Cells expressing anti-CD70 chimeric receptors and uses thereof

Abstract

The presently disclosed subject matter provides cells, compositions and methods for enhancing an immune response against tumor antigens. It relates to a cell comprising a first antigen recognition receptor targeting CD70. These cells have improved activity and / or efficiency.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 380,482, filed on October 21, 2022, the contents of which are incorporated by reference in their entirety, and claims priority to which.

[0003] Sequence Listing

[0004] The sequence listing in accordance with the rules of WIPO Standard ST.26 is hereby incorporated by reference. The sequence listing has been submitted via PatentCenter in the form of an electronic document, encoded as XML in UTF-8 text. The electronic document created on October 18, 2023 is named "072734_1498_SL" and is 102,400 bytes in size. Technical Field

[0005] The subject matter disclosed in this application provides cells, compositions and methods for enhancing immune responses against tumor antigens. It relates to cells comprising antigen recognition receptors (e.g., TCR-like fusion molecules) targeting CD70. The cells disclosed in this application have improved activity against solid tumors (e.g., renal cell carcinoma). Background Art

[0006] 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 by introducing genetic material encoding for antigen recognition receptors (e.g., TCR-like fusion molecules). Patient-engineered CAR T cells have shown significant efficacy against a range of liquid and solid malignancies. However, treatment failure and relapse occur in a significant proportion of patients. Therefore, there remains a need for improved immunotherapy. Summary of the invention

[0007] The subject matter disclosed in the present application provides a method of reducing tumor load and / or preventing and / or treating a tumor in a subject suffering from renal cell carcinoma, pancreatic cancer or ovarian cancer. Additionally or alternatively, the subject matter disclosed in the present application provides a method of reducing tumor load and / or preventing and / or treating a tumor in a subject, wherein the tumor is renal cell carcinoma, pancreatic cancer or ovarian cancer.

[0008] In certain embodiments, the method comprises: administering to a subject an effective amount of cells comprising a TCR-like fusion molecule targeting CD70. In certain embodiments, the method reduces the number of tumor cells, reduces the size of the tumor, and / or eradicates the tumor in the subject.

[0009] The subject matter disclosed in the present application further provides a method for preventing and / or treating a tumor in a subject in need thereof, the method comprising: obtaining a tumor sample having undetectable levels of CD70 polypeptide from the subject, detecting CD70 polynucleotides by FISH, and if CD70 polynucleotides are detected, administering to the subject an effective amount of cells comprising a TCR-like fusion molecule targeting CD70. In addition, the subject matter disclosed in the present application further provides a method for preventing and / or treating a tumor in a subject in need thereof, the method comprising: obtaining a tumor sample having undetectable levels of CD70 polypeptide from the subject, b) contacting the sample with an Ezh2 inhibitor, and if CD70 polypeptide is detected in the sample, administering to the subject an effective amount of cells comprising a TCR-like fusion molecule targeting CD70.

[0010] In certain embodiments, the TCR-like fusion molecule comprises: i) a first antigen-binding chain comprising an antigen-binding fragment of the heavy chain variable region (VH) of an antibody; and ii) a second antigen-binding chain comprising an antigen-binding fragment of the light chain variable region (VL) of an antibody; wherein the first antigen-binding chain and the second antigen-binding chain a) each comprise a TRAC polypeptide or a TRBC polypeptide, and b) bind to a second antigen, wherein the TCR-like fusion molecule binds to the second antigen in an HLA-independent manner.

[0011] In certain embodiments, at least one of the TRAC polypeptide and the TRBC polypeptide is endogenous. In certain embodiments, the first antigen-binding chain and the second antigen-binding chain are expressed in an amount of about 1 × 10 -8 M or less dissociation constant (KD) and binds to the second antigen. In certain embodiments, the first antigen binding chain and the second antigen binding chain bind to the second antigen with a dissociation constant (KD) of about 5 × 10 -9 The protein binds to the second antigen with a dissociation constant (KD) of M or less.

[0012] In certain embodiments, the first antigen-binding chain comprises an antigen-binding fragment of the VH of the antibody and a TRBC polypeptide, and the second antigen-binding chain comprises an antigen-binding fragment of the VL of the antibody and a TRAC polypeptide. In certain embodiments, the first antigen-binding chain comprises an antigen-binding fragment of the VH of the antibody and a TRAC polypeptide, and the second antigen-binding chain comprises an antigen-binding fragment of the VL of the antibody and a TRBC polypeptide.

[0013] In certain embodiments, i) the first antigen-binding chain comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 34, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 35, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 36, and the second antigen-binding chain comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 37, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 38, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 39. In certain embodiments, the first antigen-binding chain comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 37, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 38, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 39, and the second antigen-binding chain comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 34, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 35, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 36.

