recognizing receptor can include the natural or modified transmembrane domain of a CD8 polypeptide, a CD28 polypeptide, a CD3ζ polypeptide, a CD40 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, a CD84 polypeptide, a CD166 polypeptide, a CD8a polypeptide, a CD8b polypeptide, an ICOS polypeptide, an ICAM-1 polypeptide, a CTLA-4 polypeptide, a CD27 polypeptide, a CD40 polypeptide, an NKG2D polypeptide, a synthetic polypeptide (not based on a protein related to immune response), or a combination thereof.

[0149] In certain embodiments, the transmembrane domain of the CAR comprises a CD28 polypeptide (e.g., the transmembrane domain of CD28 or a portion thereof). In certain embodiments, the transmembrane domain of the CAR comprises the transmembrane domain of human CD28 or a portion thereof. In certain embodiments, the CD28 polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of the amino acid sequence having NCBI Reference Number: NP_006130 (SEQ ID NO: 52) that is at least about 20, or at least about 25, or at least about 30, and / or up to about 220 amino acids in length. In certain embodiments, the CD28 polypeptide comprises or consists of the amino acid sequence of amino acids 1-220, 1-50, 50-100, 100-150, 114-220, 150-200, 153-179, or 200-220 of SEQ ID NO: 52. In certain embodiments, the transmembrane domain of the CAR comprises a CD28 polypeptide comprising or consisting of amino acids 153-179 of SEQ ID NO: 52. SEQ ID NO: 52 is provided below. MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS [SEQ ID NO: 52]

[0150] In certain embodiments, the second antigen-recognizing receptor is a CAR that further comprises a hinge / spacer region that links the extracellular antigen-binding domain to the transmembrane domain. The hinge / spacer region may be flexible enough to allow the antigen-binding domain to orient in different directions to facilitate antigen recognition. In certain embodiments, the hinge / spacer region of the CAR may comprise a natural or modified hinge region of a CD8 polypeptide, a CD28 polypeptide, a CD3ζ polypeptide, a CD40 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, a CD84 polypeptide, a CD166 polypeptide, a CD8a polypeptide, a CD8b polypeptide, an ICOS polypeptide, an ICAM-1 polypeptide, a CTLA-4 polypeptide, a CD27 polypeptide, a CD40 polypeptide, an NKG2D polypeptide, a synthetic polypeptide (not based on a protein associated with an immune response), or a combination thereof. The hinge / spacer region can be a hinge region from IgG1, or an immunoglobulin CH2CH3 region, and a portion of CD3, a portion of a CD28 polypeptide (e.g., a portion of SEQ ID NO: 52), a portion of a CD8 polypeptide, or a synthetic spacer sequence.

[0151] In certain embodiments, the second antigen-recognizing receptor is a CAR that further comprises a hinge / spacer region comprising a native or modified hinge region of a CD28 polypeptide. In certain embodiments, the hinge / spacer region of the first antigen-recognizing receptor (e.g., a CAR) comprises a CD28 polypeptide comprising or consisting of amino acids 114 to 152 of SEQ ID NO:52.

[0152] In certain embodiments, the hinge / spacer region is located between the extracellular antigen-binding domain and the transmembrane domain. In certain embodiments, the hinge / spacer region comprises a CD8 polypeptide, a CD28 polypeptide, a CD3ζ polypeptide, a CD4 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, a CD166 polypeptide, a CD8a polypeptide, a CD8b polypeptide, an ICOS polypeptide, an ICAM-1 polypeptide, a CTLA-4 polypeptide, a CD27 polypeptide, a CD40 polypeptide, an NKG2D polypeptide, a synthetic polypeptide (not based on a protein associated with an immune response), or a combination thereof. In certain embodiments, the transmembrane domain comprises a CD8 polypeptide, a CD28 polypeptide, a CD3ζ polypeptide, a CD4 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, a CD166 polypeptide, a CD8a polypeptide, a CD8b polypeptide, an ICOS polypeptide, an ICAM-1 polypeptide, a CTLA-4 polypeptide, a CD27 polypeptide, a CD40 polypeptide, an NKG2D polypeptide, a synthetic polypeptide (not based on a protein associated with an immune response), or a combination thereof.

[0153] In certain embodiments, the transmembrane domain and hinge / spacer region are derived from the same molecule. In certain embodiments, the transmembrane domain and hinge / spacer region are derived from different molecules. In certain embodiments, the hinge / spacer region comprises a CD28 polypeptide, and the transmembrane domain comprises a CD28 polypeptide. In certain embodiments, the hinge / spacer region comprises a CD28 polypeptide, and the transmembrane domain comprises a CD28 polypeptide. In certain embodiments, the hinge / spacer region comprises a CD84 polypeptide, and the transmembrane domain comprises a CD84 polypeptide. In certain embodiments, the hinge / spacer region comprises a CD166 polypeptide, and the transmembrane domain comprises a CD166 polypeptide. In certain embodiments, the hinge / spacer region comprises a CD8a polypeptide, and the transmembrane domain comprises a CD8a polypeptide. In certain embodiments, the hinge / spacer region comprises a CD8b polypeptide, and the transmembrane domain comprises a CD8b polypeptide. In certain embodiments, the hinge / spacer region comprises a CD28 polypeptide and the transmembrane domain comprises an ICOS polypeptide.

[0154] In certain embodiments, the second antigen-recognizing receptor is a CAR comprising an intracellular signaling domain. In certain embodiments, the intracellular signaling domain of the CAR comprises a CD3ζ polypeptide. CD3ζ can activate or stimulate cells (e.g., lymphoid cells, e.g., T cells). Wild-type ("native") CD3ζ comprises three functional immunoreceptor tyrosine-based activation motifs (ITAMs) and three functional basic-rich extension (BRS) regions (BRS1, BRS2, ​​and BRS3). CD3ζ transmits activation signals to cells (e.g., lymphoid cells, e.g., T cells) after antigen binding. The intracellular signaling domain of the CD3ζ chain is the primary transmitter of signals from endogenous TCRs.

[0155] In certain embodiments, the intracellular signaling domain of the CAR comprises native CD3ζ. In certain embodiments, native CD3ζ comprises or consists of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical or homologous to the amino acid sequence having NCBI Reference Number: NP_932170 (SEQ ID NO: 53), or a fragment thereof, and / or may optionally contain up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the CD3ζ polypeptide comprises or consists of an amino acid sequence that is at least about 20, or at least about 30, or at least about 40, or at least about 50, and up to about 164 amino acids in length. In certain embodiments, native CD3ζ comprises or consists of the amino acid sequence of amino acids 1-164, 1-50, 50-100, 52-164, 100-150, or 150-164 of SEQ ID NO: 53. In certain embodiments, the intracellular signaling domain of the CAR comprises native CD3ζ comprising or consisting of the amino acid sequence of amino acids 52-164 of SEQ ID NO: 53. SEQ ID NO: 53 is provided below. MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR [SEQ ID NO: 53]

[0156] In certain embodiments, native CD3ζ comprises or consists of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical or homologous to the amino acid sequence set forth in SEQ ID NO: 54. SEQ ID NO: 54 is provided below. RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR [SEQ ID NO: 54]

[0157] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide. In certain embodiments, the modified CD3ζ polypeptide comprises one, two, or three ITAMs. In certain embodiments, the modified CD3ζ polypeptide comprises a native ITAM1. In certain embodiments, the native ITAM1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 55, as provided below. QNQLYNELNLGRREEYDVLDKR [SEQ ID NO: 55]

[0158] An exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:55 is set forth in SEQ ID NO:56, provided below. CAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGA [SEQ ID NO: 56]

[0159] In certain embodiments, the modified CD3ζ polypeptide comprises an ITAM1 variant comprising one or more loss-of-function mutations. In certain embodiments, the ITAM1 variant comprises or consists of two loss-of-function mutations. In certain embodiments, each of the one or more (e.g., two) loss-of-function mutations comprises a mutation of a tyrosine residue in ITAM1. In certain embodiments, the ITAM1 variant consists of two loss-of-function mutations. In certain embodiments, the ITAM1 variant comprises or consists of the amino acid sequence set forth in SEQ ID NO: 57, provided below. QNQLFNELNLGRREEFDVLDKR [SEQ ID NO: 57]

[0160] An exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:57 is set forth in SEQ ID NO:58, provided below. CAGAACCAGCTCTTTAACGAGCTCAATCTAGGACGAAGAGAGGAGTTCGATGTTTTGGACAAGAGA [SEQ ID NO: 58]

[0161] In certain embodiments, the modified CD3ζ polypeptide comprises a native ITAM2, which comprises or consists of the amino acid sequence set forth in SEQ ID NO:59, provided below. QEGLYNELQKDKMAEAYSEIGMK [SEQ ID NO: 59]

[0162] An exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:59 is set forth in SEQ ID NO:60, provided below. CAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAA [SEQ ID NO: 60]

[0163] In certain embodiments, the modified CD3ζ polypeptide comprises an ITAM2 variant. In certain embodiments, the ITAM2 variant comprises or consists of one or more loss-of-function mutations. In certain embodiments, the ITAM2 variant comprises or consists of two loss-of-function mutations. In certain embodiments, each of the one or more (e.g., two) loss-of-function mutations comprises a mutation of a tyrosine residue in ITAM2. In certain embodiments, the ITAM1 variant consists of two loss-of-function mutations. In certain embodiments, the ITAM2 variant comprises or consists of the amino acid sequence set forth in SEQ ID NO: 61, provided below. QEGLFNELQKDKMAEAFSEIGMK [SEQ ID NO: 61]

[0164] An exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:61 is set forth in SEQ ID NO:62, provided below. CAGGAAGGCCTGTTCAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTTCAGTGAGATTGGGATGAAA [SEQ ID NO: 62]

[0165] In certain embodiments, the modified CD3ζ polypeptide comprises a native ITAM3, which comprises or consists of the amino acid sequence set forth in SEQ ID NO:63, provided below. HDGLYQGLSTATKDTYDALHMQ [SEQ ID NO: 63]

[0166] An exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:63 is set forth in SEQ ID NO:64, provided below. CACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAG [SEQ ID NO: 64]

[0167] In certain embodiments, the modified CD3ζ polypeptide comprises an ITAM3 variant. In certain embodiments, the ITAM3 variant comprises or consists of two loss-of-function mutations. In certain embodiments, each of the one or more (e.g., two) loss-of-function mutations comprises a mutation of a tyrosine residue in ITAM3. In certain embodiments, the ITAM3 variant comprises or consists of two loss-of-function mutations. In certain embodiments, the ITAM3 variant comprises or consists of the amino acid sequence set forth in SEQ ID NO: 65, provided below. HDGLFQGLSTATKDTFDALHMQ [SEQ ID NO: 65]

[0168] An exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:65 is set forth in SEQ ID NO:66, provided below. CACGATGGCCTTTTCCAGGGGCTCAGTACAGCCACCAAGGACACCTTCGACGCCCTTCACATGCAG[SEQ ID NO: 66]

[0169] Various modified CD3ζ polypeptides and CARs comprising modified CD3ζ polypeptides are disclosed in International Patent Application Publication No. 2019 / 133969, the entire contents of which are incorporated herein by reference.

[0170] In certain embodiments, the intracellular signaling domain of a CAR comprises a modified CD3ζ polypeptide comprising a native ITAM1, an ITAM2 variant comprising or consisting of one or more (e.g., two) loss-of-function mutations, and an ITAM3 variant comprising or consisting of one or more (e.g., two) loss-of-function mutations. In certain embodiments, the intracellular signaling domain of a CAR comprises a modified CD3ζ polypeptide comprising a native ITAM1, an ITAM2 variant consisting of two loss-of-function mutations, and an ITAM3 variant consisting of two loss-of-function mutations. In certain embodiments, the intracellular signaling domain of a CAR comprises a modified CD3ζ polypeptide comprising a native ITAM1 consisting of the amino acid sequence set forth in SEQ ID NO:55, an ITAM2 variant consisting of the amino acid sequence set forth in SEQ ID NO:59, and an ITAM3 variant consisting of the amino acid sequence set forth in SEQ ID NO:63. In certain embodiments, a CAR is referred to as "1XX." In certain embodiments, the modified CD3ζ polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO:67. SEQ ID NO:67 is provided below. RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR [SEQ ID NO: 67]

[0171] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide comprising, or consisting of, an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% identical to SEQ ID NO: 67, or a fragment thereof, and / or optionally comprises up to one, or up to two, or up to three conservative amino acid substitutions.

[0172] An exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:67 is set forth in SEQ ID NO:68, provided below. AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCC TGTTCAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTTCAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTTCCAGGGGCTCAGTACAGCCACCAAGGACACCTTCGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC [SEQ ID NO: 68]

[0173] In certain embodiments, the intracellular signaling domain of the CAR further comprises at least one costimulatory signaling region. In certain embodiments, the at least one costimulatory region comprises a costimulatory molecule or a portion thereof. In certain embodiments, the at least one costimulatory region comprises at least the intracellular domain of at least one costimulatory molecule or a portion thereof. Non-limiting examples of costimulatory molecules include CD28, 4-1BB, OX40, CD27, CD40, CD154, CD97, CD11a / CD18, ICOS, DAP-10, CD2, CD150, CD226, and NKG2D.

[0174] In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising the intracellular domain of a CD28 polypeptide, e.g., CD28, or a portion thereof. In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising the intracellular domain of human CD28, or a portion thereof.

[0175] In certain embodiments, the CD28 polypeptide comprised in the costimulatory signaling region of the second antigen recognizing receptor comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or at least about 100% identical or homologous to the amino acid sequence set forth in SEQ ID NO: 52, or a fragment thereof, and / or may optionally comprise up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the CD28 polypeptide comprised in the costimulatory signaling region of the CAR comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 52 that is at least about 20, or at least about 30, or at least about 40, or at least about 50, and up to about 220 amino acids in length. Alternatively or additionally, in certain embodiments, a CD28 polypeptide comprised in a costimulatory signaling region of the CAR comprises or consists of amino acids 1-220, 1-50, 50-100, 100-150, 114-220, 150-200, 180-220, or 200-220 of SEQ ID NO: 52. In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising a CD28 polypeptide comprising or consisting of amino acids 180-220 of SEQ ID NO: 52.

[0176] An exemplary nucleic acid sequence encoding the amino acid sequence of amino acids 180-220 of SEQ ID NO:52 is set forth in SEQ ID NO:69, provided below. AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC [SEQ ID NO: 69]

[0177] In certain embodiments, the intracellular signaling domain of the second antigen-recognizing receptor comprises a costimulatory signaling region comprising the intracellular domain of mouse CD28 or a portion thereof. In certain embodiments, the CD28 polypeptide comprised in the costimulatory signaling region comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or at least about 100% identical or homologous to the amino acid sequence having NCBI Reference Number: NP_031668.3 (or SEQ ID NO: 70), or a fragment thereof, and / or may optionally comprise up to one, up to two, or up to three conservative amino acid substitutions. In certain embodiments, the CD28 polypeptide comprised in the costimulatory signaling region of the CAR comprises or consists of an amino acid sequence that is at least about 20, or at least about 30, or at least about 40, or at least about 50, and up to 218 amino acids in length. In certain embodiments, the CD28 polypeptide comprised in the costimulatory signaling region of the CAR comprises or consists of the amino acid sequence of amino acids 1-218, 1-50, 50-100, 100-150, 150-218, 178-218, or 200-218 of SEQ ID NO: 70. In certain embodiments, the costimulatory signaling region of the CAR comprises a CD28 polypeptide comprising or consisting of amino acids 178-218 of SEQ ID NO: 70. SEQ ID NO: 70 is provided below. MTLRLLFLAL NFFSVQVTEN KILVKQSPLL VVDSNEVSLS CRYSYNLLAK EFRASLYKGV NSDVEVCVGN GNFTYQPQFR SNAEFNCDGD FDNETVTFRL WNLHVNHTDI YFCKIEFMYP PPYLDNERSN GTIIHIKEKH LCHTQSSPKL FWALVVVAGV LFCYGLLVTV ALCVIWTNSR RNRLLQSDYM NMTPRRPGLT RKPYQPYAPA RDFAAYRP [SEQ ID NO: 70]

[0178] In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising a 4-1BB polypeptide, e.g., the intracellular domain of 4-1BB, or a portion thereof. In certain embodiments, the costimulatory signaling region comprises the intracellular domain of human 4-1BB, or a portion thereof. In certain embodiments, the 4-1BB comprised in the costimulatory signaling region of the CAR comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence having NCBI Reference Number: NP_001552 (SEQ ID NO: 71), or a fragment thereof, and / or may optionally comprise up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the 4-1BB included in the costimulatory signaling region of the CAR comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 71 that is at least about 20, or at least about 30, or at least about 40, or at least about 50, and / or up to about 50, up to about 60, up to about 70, up to about 80, up to about 90, up to about 100, up to about 200, or up to about 255 amino acids in length. In certain embodiments, the costimulatory signaling region of the CAR comprises a 4-1BB polypeptide that comprises or consists of the amino acid sequence of amino acids 1-255, 1-50, 50-100, 100-150, 150-200, or 200-255 of SEQ ID NO: 71. In certain embodiments, the costimulatory signaling region of the CAR comprises a 4-1BB polypeptide that comprises or consists of the amino acid sequence of amino acids 214-255 of SEQ ID NO: 71. SEQ ID NO: 71 is provided below. MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL [SEQ ID NO: 71]

[0179] In certain embodiments, the intracellular signaling domain of the CAR comprises two costimulatory signaling regions, wherein the first costimulatory signaling region comprises the intracellular domain of a first costimulatory molecule or a portion thereof, and the second costimulatory signaling region comprises the intracellular domain of a second costimulatory molecule or a portion thereof. The first and second costimulatory molecules are independently selected from the group consisting of CD28, 4-1BB, OX40, CD27, CD40, CD154, CD97, CD11a / CD18, ICOS, DAP-10, CD2, CD150, CD226, and NKG2D. In certain embodiments, the intracellular signaling domain of the CAR comprises two costimulatory signaling regions, wherein the first costimulatory signaling region comprises the intracellular domain of CD28 or a portion thereof, and the second costimulatory signaling region comprises the intracellular domain of 4-1BB or a portion thereof.