[0014] In certain embodiments, the first antigen binding chain comprises the CDR1, CDR2 and CDR3 of the heavy chain variable region shown in SEQ ID NO: 40, and the second antigen binding chain comprises the CDR1, CDR2 and CDR3 of the light chain variable region shown in SEQ ID NO: 42. In certain embodiments, the first antigen binding chain comprises the CDR1, CDR2 and CDR3 of the light chain variable region shown in SEQ ID NO: 42, and the second antigen binding chain comprises the CDR1, CDR2 and CDR3 of the heavy chain variable region shown in SEQ ID NO: 40. In certain embodiments, the first antigen binding chain comprises the heavy chain variable region shown in SEQ ID NO: 40, and the second antigen binding chain comprises the CDR1, CDR2 and CDR3 of the light chain variable region shown in SEQ ID NO: 42. In certain embodiments, the first antigen binding chain comprises the light chain variable region shown in SEQ ID NO: 42; and the second antigen binding chain comprises the heavy chain variable region shown in SEQ ID NO: 40.

[0015] In certain embodiments, the first antigen binding chain and the second antigen binding chain are capable of associating with a CD3 zeta polypeptide. In certain embodiments, the first antigen binding chain and the second antigen binding chain are capable of activating a CD3 zeta polypeptide when bound to a second antigen. In certain embodiments, activation of a CD3 zeta polypeptide is capable of activating a cell.

[0016] In certain embodiments, the cell further comprises a gene disruption to the TRAC locus. In certain embodiments, the cell further comprises a gene disruption to the CD70 locus. In certain embodiments, the cell further comprises a gene disruption to the TRAC locus and CD70.

[0017] In certain embodiments, the tumor comprises tumor cells with low antigen density of CD70. In certain embodiments, the tumor has CD70+ tumor cells with low tumor cell frequency. In certain embodiments, the tumor comprises a CD70 polypeptide that cannot be detected by the following: immunodiffusion, immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, Western blot, conjugate-ligand assay, immunohistochemical technique, agglutination, complement assay, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), superdiffusion chromatography, or a combination thereof. In certain embodiments, the tumor and / or neoplasm comprises a CD70 polypeptide that cannot be detected by immunohistochemistry (IHC). In certain embodiments, the tumor comprises a CD70 polypeptide that cannot be detected by immunohistochemistry (IHC).

[0018] In certain embodiments, the cell is a lymphocyte or a myeloid cell. In certain embodiments, the lymphocyte 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 + T cells are independent of CD4. 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.

[0019] In certain embodiments, the cell further comprises a chimeric antigen receptor (CAR) targeting a second antigen. In certain embodiments, CAR comprises an extracellular antigen binding domain that binds to a first antigen, and an intracellular signaling domain that can deliver an activation signal to a cell. In certain embodiments, the intracellular signaling domain of CAR comprises a CD3 ζ polypeptide. In certain embodiments, the CD3 ζ polypeptide is a natural CD3 ζ polypeptide or a modified CD3 ζ polypeptide. In certain embodiments, the modified CD3 ζ polypeptide comprises a natural 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 CAR further comprises at least one costimulatory signaling region. In certain embodiments, the at least one costimulatory signaling region comprises at least an intracellular domain of a costimulatory molecule or a portion thereof. In certain embodiments, the co-stimulatory 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.

[0020] In certain embodiments, cell further comprises chimeric costimulatory receptor (CCR).In certain embodiments, CCR comprises the extracellular antigen binding domain combined with the third antigen, and can deliver costimulatory signal to cell but not deliver activation signal to cell alone Intracellular domain.In certain embodiments, the intracellular domain of CCR comprises at least the intracellular domain of costimulatory molecule or a part thereof.In certain embodiments, costimulatory molecule is selected from the group consisting of the following items: CD28, 4-1BB, OX40, CD27, CD40, CD154, CD97, CD11a / CD18, ICOS, DAP-10, CD2, CD150, CD226 and NKG2D.

[0021] In certain embodiments, the second antigen is a tumor antigen or a pathogen antigen. In certain embodiments, the tumor antigen is selected from the group consisting of: CD19, IL1RAP, ABCG2, AChR, ACKR6, ADAMTS13, ADGRE2, ADGRE2 (EMR2), ADORA3, ADRA1D, AGER, ALS2, antigen of cytomegalovirus (CMV)-infected cells (e.g., cell surface antigen), ANO9, AQP2, ASIC3, ASPRV1, ATP6V0A4, B3GNT4, B7-H3, BCMA, BEST4, C3orf35, CADM3, CAIX, CAPN3, CCDC155, CCR1, CD10, CD117, CD123, 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, COL15A1, COLEC12, 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-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, kappa-light chain, L1CAM, LAX1, LEPR, LewisY (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 ligand, NLGN3, NPAS2, NY-ESO-1, carcinoembryonic 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, SCIN, SCN11A, SCN2A, SCNN1D, SEC31B, SEMA4A, SH3PXD2A, SIGLEC11, SIRPB1, SLC16A6, SLC19A1, SLC22A5, SLC25A3 6. SLC25A41, SLC30A1, SLC34A3, SLC43A3, SLC44A1, SLC44A3, SLC45A3, SLC6A16, SLC6A6, SLC8A3, SLC9A1, SLCO2B1, SPAG17, STC1, STON2, SUN3, survivin, 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.

[0022] In certain embodiments, the cell further comprises at least one exogenous co-stimulatory ligand. In certain embodiments, the at least one exogenous co-stimulatory ligand is selected from the group consisting of tumor necrosis factor (TNF) family members, immunoglobulin (Ig) superfamily members, and combinations thereof. In certain embodiments, the TNF family members are selected from the group consisting of 4-1BBL, OX40L, CD70, FasL, GITRL, TNF-related apoptosis-inducing ligand (TRAIL), CD30L, LIGHT (TNFSF14), CD40L.

[0023] In certain embodiments, the Ig superfamily members are selected from the group consisting of CD80, CD86, ICOSLG, and combinations thereof. In certain embodiments, the at least one exogenous co-stimulatory ligand comprises CD80. In certain embodiments, the at least one exogenous co-stimulatory ligand comprises 4-1BBL. In certain embodiments, the cell comprises two exogenous co-stimulatory ligands. In certain embodiments, the at least two exogenous co-stimulatory ligands comprise CD80 and 4-1BBL.

[0024] In certain embodiments, the cell further comprises a fusion polypeptide, and the fusion polypeptide comprises: a) an extracellular domain and a transmembrane domain of a costimulatory ligand, and b) an intracellular domain of a first costimulatory molecule. In certain embodiments, the costimulatory ligand is selected from the group consisting of the following items: tumor necrosis factor (TNF) family members, immunoglobulin (Ig) superfamily members, and combinations thereof. In certain embodiments, TNF family members are selected from the group consisting of the following items: 4-1BBL, OX40L, CD70, GITRL, CD40L, and combinations thereof. In certain embodiments, Ig superfamily members are selected from the group consisting of the following items: CD80, CD86, ICOSLG, and combinations thereof. In certain embodiments, the costimulatory ligand is CD80. In certain embodiments, the first costimulatory molecule is selected from the group consisting of the following items: CD28, 4-1BB, OX40, ICOS, DAP-10, CD27, CD40, NKG2D, CD2, and combinations 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 the second costimulatory molecule. In certain embodiments, the second costimulatory molecule is selected from the group consisting of the following items: 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.

[0025] In certain embodiments, the cells are autologous. In certain embodiments, the cells are allogeneic. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The following detailed description, which is given by way of example and is not intended to limit the presently disclosed subject matter to the specific embodiments described, may be read in conjunction with the accompanying drawings.

[0027] Figure 1 A picture illustrating the challenges facing CAR in solid tumors is depicted.

[0028] Figure 2A Characteristics (histology, prior treatment, and genetic mutations) of two RCC PDX models, “K5” and “K7”, are shown. Figure 2B Shown is the expression of CD70 in K5 and K7 cells in vitro. Figure 2C A schematic diagram of a CD70-28z1XX CAR with a CD28 co-stimulatory domain and an attenuated zeta chain signaling domain (referred to as "1XX") is shown.

[0029] Figure 3A Figure 3E depicts the establishment of a renal cell carcinoma PDX model. Figure 3A CD70 CAR-mediated killing in vitro is shown. Figure 3B Shown is the K7 RCC PDX lung metastasis model obtained by tail vein administration. Figure 3C Shown is a K5 RCC PDX lung metastasis model obtained by tail vein administration. Figure 3D Shown are K5 and K7 RCC PDX primary site models obtained by orthotopic renal administration.

[0030] Figure 4A and Figure 4B Delineated the effects of anti-CD70 CAR T cells in vivo. Figure 4A Shown is the effect of anti-CD70 CAR T cells in the K7 RCC PDX lung metastasis model obtained by tail vein administration. Figure 4B Shown is the effect of anti-CD70 CAR T cells in the K5 RCC PDX model obtained by orthotopic renal administration. Figure 4C Shown is the effect of anti-CD70 CAR T cells in the K5 RCC PDX lung metastasis model obtained by tail vein administration. Figure 4D Shown is the effect of anti-CD70 CAR T cells in the K7 RCC PDX primary site model obtained by orthotopic renal administration.