[0180] 2.1.4.3. Chimeric Ligand Receptors In certain embodiments, the second antigen-recognizing receptor is a chimeric ligand receptor comprising a ligand or a portion thereof that binds to the first antigen, hi certain embodiments, the chimeric ligand receptor further comprises a transmembrane domain and an intracellular signaling domain.

[0181] In certain embodiments, the transmembrane domain is fused to a ligand or portion thereof. In certain embodiments, the transmembrane domain is fused to an intracellular signaling domain. In certain embodiments, the transmembrane domain is located between the ligand or portion thereof and the intracellular signaling domain. In certain embodiments, the transmembrane domain of the chimeric ligand receptor is a transmembrane domain disclosed in Section 2.1.5.2. In certain embodiments, the intracellular signaling domain of the chimeric ligand receptor comprises a CD3ζ polypeptide (e.g., as disclosed in Section 2.1.5.2).

[0182] Further information regarding the chimeric ligand receptors disclosed herein can be found in Sauer et al., Blood (2021) 138(4):318-330, the contents of which are incorporated by reference in their entirety.

[0183] 2.1.4.4.CCR In certain embodiments, the cells disclosed herein comprising a first antigen-recognizing receptor further comprise a CCR. The term "chimeric costimulatory receptor" or "CCR" refers to a chimeric receptor that binds to an antigen and provides a costimulatory signal but does not provide a T cell activation signal to a CCR-containing cell. Various CCRs are described in US2002 / 0018783, the contents of which are incorporated by reference in their entirety. CCRs mimic costimulatory signals but, unlike CARs, do not provide a T cell activation signal. In certain embodiments, the CCR lacks the CD3ζ polypeptide.

[0184] CCRs provide costimulatory signals (e.g., CD28-like or 4-1BB-like signals) in the absence of natural costimulatory ligands on antigen-presenting cells. Combinatorial antigen recognition, i.e., the use of CCRs in combination with CARs, can enhance T cell reactivity against dual-antigen-expressing T cells, thereby improving selective tumor targeting. Kloss et al. have described a strategy that incorporates combinatorial antigen recognition, split signaling, and, importantly, balanced strength of T cell activation and costimulation to generate T cells that eliminate target cells expressing a combination of antigens while sparing cells expressing each antigen individually (Kloss et al., Nature Biotechnology (2013); 31(1):71-75, the contents of which are incorporated by reference in their entirety). In this approach, T cell activation requires CAR-mediated recognition of one antigen, while costimulation is independently mediated by a CCR specific for a second antigen. To achieve tumor selectivity, combinatorial antigen recognition approaches reduce the efficiency of T cell activation to a level that is ineffective without the rescue provided by simultaneous CCR recognition of a second antigen.

[0185] In certain embodiments, the CCR comprises an extracellular antigen-binding domain that binds to a third antigen and an intracellular domain that can deliver a costimulatory signal to a cell but does not alone deliver an activation signal to a cell. In certain embodiments, the CCR further comprises a transmembrane domain. In certain embodiments, the intracellular domain of the CCR comprises at least the intracellular domain of a costimulatory molecule, or a portion thereof. In certain embodiments, the costimulatory molecule is selected from the group consisting of CD28, 4-1BB, OX40, CD27, CD40, CD154, CD97, CD11a / CD18, ICOS, DAP-10, CD2, CD150, CD226, and NKG2D.

[0186] In certain embodiments, the CCR comprises the intracellular domain of CD28 or a portion thereof. In certain embodiments, the CCR comprises the intracellular domain of 4-1BB or a portion thereof. In certain embodiments, the CCR comprises the intracellular domain of CD28 or a portion thereof and the intracellular domain of 4-1BB or a portion thereof.

[0187] In certain embodiments, the second antigen is selected such that expression of both the first and second antigens is restricted to the target cell (e.g., cancer tissue or cancer cell). Similar to the CAR, the extracellular antigen-binding domain can be an scFv, Fab, F(ab)2, or a fusion protein with heterologous sequences to form the extracellular antigen-binding domain.

[0188] In certain embodiments, cells comprising a first antigen-recognizing receptor and a CCR exhibit a higher degree of cytolytic activity against cells positive for both the first / second antigen and the third antigen compared to cells positive for the first / second antigen alone, hi certain embodiments, cells comprising a first antigen-recognizing receptor and a CCR exhibit substantially no or negligible cytolytic activity against cells positive for the first / second antigen alone.

[0189] In certain embodiments, the first antigen-recognizing receptor (e.g., those disclosed in Section 2.1.2) has a low binding affinity, e.g., about 1×10 -8 M or more, approximately 5 x 10 -8 M or more, approximately 1 x 10 -7 M or more, approximately 5 x 10 -7 M or more, or approximately 1 x 10 -6 M or more, or approximately 1 x 10 -8 M ~ approx. 1×10 -6 Dissociation constant of M (K D) binds to the antigen. In certain embodiments, the antigen recognizing receptor (e.g., a CAR, TCR, or TCR-like fusion molecule) binds to the antigen with low binding affinity. In certain embodiments, the antigen recognizing receptor (e.g., a TCR-like fusion molecule) binds to the antigen at an epitope with low accessibility. In certain embodiments, the antigen recognizing receptor (e.g., a TCR-like fusion molecule) binds to the antigen with a lower binding affinity compared to the binding affinity with which a second antigen recognizing receptor (e.g., a CCR) binds to the second antigen. In certain embodiments, the CCR binds to the antigen with a binding affinity of about 1 x 10 -9 M ~ approx. 1×10 -7 M, e.g., about 1 x 10 -7 M or less, approximately 1×10 -8 M or less, or about 1 x 10 -9 Binding affinity K below M D binds to a second antigen.

[0190] T cell receptor (TCR) In certain embodiments, the cells disclosed herein comprising a first antigen-recognizing receptor further comprise a TCR. A TCR is a disulfide-linked heterodimeric protein consisting of two variable chains expressed as part of a complex with an invariant CD3 chain molecule. TCRs are found on the surface of T cells and are involved in recognizing antigens as peptides bound to major histocompatibility complex (MHC) molecules. In certain embodiments, the TCR comprises an alpha chain and a beta chain (encoded by TRA and TRB, respectively). In certain embodiments, the TCR comprises a gamma chain and a delta chain (encoded by TRG and TRD, respectively).

[0191] Each chain of the TCR is composed of two extracellular domains: a variable (V) region and a constant (C) region. The constant region is proximal to the cell membrane and is followed by a transmembrane region and a short cytoplasmic tail. The variable region binds to the peptide / MHC complex. The variable domains of both chains each have three complementarity-determining regions (CDRs).

[0192] In certain embodiments, a TCR can form a receptor complex with three dimeric signaling modules, CD3δ / ε, CD3γ / ε, and CD247ζ / ζ or ζ / η. Engagement of the TCR complex with its antigen and MHC (peptide / MHC) activates a T cell expressing the TCR complex.

[0193] In certain embodiments, the TCR is an endogenous TCR. In certain embodiments, the TCR is a naturally occurring TCR.

[0194] In certain embodiments, the TCR is an exogenous TCR. In certain embodiments, the TCR is a recombinant TCR. In certain embodiments, the TCR is a non-naturally occurring TCR. In certain embodiments, a non-naturally occurring TCR differs from any naturally occurring TCR by at least one amino acid residue. In certain embodiments, a non-naturally occurring TCR differs from any naturally occurring TCR by at least about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 20, about 25, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, or more amino acid residues. In certain embodiments, a non-naturally occurring TCR is modified from a naturally occurring TCR by at least one amino acid residue. In certain embodiments, a non-naturally occurring TCR has at least about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 20, about 25, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, or more amino acid residues modified from a naturally occurring TCR.

[0195] 2.1.5.6. Delivery of a Second Antigen-Recognizing Receptor In certain embodiments, the second antigen-recognizing receptor is delivered to the cell by a viral method. In certain embodiments, the viral method includes a viral vector. In certain embodiments, the viral vector is a retroviral vector (e.g., a gamma retroviral vector or a lentiviral vector). Other viral vectors include adenoviral vectors, adeno-associated viral vectors, vaccinia virus, bovine papillomavirus, and herpesviruses (e.g., Epstein-Barr virus).

[0196] In certain embodiments, the second antigen-recognizing receptor is delivered to cells by a non-viral method. Any targeted genome editing method can also be used to deliver the second antigen-recognizing receptor to cells. In certain embodiments, the second antigen-recognizing receptor is delivered to cells by a method comprising homologous recombination, zinc finger nuclease, meganuclease, transcription activator-like effector nuclease (TALEN), clustered regularly interspaced short palindromic repeats (CRISPR) system, or a combination thereof. In certain embodiments, the CRISPR system is used to deliver the second antigen-recognizing receptor to cells.

[0197] In certain embodiments, the cell is a T cell, and the second antigen-recognizing receptor is integrated into a locus within the genome of the T cell. Non-limiting examples of loci include the TRAC locus, the TRBC locus, the TRDC locus, and the TRGC locus. In certain embodiments, the locus is the TRAC locus or the TRBC locus. In certain embodiments, the cell is a T cell, and the second antigen-recognizing receptor is integrated into the TRAC locus.

[0198] 2.2.Co-stimulatory Ligands In certain embodiments, a cell disclosed herein comprising an antigen-recognizing receptor (e.g., a first antigen-recognizing receptor, e.g., one disclosed in Section 2.1.2) further comprises at least one recombinant or exogenous costimulatory ligand. For example, a cell disclosed herein can be further transduced with at least one costimulatory ligand such that the cell expresses, or is induced to express, the first antigen-recognizing receptor, a second antigen-recognizing receptor, and at least one costimulatory ligand. The at least one costimulatory ligand provides a costimulatory signal to the cell.

[0199] Non-limiting examples of costimulatory ligands include, but are not limited to, members of the tumor necrosis factor (TNF) superfamily and immunoglobulin (Ig) superfamily ligands. TNF is a cytokine involved in systemic inflammation and stimulates the acute phase response. Its primary role is in regulating immune cells. Members of the TNF superfamily share several common characteristics. Most TNF superfamily members are synthesized as type II transmembrane proteins (extracellular C-terminus) containing a short cytoplasmic segment and a relatively long extracellular region. Non-limiting examples of TNF superfamily members include nerve growth factor (NGF), CD40L (also known as "CD154"), 4-1BBL, TNF-α, OX40L, CD70, Fas ligand (FasL), CD30L, tumor necrosis factor beta (TNFβ) / lymphotoxin-alpha (LTα), lymphotoxin-beta (LTβ), CD257 / B cell activating factor (BAFF) / Blys / THANK / Tall-1, glucocorticoid-induced TNF receptor ligand (GITRL), TNF-related apoptosis-inducing ligand (TRAIL), and LIGHT (TNFSF14). The immunoglobulin (Ig) superfamily is a large group of cell surface and soluble proteins involved in cell recognition, binding, or adhesion processes. These proteins share structural features with immunoglobulins and possess immunoglobulin domains (Ig domains). Non-limiting examples of immunoglobulin superfamily ligands include CD80, CD86, and ICOSLG. In certain embodiments, the at least one costimulatory ligand is selected from the group consisting of 4-1BBL, CD80, CD86, CD70, GITRL, CD40L, OX40L, CD30L, TNFRSF14, ICOSLG, TRAIL, and combinations thereof.

[0200] In certain embodiments, the cells further comprise one exogenous costimulatory ligand that is 4-1BBL. In certain embodiments, the costimulatory ligand is human 4-1BBL. In certain embodiments, 4-1BBL comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or at least about 100% homologous or identical to an amino acid sequence having Uniprot Reference Number: P41273-1 (SEQ ID NO: 72) or a fragment thereof, and / or may optionally contain up to one, up to two, or up to three conservative amino acid substitutions. In certain embodiments, 4-1BBL comprises or consists of an amino acid sequence that is a contiguous portion of the amino acid sequence of SEQ ID NO: 72. SEQ ID NO: 72 is provided below. MEYASDASLDPEAPWPPAPRARACRVLPWALVAGLLLLLLLAAACAVFLACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE [SEQ ID NO: 72]

[0201] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO:72 is set forth in SEQ ID NO:73. [query number 73]

[0202] In certain embodiments, the cells further comprise one exogenous costimulatory ligand that is CD80. In certain embodiments, the costimulatory ligand is human CD80. In certain embodiments, the CD80 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence having NCBI Reference Number: NP_005182 (SEQ ID NO: 74) or a fragment thereof, and / or may optionally contain up to one, up to two, or up to three conservative amino acid substitutions. In certain embodiments, the CD80 comprises or consists of an amino acid sequence that is a contiguous portion of the amino acid sequence of SEQ ID NO: 74. SEQ ID NO: 74 is provided below. MGHTRRQGTSPSKCPYLNFFQLLVLAGLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV [SEQ ID NO: 74]

[0203] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 74 is set forth in SEQ ID NO: 75. SEQ ID NO: 75 is provided below. ATGGGCCACACACGGAGGCAGGGAACATCACCATCCAAGTGTCCATACCTCAATTTCTTTCAGCTCTTGGTGCTGGCTGGTCTTTCTCACTTCTGTTCAGGTGTTATCCACGTGACCAAGGAAGTGAAAGAAGTGGCAACGCTGTCCTGTGGTCACAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCTACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATCCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGAAGTATGAAAAAGACGCTTTCAAGCGGGAACACCTGGCTGAAGTGACGTTATCAGTCAAAGCTGACTTCCCTACACCTAGTATATCTGACTTTGAAATTCCAACTTCTAATATTAGAAGGATAATTTGCTCAACCTCTGGAGGTTTTCCAGAGCCTCACCTCTCCTGGTTGGAAAATGGAGAAGAATTAAATGCCATCAACACAACAGTTTCCCAAGATCCTGAAACTGAGCTCTATGCTGTTAGCAGCAAACTGGATTTCAATATGACAACCAACCACAGCTTCATGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACAACCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCTTAATCTCAGTAAATGGAATTTTTGTGATATGCTGCCTGACCTACTGCTTTGCCCCAAGATGCAGAGAGAGAAGGAGGAATGAGAGATTGAGAAGGGAAAGTGTACGCCCTGTA [SEQ ID NO: 75]

[0204] In certain embodiments, the cells further comprise two exogenous costimulatory ligands, 4-1BBL and CD80. In certain embodiments, the cells further comprise two exogenous costimulatory ligands, 4-1BBL and CD80, wherein 4-1BBL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 72 and CD80 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 74.

[0205] Cells containing a receptor that includes at least one exogenous costimulatory ligand are described in US Pat. No. 8,389,282, which is incorporated by reference in its entirety.

[0206] 2.3. Fusion Polypeptides In certain embodiments, a cell disclosed herein comprising an antigen-recognizing receptor (e.g., a first antigen-recognizing receptor, e.g., one disclosed in Section 2.1.2) further comprises a fusion polypeptide. For example, a cell disclosed herein can be further transduced with a fusion polypeptide such that the cell expresses or is induced to express the first antigen-recognizing receptor, the second antigen-recognizing receptor, and the fusion polypeptide. The fusion polypeptide provides a costimulatory signal to the cell. The fusion polypeptide can enhance the activity and / or efficacy of a cell comprising the first antigen-recognizing receptor (e.g., a CAR or TCR-like fusion molecule). In certain embodiments, the fusion polypeptide comprises a) the extracellular domain and transmembrane domain of a costimulatory ligand, and b) the intracellular domain of a first costimulatory molecule.

[0207] Non-limiting examples of costimulatory ligands include tumor necrosis factor (TNF) family members, immunoglobulin (Ig) superfamily members, and combinations thereof. TNF family members can be selected from the group consisting of 4-1BBL, OX40L, CD70, GITRL, CD40L, and combinations thereof. Ig superfamily members can be selected from the group consisting of CD80, CD86, ICOS ligand (ICOSLG (also known as "CD275"), and combinations thereof. In certain embodiments, the costimulatory ligand is selected from the group consisting of 4-1BBL, OX40L, CD70, GITRL, CD40L, CD80, CD86, ICOSLG, and combinations thereof.

[0208] In certain embodiments, the fusion polypeptide comprises the extracellular domain and transmembrane domain of a costimulatory ligand that is CD80. In certain embodiments, the costimulatory ligand is human CD80. In certain embodiments, the CD80 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 69 or a fragment thereof, and / or may optionally contain up to one, up to two, or up to three conservative amino acid substitutions. In certain embodiments, the CD80 comprises or consists of an amino acid sequence that is a contiguous portion of the amino acid sequence of SEQ ID NO: 74.

[0209] In certain embodiments, the extracellular domain of CD80 comprises or consists of an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to amino acids 1-242 of SEQ ID NO: 74. In certain embodiments, the extracellular domain of CD80 comprises or consists of amino acids 1-242 of SEQ ID NO: 74, or a functional fragment thereof. A functional fragment can be a contiguous portion of amino acids 1-242 of SEQ ID NO:74 that is at least about 50, at least about 75, at least about 100, at least about 125, at least about 150, at least about 175, or at least about 200, or at least about 220 amino acids in length. In certain embodiments, a functional fragment retains at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% of the primary functions of the extracellular domain of CD80. Non-limiting examples of the primary functions of the extracellular domain of CD80 include binding to / interacting with CD28, binding to / interacting with CTLA-4, binding to / interacting with PD-L1, and contributing to CD80 homodimerization. In certain embodiments, the extracellular domain of CD80 comprises or consists of amino acids 1-242 of SEQ ID NO:74.

[0210] In certain embodiments, the transmembrane domain of CD80 comprises or consists of an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to amino acids 243-263 of SEQ ID NO: 74. In certain embodiments, the transmembrane domain of CD80 comprises or consists of amino acids 243-263 of SEQ ID NO: 74 or a fragment thereof. Such a fragment can be at least about 5, at least about 10, at least about 15, or at least about 20 amino acids in length. In certain embodiments, the transmembrane domain of CD80 comprises or consists of amino acids 243 to 263 of SEQ ID NO:74.

[0211] Non-limiting examples of costimulatory molecules include CD28, 4-1BB, OX40, ICOS, DAP-10, CD27, CD40, NKG2D, CD2, and combinations thereof.

[0212] In certain embodiments, the fusion polypeptide comprises the extracellular domain and transmembrane domain of a costimulatory molecule that is 4-1BB. In certain embodiments, the costimulatory molecule is human 4-1BB. In certain embodiments, 4-1BB comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:71 or a fragment thereof, and / or may optionally contain up to one, up to two, or up to three conservative amino acid substitutions. In certain embodiments, 4-1BB comprises or consists of an amino acid sequence that is a continuous portion of the amino acid sequence of SEQ ID NO:71. In certain embodiments, the intracellular domain of 4-1BB comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or at least about 100% homologous or identical to amino acids 214-255 of SEQ ID NO: 71, or a fragment thereof, and / or may optionally contain up to one, up to two, or up to three conservative amino acid substitutions. In certain embodiments, the intracellular domain of 4-1BB comprises or consists of amino acids 214-255 of SEQ ID NO: 71, or a functional fragment thereof. Such a functional fragment can be a contiguous portion of amino acids 214-255 of SEQ ID NO: 71 that is at least about 20, at least about 25, at least about 30, at least about 35, or at least about 40 amino acids in length. In certain embodiments, a functional fragment of amino acids 214-255 of SEQ ID NO: 71 retains at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% of the primary function of the intracellular domain of 4-1BB, non-limiting examples of which include providing costimulatory signaling for the activation and proliferation of immunoresponsive cells (e.g., T cells) and interacting with and activating downstream adaptors (e.g., TRAFs).In certain embodiments, the intracellular domain of 4-1BB comprises or consists of amino acids 214 to 255 of SEQ ID NO:71.

[0213] In certain embodiments, the costimulatory molecule is CD28. In certain embodiments, the costimulatory molecule is human CD28. In certain embodiments, CD28 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 52 or a fragment thereof, and / or may optionally contain up to one, up to two, or up to three conservative amino acid substitutions. In certain embodiments, CD28 comprises or consists of an amino acid sequence that is a contiguous portion of the amino acid sequence of SEQ ID NO: 52. In certain embodiments, the intracellular domain of CD28 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or at least about 100% homologous or identical to amino acids 180-219 of SEQ ID NO: 52, or a fragment thereof, and / or may optionally contain up to one, up to two, or up to three conservative amino acid substitutions. In certain embodiments, the intracellular domain of CD28 comprises or consists of amino acids 180-219 of SEQ ID NO: 52, or a functional fragment thereof. A functional fragment of amino acids 180-219 of SEQ ID NO: 52 can be a contiguous portion of amino acids 180-219 of SEQ ID NO: 52 that is at least about 20, at least about 25, at least about 30, or at least about 35 amino acids in length. In certain embodiments, such functional fragments retain at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% of the primary functions of the intracellular domain of CD28, non-limiting examples of which include providing costimulatory signaling for the activation and proliferation of immunoresponsive cells (e.g., T cells) and interacting with protein adaptors (e.g., PI3K, GRB2, and LCK).In certain embodiments, the intracellular domain of CD28 comprises or consists of amino acids 180 to 219 of SEQ ID NO:52.

[0214] In certain embodiments, the fusion polypeptide comprises the intracellular domain of a second costimulatory molecule. In certain embodiments, the fusion polypeptide comprises the intracellular domain of a third costimulatory molecule. In certain embodiments, the fusion polypeptide comprises the intracellular domain of a fourth costimulatory molecule. In certain embodiments, the fusion polypeptide comprises the intracellular domain of a fifth costimulatory molecule. In certain embodiments, the first, second, third, fourth, and fifth costimulatory molecules can be the same or different from one another.

[0215] In certain embodiments, the fusion polypeptide comprises the extracellular domain and transmembrane domain of a costimulatory ligand that is CD80 and the intracellular domain of a costimulatory molecule that is 4-1BB. In certain embodiments, the fusion polypeptide comprises or consists of an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 76. In certain embodiments, the fusion polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 76. SEQ ID NO: 76 is provided below. MGHTRRQGTSPSKCPYLNFFQLLVLAGLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL [SEQ ID NO: 76]

[0216] In certain embodiments, the fusion polypeptide comprises the extracellular domain and transmembrane domain of a costimulatory ligand that is CD80, the intracellular domain of a first costimulatory molecule that is 4-1BB, and the intracellular domain of a second costimulatory molecule that is CD28.

[0217] In certain embodiments, the fusion polypeptide comprises an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 77. In certain embodiments, the fusion polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 77. SEQ ID NO: 77 is provided below. MGHTRRQGTSPSKCPYLNFFQLLVLAGLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL [SEQ ID NO: 77]

[0218] Various modified fusion polypeptides are disclosed in International Patent Application No. PCT / US20 / 42753, which is incorporated herein by reference in its entirety.

[0219] 2.4. Gene disruption and gene modification In certain embodiments, the cells disclosed herein comprising an antigen-recognizing receptor (e.g., a first antigen-recognizing receptor, e.g., one disclosed in Section 2.1.2) further comprise a genetic disruption of the CD70 locus. Genetic disruption of the CD70 locus can result in a non-functional CD70 protein or knockout of CD70 gene expression. In certain embodiments, genetic disruption of the CD70 locus results in knockout of CD70 gene expression.

[0220] Non-limiting examples of gene disruptions include substitutions, deletions, insertions, or combinations thereof. In certain embodiments, the mutation comprises a missense mutation, a nonsense mutation, or a combination thereof. In certain embodiments, the deletion comprises a non-frameshift deletion, a frameshift deletion, or a combination thereof. In certain embodiments, the insertion comprises a non-frameshift insertion, a frameshift insertion, or a combination thereof.

[0221] In certain embodiments, the CD70 locus is a human CD70 locus. A gene disruption of the CD70 locus can be generated by any suitable gene editing method. In certain embodiments, a gene disruption of the CD70 locus (e.g., a knockout of the CD70 locus) is generated using a viral method. In certain embodiments, the viral method comprises a viral vector. In certain embodiments, the viral vector is a retroviral vector (e.g., a gamma retroviral vector or a lentiviral vector). Other viral vectors include adenoviral vectors, adena-associated viral vectors, vaccinia virus, bovine papillomavirus, and herpesviruses (e.g., Epstein-Barr virus).

[0222] In certain embodiments, a genetic disruption of the CD70 locus (e.g., a knockout of the CD70 locus) is generated using non-viral methods. Non-viral approaches can also be used for the genetic modification of cells. For example, nucleic acid molecules can be delivered by lipofection (Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413, 1987; Ono et al., Neuroscience Letters 17:259, 1990; Brigham et al., Am. J. Med. Sci. 298:278, 1989; Staubinger et al., Methods in Enzymology 101:512, 1983), administration of nucleic acids in the presence of asialoorosomucoid-polylysine conjugation (Wu et al., Journal of Biological Chemistry 263:14621, 1988; Wu et al., Journal of Biological Chemistry 264:16985, 1989), or by microinjection under surgical conditions (Wolff et al., Science 247:1465, 1990). Other non-viral means for gene transfer include in vitro transfection using calcium phosphate, DEAE-dextran, electroporation, and protoplast fusion. Liposomes can also potentially be useful for delivering DNA to cells. Transplantation of normal genes into diseased tissues of a subject can also be achieved by transferring normal nucleic acids into ex vivo culturable cell types (e.g., autologous or heterologous primary cells or their progeny), followed by injection of the cells (or their progeny) into the target tissue or systemically injecting them. Recombinant receptors can also be induced or obtained using transposases or targeted nucleases (e.g., zinc finger nucleases, meganucleases, or TALE nucleases, CRISPR). Transient expression can be obtained by RNA electroporation.

[0223] Any targeted genome editing method can be used to generate a gene disruption at the CD70 locus. In certain embodiments, the gene disruption at the CD70 locus is generated by a method including homologous recombination, zinc finger nucleases, meganucleases, transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR) systems, or a combination thereof.

[0224] In certain embodiments, a CRISPR system is used to generate a gene disruption of the CD70 locus.

[0225] The clustered regularly interspaced short palindromic repeats (CRISPR) system is a genome editing tool discovered in prokaryotic cells. When used for genome editing, the CRISPR system includes Cas9 (a protein that can modify DNA using crRNA as a guide), CRISPR RNA (crRNA, which contains an RNA used by Cas9 to guide Cas9 to the correct part of the host DNA, along with a region that binds to tracrRNA (generally in the form of a hairpin loop) that forms an active complex with Cas9), transactivating crRNA (tracrRNA, which binds to crRNA and forms an active complex with Cas9), and an optional portion of a DNA repair template (DNA that guides the cellular repair process, allowing for the insertion of a specific DNA sequence). CRISPR / Cas9 is often transfected using a plasmid into target cells. The crRNA is the sequence used by Cas9 to identify and directly bind to the target DNA in the cell, so it must be designed for each application. The repair template carrying the CAR expression cassette must also be designed for each application because it must overlap with the sequences on both sides of the cut and encode the insertion sequence. Multiple crRNAs and tracrRNAs can be packaged together to form a single guide RNA (sgRNA). This sgRNA can be spliced ​​together with a Cas9 gene and made into a plasmid for transfection into cells. In certain embodiments, the CRISPR system comprises a base editor. In certain embodiments, the CRISPR system comprises a transposase / recombinase. In certain embodiments, the CRISPR system comprises a prime editor. In certain embodiments, the CRISPR system comprises an epigenetic regulator. In certain embodiments, the CRISPR system comprises a CRISPRoff system. Further details regarding the CRISPR systems of the subject matter disclosed herein can be found in Anzalone et al., Nature biotechnology 38.7(2020):824-844 and Nunez et al., Cell 184.9(2021):2503-2519, the contents of each of which are incorporated by reference in their entirety.

[0226] In certain embodiments, a gRNA molecule is used to disrupt the CD70 locus to knock out expression of CD70. The gRNA molecule can target a coding sequence of a CD70 gene (e.g., a human CD70 gene) or a non-coding sequence of a CD70 gene (e.g., a human CD70 gene). In certain embodiments, the gRNA molecule targets a coding sequence of a CD70 gene (e.g., a human CD70 gene). In certain embodiments, the gRNA molecule targets a target sequence within the human CD70 gene.

[0227] In certain embodiments, zinc finger nucleases are used to generate gene disruptions in the CD70 locus. Zinc finger nucleases (ZFNs) are artificial restriction enzymes generated by combining a zinc finger DNA-binding domain with a DNA cleavage domain. The zinc finger domain can be engineered to target specific DNA sequences, allowing the zinc finger domain to target desired sequences within the genome. The DNA-binding domain of an individual ZFN typically contains multiple individual zinc finger repeats, each capable of recognizing multiple base pairs. The most common method for generating new zinc finger domains is to combine zinc finger "modules" of known smaller specificity. The most common cleavage domain in ZFNs is the non-specific cleavage domain from the type II restriction endonuclease FokI. Using the endogenous homologous recombination (HR) machinery and a homologous DNA template carrying the CAR expression cassette, ZFNs can be used to insert a CAR expression cassette into the genome. When the target sequence is cleaved by the ZFN, the HR machinery searches for homology between the damaged chromosome and the homologous DNA template, and then copies the sequence of the template between the two broken ends of the chromosome, thereby integrating the homologous DNA template into the genome.

[0228] In certain embodiments, a TALEN system is used to generate gene disruptions at the CD70 locus. Transcription activator-like effector nucleases (TALENs) are restriction enzymes that can be engineered to cleave specific sequences in DNA. TALEN systems operate on roughly the same principle as ZFNs. They are generated by combining a transcription activator-like effector DNA-binding domain with a DNA-cleavage domain. Transcription activator-like effectors (TALEs) consist of a 33-34 amino acid repeat motif with two variable positions that strongly recognize specific nucleotides. By assembling an array of these TALEs, the TALE DNA-binding domain can be engineered to bind to the desired DNA sequence, thereby directing the nuclease to cleave at a specific location in the genome. cDNA expression for use in polynucleotide therapy methods can be derived from any suitable promoter (e.g., human cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein promoter) and regulated by any appropriate mammalian regulatory element or intron (e.g., elongation factor 1a enhancer / promoter / intron structure). For example, if desired, enhancers known to preferentially direct gene expression in specific cell types can be used to direct nucleic acid expression. Enhancers used can include, but are not limited to, those characterized as tissue- or cell-specific enhancers. Alternatively, if a genomic clone is used as a therapeutic construct, regulation can be mediated by cognate regulatory sequences or, if desired, by regulatory sequences derived from a heterologous source, including any of the promoters or regulatory elements described above.

[0229] The method for delivering the genome editing agent / system can vary depending on the need. In certain embodiments, the components of the selected genome editing method are delivered as a DNA construct in one or more plasmids. In certain embodiments, the components are delivered via a viral vector. Common delivery methods include, but are not limited to, electroporation, microinjection, gene gun, imparefection, hydrostatic pressure, continuous injection, sonication, magnetofection, adeno-associated virus, envelope protein pseudotyped viral vectors, replication-competent vector cis- and trans-acting elements, herpes simplex virus, and chemical vehicles (e.g., oligonucleotides, lipoplexes, polymersomes, polyplexes, dendrimers, inorganic nanoparticles, and cell-penetrating peptides).

[0230] In certain embodiments, the gene disruption of the CD70 locus can be a disruption of the coding region of the CD70 locus and / or a disruption of a non-coding region of the CD70 locus. In certain embodiments, the gene disruption of the CD70 locus comprises a disruption of the coding region of the CD70 locus. In certain embodiments, the gene disruption of the CD70 locus comprises an insertion in the coding region of the CD70 locus. The human CD70 protein comprises three exons: exon 1, exon 2, and exon 3. In certain embodiments, the gene disruption of the CD70 locus comprises a disruption in one or more of exon 1, exon 2, and exon 3 of the CD70 locus. In certain embodiments, the gene disruption of the CD70 locus comprises a disruption in exon 1 of the CD70 locus. In certain embodiments, the gene disruption of the CD70 locus comprises an insertion in exon 1 of the CD70 locus.

[0231] In certain embodiments, the cells disclosed herein comprising an antigen-recognizing receptor (e.g., a first antigen-recognizing receptor, e.g., one disclosed in Section 2.1.2) further comprise a genetic disruption of the TRAC locus. In certain embodiments, the genetic disruption of the TRAC locus results in a non-functional TCR. In certain embodiments, the genetic disruption of the TRAC locus results in knockout of TCR gene expression.

[0232] Any of the methods for generating a gene disruption in the CD70 locus disclosed above can be used to generate a gene disruption in the TRAC locus. In certain embodiments, the gene disruption in the TRAC locus is generated by a method comprising a gene editing method comprising homologous recombination, zinc finger nucleases, meganucleases, transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR) systems, or a combination thereof.

[0233] In certain embodiments, the cells disclosed herein further comprise a genetic modification of the CD70 gene. The genetic modification of the CD70 gene can result in a non-functional CD70 protein or knockdown of CD70 gene expression. In certain embodiments, the genetic modification of the CD70 gene results in knockout of CD70 gene expression.

[0234] In certain embodiments, modification of the CD70 gene involves the use of an RNAi agent, including, but not limited to, shRNA, siRNA, LNA, dsRNA, and miRNA. In certain embodiments, the RNAi agent comprises an shRNA. In certain embodiments, the RNAi agent (e.g., shRNA) targets one or more isoforms of the CD70 gene, thereby reducing or eliminating expression of the CD70 gene or CD70 protein. In certain embodiments, the RNAi agent (e.g., shRNA) is expressed from the same construct that expresses a first antigen-recognizing receptor and / or a second antigen-recognizing receptor disclosed herein. In certain embodiments, expression of the RNAi agent (e.g., shRNA), the first antigen-recognizing receptor, and the second antigen-recognizing receptor is driven by the same promoter (e.g., the same promoter). In certain embodiments, expression of the shRNA disclosed herein, the first antigen-recognizing receptor, and the second antigen-recognizing receptor is driven by different promoters.

[0235] In certain embodiments, an RNAi agent (e.g., shRNA) comprises a nucleotide sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% homologous or identical to at least a portion of a CD70 nucleic acid sequence. In certain embodiments, an RNAi agent (e.g., shRNA) comprises a nucleotide sequence complementary to a CD70 gene that is at least about 10 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, or at least about 30 nucleotides. In certain embodiments, an RNAi agent (e.g., an shRNA) comprises a nucleotide sequence of at most 15 nucleotides, at most 20 nucleotides, at most 25 nucleotides, at most 30 nucleotides, at most 35 nucleotides, at most 40 nucleotides, at most 55 nucleotides, at most 60 nucleotides, at most 65 nucleotides, at most 70 nucleotides, at most 75 nucleotides, at most 80 nucleotides, at most 85 nucleotides, at most 90 nucleotides, at most 95 nucleotides, or at most 100 nucleotides in length. In certain embodiments, an RNAi agent comprises DNA or an atypical or non-naturally occurring residue, such as, but not limited to, a phosphorothioate residue.

[0236] In certain embodiments, the RNAi agent reduces expression (e.g., endogenous expression) of CD70 by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 100%, or any intermediate value or range therein.

[0237] In certain embodiments, the gene disruption at the TRAC locus can be a disruption of the coding region of the TRAC locus and / or a disruption of a non-coding region of the TRAC locus. In certain embodiments, the gene disruption at the TRAC locus comprises a disruption of the coding region of the TRAC locus. In certain embodiments, the gene disruption at the TRAC locus comprises an insertion in the coding region of the TRAC locus. The human TRAC protein comprises four exons: exon 1, exon 2, exon 3, and exon 4. In certain embodiments, the coding region of the TRAC locus comprises exon 1, exon 2, exon 3, and exon 4. In certain embodiments, the gene disruption at the TRAC locus comprises a disruption in one or more of exons 1 through 4 of the TRAC locus. In certain embodiments, the gene disruption at the TRAC locus comprises a disruption in exon 1 of the TRAC locus. In certain embodiments, the gene disruption at the TRAC locus comprises an insertion in exon 1 of the TRAC locus.

[0238] The cells disclosed herein can be isolated and activated using CD3 / CD28 antibodies prior to generating the gene disruption. In certain embodiments, the cells disclosed herein comprising an antigen-recognizing receptor (e.g., a first antigen-recognizing receptor, such as those disclosed in Section 2.1.2) further comprise a gene disruption of the TRAC locus and the CD70 locus.

[0239] In certain embodiments, the gene disruption of the TRAC locus and the gene disruption of the CD70 locus are generated after isolation and activation of the cells (e.g., T cells). In certain embodiments, the gene disruption of the TRAC locus and the gene disruption of the CD70 locus are generated before isolation and activation of the cells (e.g., T cells).