[0031] Figure 5 The distribution of RCC tumors (labeled with firefly luciferase) and CAR T cells (labeled with Gaussia luciferase) in a lung metastasis model, an orthotopic model, and in mice with tumors at both lung and kidney tumor sites is shown, indicating that although tumors are cleared from the lungs but not the kidneys, CAR T cells are trafficked to both sites.

[0032] Figure 6 Depicted is a FACS analysis of CD70 expressed in the lung and kidney of untreated mice. CD70 is homogenous and high in the lung, thus enabling tumor eradication in the lung metastasis model by CD70 CAR T cells. CD70 expression is heterogeneous at the orthotopic site in the kidney (MFI negative in the CD70 negative population), explaining the inability to achieve tumor eradication by CD70 CAR T cells.

[0033] Figure 7 Figure 3 shows the expression of CD70lo or CD70 in lungs from untreated mice. +Sorting and bulk RNA sequencing of renal tumors. Bulk RNA sequencing showed very low levels of CD70 expression by transcripts in the CD70lo renal tumor population. Other genes that were not expressed in RCC tumors are listed for comparison.

[0034] Fig. 8A and Figure 8B Depicted is FACS analysis of in vitro cell cultures in which CD70 expression was restored. Fig. 8A It is shown that cultures of untreated K5 renal tumors, which had a low density of CD70 (CD70 MFI negative population) in vivo renal population, restored CD70 expression in vitro. Figure 8B It was shown that cultures of untreated K7, which had a low density of CD70 (CD70 MFI negative population) in vivo kidney population, restored CD70 expression in vitro.

[0035] 9A to 9D Epigenetic analysis is depicted. Fig.9A Epigenetic analysis of the CD70 locus in the indicated cell types is shown. Fig. 9B Epigenetic analysis of the CAIX locus in the indicated cell types is shown. Fig. 9C Interaction of certain methylation regulators (Ezh2, H3K4me3, H3K27me3) with the CD70 locus is shown. Fig.9D Shown are the effects of tazemetostat, an Ezh2 inhibitor, on ex vivo cultures of sorted heterogeneous renal tumors and treatment with the Ezh2 inhibitor for up to 8 days.

[0036] FIG. 10A to FIG. 10E The effect of CD70 overexpression on tumor clearance is depicted. Fig. 10A Constructs including mCeruleanCD70 and their expression are shown. Fig. 10B Shown is the effect of overexpression of mCerulean CD70 fusion protein on NALM6. Fig. 10C It was shown that overexpression of mCerulean CD70 fusion protein on NALM6 did not impair in vitro killing in 18 h CTLs. Fig. 10D and Fig. 10E It was shown that overexpression of CD70 on K5 and K7 PDX orthotopic models resulted in tumor clearance in vivo.

[0037] FIG. 11A to FIG. 11C The expression levels of CAIX are depicted. Fig.11A Shown is FACS analysis of in vitro CAIX expression. Fig. 11B CD70 / CAIX expression in K5 untreated mice is shown. Fig. 11C CD70 / CAIX expression in K7 untreated mice is shown.

[0038] FIG. 12A to FIG. 12I A dual targeting approach using CD70 and CAIX is depicted. Fig. 12A CAIX28z1XXCAR T killing in vitro is shown. Fig. 12B Dual targeting of CD70 and CAIX was shown to improve tumor control against K5. Fig. 12C Shown is the residual CD70-positive population after CAIX 28z1XX CAR treatment. Fig.12D and Fig.12E It was shown that dual targeting of CD70 and CAIX did not result in tumor clearance of the residual low-density antigen population. Fig.12F Shown is a FACS analysis illustrating the comparison of tumor profiles. FIG. 12G to FIG. 12I Shown are the effects of anti-CD70 CAR, anti-CAIX CAR, and dual-transduced T cells on tumor models.