[0240] In certain embodiments, the gene disruption of the TRAC locus is generated prior to isolation and activation of the cells (e.g., T cells), and the gene disruption of the CD70 locus is generated after isolation and activation of the cells (e.g., T cells). In certain embodiments, the gene disruption of the CD70 locus is generated prior to isolation and activation of the cells (e.g., T cells), and the gene disruption of the TRAC locus is generated after isolation and activation of the cells (e.g., T cells).

[0241] 3. Nucleic Acid Compositions and Vectors The presently disclosed subject matter provides nucleic acid compositions comprising a first polynucleotide encoding an antigen-recognizing receptor disclosed herein (e.g., as disclosed in Section 2.1). Also provided are cells comprising such nucleic acid compositions. In certain embodiments, the nucleic acid composition further comprises a promoter operably linked to the antigen-recognizing receptor. In certain embodiments, the nucleic acid composition further comprises a second promoter operably linked to a second antigen-recognizing receptor (e.g., as disclosed in Section 2.1).

[0242] In addition, the presently disclosed subject matter provides a nucleic acid composition comprising a polynucleotide encoding an antigen-recognizing receptor disclosed herein (e.g., as disclosed in Section 2.1). Also provided is a cell comprising such a nucleic acid composition. In certain embodiments, the nucleic acid composition further comprises a second promoter operably linked to a second antigen-recognizing receptor.

[0243] In certain embodiments, the presently disclosed subject matter provides a nucleic acid composition comprising a polynucleotide encoding an antigen-recognizing receptor disclosed herein (e.g., as disclosed in Section 2.1) and a costimulatory ligand (e.g., as disclosed in Section 2.2). In certain embodiments, the nucleic acid composition further comprises a promoter operably linked to the antigen-recognizing receptor. In certain embodiments, the nucleic acid composition further comprises a second promoter operably linked to a costimulatory ligand (e.g., as disclosed in Section 2.2). In certain embodiments, the nucleic acid composition further comprises a third promoter operably linked to a second costimulatory ligand (e.g., as disclosed in Section 2.2).

[0244] Additionally or alternatively, the presently disclosed subject matter provides nucleic acid compositions comprising a polynucleotide encoding an antigen-recognizing receptor disclosed herein (e.g., as disclosed in Section 2.1) and a fusion polypeptide (e.g., as disclosed in Section 2.3). In certain embodiments, the nucleic acid composition further comprises a first promoter operably linked to the antigen-recognizing receptor. In certain embodiments, the nucleic acid composition further comprises a second promoter operably linked to the fusion polypeptide.

[0245] In certain embodiments, one or both of the first and second promoters are endogenous or exogenous.

[0246] In certain embodiments, the exogenous promoter is selected from an elongation factor (EF)-1 promoter, a CMV promoter, an SV40 promoter, a PGK promoter, and a metallothionein promoter. In certain embodiments, one or both of the first and second promoters are inducible promoters. In certain embodiments, the inducible promoter is selected from an NFAT transcription response element (TRE) promoter, a CD69 promoter, a CD25 promoter, and an IL-2 promoter.

[0247] In certain embodiments, the first and / or second antigen-recognizing receptor is integrated into a locus in the genome of the T cell, such as the TRAC locus, TRBC locus, TRDC locus, or TRGC locus. In certain embodiments, the locus is the TRAC locus. In certain embodiments, the expression of the first and / or second antigen-recognizing receptor is under the control of an endogenous promoter. Non-limiting examples of endogenous promoters include the endogenous TRAC promoter, endogenous TRBC promoter, endogenous TRDC promoter, and endogenous TRGC promoter. In certain embodiments, the endogenous promoter is the endogenous TRAC promoter.

[0248] In certain embodiments, the nucleic acid composition is a vector. In certain embodiments, the vector is a retroviral vector (e.g., a gammaretroviral vector or a lentiviral vector). In certain embodiments, the vector is a viral vector selected from the group consisting of an adenoviral vector, an adena-associated viral vector, a vaccinia virus, a bovine papillomavirus, and a herpesvirus (e.g., an Epstein-Barr virus).

[0249] Additionally, nucleic acid compositions can be administered to a subject and / or delivered to cells by methods known in the art or as described herein. Genetic modification of cells (e.g., T cells or NK cells) can be achieved by transducing a substantially homogeneous cell composition with a recombinant DNA construct. In certain embodiments, retroviral vectors (either gammaretroviruses or lentiviruses) are used to introduce nucleic acid compositions into cells. For example, a first polynucleotide and a second polynucleotide can be cloned into a retroviral vector, and expression can be driven from their endogenous promoters, retroviral long terminal repeats, or promoters specific to the target cell type of interest. Non-viral vectors can also be used.

[0250] The first polynucleotide and the second polynucleotide can be constructed within a single multicistronic expression cassette, within multiple expression cassettes within a single vector, or within multiple vectors. Examples of elements that create polycistronic expression cassettes include, but are not limited to, various viral and non-viral internal ribosome entry sites (IRESs, e.g., FGF-1 IRES, FGF-2 IRES, VEGF IRES, IGF-II IRES, NF-κB IRES, RUNX1 IRES, p53 IRES, Hepatitis A IRES, Hepatitis C IRES, Plague virus IRES, Aphthovirus IRES, Picornavirus IRES, Poliovirus IRES, and Encephalomyocarditis virus IRES), and cleavable linkers (e.g., 2A peptides, e.g., P2A, T2A, E2A, and F2A peptides). Retroviral vectors in combination with appropriate packaging lines are also suitable, and the capsid proteins will function to infect human cells. Various amphotropic virus-producing cell lines are known, including, but not limited to, PA12 (Miller, et al. (1985) Mol. Cell. Biol. 5:431-437), PA317 (Miller, et al. (1986) Mol. Cell. Biol. 6:2895-2902), and CRIP (Danos, et al. (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464). Non-amphotropic particles, such as particles pseudotyped with VSVG, RD114, or GALV envelopes and any others known in the art, are also suitable.

[0251] Possible transduction methods also include direct co-culturing of cells with producer cells, e.g., by the method of Bregni, et al. (1992) Blood 80:1418-1422, or culturing with viral supernatant alone or concentrated vector stocks with or without appropriate growth factors and polycations, e.g., by the method of Xu, et al. (1994) Exp. Hemat. 22:223-230, and Hughes, et al. (1992) J. Clin. Invest. 89:1817.

[0252] Other transducing viral vectors can be used to modify cells. In certain embodiments, the selected vector exhibits high infection efficiency and stable integration and expression (see, e.g., Cayouette et al., Human Gene Therapy 8:423-430, 1997; Kido et al., Current Eye Research 15:833-844, 1996; Bloomer et al., Journal of Virology 71:6641-6649, 1997; Naldini et al., Science 272:263-267, 1996; and Miyoshi et al., Proc. Natl. Acad. Sci. USA 94:10319, 1997). Other viral vectors that can be used include, for example, adenovirus, lentivirus, and adena-associated virus vectors, vaccinia virus, bovine papilloma virus, or herpes viruses such as Epstein-Barr virus (see also, e.g., Miller, Human Gene Therapy 15-14, 1990; Friedman, Science 244:1275-1281, 1989; Eglitis et al., BioTechniques 6:608-614, 1988; Tolstoshev et al., Current Opinion in Biotechnology 1:55-61, 1990; Sharp, The Lancet 337:1277-1278, 1991; Cornetta et al., Nucleic Acid Research and Molecular Biology 36:311-322, 1987; Anderson, Science 226:401-409, 1984; Moen, Blood Cells 17:407-416, 1991; Miller et al., Biotechnology 7:980-990, 1989; LeGal La Salle et al., Science 259:988-990, 1993; and Johnson, Chest 107:77S-83S, 1995).Retroviral vectors are particularly well developed and are used in clinical settings (Rosenberg et al., N. Engl. J. Med 323:370, 1990; Anderson et al., U.S. Pat. No. 5,399,346).

[0253] Non-viral approaches can also be used for the genetic modification of cells. For example, nucleic acid molecules can be delivered by lipofection (Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413, 1987; Ono et al., Neuroscience Letters 17:259, 1990; Brigham et al., Am. J. Med. Sci. 298:278, 1989; Staubinger et al., Methods in Enzymology 101:512, 1983), administration of nucleic acids in the presence of asialoorosomucoid-polylysine conjugation (Wu et al., Journal of Biological Chemistry 263:14621, 1988; Wu et al., Journal of Biological Chemistry 264:16985, 1989), or by microinjection under surgical conditions (Wolff et al., Science 247:1465, 1990). Other non-viral means for gene transfer include in vitro transfection using calcium phosphate, DEAE-dextran, electroporation, and protoplast fusion. Liposomes may also be potentially useful for delivering DNA to cells. Transplantation of normal genes into diseased tissues of a subject can also be achieved by transferring normal nucleic acids into ex vivo culturable cell types (e.g., autologous or heterologous primary cells or their progeny), followed by injection of the cells (or their descendants) into the target tissue or systemically. Transient expression can be obtained by RNA electroporation.

[0254] The method for delivering the genome editing agent / system can vary depending on the need. In certain embodiments, the components of the selected genome editing method are delivered as a DNA construct in one or more plasmids. In certain embodiments, the components are delivered via a viral vector. Common delivery methods include, but are not limited to, electroporation, microinjection, gene gun, imparefection, hydrostatic pressure, continuous injection, sonication, magnetofection, adeno-associated virus, envelope protein pseudotyped viral vectors, replication-competent vector cis- and trans-acting elements, herpes simplex virus, and chemical vehicles (e.g., oligonucleotides, lipoplexes, polymersomes, polyplexes, dendrimers, inorganic nanoparticles, and cell-penetrating peptides).

[0255] 3.1. Delivery method The method for delivering the genome editing agent / system can vary depending on the need. In certain embodiments, the components of the selected genome editing method are delivered as a DNA construct in one or more plasmids. In certain embodiments, the components are delivered via a viral vector. Common delivery methods include, but are not limited to, electroporation, microinjection, gene gun, imparefection, hydrostatic pressure, continuous injection, sonication, magnetofection, adeno-associated virus, envelope protein pseudotyped viral vectors, replication-competent vector cis- and trans-acting elements, herpes simplex virus, and chemical vehicles (e.g., oligonucleotides, lipoplexes, polymersomes, polyplexes, dendrimers, inorganic nanoparticles, and cell-penetrating peptides).

[0256] In certain embodiments, the delivery method involves the use of a colloid. As used herein, the term "colloid" refers to a system in which there are two or more phases, where one phase (e.g., a dispersed phase) is distributed in another phase (e.g., a continuous phase). Furthermore, at least one of the phases has a small dimension (about 10 -9 ~about 10 -6Non-limiting examples of colloids encompassed by the presently disclosed subject matter include macromolecule complexes, nanocapsules, microparticles, beads, and lipid-based systems (e.g., micelles, liposomes, and lipid nanoparticles).

[0257] In certain embodiments, the delivery method involves the use of liposomes. As used herein, the term "liposome" refers to a spherical lipid bilayer structure, either monolayer or multilayer, formed from lipids dissolved in an organic solvent and then dispersed in an aqueous medium. Used experimentally and therapeutically to deliver active pharmaceutical ingredients (e.g., nucleic acid compositions disclosed herein) to cells, liposomes fuse with the cell membrane, thereby transferring their contents into the cytoplasm.

[0258] In certain embodiments, the delivery method involves the use of lipid nanoparticles. As used herein, the term "lipid nanoparticle" refers to a particle having at least one dimension on the order of a nanometer (e.g., about 1 nm to about 1,000 nm) and comprising at least one lipid. In certain embodiments, the lipid nanoparticle may contain an active pharmaceutical ingredient (e.g., a nucleic acid composition disclosed herein) for delivery to a cell. The morphology of lipid nanoparticles may differ from that of liposomes. While liposomes are characterized by a lipid bilayer surrounding a hydrophilic core, lipid nanoparticles have an electron-dense core in which cationic lipids and / or ionizable lipids are organized into inverted micelles around the active pharmaceutical ingredient (e.g., a nucleic acid composition disclosed herein). Further information on the morphology and properties of lipid nanoparticles and liposomes can be found in Wilczewska, et al., Pharmacological reports 64, no. 5 (2012): 1020-1037; Eygeris et al., Accounts of Chemical Research 55, no. 1 (2021): 2-12; Zhang et al., Chemical Reviews 121, no. 20 (2021): 12181-12277; and Fan et al., Journal of pharmaceutical and biomedical analysis 192 (2021): 113642.

[0259] In certain embodiments, the lipid nanoparticles have an average diameter of about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 nm to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm.

[0260] In certain embodiments, lipid nanoparticles may comprise cationic lipids or ionizable lipids. The term "cationic lipid" refers to lipids that comprise a head group with a permanent positive charge. Non-limiting examples of cationic lipids encompassed by the subject matter of the present disclosure include 1,2-di-O-octadecenyl-3-trimethylammonium-propane (DOTMA), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), and ethylphosphatidylcholine (ePC).

[0261] As used herein, the term " ionizable lipid " refers to lipids that are protonated at low pH and neutral at physiological pH.The pH sensitivity of ionizable lipids is particularly beneficial for in vivo delivery (such as delivery of the nucleic acid composition disclosed herein), because neutral lipids interact less with the anionic membrane of blood cells, thus improving the biocompatibility of lipid nanoparticles.When trapped in endosomes, ionizable lipids are protonated, promoting membrane destabilization and allowing nanoparticles to escape from endosomes.Non-limiting examples of ionizable lipids encompassed by the presently disclosed subject matter include tetrakis(8-methylnonyl)3,3',3'',3'''-(((methylazanediyl)bis(propane-3,1-diyl))bis(azanetriyl))tetrapropionate, decyl(2-(dioctylammonio)ethyl)phosphate, ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), bis(2-(dodecyldiphenyl)propionate), bis(2-(dioctylammonio)ethyl)phosphate, ... sulfanyl)ethyl)3,3'-((3-methyl-9-oxo-10-oxa-13,14-dithia-3,6-diazahexacosyl)azanediyl)dipropionate, 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol), cKK-E12, 3,6-bis(4-(bis(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol), Decyl)amino)butyl)piperazine-2,5-dione, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, hexa(octan-3-yl)9,9',9'',9''',9'''',9'''''-((((benzene-1,3,5-tricarbonyl)iris(azanediyl))tris(propane-3,1-diyl))tris(azanetriyl))hexanonanoate , heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate, and (((3,6-dioxopiperazine-2,5-diyl)bis(butane-4,1-diyl)bis(azanetriyl))tetrakis(ethane-2,1-diyl)(9Z,9'Z,9''Z,9'''Z,12Z,12'Z,12''Z,12'''Z)-tetrakis(octadeca-9,12-dienoate).

[0262] Additionally, in certain embodiments, lipid nanoparticles may contain other lipids. For example, but not limited to, lipid nanoparticles of the presently disclosed subject matter may contain phospholipids, cholesterol, polyethylene glycol (PEG)-functionalized lipids (PEG-lipids). These lipids can improve certain properties of lipid nanoparticles (e.g., stability, biodistribution, etc.). For example, cholesterol enhances the stability of lipid nanoparticles by adjusting their integrity and rigidity. Non-limiting examples of other lipids present in the lipid nanoparticles include cholesterol, DC-cholesterol, β-sitosterol, BHEM-cholesterol, ALC-0159, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine ( Examples of phosphatidylethanolamines include dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearioyl-2-oleoyl-phosphatidiethanolamine (SOPE), and 1,2-dielideyl-sn-glycero-3-phosphoethanolamine (transDOPE).

[0263] In certain embodiments, the lipid nanoparticles may include a targeting moiety that binds to a ligand. The use of a targeting moiety enables selective delivery of an active pharmaceutical ingredient (e.g., a nucleic acid composition disclosed herein) to a target cell that expresses the ligand (e.g., a T cell). In certain embodiments, the targeting moiety may be an antibody or antigen-binding fragment thereof that binds to a cell surface receptor. For example, but not limited to, the targeting domain may be an antibody or antigen-binding fragment thereof that binds to a receptor expressed on the surface of a T cell (e.g., CD3, CD4, CD8, CD16, CD40L, CD95, FasL, CTLA-4, OX40, GITR, LAG3, ICOS, and PD-1).

[0264] In certain embodiments, the delivery method is an in vivo delivery method. In certain embodiments, the delivery method is an ex vivo delivery method.

[0265] 4. Formulation and Administration The presently disclosed subject matter provides cells, including cells disclosed herein (e.g., as disclosed in Section 2). In certain embodiments, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable excipient.

[0266] Compositions containing the cells disclosed herein can be conveniently provided as sterile liquid preparations, such as isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may be buffered to a selected pH. Liquid preparations are generally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, particularly by injection. Viscous compositions, on the other hand, can be formulated within an appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can include a carrier, which can be a solvent or dispersion medium containing, for example, water, saline, phosphate-buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof.

[0267] Compositions comprising the cells disclosed herein can be provided systemically or directly to a subject to induce and / or enhance an immune response to an antigen and / or treat and / or prevent a neoplasm. In certain embodiments, the cells disclosed herein or compositions comprising them are directly injected into an organ of interest (e.g., an organ affected by a neoplasm). Alternatively, the cells disclosed herein or compositions comprising them are provided indirectly to an organ of interest, for example, by administration to the circulatory system (e.g., tumor vasculature). Expansion and differentiation agents can be provided before, during, or after administration of the cells or compositions to increase cell production in vitro or in vivo.

[0268] The amount of cells administered can vary depending on the subject being treated. In certain embodiments, about 10 4 ~about 10 10 , about 10 4 ~about 10 7 , about 10 5 ~about 10 7 , about 10 5 ~about 10 9 , or about 10 6 ~about 10 8 In certain embodiments, about 10 cells of the present disclosure are administered to a subject. 5 pieces ~ about 10 7 cells disclosed herein are administered to a subject. More effective cells can be administered in even smaller numbers. Typically, at least about 1 x 10 5 cells are administered, and ultimately approximately l × l0 10 In certain embodiments, at least about 1 x 10 5 pieces, about 5×10 5 pieces, about 1×10 6 pieces, about 5×10 6 pieces, about 1×10 7 pieces, about 5×10 7 pieces, about 1×10 8 pieces, or approximately 5 x 10 8 In certain embodiments, about 1 x 10 cells disclosed herein are administered to a subject. 5 In certain embodiments, about 5×10 cells of the present disclosure are administered to a subject.5 In certain embodiments, about 1 x 10 cells of the present disclosure are administered to a subject. 6 The cells of the present disclosure are administered to a subject. The precise determination of what is considered an effective dose may be based on factors individual to each subject, including the size, age, sex, weight, and condition of the particular subject. Dosage amounts can be readily ascertained by one skilled in the art from this disclosure and knowledge in the art.

[0269] The cells and compositions disclosed herein can be administered by any method known in the art, including, but not limited to, intravenous administration, subcutaneous administration, intranodal administration, intratumoral administration, intrathecal administration, intrapleural administration, intraosseous administration, intraperitoneal administration, pleural administration, and direct administration to a subject. The cells disclosed herein can be administered in any physiologically acceptable vehicle, usually intravascularly, but they may also be introduced into bone or other convenient sites where the cells can find a suitable site for regeneration and differentiation (e.g., the thymus). Cells can be introduced by injection, catheter, etc.

[0270] Compositions comprising the cells disclosed herein can be provided systemically or directly to a subject to induce and / or enhance an immune response to an antigen and / or treat and / or prevent a neoplasm (e.g., cancer), pathogen infection, or infectious disease. In certain embodiments, the cells, compositions, or nucleic acid compositions disclosed herein are directly injected into an organ of interest (e.g., an organ affected by a neoplasm). Alternatively, the cells, compositions, or nucleic acid compositions disclosed herein are provided indirectly to an organ of interest, for example, by administration to the circulatory system (e.g., tumor vasculature). Expansion and differentiation agents can be provided before, during, or after administration of the cells, compositions, or nucleic acid compositions to increase production of cells (e.g., T cells (e.g., CTL cells) or NK cells) in vitro or in vivo.

[0271] The compositions disclosed herein can be pharmaceutical compositions comprising the cells disclosed herein or their precursors and a pharmaceutically acceptable carrier. Administration can be autologous or xenogeneic. For example, cells or precursors can be obtained from one subject and administered to the same subject or a different compatible subject. Peripheral blood-derived cells or their progeny (e.g., derived in vivo, ex vivo, or in vitro) can be administered via catheter administration, systemic injection, local injection, intravenous injection, or local injection, including parenteral administration. When administering a therapeutic composition of the subject matter disclosed herein (e.g., a pharmaceutical composition comprising the cells disclosed herein), it can be formulated in a unit-dosage injectable form (solution, suspension, emulsion).

[0272] 5.Treatment method The subject matter disclosed herein provides various methods of using the cells disclosed herein or compositions comprising same. The cells disclosed herein and compositions comprising same can be used in therapy or medicine. For example, the subject matter disclosed herein provides methods for inducing and / or increasing an immune response in a subject in need thereof. The cells disclosed herein and compositions comprising same can be used to reduce tumor burden in a subject. The cells disclosed herein and compositions comprising same can reduce the number of tumor cells, reduce tumor size, and / or eradicate tumors in a subject. The cells disclosed herein and compositions comprising same can be used to treat and / or prevent tumors (or neoplasms) in a subject. The cells disclosed herein and compositions comprising same can be used to extend the survival time of a subject suffering from a tumor. In certain embodiments, the tumor is cancer. The cells, compositions, and nucleic acid compositions disclosed herein can also be used to treat and / or prevent pathogen infection or other infectious diseases in a subject, such as an immunocompromised human subject. The cells, compositions, and nucleic acid compositions disclosed herein can also be used to treat and / or prevent autoimmune diseases in a subject. In certain embodiments, each of the above methods includes administering the cells disclosed herein or a composition comprising the same (e.g., a pharmaceutical composition) to achieve a desired effect, such as alleviating an existing condition or preventing recurrence. For treatment, the dosage is an amount effective to produce the desired effect. An effective amount can be provided in a single administration or a series of administrations. An effective amount can be provided in a bolus or by continuous perfusion.

[0273] Non-limiting examples of tumors (or neoplasms) include blood cancers (e.g., leukemia, lymphoma, and myeloma), ovarian cancer, breast cancer, bladder cancer, brain cancer, colon cancer, intestinal cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, glioblastoma, pharyngeal cancer, melanoma, neuroblastoma, adenocarcinoma, glioma, soft tissue sarcoma, and various carcinomas (including prostate and small cell lung cancer). Preferred carcinomas include astrocytoma, fibrosarcoma, myxosarcoma, liposarcoma, oligodendroglioma, ependymoma, medulloblastoma, primitive neuroectodermal tumor (PNET), chondrosarcoma, osteogenic sarcoma, pancreatic ductal adenocarcinoma, small cell and large cell lung adenocarcinoma, chordoma, angiosarcoma, endothelial sarcoma, squamous cell carcinoma, bronchoalveolar carcinoma, epithelial adenocarcinoma and its liver metastases, lymphangiosarcoma, lymphangioendothelial sarcoma, hepatocellular carcinoma, cholangiocarcinoma, synovium, mesothelioma, Ewing's tumor, rhabdomyosarcoma, colon carcinoma, basal cell carcinoma, sweat gland carcinoma, papillary carcinoma, sebaceous gland carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, and pulmonary carcinoma. The neoplasm further includes any known in the field of oncology, including, but not limited to, leukemia, Wilms' tumor, testicular tumor, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, leukemia, multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease, breast tumors such as ductal and lobular adenocarcinoma, squamous cell and adenocarcinoma of the cervix, epithelial carcinoma of the uterus and ovary, prostate adenocarcinoma, transitional squamous cell carcinoma of the bladder, B- and T-cell lymphoma (nodular and diffuse), plasmacytoma, acute and chronic leukemia, malignant melanoma, soft tissue sarcoma, and leiomyosarcoma. In certain embodiments, the neoplasm is cancer.

[0274] In certain embodiments, the tumor and / or neoplasm is a solid tumor. Non-limiting examples of solid tumors include renal cell carcinoma, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, lung neuroendocrine carcinoma, small cell lung cancer, pancreatic cancer, breast cancer, astrocytoma, glioblastoma, laryngeal / pharyngeal cancer, EBV-associated nasopharyngeal carcinoma, and ovarian cancer.

[0275] In certain embodiments, the tumor and / or neoplasm is renal cell carcinoma. In certain embodiments, the tumor and / or neoplasm is ovarian cancer. In certain embodiments, the tumor and / or neoplasm is pancreatic cancer.

[0276] In certain embodiments, tumors and / or neoplasms comprise tumor cells with low CD70 antigen density. In certain embodiments, cells with low CD70 antigen density comprise a cell surface density of CD70 that is less than about 5,000 molecules per cell, less than about 4,000 molecules per cell, less than about 3,000 molecules per cell, less than about 2,000 molecules per cell, less than about 1,500 molecules per cell, less than about 1,000 molecules per cell, less than about 500 molecules per cell, less than about 200 molecules per cell, or less than about 100 molecules per cell.

[0277] In certain embodiments, tumors and / or neoplasms have a low tumor cell frequency and / or a CD70 + In certain embodiments, the CD70 + The tumor cells have a frequency of less than about 50% per tumor, less than about 40% per tumor, less than about 30% per tumor, less than about 20% per tumor, less than about 15% per tumor, less than about 10% per tumor, less than about 5% per tumor, less than about 2% per tumor, or less than about 1% per tumor.

[0278] As illustrated in Figure 20A, low antigen density and / or low tumor cell frequency can result in undetectable CD70 polypeptide levels using certain methods known in the art, including, for example, but not limited to, immunodiffusion, immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, Western blotting, binder-ligand assay, immunohistochemical techniques, agglutination, complement assay, high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), hyperdiffusion chromatography, etc. (e.g., Basic and Clinical Immunology, Sites and Terr, eds., Appleton and Lange, Norwalk, Conn., pp. 217-262, 1991). Undetectable CD70 polypeptide levels can generate false-negative results and result in the lack of treatment for certain patients.

[0279] In certain embodiments, tumors and / or neoplasms comprise a CD70 polypeptide that is not detectable by immunodiffusion, immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, Western blotting, binder-ligand assay, immunohistochemical techniques, agglutination, complement assay, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, or a combination thereof. In certain embodiments, tumors and / or neoplasms comprise a CD70 polypeptide that is not detectable by immunohistochemistry (IHC).

[0280] Furthermore, Figure 20A shows that CD70 polynucleotide levels allow for the detection of low antigen density and / or low tumor cell frequency. For example, without limitation, CD70 polynucleotides are detectable by RNA-seq, single-cell RNA-seq, quantitative RT-PCR, single-cell qPCR, fluorescent in situ hybridization (FISH), RNA-FISH, MERFISH (multiple (in situ) RNA FISH), by in situ hybridization, or a combination thereof. In certain embodiments, tumors and / or neoplasms comprise CD70 polynucleotides that are detectable by RNA-seq, single-cell RNA-seq, quantitative RT-PCR, single-cell qPCR, fluorescent in situ hybridization (FISH), RNA-FISH, MERFISH (multiple (in situ) RNA FISH), by in situ hybridization, or a combination thereof. In certain embodiments, tumors and / or neoplasms comprise CD70 polynucleotides that are detectable by fluorescent in situ hybridization (FISH).

[0281] Additionally or alternatively, the presently disclosed subject matter provides methods for inducing and / or increasing an immune response, reducing tumor burden, treating and / or preventing a tumor (or neoplasm), and / or extending survival time in a subject having a tumor and / or neoplasm with undetectable CD70 polypeptide levels. In certain embodiments, the methods include obtaining a sample from the subject. In certain embodiments, the sample is a tumor sample.

[0282] In certain embodiments, the sample contains a CD70 polynucleotide that is detectable by FISH. In certain embodiments, if a CD70 polynucleotide is detected in the sample by FISH, the method comprises administering a cell disclosed herein or a composition comprising the same (e.g., a pharmaceutical composition) to achieve a desired effect, e.g., alleviation of an existing condition or prevention of recurrence.

[0283] As illustrated in Figures 17C and 17D, Ezh2 inhibitors can increase CD70 polypeptide levels, making them detectable. Thus, the use of Ezh2 inhibitors can identify subjects with undetectable CD70 polypeptide levels. In certain embodiments, a sample (e.g., a tumor sample) is contacted with an Ezh2 inhibitor. Non-limiting examples of Ezh2 inhibitors include tazemetostat, 3-deazaneplanocin A (DZNep), EPZ005687, EI1, GSK126, and UNC1999. In certain embodiments, the Ezh2 inhibitor is tazemetostat. In certain embodiments, the sample is contacted with the Ezh2 inhibitor for at least about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 36 hours, or about 48 hours. In certain embodiments, the sample is contacted with the Ezh2 inhibitor for up to about 1 day, up to about 2 days, up to about 3 days, up to about 4 days, up to about 5 days, up to about 6 days, up to about 7 days, up to about 8 days, up to about 9 days, up to about 10 days, up to about 12 days, up to about 13 days, or up to about 14 days. In certain embodiments, contacting the sample with the Ezh2 inhibitor increases CD70 polypeptide levels (e.g., detectable by IHC, immunodiffusion, immunoelectrophoresis, RIA, ELISA, immunofluorescence assay, Western blotting, binder-ligand assay, immunohistochemistry, agglutination, complement assay, HPLC, TLC, hyperdiffusion chromatography, or a combination thereof). In certain embodiments, if the Ezh2 inhibitor increases CD70 polypeptide levels in the sample, the method comprises administering a cell disclosed herein or a composition comprising the same (e.g., a pharmaceutical composition) to achieve a desired effect, e.g., alleviation of an existing condition or prevention of recurrence.

[0284] In certain embodiments, the methods disclosed herein include: a) obtaining a sample from a subject having an undetectable CD70 polypeptide level; b) detecting a CD70 polynucleotide by FISH; and c) administering a cell disclosed herein or a composition comprising the same (e.g., a pharmaceutical composition) if the CD70 polynucleotide is detected (e.g., detected by FISH).

[0285] In certain embodiments, the methods disclosed herein include: a) obtaining a sample from a subject having an undetectable CD70 polypeptide level; b) contacting the sample with an Ezh2 inhibitor; and c) administering a cell disclosed herein or a composition comprising the same (e.g., a pharmaceutical composition) if the CD70 polypeptide level is increased.

[0286] In certain embodiments, the methods disclosed herein include: a) obtaining a sample from a subject having undetectable CD70 polypeptide levels; b) contacting the sample with an Ezh2 inhibitor; and c) administering a cell disclosed herein or a composition comprising the same (e.g., a pharmaceutical composition) if CD70 polypeptide is detected (e.g., detected by IHC).

[0287] The presently disclosed subject matter provides methods for treating and / or preventing a viral infection in a subject. The method may include administering an effective amount of a cell, a composition, or a nucleic acid composition disclosed herein to a subject with a viral infection. Non-limiting examples of viral infections include those caused by cytomegalovirus (CMV), Epstein-Barr virus (EBV), hepatitis A, B, C, D, E, F, or G, human immunodeficiency virus (HIV), adenovirus, BK polyomavirus, coronavirus, coxsackievirus, poliovirus, herpes simplex 1, herpes simplex 2, human cytomegalovirus, human herpesvirus 8, varicella-zoster virus, influenza virus, measles virus, mumps virus, parainfluenza virus, respiratory syncytial virus, papillomavirus, rabies virus, and rubella virus.Other viral targets include Paramyxoviridae (e.g., Pneumonia virus, Measles virus, Metapneumovirus, Respirovirus, or Rubulavirus), Adenoviridae (e.g., Adenovirus), Arenaviridae (e.g., Arenaviruses such as Lymphocytic Choriomeningitis virus), Arteriviridae (e.g., Porcine Respiratory and Reproductive Syndrome virus, or Equine Arteritis virus), Bunyaviridae (e.g., Phlebovirus or Hantavirus), Caliciviridae (e.g., Norwalk virus), Coronaviridae (e.g., Coronavirus or Torovirus), Filoviridae (e.g., Ebola-like virus), Flaviviridae (e.g., Hepacivirus or Flavivirus), Herpesviridae (e.g., Simplex virus, Varicellovirus, Cytomegalovirus, Roseolovirus, or Lymphocryptovirus), Orthomyxoviridae (e.g., Influenza virus, or Thogotovirus), Parvoviridae (e.g., Parvovirus), Picomaviridae (e.g., Enterovirus or Hepatovirus), Poxviridae (e.g., Orthopoxvirus, Avipoxvirus, or Leporipoxvirus), Retroviridae (e.g., Lentivirus or Spumavirus), Reoviridae (e.g., Rotavirus), Rhabdoviridae (e.g., Lyssavirus, Nobilavudovirus, or Vesiculovirus), and Togaviridae (e.g., Alphavirus or Rubivirus). In certain embodiments, viral infections include human respiratory coronavirus, influenza viruses A-C, hepatitis viruses A-G, and herpes simplex viruses 1-9. In certain embodiments, the subject has an immunodeficiency.

[0288] The presently disclosed subject matter provides methods for treating and / or preventing a bacterial infection in a subject. The methods can include administering an effective amount of a cell disclosed herein, a composition disclosed herein, or a nucleic acid composition disclosed herein to a subject having a bacterial infection. Bacterial infections include Mycobacteria, Rickettsia, Mycoplasma, Neisseria meningitides, Neisseria gonorrheoeae, Legionella, Vibrio cholerae, Streptococci, Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Corynobacteria diphtheriae, Clostridium. spp., enterotoxigenic Escherichia coli, Bacillus anthracis, Rickettsia, Bartonella henselae, Bartonella quintana, Coxiella burnetii, chlamydia, Mycobacterium leprae, Salmonella, shigella, Yersinia enterocolitica, Yersinia pseudotuberculosis; Legionella pneumophila; Mycobacterium tuberculosis;Listeria monocytogenes;Mycoplasma spp., Pseudomonas fluorescens, Vibrio cholerae, Haemophilus influenzae, Bacillus anthracis, Treponema pallidum, Leptospira, Borrelia, Corynebacterium diphtheriae, Francisella, Brucella melitensis, Campylobacter jejuni, Enterobacter, Proteus mirabilis, Proteus, and Klebsiella pneumoniae.

[0289] The presently disclosed subject matter provides methods for treating and / or preventing an autoimmune disease in a subject. The method may include administering to a subject having an autoimmune disease an effective amount of a cell disclosed herein, a composition disclosed herein, or a nucleic acid composition disclosed herein.

[0290] The presently disclosed subject matter provides methods for treating and / or preventing an infectious disease in a subject. The method can include administering an effective amount of a cell disclosed herein, a composition disclosed herein, or a nucleic acid composition disclosed herein to a subject with the infectious disease.

[0291] Non-limiting examples of autoimmune and inflammatory diseases or conditions thereof include arthritis, e.g., rheumatoid arthritis (RA), type 1 diabetes, systemic lupus erythematosus (SLE), inflammatory bowel disease, ulcerative colitis, psoriasis, psoriatic arthritis, scleroderma, autoimmune thyroid disease, Graves' disease, Crohn's disease, multiple sclerosis, systemic sclerosis, asthma, organ transplant rejection, transplant-related diseases or conditions, Takayasu's arteritis, giant cell arteritis, Kawasaki disease, polyarteritis nodosa, Behcet's syndrome, Wegener's granulomatosis, ANCA-vasculitis, Churg-Strauss syndrome, microscopic polyangiitis, vasculitis of the connective tissue diseases, Henoch-Schonlein purpura, cryoglobulinemic vasculitis, cutaneous leukocytoclastic vasculitis, sarcoidosis, Cogan's syndrome, Wiskott-Aldrich syndrome, primary hematogenous vasculitis of the CNS, and vasculitis of the urinary tract. autoimmune thrombocytopenia, autoimmune neutropenia, autoimmune hemolytic anemia, autoimmune lymphopenia, chronic autoimmune thyroiditis, autoimmune hepatitis, Hashimoto's thyroiditis, atopic thyroiditis, Graves' disease, autoimmune polyglandular syndrome, autoimmune Addison's syndrome, and / or myasthenia gravis. In accordance with the presently disclosed subject matter, the various methods described above can include administering a checkpoint immune blockade agent to the subject.

[0292] The subject may have an advanced form of the disease, in which case the treatment objectives may include slowing or reversing disease progression and / or ameliorating side effects. The subject may have a history of a condition that has already been treated, in which case the treatment objectives typically include reducing or delaying the risk of recurrence.

[0293] Further modifications can be introduced into the cells disclosed herein to avoid or minimize the risk of immunological complications (known as "malignant T-cell transformation"), such as graft-versus-host disease (GvHD), or when healthy tissue expresses the same target antigen as tumor cells, resulting in an outcome similar to GvHD. A potential solution to this problem is to engineer a suicide gene into the cells disclosed herein. Suitable suicide genes include, but are not limited to, herpes simplex virus thymidine kinase (hsv-tk), inducible caspase-9 suicide gene (iCasp-9), and truncated human epidermal growth factor receptor (EGFRt) polypeptide. In certain embodiments, the suicide gene is an EGFRt polypeptide. EGFRt polypeptide can enable T-cell elimination by administering an anti-EGFR monoclonal antibody (e.g., cetuximab). EGFRt can be covalently linked upstream of an antigen-recognition receptor. The suicide gene can be included in a vector containing a nucleic acid encoding the antigen-recognition receptor disclosed herein. In this manner, administration of a prodrug (e.g., AP1903, which can activate iCasp-9) designed to activate a suicide gene during malignant T cell transformation (e.g., GVHD) induces apoptosis in suicide gene-activated cells expressing the antigen recognition receptors disclosed herein. Incorporation of a suicide gene into the antigen recognition receptors disclosed herein provides an additional level of safety with the ability to eliminate most receptor-expressing cells within a very short period of time. Cells disclosed herein that incorporate a suicide gene can be preemptively eliminated at a given time point after cell infusion or eradicated at the earliest sign of toxicity.

[0294] 6. Kit The presently disclosed subject matter provides kits for inducing and / or enhancing an immune response in a subject and / or treating and / or preventing a neoplasm or pathogen infection (e.g., an autoimmune disease or an infectious disease). In certain embodiments, the kit comprises an effective amount of a cell disclosed herein, a composition disclosed herein, or a nucleic acid composition disclosed herein. In certain embodiments, the kit comprises a sterile container, which may be a box, an ampoule, a bottle, a vial, a tube, a bag, a pouch, a blister pack, or other suitable container form known in the art. Such containers may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding pharmaceutical products. In certain non-limiting embodiments, the kit comprises an isolated nucleic acid molecule encoding an antigen-recognition receptor (e.g., a TCR-like fusion molecule) directed against an antigen of interest in an expressible form, which may optionally be contained in the same or a different vector.

[0295] Optionally, the cells, compositions, or nucleic acid compositions are provided with instructions for administering the cells, compositions, or nucleic acid compositions to a subject having or at risk of developing a tumor (e.g., cancer), or a pathogen infection (e.g., an infectious disease), or an immune disorder (e.g., an autoimmune disease). The instructions generally include information regarding the use of the cells, compositions, or nucleic acid compositions for the treatment and / or prevention of a neoplasm, or a pathogen infection (e.g., an infectious disease), or an immune disorder (e.g., an autoimmune disease). In certain embodiments, the instructions include at least one of the following: a description of the therapeutic agent; dosing schedules and administration for the treatment or prevention of a neoplasm, a pathogen infection (e.g., an infectious disease), or an immune disorder (e.g., an autoimmune disease), or symptoms thereof; precautions; warnings; indications; counterindications; overdosage information; adverse reactions; animal pharmacology; clinical studies; and / or reference matters. The instructions may be printed directly on the container (if present), as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.

[0296] 7. Exemplary Embodiments Embodiment 1. A method of reducing tumor burden in a subject having renal cell carcinoma, pancreatic cancer, or ovarian cancer, the method comprising administering to the subject an effective amount of cells comprising a TCR-like fusion molecule that targets CD70.

[0297] Embodiment 2. The method of embodiment 2, wherein the method reduces the number of tumor cells, reduces tumor size, and / or eradicates tumors in the subject.

[0298] Embodiment 3. A method of reducing tumor burden in a subject, the method comprising administering to the subject an effective amount of cells comprising a TCR-like fusion molecule that targets CD70, wherein the tumor is renal cell carcinoma, pancreatic cancer, or ovarian cancer.

[0299] Embodiment 4. The method of embodiment 3, wherein the method reduces the number of tumor cells, reduces tumor size, and / or eradicates tumors in the subject.

[0300] Embodiment 5. A method of preventing and / or treating a tumor in a subject having a renal cell carcinoma neoplasm, renal cell carcinoma, pancreatic cancer, or ovarian cancer, the method comprising administering to the subject an effective amount of cells comprising a TCR-like fusion molecule that targets CD70.

[0301] Embodiment 6. A method of preventing and / or treating a tumor in a subject, the method comprising administering to the subject an effective amount of cells comprising a TCR-like fusion molecule that targets CD70, wherein the tumor is a renal cell carcinoma neoplasm, renal cell carcinoma, pancreatic cancer, or ovarian cancer.

[0302] Embodiment 7. A method of preventing and / or treating a tumor in a subject in need thereof, the method comprising: a) obtaining a tumor sample having an undetectable CD70 polypeptide level from the subject; b) detecting a CD70 polynucleotide by FISH; and c) administering to the subject an effective amount of cells comprising a TCR-like fusion molecule that targets CD70 if the CD70 polynucleotide is detected.

[0303] Embodiment 8. A method of preventing and / or treating a tumor in a subject in need thereof, the method comprising: a) obtaining a tumor sample from the subject having an undetectable level of CD70 polypeptide; b) contacting the sample with an Ezh2 inhibitor; and c) administering to the subject an effective amount of cells comprising a TCR-like fusion molecule that targets CD70 if CD70 polypeptide is detected in the sample.

[0304] Embodiment 9. The method of any one of embodiments 1 to 8, wherein the TCR-like fusion molecule comprises i) a first antigen-binding chain comprising an antigen-binding fragment of an antibody heavy chain variable region (VH), and ii) a second antigen-binding chain comprising an antigen-binding fragment of an antibody light chain variable region (VL), wherein the first and second antigen-binding chains a) each comprise a TRAC polypeptide or a TRBC polypeptide, and b) bind to a second antigen, and wherein the TCR-like fusion molecule binds to the second antigen in an HLA-independent manner.

[0305] Embodiment 10 The method of embodiment 9, wherein at least one of the TRAC polypeptide and the TRBC polypeptide is endogenous.

[0306] Embodiment 11. The first and second antigen-binding chains are about 1 x 10 -8 11. The method of embodiment 9 or 10, wherein the antibody binds to the second antigen with a dissociation constant (KD) of M or less.

[0307] Embodiment 12. The first and second antigen-binding chains are about 5 x 10 -912. The method of any one of embodiments 9-11, wherein the antibody binds to the second antigen with a dissociation constant (KD) of M or less.

[0308] Embodiment 13. The method of any one of embodiments 9 to 12, wherein the first antigen binding chain comprises an antigen-binding fragment of the VH of an antibody and a TRBC polypeptide, and the second antigen binding chain comprises an antigen-binding fragment of the VL of an antibody and a TRAC polypeptide.

[0309] Embodiment 14. The method of any one of embodiments 9 to 13, wherein the first antigen binding chain comprises an antigen-binding fragment of the VH of an antibody and a TRAC polypeptide, and the second antigen binding chain comprises an antigen-binding fragment of the VL of an antibody and a TRBC polypeptide.

[0310] Embodiment 15. The method of any one of Embodiments 9 to 14, wherein i) the first antigen binding chain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 34, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 35, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 36, and the second antigen binding chain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 37, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 38, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 39, or ii) the first antigen binding chain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 37, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 38, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 39, and the second antigen binding chain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 34, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 35, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 36.

[0311] Embodiment 16. The method according to any one of embodiments 9 to 15, wherein i) the first antigen binding chain comprises CDR1, CDR2, and CDR3 of the heavy chain variable region set forth in SEQ ID NO: 40, and the second antigen binding chain comprises CDR1, CDR2, and CDR3 of the light chain variable region set forth in SEQ ID NO: 42, or ii) the first antigen binding chain comprises CDR1, CDR2, and CDR3 of the light chain variable region set forth in SEQ ID NO: 42, and the second antigen binding chain comprises CDR1, CDR2, and CDR3 of the heavy chain variable region set forth in SEQ ID NO: 40.

[0312] Embodiment 17. The method of any one of embodiments 9 to 15, wherein i) the first antigen-binding chain comprises the heavy chain variable region set forth in SEQ ID NO: 40, and the second antigen-binding chain comprises CDR1, CDR2, and CDR3 of the light chain variable region set forth in SEQ ID NO: 42, or ii) the first antigen-binding chain light chain variable region set forth in SEQ ID NO: 42, and the second antigen-binding chain comprises the heavy chain variable region set forth in SEQ ID NO: 40.

[0313] Embodiment 18. The method of any one of embodiments 9 to 17, wherein the first and second antigen-binding chains are capable of associating with a CD3ζ polypeptide.

[0314] Embodiment 19. The method of embodiment 18, wherein the first and second antigen-binding chains are capable of activating the CD3ζ polypeptide upon binding to the second antigen.

[0315] Embodiment 20. The method of embodiment 19, wherein activation of the CD3ζ polypeptide is capable of activating the cell.

[0316] Embodiment 21. The method of any one of embodiments 9 to 20, wherein the cell further comprises a gene disruption at the TRAC locus.

[0317] Embodiment 22. The method of any one of embodiments 9 to 20, wherein the cell further comprises a gene disruption of the CD70 locus.

[0318] Embodiment 23. The method of any one of embodiments 9 to 20, wherein the cell further comprises a gene disruption of the TRAC locus and CD70.

[0319] Embodiment 24. The method of any one of embodiments 1 to 23, wherein the tumor comprises tumor cells with low CD70 antigen density.

[0320] Embodiment 25. A tumor having a low tumor cell frequency of CD70+ tumor cells, the method of any one of embodiments 1 to 24.

[0321] Embodiment 26. The method of any one of embodiments 1 to 25, wherein the tumor comprises a CD70 polypeptide that is not detectable by immunodiffusion, immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, Western blotting, binder-ligand assay, immunohistochemical techniques, agglutination, complement assay, high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), hyperdiffusion chromatography, or a combination thereof. In certain embodiments, the tumor and / or neoplasm comprises a CD70 polypeptide that is not detectable by immunohistochemistry (IHC).

[0322] Embodiment 27. The method of any one of embodiments 1 to 26, wherein the tumor comprises a CD70 polypeptide by immunohistochemistry (IHC).

[0323] Embodiment 28. The method of any one of embodiments 1 to 27, wherein the cell is a cell of the lymphoid lineage or a cell of the myeloid lineage.

[0324] Embodiment 29. The method of embodiment 28, wherein the cells of the lymphoid lineage are selected from the group consisting of T cells, B cells, natural killer (NK) cells, and dendritic cells.

[0325] Embodiment 30. The method of any one of embodiments 1 to 29, wherein the cell is a T cell.

[0326] Embodiment 31 The method of embodiment 30, wherein the T cells are derived from induced pluripotent stem cells.

[0327] Embodiment 32. The method of embodiment 30 or 31, wherein the T cells are CD8+ T cells.

[0328] Embodiment 33 The method of embodiment 32, wherein the CD8+ T cells are CD4-independent.

[0329] Embodiment 34. The method of any one of embodiments 30-33, wherein the T cells are selected from the group consisting of cytotoxic T lymphocytes (CTLs), gamma delta T cells, tumor-infiltrating lymphocytes (TILs), regulatory T cells, and natural killer T (NKT) cells.

[0330] Embodiment 35. The method of any one of embodiments 1 to 34, wherein the cell further comprises a chimeric antigen receptor (CAR) that targets a second antigen.

[0331] Embodiment 36. The method of embodiment 35, wherein the CAR comprises an extracellular antigen-binding domain that binds to a first antigen and an intracellular signaling domain that can deliver an activating signal to a cell.

[0332] Embodiment 37. The method of embodiment 36, wherein the intracellular signaling domain of the CAR comprises a CD3ζ polypeptide.

[0333] Embodiment 38. The method of embodiment 37, wherein the CD3ζ polypeptide is a native CD3ζ polypeptide or a modified CD3ζ polypeptide.

[0334] Embodiment 39. The method of embodiment 38, wherein the modified CD3ζ polypeptide comprises a native ITAM1, an ITAM2 variant consisting of two loss-of-function mutations, and an ITAM3 variant consisting of two loss-of-function mutations.

[0335] Embodiment 40. The method of any one of embodiments 36-39, wherein the intracellular signaling domain of the CAR further comprises at least one costimulatory signaling region.

[0336] Embodiment 41 The method of embodiment 40, wherein at least one costimulatory signaling region comprises at least the intracellular domain of a costimulatory molecule or a portion thereof.

[0337] Embodiment 42. The method of embodiment 41, wherein the costimulatory molecule is selected from the group consisting of CD28, 4-1BB, OX40, CD27, CD40, CD154, CD97, CD11a / CD18, ICOS, DAP-10, CD2, CD150, CD226, and NKG2D.

[0338] Embodiment 43. The method of any one of embodiments 36 to 42, wherein the CAR comprises a transmembrane domain.

[0339] Embodiment 44. The method of any one of embodiments 1 to 43, wherein the cell further comprises a chimeric costimulatory receptor (CCR).

[0340] Embodiment 45. The method of embodiment 44, wherein the CCR comprises an extracellular antigen-binding domain that binds to a third antigen and an intracellular domain that can deliver a costimulatory signal to the cell but does not alone deliver an activating signal to the cell.

[0341] Embodiment 46 The method of embodiment 45, wherein the intracellular domain of the CCR comprises at least the intracellular domain of a costimulatory molecule or a portion thereof.

[0342] Embodiment 47. The method of embodiment 46, wherein the costimulatory molecule is selected from the group consisting of CD28, 4-1BB, OX40, CD27, CD40, CD154, CD97, CD11a / CD18, ICOS, DAP-10, CD2, CD150, CD226, and NKG2D.

[0343] Embodiment 48. The method of any one of embodiments 35 to 47, wherein the second antigen is a tumor antigen or a pathogen antigen.

[0344] Embodiment 49. The tumor antigen is CD19, IL1RAP, ABCG2, AChR, ACKR6, ADAMTS13, ADGRE2, ADGRE2 (EMR2), ADORA3, ADRA1D, AGER, ALS2, an antigen of a cytomegalovirus (CMV)-infected cell (e.g., a cell surface antigen), ANO9, AQP2, ASIC3, ASPRV1, ATP6V0A4, B3GNT4, B7-H3, BCMA, BEST4, C3orf35, CADM3, CAIX, CAPN3, CCDC155, CCR1, CD10, CD117, CD123, CD133, CD1 35(FLT3), CD138, CD20, CD22, CD244(2B4), CD25, CD26, CD30, CD300LF, CD312, CD32, CD321, CD33, CD34, CD36, CD38, CD41, CD44, CD44V6, CD47, CD49f, C D56, CD7, CD71, CD74, CD8, CD82, CD96, CD98, CD99, CDH13, CDHR1, CEA, CEACAM6, CHST3, CLEC12A, CLEC1A, CLL1, CNIH2, COL15A1, COLEC12, CPM, CR1, CX3 CR1, CXCR4, CYP4F11, DAGLB, DARC, DFNB31, DGKI, EGF1R, EGFR-VIII, EGP-2, EGP-40, ELOVL6, EMB, EMC10, EMR2, ENG, EpCAM, EphA2, EPHA4, ERBB, ERBB2, Erb-B3, Erb-B4, E-selectin, EXOC3L4, EXTL3, FAM186B, FBP, FCGR1A, FKBP1B, FLRT1, folate receptor-α, FOLR2, FRMD5, GABRB2, GAS2, GD2, GD3, GDPD3, GNA14, GNAZ, GPR153, GPR56, GYPA, HEPHL1, HER-2, hERT, HILPDA, HLA-DR, HOOK1, hTERT, HTR2A, ICAM1, IGFBP3, IL10RB, IL20RB, IL23R, ILDR1, interleukin-13 receptor subunit alpha-2 (IL-13Rα2), ITFG3, ITGA4, ITGA5, ITGA8, ITGAX, ITGB5, ITGB8, JAM3, KCND1, KCNJ5, KCNK13, KCNN4, KCNV2, KDR, KIF19, KIF26B, κ-light chain, L1CAM,LAX1, LEPR, Lewis Y (CD174), Lewis Y (LeY), LILRA2, LILRA6, LILRB2, LILRB3, LILRB4, LOXL4, LPAR2, LRRC37A3, LRRC8E, LRRN2, LRRTM2, LTB4R, MAGE-A1, MAGEA3, MANSC1, MART1, GP100, MBOAT1, MBOAT7, melanoma antigen family A, mesothelin (MSLN), MFAP3L, MMP25, MRP1, MT-ND1, mucin 1 (MUC1), mucin 16 (MUC16), MYADM, MYADML2, NGFR, NKCS1, NKG2D Ligands, NLGN3, NPAS2, NY-ESO-1, oncofetal antigen (h5T4), OTOA, P2RY13, p53, PDE3A, PEAR1, PIEZO1, PLXNA4, PLXNC1, PNPLA3, PPFIA4, PPP2R5B, PRAME, PRAME, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), proteinase 3 (PR1), PSD2, PTPRJ, RDH16, receptor tyrosine-protein kinase Erb-B2, RHBDL3, RNF173, RNF183, ROR1, RYR2, SC IN, SCN11A, SCN2A, SCNN1D, SEC31B, SEMA4A, SH3PXD2A, SIGLEC11, SIRPB1, SLC16A6, SLC19A1, SLC22A5, SLC25A36, SLC25A41, SLC30A1, SLC34A3, SLC43A3, SLC44A1, SLC44A3, SLC45A3, SLC6A16, SLC6A6, SLC8A3, SLC9A1, SLCO2B1, SPAG17, STC1, STON2, SUN3, Survivin, SUSD2, SYNC, TACSTD2, TAS 1R3, TEX29, TFR2, TIM-3 (HAVCR2), TLR2, TMEFF2, TMEM145, TMEM27, TMEM40, TMEM59L, TMEM89, TMPRSS5, TNFRSF14, TNFRSFlB, TRIM55, TSPEAR, TTYH3, tumor-associated glycoprotein 72 (TAG-72), tyrosinase, vascular endothelial growth factor R2 (VEGF-R2), VLA-4, Wilms tumor protein (WT-1), WNT4, WT1, and ZDHHC11.

[0345] Embodiment 50. The method of any one of embodiments 1 to 49, wherein the cells further comprise at least one exogenous costimulatory ligand.

[0346] Embodiment 51. The method of embodiment 50, wherein the at least one exogenous costimulatory ligand is selected from the group consisting of tumor necrosis factor (TNF) family members, immunoglobulin (Ig) superfamily members, and combinations thereof.

[0347] Embodiment 52. The method of embodiment 51, wherein the TNF family member is selected from the group consisting of 4-1BBL, OX40L, CD70, FasL, GITRL, TNF-related apoptosis-inducing ligand (TRAIL), CD30L, LIGHT (TNFSF14), CD40L.

[0348] Embodiment 53. The method of embodiment 51 or 52, wherein the Ig superfamily member is selected from the group consisting of CD80, CD86, ICOSLG, and combinations thereof.

[0349] Embodiment 54. The method of any one of embodiments 50 to 53, wherein at least one exogenous costimulatory ligand comprises CD80.

[0350] Embodiment 55. The method of any one of embodiments 50 to 53, wherein at least one exogenous costimulatory ligand comprises 4-1BBL.

[0351] Embodiment 56. The method of any one of embodiments 50 to 53, wherein the cell comprises two exogenous costimulatory ligands.

[0352] Embodiment 57. The method of embodiment 56, wherein the at least two exogenous costimulatory ligands comprise CD80 and 4-1BBL.

[0353] Embodiment 58. The method of any one of embodiments 1 to 57, wherein the cell further comprises a fusion polypeptide comprising a) the extracellular domain and transmembrane domain of a costimulatory ligand, and b) the intracellular domain of a first costimulatory molecule.

[0354] Embodiment 59. The method of embodiment 58, wherein the costimulatory ligand is selected from the group consisting of tumor necrosis factor (TNF) family members, immunoglobulin (Ig) superfamily members, and combinations thereof.

[0355] Embodiment 60. The method of embodiment 59, wherein the TNF family member is selected from the group consisting of 4-1BBL, OX40L, CD70, GITRL, CD40L, and combinations thereof.

[0356] Embodiment 61. The method of embodiment 59 or 60, wherein the Ig superfamily member is selected from the group consisting of CD80, CD86, ICOSLG, and combinations thereof.

[0357] Embodiment 62. The method of any one of embodiments 58 to 61, wherein the costimulatory ligand is CD80.

[0358] Embodiment 63. The method of any one of embodiments 58 to 62, wherein the first costimulatory molecule is selected from the group consisting of CD28, 4-1BB, OX40, ICOS, DAP-10, CD27, CD40, NKG2D, CD2, and combinations thereof.

[0359] Embodiment 64. The method of embodiment 63, wherein the first costimulatory molecule is 4-1BB.

[0360] Embodiment 65. The method of any one of embodiments 58 to 64, wherein the costimulatory ligand is CD80 and the first costimulatory molecule is 4-1BB.

[0361] Embodiment 66 The method of any one of embodiments 58 to 65, wherein the fusion polypeptide further comprises the intracellular domain of a second costimulatory molecule.

[0362] Embodiment 67. The method of embodiment 66, wherein the second costimulatory molecule is selected from the group consisting of CD28, 4-1BB, OX40, ICOS, DAP-10, CD27, CD40, NKG2D, CD2, and combinations thereof.

[0363] Embodiment 68. The method of embodiment 66 or 67, wherein the second costimulatory molecule is CD28.

[0364] Embodiment 69. The method of any one of embodiments 61 to 68, wherein the costimulatory ligand is CD80, the first costimulatory molecule is 4-1BB, and the second costimulatory molecule is CD28.

[0365] Embodiment 70. The method of any one of embodiments 1 to 69, wherein the cells are autologous.

[0366] Embodiment 71. The method of any one of embodiments 1 to 69, wherein the cells are allogeneic. [Example]

[0367] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, well within the skill of one in the art. Such techniques are fully explained in such publications as "Molecular Cloning: A Laboratory Manual," second edition (Sambrook, 1989), "Oligonucleotide Synthesis" (Gait, 1984), "Animal Cell Culture" (Freshney, 1987), "Methods in Enzymology," "Handbook of Experimental Immunology" (Weir, 1996), "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987), "Current Protocols in Molecular Biology" (Ausubel, 1987), "PCR: The Polymerase Chain Reaction" (Mullis, 1994), and "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides disclosed herein and, as such, may be considered in making and practicing the presently disclosed subject matter. Particularly useful techniques for particular embodiments will be described in the following sections.

[0368] The following examples are put forward so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the cells and compositions disclosed herein, and are not intended to limit the scope of what the inventors regard as the invention.

[0369] Example 1 CAR-T cells have not been effective in eradicating solid tumors. Early-stage clinical trials using CAIX-CAR-T for renal cell carcinoma (RCC) have failed, and current clinical trials using CD70 CAR-T for RCC have shown limited therapeutic responses. Other clinical trials for prostate cancer, breast cancer, and lung cancer (to name a few solid tumors) have failed to demonstrate favorable tumor responses. The primary reason for the failure of CAR-T cells is the heterogeneity of tumor antigens (see Figure 1). It is difficult to find a tumor target that is expressed on all tumor cells and not on normal tissues. In RCC, CD70 is expressed on tumors. To date, approaches involving either CD70 CARs or CAIX CARs, or a combination thereof, have shown limited efficacy.

[0370] To study renal cell carcinoma (RCC), the presently disclosed subject matter developed two patient-derived xenograft models (K5 and K7) from patients with clear cell RCC.

[0371] As shown in Figure 2B, CD70 expression is highly maintained in an in vitro setting. Furthermore, T cells expressing anti-CD70 CARs can activate cytotoxic effects in an in vitro model. See Figure 3A. Next, it was shown that anti-CD70 CAR T cells can induce cytotoxicity in an established RCC model by tail vein administration, but not by orthotopic kidney administration. See Figures 4A-4D.

[0372] Next, we determined whether antigen downregulation was a possible mechanism for the observed resistance. As shown in Figure 6, CD70 was nearly 100% positive in the lungs, but only a portion of the kidney tumors were CD70 positive. We then determined that in vitro culture of untreated K5 and K7 cells, which have reduced expression of CD70, restored antigen expression levels. See Figures 8A and 8B. Therefore, we investigated whether expression of CD70 fusion proteins would improve tumor clearance in an in vivo model. As seen in Figures 10A-10E, overexpression of CD70 was sufficient to result in tumor clearance in an in vivo model. Overall, these data demonstrate that i) CD70 was downregulated in the kidney, but not in the lung, in vivo in untreated mice, ii) the in vivo downregulation of CD70 was reversed by culturing "CD70-low" cells in vitro, and iii) lentiviral overexpression of CD70 led to kidney tumor clearance by SFG CD70 28z1xx in K5 and K7, but rapid recurrence in K7.

[0373] Because carbonic anhydrase IX (CAIX) is expressed in clear cell renal cell carcinoma and sarcomatoid renal cell carcinoma, we determined whether this antigen was regulated and heterogeneously expressed as CD70. Figures 11A-11C show that CAIX is heterogeneously expressed in K5 and K7 RCC cells and that it is upregulated by hypoxia (2% O2). Transcriptomic and proteomic analyses of sorted CD70 / CAIX double-negative, double-positive, and single-positive untreated kidney tumors revealed that the CD70 / CAIX double-negative population exhibited very low CD70 expression. Thus, CAIX and CD70 are regulated target antigens in RCC.

[0374] Next, we used a dual-targeting approach (pooling two CAR-T cells or a dual-transduction approach) using the SFG-CD70 28z1xx CAR and the SFG CAIX 28z1xx CAR. The dual-targeting approach, including anti-CAIX CAR T cells and anti-CD70 CAR T cells, was capable of in vitro killing. See Figures 12A and 12B. However, in vivo studies showed that CD70 and CAIX dual targeting did not result in tumor clearance due to a residual low-density antigen population. See Figures 12C-12I. In conclusion, CD70 / CAIX dual targeting improved tumor control in K5 cells but not in K7 cells. Furthermore, a significant amount of cells bearing low-density antigens was observed, and although there was a modest improvement in tumor control, a "CD70 / CAIX double-negative" population remained.

[0375] The presently disclosed subject matter has discovered that RCC contains a "low-density antigen" population of tumor cells with low CD70 density (500-2,000 molecules per cell) that can be targeted by HIT CD70. RNA sequencing has demonstrated that this "CD70lo" population of tumor cells may represent a more aggressive fraction of tumors enriched for cancer stem cell pathways. In addition, dynamic in vivo regulation of CD70 expression allows the low population to differentiate into a high-density population. See Figure 13E.

[0376] Therefore, we hypothesized that 70-HIT could completely eradicate tumors by targeting more aggressive tumor / cancer stem cell-like populations. Indeed, as shown in Figures 14A-14E, 70-HIT cells co-expressing costimulatory ligands (e.g., CD80 polypeptide and 4-1BBL, as disclosed in Section 2.2) were able to address a broad range of expression (e.g., low-density antigens). This approach successfully overcomes the antigenic heterogeneity observed in RCC.

[0377] The 70-HIT disclosed herein can be used to treat other solid tumors that express CD70 and have heterogeneous CD70 expression (e.g., glioblastoma, ovarian cancer, and pancreatic cancer).

[0378] The presently disclosed subject matter now demonstrates that HIT is a superior strategy to CAR for targeting heterologous targets in solid tumors. Other tumor targets, such as PMSA, mesothelin, and CEA, can now potentially be targeted with the knowledge that expression is no longer positive vs. negative, but a spectrum of expression.

[0379] In the context of RCC and other solid tumors, HIT+SFG 80 / 41BBL was effective against K5 and K7 tumors. The 70-HIT disclosed herein, which co-expresses costimulatory ligands (including, for example, CD80 polypeptide and 4-1BBL), represents a safe and effective therapy for RCC and other solid tumors.

[0380] Example 2 CD70 is a cancer antigen expressed on the cell surface membrane of clear cell RCC (ccRCC) tumors, but not in normal kidneys (Jilaveanu et al., Hum. Path. 2012), and is isolated by FACS on day 25. Importantly, its expression is retained in metastatic tissues. However, CD70 expression is heterogeneous. IHC ccRCC tumor microarrays identified that 22% of ccRCC cases had more than 50% of tumor cells positive for CD70 expression (Ye et al., J. Clinical Onc. 2022). Therefore, the presently disclosed subject matter determined whether CD70 represents an immunotherapy target in RCC.

[0381] To study renal cell carcinoma (RCC), two patient-derived xenograft models (K5 and K7) were developed from patients with clear cell RCC. Details of the K5 and K7 cell lines are listed in the table below and in Figure 2A, while CD70 expression levels are shown in Figure 2B. [Table 2]

[0382] Injection of these two cell lines in the tail vein resulted in engraftment in the lungs and later in the liver (Figures 3B and 3C).

[0383] Next, we determined the ability of T cells expressing an anti-CD70 CAR to kill K5 and K7 cells in vitro. Briefly, T cells were engineered to overexpress a CAR targeting CD70 and containing an IXX domain (see Figure 2C and section 2.1 for details). As seen in Figure 3B, anti-CD70 CAR T cells were able to induce the killing of K5 and K7 cells in vitro. However, although the in vivo killing effect of anti-CD70 CAR T cells was observed in established lung tumors, no effect was observed in a primary site orthotopic RCC model (Figures 4A-4D). Figure 5 shows that this effect was not due to differences in the ability to reach the tumor site. Thus, differential CD70 expression results in differential killing between tumor sites.

[0384] Next, the presently disclosed subject matter determined the mechanism behind the observed resistance. As seen in Figures 4A-4D, tumors established at orthotopic sites were not inherently resistant to CAR therapy. Therefore, we hypothesized that CD70 may have a role in the observed resistance. FACS analysis showed differential expression of CD70 between untreated lung and kidney tumors established with the K5 and K7 cell lines (Figure 6). These tumors expressed low levels of CD70 (CD70lo). - ) expressing CD70lo (Figure 7), these cells were cultured in vitro to study their expression profile. -The cells recovered CD70 expression over time (Figures 8A and 8B). Epigenetic analysis showed that the CD70 locus, but not the CAIX promoter locus, was epigenetically regulated (Figures 9A and 9B), and that the CD70 promoter was accessible by Ezh2 protein (Figure 9C). Therefore, we determined whether inhibition of Ezh2 could regulate CD70 expression. As seen in Figure 9D, treatment with tazemetostat significantly reduced CD70 locus expression. - Thus, the presently disclosed subject matter demonstrates that CD70 expression is epigenetically regulated by Ezh2-mediated H3K27me3 inhibition at different organ sites (e.g., primary vs. metastatic) within the same mouse, and that in vitro inhibition of Ezh2 results in restoration of CD70 in CD70lo kidney tumors.

[0385] To further confirm the observed data, K5 and K7 cells were engineered to overexpress CD70 and engrafted to establish tumors. As seen in Figures 10D and 10E, overexpression of CD70 in an orthotopic model resulted in tumor clearance. Overall, these data demonstrate that i) differential CD70 expression results in differential killing between sites, ii) CD70 expression is epigenetically regulated at different organ sites (primary vs. metastatic) within the same mouse by Ezh2-mediated H3K27me3 inhibition, iii) in vitro inhibition of Ezh2 results in the restoration of CD70 in CD70lo kidney tumors, iv) CD70 CAR T cells are unable to eliminate orthotopic sites because CD70 is downregulated in vivo below the detection threshold of CAR T cells, and v) exogenous CD70 overexpression enables CAR killing. Carbonic anhydrase 9 (CAIX) is a tumor antigen expressed in several solid tumors, including ccRCC, GBM, ovarian, and colorectal cancer (Campos, NSPd et al. Cancers 2022). CAIX is induced by hypoxia, and its expression on normal tissues is observed in the intrahepatic bile duct, gastric mucosa, and duodenum. First-generation anti-CAIX CAR T cell therapy for metastatic ccRCC showed no clinical response and no toxicity (Lamers et al. 2016). Given these characteristics, the roles of CAIX and CD70 were further investigated.

[0386] In light of these findings, the inventors of the presently disclosed subject matter desired to determine whether the inclusion of an alternative antigen-recognition receptor and a fusion polypeptide capable of providing costimulatory signals to the cells could reverse the resistance of kidney orthotopic tumors derived from K5 and K7. T cells were engineered to target CD70 and express a CAR containing a 1XX domain or a HIT receptor targeting CD70 using the same antigen-binding fragment of the CAR. In addition, as disclosed in Section 2.2, certain T cells were further engineered to contain a CD80 polypeptide and 4-1BBL. As seen in Figures 14A-14E and 15, T cells expressing a HIT receptor targeting CD70 and a fusion protein (e.g., having SEQ ID NO: 76) were able to induce complete responses in kidney orthotopic tumors derived from K5 and K7. This effect was attributed to the depletion of CAR T cells, as indicated by the higher expression profile of PD1, TIM3, and LAG3 observed in CAR T cells compared to HIT cells (Figure 15).

[0387] Furthermore, the presently disclosed subject matter determined that the efficacy of HIT CD70 T cells was not due to bystander killing of CD70-negative tumors, as knocking out CD70 on the K5 or K7 PDX lines resulted in tumors resistant to HIT CD70 killing (Figures 16A-16C). Overall, the data disclosed herein demonstrate that i) CD70 expression is not binary but rather a spectrum ranging from high to very low expression, ii) highly sensitive CD70 HIT T cells can effectively target this very low CD70 population, iii) CAR T cells were up to 40-50% positive for three exhaustion markers at days 7 and 14, compared with less than 15% for HIT T cells, and iv) the efficacy of HIT CD70 T cells was not due to bystander killing of CD70-negative tumors.

[0388] Recently, adoptive cell therapy involving anti-CD70 CARs has been developed, with responses observed only in tumors expressing high levels of CD70 (Srour et al., Cancer Research 83.8_Supplement(2023):CT011-CT011). Thus, the subject matter disclosed herein is directed to the development of CD70 lo - To investigate whether these cells have been observed in other tumors, SK-OV3 is a cell line capable of developing ovarian orthotopic and intraperitoneal carcinomas that express CD70 (Figures 19A and 19B). Importantly, administration of cells expressing HIT receptors and fusion proteins (e.g., those with SEQ ID NO: 76) targeting CD70 induced a complete and durable response, overcoming the tumor (Figures 19C-19E).

[0389] Next, we determined whether cells expressing HIT receptors and fusion proteins (e.g., those with SEQ ID NO: 76) targeting CD70 could reverse the effects of resistant pancreatic cancer. PANC-1 cell lines were analyzed for their CD70 expression profile and ability to establish orthotopic tumors (Figure 18A). Notably, cells expressing HIT receptors and fusion proteins (e.g., those with SEQ ID NO: 76) targeting CD70 were able to induce complete responses in pancreatic cancer and overcome tumor challenge, indicating that CD70 lo -This was achieved due to the presence of CD70 T cells (Figure 18B). Furthermore, pancreatic ductal adenocarcinoma PDX (PDAC2) were analyzed for CD70 expression. Notably, in vitro, these aggressive pancreatic PDX were heterogeneous for CD70, with approximately 23% positive CD70 expression in vitro and only 30% positive CD70 in vivo at orthotopic sites in the pancreas (Figures 17A-17B). Interestingly, cells expressing HIT receptors and fusion proteins (e.g., those with SEQ ID NO: 76) targeting CD70 were able to induce complete responses in a pancreatic PDAC2 orthotopic model, and this effect was specific to CD70, as knockout of CD70 in PDAC2 tumors rendered the tumors resistant to HIT CD70 T cell killing (Figure 17E). The CD70 locus was confirmed to be epigenetically regulated in PDAC2 (an alternative pancreatic cancer cell type; see Figures 17C and 17D). These data confirm the heterogeneity of CD70 in pancreatic, ovarian, and renal cancers and its epigenetic regulation.

[0390] Finally, traditional diagnostic methods (e.g., IHC) have not demonstrated the ability to detect CD70lo - We then determined whether we could identify these tumors that contained CD70lo cells. As can be seen in Figure 20A, - Tumors containing the cells yielded negative IHC detection, highlighting the limitations of conventional diagnostic methods.

[0391] In conclusion, the subject matter disclosed herein demonstrates that i) HIT is superior to CAR in RCC, pancreatic, and ovarian cancers with heterogeneous CD70 expression in vivo; ii) the mechanism of CD70 downregulation by Ezh2 inhibition may be conserved across CD70-heterogeneous tumors; iii) in CD70-heterogeneous PDAC2 tumors, Ezh2 inhibition in vitro can restore CD70 expression, demonstrating that partially CD70-positive tumors can negate tumors with very low CD70 expression that can be revealed by Ezh2 inhibition; iv) conventional IHC does not capture very low CD70 expression, and RNA FISH approaches can be applied as clinical diagnostic tools; v) it may now be necessary to re-stratify patients who may benefit from treatments that would otherwise be discounted based on partial / negative CD70 expression; and vi) identifying that CD70-negative tumors may have very low CD70 expression paves the way for HIT CD70 as the basis for either single- or dual-targeting approaches.

[0392] Embodiments of the presently disclosed subject matter From the above description, it will be apparent that variations and modifications can be made to the subject matter disclosed herein to employ the subject matter in a variety of applications and conditions, and such embodiments also fall within the scope of the following claims.

[0393] The recitation of a list of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of the listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0394] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each individual patent and publication was specifically and individually indicated to be incorporated by reference.

Claims

1. 1. A method of reducing tumor burden in a subject having renal cell carcinoma, pancreatic cancer, or ovarian cancer, the method comprising administering to the subject an effective amount of cells comprising a TCR-like fusion molecule that targets CD70.

2. 3. The method of claim 2, wherein the method reduces the number of tumor cells, reduces tumor size, and / or eradicates tumors in the subject.

3. 1. A method of reducing tumor burden in a subject, the method comprising administering to the subject an effective amount of cells comprising a TCR-like fusion molecule that targets CD70, wherein the tumor is renal cell carcinoma, pancreatic cancer, or ovarian cancer.

4. 4. The method of claim 3, wherein the method reduces the number of tumor cells, reduces tumor size, and / or eradicates the tumor in the subject.

5. 1. A method for preventing and / or treating a tumor in a subject having a renal cell carcinoma neoplasm, renal cell carcinoma, pancreatic cancer, or ovarian cancer, the method comprising administering to the subject an effective amount of cells comprising a TCR-like fusion molecule that targets CD70.

6. 1. A method for preventing and / or treating a tumor in a subject, the method comprising administering to the subject an effective amount of cells comprising a TCR-like fusion molecule that targets CD70, wherein the tumor is a renal cell carcinoma neoplasm, renal cell carcinoma, pancreatic cancer, or ovarian cancer.

7. 1. A method for preventing and / or treating a tumor in a subject in need thereof, said method comprising: a) obtaining a tumor sample from said subject having an undetectable CD70 polypeptide level; b) detecting CD70 polynucleotides by FISH; c) if said CD70 polynucleotide is detected, administering to said subject an effective amount of cells comprising a TCR-like fusion molecule that targets CD70.

8. 1. A method for preventing and / or treating a tumor in a subject in need thereof, said method comprising: a) obtaining a tumor sample from said subject having an undetectable CD70 polypeptide level; b) contacting the sample with an Ezh2 inhibitor; c) administering to said subject an effective amount of cells comprising a TCR-like fusion molecule that targets CD70 if said CD70 polypeptide is detected in said sample.

9. 9. The method of claim 1, wherein the TCR-like fusion molecule comprises i) a first antigen-binding chain comprising an antigen-binding fragment of an antibody heavy chain variable region (VH), and ii) a second antigen-binding chain comprising an antigen-binding fragment of an antibody light chain variable region (VL), wherein the first and second antigen-binding chains a) each comprise a TRAC polypeptide or a TRBC polypeptide, and b) bind to a second antigen, and wherein the TCR-like fusion molecule binds to the second antigen in an HLA-independent manner.

10. 10. The method of claim 9, wherein at least one of the TRAC polypeptide and the TRBC polypeptide is endogenous.

11. The first and second antigen-binding chains are about 1 x 10 －８ 10. The method of claim 9, wherein the antibody binds to the second antigen with a dissociation constant (KD) of M or less.

12. The first and second antigen-binding chains are about 5 x 10 －９ 10. The method of claim 9, wherein the antibody binds to the second antigen with a dissociation constant (KD) of M or less.

13. 10. The method of claim 9, wherein the first antigen binding chain comprises an antigen binding fragment of a VH of an antibody and a TRBC polypeptide, and the second antigen binding chain comprises an antigen binding fragment of a VL of the antibody and a TRAC polypeptide.

14. 10. The method of claim 9, wherein the first antigen binding chain comprises an antigen binding fragment of a VH of an antibody and a TRAC polypeptide, and the second antigen binding chain comprises an antigen binding fragment of a VL of the antibody and a TRBC polypeptide.

15. i) the first antigen binding chain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 34, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 35, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 36, and the second antigen binding chain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 37, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 38, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 39; or ii) the first antigen binding chain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 37, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 38, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 39, and the second antigen binding chain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 34, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 35, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

36.

16. i) the first antigen-binding chain comprises CDR1, CDR2, and CDR3 of a heavy chain variable region set forth in SEQ ID NO: 40, and the second antigen-binding chain comprises CDR1, CDR2, and CDR3 of a light chain variable region set forth in SEQ ID NO: 42; or ii) the first antigen-binding chain comprises CDR1, CDR2, and CDR3 of a light chain variable region set forth in SEQ ID NO: 42, and the second antigen-binding chain comprises CDR1, CDR2, and CDR3 of a heavy chain variable region set forth in SEQ ID NO:

40.

17. i) the first antigen-binding chain comprises a heavy chain variable region set forth in SEQ ID NO: 40, and the second antigen-binding chain comprises CDR1, CDR2, and CDR3 of a light chain variable region set forth in SEQ ID NO: 42; or ii) the first antigen-binding chain light chain variable region set forth in SEQ ID NO: 42, and the second antigen-binding chain comprises a heavy chain variable region set forth in SEQ ID NO:

40.

18. The method of claim 9, wherein the first and second antigen-binding chains are capable of associating with a CD3ζ polypeptide.

19. The method of claim 18, wherein the first and second antigen-binding chains are capable of activating the CD3ζ polypeptide when bound to the second antigen.

20. The method of claim 19, wherein said activation of said CD3ζ polypeptide can activate said cell.

21. 10. The method of claim 9, wherein the cell further comprises a gene disruption at the TRAC locus.

22. The method of claim 9, wherein the cell further comprises a gene disruption of the CD70 locus.

23. 10. The method of claim 9, wherein the cells further comprise a gene disruption of the TRAC locus and CD70.

24. The method of claim 9, wherein the tumor comprises tumor cells with low CD70 antigen density.

25. The tumor expresses a low frequency of CD70 ＋ The method of claim 9 comprising tumor cells.

26. 10. The method of claim 9, wherein the tumor comprises a CD70 polypeptide that is undetectable by immunodiffusion, immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, Western blotting, binder-ligand assay, immunohistochemical techniques, agglutination, complement assay, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, or a combination thereof.

27. 27. The method of claim 26, wherein the tumor comprises a CD70 polypeptide that is not detectable by immunohistochemistry (IHC).

28. The method of any one of claims 1 to 27, wherein the cell is a cell of the lymphoid lineage or a cell of the myeloid lineage.

29. 29. The method of claim 28, wherein the cells of the lymphoid lineage are selected from the group consisting of T cells, B cells, natural killer (NK) cells, and dendritic cells.

30. The method of any one of claims 1 to 29, wherein the cell is a T cell.

31. 31. The method of claim 30, wherein the T cells are derived from induced pluripotent stem cells.

32. The T cell is CD8 ＋ 32. The method of claim 30 or 31, wherein the cell is a T cell.

33. The CD8 ＋ 33. The method of claim 32, wherein the T cells are CD4-independent.

34. 34. The method of any one of claims 30 to 33, wherein the T cells are selected from the group consisting of cytotoxic T lymphocytes (CTLs), gamma delta T cells, tumor infiltrating lymphocytes (TILs), regulatory T cells, and natural killer T (NKT) cells.

35. 35. The method of any one of claims 1 to 34, wherein the cell further comprises a chimeric antigen receptor (CAR) that targets a second antigen.

36. 36. The method of claim 35, wherein the CAR comprises an extracellular antigen-binding domain that binds to the first antigen, and an intracellular signaling domain that is capable of delivering an activating signal to the cell.

37. 37. The method of Claim 36, wherein the intracellular signaling domain of the CAR comprises a CD3ζ polypeptide.

38. The method of claim 37, wherein the CD3ζ polypeptide is a natural CD3ζ polypeptide or a modified CD3ζ polypeptide.

39. The method of claim 38, wherein the modified CD3ζ polypeptide comprises a native ITAM1, an ITAM2 variant consisting of two loss-of-function mutations, and an ITAM3 variant consisting of two loss-of-function mutations.

40. 40. The method of any one of claims 36-39, wherein the intracellular signaling domain of the CAR further comprises at least one costimulatory signaling region.

41. 41. The method of claim 40, wherein the at least one costimulatory signaling region comprises at least the intracellular domain of a costimulatory molecule or a portion thereof.

42. 42. The method of claim 41, wherein the costimulatory molecule is selected from the group consisting of CD28, 4-1BB, OX40, CD27, CD40, CD154, CD97, CD11a / CD18, ICOS, DAP-10, CD2, CD150, CD226, and NKG2D.

43. The method of any one of claims 36 to 42, wherein the CAR comprises a transmembrane domain.

44. 44. The method of any one of claims 1 to 43, wherein the cells further comprise a chimeric costimulatory receptor (CCR).

45. 45. The method of claim 44, wherein the CCR comprises an extracellular antigen-binding domain that binds to a third antigen and an intracellular domain that is capable of delivering a costimulatory signal to the cell but does not alone deliver an activating signal to the cell.

46. 46. ​​The method of claim 45, wherein the intracellular domain of the CCR comprises at least the intracellular domain of a costimulatory molecule or a portion thereof.

47. 47. The method of claim 46, wherein the costimulatory molecule is selected from the group consisting of CD28, 4-1BB, OX40, CD27, CD40, CD154, CD97, CD11a / CD18, ICOS, DAP-10, CD2, CD150, CD226, and NKG2D.

48. 48. The method of any one of claims 35 to 47, wherein the second antigen is a tumor antigen or a pathogen antigen.

49. The tumor antigen is selected from the group consisting of CD19, IL1RAP, ABCG2, AChR, ACKR6, ADAMTS13, ADGRE2, ADGRE2 (EMR2), ADORA3, ADRA1D, AGER, ALS2, an antigen of a cytomegalovirus (CMV)-infected cell (e.g., a cell surface antigen), ANO9, AQP2, ASIC3, ASPRV1, ATP6V0A4, B3GNT4, B7-H3, BCMA, BEST4, C3orf35, CADM3, CAIX, CAPN3, CCDC155, CCR1, CD10, CD117, CD123, CD133, CD135 (FL T3), CD138, CD20, CD22, CD244 (2B4), CD25, CD26, CD30, CD300LF, CD312, CD 32, CD321, CD33, CD34, CD36, CD38, CD41, CD44, CD44V6, CD47, CD49f, CD56, C D7, CD71, CD74, CD8, CD82, CD96, CD98, CD99, CDH13, CDHR1, CEA, CEACAM6, C HST3, CLEC12A, CLEC1A, CLL1, CNIH2, COL15A1, COLEC12, CPM, CR1, CX3CR1, C XCR4, CYP4F11, DAGLB, DARC, DFNB31, DGKI, EGF1R, EGFR-VIII, EGP-2, EGP- 40, ELOVL6, EMB, EMC10, EMR2, ENG, EpCAM, EphA2, EPHA4, ERBB, ERBB2, Erb-B 3, Erb-B4, E-selectin, EXOC3L4, EXTL3, FAM186B, FBP, FCGR1A, FKBP1B, FLRT1 , folate receptor-α, FOLR2, FRMD5, GABRB2, GAS2, GD2, GD3, GDPD3, GNA14, GNAZ, GPR15 3, GPR56, GYPA, HEPHL1, HER-2, hERT, HILPDA, HLA-DR, HOOK1, hTERT, HTR2A, ICAM1, IGFBP3, IL10RB, IL20RB, IL23R, ILDR1, interleukin-13 receptor subunit alpha-2 (IL-13Rα2), ITFG3, ITGA4, ITGA5, ITGA8, ITGAX, ITGB5, ITGB8, JAM3, KCND1, KCNJ5, KCNK13, KCNN4, KCNV2, KDR, KIF19, KIF26B, κ-light chain, L1CAM, LAX1,LEPR, Lewis Y (CD174), Lewis Y (LeY), LILRA2, LILRA6, LILRB2, LILRB3, LILRB4, LOXL4, LPAR2, LRRC37A3, LRRC8E, LRRN2, LRRTM2, LTB4R, MAGE-A1, MAGEA3, MANSC1, MART1, GP100, MBOAT1, MBOAT7, melanoma antigen family A, mesothelin (MSLN), MFAP3L, MMP25, MRP1, MT-ND1, mucin 1 (MUC1), mucin 16 (MUC16), MYADM, MYADML2, NGFR, NKCS1, NKG2D Ligands, NLGN3, NPAS2, NY-ESO-1, oncofetal antigen (h5T4), OTOA, P2RY13, p53, PDE3A, PEAR1, PIEZO1, PLXNA4, PLXNC1, PNPLA3, PPFIA4, PPP2R5B, PRAME, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), proteinase 3 (PR1), PSD2, PTPRJ, RDH16, receptor tyrosine-protein kinase Erb-B2, RHBDL3, RNF173, RNF183, ROR1, RYR2, S CIN, SCN11A, SCN2A, SCNN1D, SEC31B, SEMA4A, SH3PXD2A, SIGLEC11, SIRP B1, SLC16A6, SLC19A1, SLC22A5, SLC25A36, SLC25A41, SLC30A1, SLC34A3 , SLC43A3, SLC44A1, SLC44A3, SLC45A3, SLC6A16, SLC6A6, SLC8A3, SLC9A 1, SLCO2B1, SPAG17, STC1, STON2, SUN3, Survivin, SUSD2, SYNC, TACSTD2, TA 49. The method of claim 48, wherein the target protein is selected from the group consisting of S1R3, TEX29, TFR2, TIM-3 (HAVCR2), TLR2, TMEFF2, TMEM145, TMEM27, TMEM40, TMEM59L, TMEM89, TMPRSS5, TNFRSF14, TNFRSF1B, TRIM55, TSPEAR, TTYH3, tumor-associated glycoprotein 72 (TAG-72), tyrosinase, vascular endothelial growth factor R2 (VEGF-R2), VLA-4, Wilms' tumor protein (WT-1), WNT4, WT1, and ZDHHC11.

50. 50. The method of any one of claims 1 to 49, wherein the cells further comprise at least one exogenous costimulatory ligand.

51. 51. The method of claim 50, wherein the at least one exogenous costimulatory ligand is selected from the group consisting of tumor necrosis factor (TNF) family members, immunoglobulin (Ig) superfamily members, and combinations thereof.

52. 52. The method of claim 51, wherein the TNF family member is selected from the group consisting of 4-1BBL, OX40L, CD70, FasL, GITRL, TNF-related apoptosis-inducing ligand (TRAIL), CD30L, LIGHT (TNFSF14), and CD40L.

53. 53. The method of claim 51 or 52, wherein the Ig superfamily member is selected from the group consisting of CD80, CD86, ICOSLG, and combinations thereof.

54. 54. The method of any one of claims 50 to 53, wherein the at least one exogenous costimulatory ligand comprises CD80.

55. 54. The method of any one of claims 50 to 53, wherein the at least one exogenous costimulatory ligand comprises 4-1BBL.

56. 54. The method of any one of claims 50 to 53, wherein the cells comprise two exogenous costimulatory ligands.

57. 57. The method of claim 56, wherein the at least two exogenous costimulatory ligands comprise CD80 and 4-1BBL.

58. 58. The method of any one of claims 1-57, wherein the cell further comprises a fusion polypeptide comprising a) the extracellular domain and transmembrane domain of a costimulatory ligand, and b) the intracellular domain of a first costimulatory molecule.

59. 59. The method of claim 58, wherein the costimulatory ligand is selected from the group consisting of a tumor necrosis factor (TNF) family member, an immunoglobulin (Ig) superfamily member, and a combination thereof.

60. 60. The method of claim 59, wherein the TNF family member is selected from the group consisting of 4-1BBL, OX40L, CD70, GITRL, CD40L, and combinations thereof.

61. 61. The method of claim 59 or 60, wherein the Ig superfamily member is selected from the group consisting of CD80, CD86, ICOSLG, and combinations thereof.

62. 62. The method of any one of claims 58 to 61, wherein the costimulatory ligand is CD80.

63. 63. The method of any one of claims 58-62, wherein the first costimulatory molecule is selected from the group consisting of CD28, 4-1BB, OX40, ICOS, DAP-10, CD27, CD40, NKG2D, CD2, and combinations thereof.

64. 64. The method of claim 63, wherein the first costimulatory molecule is 4-1BB.

65. 65. The method of any one of claims 58 to 64, wherein the costimulatory ligand is CD80 and the first costimulatory molecule is 4-1BB.

66. 66. The method of any one of claims 58 to 65, wherein the fusion polypeptide further comprises the intracellular domain of a second costimulatory molecule.

67. 67. The method of claim 66, wherein the second costimulatory molecule is selected from the group consisting of CD28, 4-1BB, OX40, ICOS, DAP-10, CD27, CD40, NKG2D, CD2, and combinations thereof.

68. 68. The method of claim 66 or 67, wherein the second costimulatory molecule is CD28.

69. 68. The method of any one of claims 61 to 67, wherein the costimulatory ligand is CD80, the first costimulatory molecule is 4-1BB, and the second costimulatory molecule is CD28.

70. 70. The method of any one of claims 1 to 69, wherein the cells are autologous.

71. 70. The method of any one of claims 1 to 69, wherein the cells are allogeneic.

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