[0039] FIG. 13A to FIG. 13I Analysis of tumor cells expressing low density CD70 and CAIX antigens is depicted. Fig.13A CD70 / CAIX expression is shown in untreated kidney tumors (sorted 4 quadrants: CD70-CAIX- / CD70+CAIX- / CD70-CAIX+ / CD70+CAIX+) and in untreated tumors at lung sites (sorted CD70+CAIX- population). Fig. 13B Shown is bulk RNA sequencing of CD70 / CAIX sorted tumor populations from kidney and lung sites of K5 RCC PDX. Fig. 13C Shown is bulk RNA sequencing of CD70 / CAIX sorted tumor populations from kidney and lung sites of K7 RCC PDX. Fig.13D Mass spectrometry analysis of CD70 / CAIX sorted tumor populations is shown. Fig.13E Shown is the time course of CD70 / CAIX expression in untreated renal tumors. Fig.13F Quantification of CD70 antigen in untreated renal tumors is shown: #mol / cell (CD70-CAIX- population is negative for CD70 by MFI and quantification). Figure 13G CAIX antigen quantification in untreated renal tumors is shown: #mol / cell. Fig.13H Shown is the quantification of CD70 antigen on day 2 of ex vivo CD70-CAIX-sorted renal tumors, which had very low levels of CD70 expression restored after 48 hours of culture. Fig.13I Shown is the quantification of CAIX antigen on day 2 of ex vivo sorted renal tumors (cultured for 48 hours).

[0040] FIG. 14A to FIG. 14E The impact of the low antigen targeting strategy is depicted. FIG. 14A to FIG. 14D It is shown that 70-HIT expressing a co-stimulatory ligand (80 / 41BBL) eliminates K5 orthotopic renal tumors. Fig.14E It is shown that 70-HIT expressing a co-stimulatory ligand (80 / 41BBL) eliminates K7 orthotopic renal tumors.

[0041] Fig.15 Depicted is the characterization of T cell phenotypes at renal tumor sites against both K5 and K7 at days 7 and 14 following CD70 HIT (expressing CD80 and 4-1BBL polypeptides) or CD7028z1XX CAR T cell treatment. The number of CAR+ or HIT+ T cells (left column), the number of tumor cells (middle column), and the expression profile of the triple inhibitory receptors PD-1, TIM-3, and LAG-3 (right column) are shown.

[0042] FIG. 16A to FIG. 16C It was shown that the in vivo efficacy of T cells expressing CD70HIT, CD80 polypeptide, and 4-1BBL polypeptide could not be explained by bystander killing of CD70-negative tumor cells. Fig.16A Depicted is an in vitro cytotoxicity assay of HIT CD70 against K5 and K7 PDX lines with CD70 knockout. Fig. 16B and Fig. 16C It was shown that CD70 HIT T cells expressing CD80 and 4-1BBL polypeptides were unable to eradicate RCC tumors with CD70 knockout in vivo. In addition, mixing 75% to 80% wild-type RCC PDX tumors with 20% to 25% CD70 knockout RCC PDX tumors did not result in bystander killing of the knockout tumors in vivo.

[0043] FIG. 17A to FIG. 17E Depicted are flow cytometric characterization of a CD70-heterogeneous pancreatic ductal adenocarcinoma (PDAC2) PDX line, the mechanism of CD70 regulation, and the cytotoxic effects of T cells expressing CD70 HIT, CD80 peptide, and 4-1BBL peptide on PDAC2-derived orthotopic pancreatic cancer. Fig.17A Characterization of the PDAC2 (pancreatic ductal adenocarcinoma) PDX line is shown. Fig. 17B Cytotoxic effects on PDAC2 cancer in vitro are shown. Fig. 17C The effects of Ezh2 inhibitors on PDAC2 cells are depicted. Fig.17D Shown are immunoblots of PDAC2 in vitro tumors treated with vehicle, 3 µM Ezh2 inhibitor, or 10 µM Ezh2 inhibitor. Fig.17EShown is the in vivo efficacy of HIT CD70 T cells expressing costimulatory (CD80 / 4-1BBL) on the PDAC2 orthotopic pancreatic PDX model.

[0044] Fig.18A and Fig.18B Describes the role of CD70 as a target for pancreatic cancer + Characterization of the PANC-1 cell model and the in vivo efficacy of HIT CD70 T cells against pancreatic cancer.

[0045] Fig.19A Shown is the expression profile of CD70 in the SK-OV3 ovarian cancer cell line. Fig.19B Shown is tumor implantation of the SK-OV3 cell line. FIG. 19C to FIG. 19E Cytotoxic effects on SK-OV3-derived orthotopic and intraperitoneal ovarian cancers are shown.

[0046] Fig. 20A and Fig. 20B Depicted are immunohistochemistry (IHC) and FISH analysis of CD70 in samples. Fig. 20A Shown are IHC and FISH analyses of CD70 in RCC renal tumor patients and K7 PDX samples. Fig. 20B Shown is FISH analysis of CD70 in PDAC2 cell samples. DETAILED DESCRIPTION

[0047] The subject matter disclosed in this application provides compositions (e.g., modified immune cells) that can be used for immunotherapy (e.g., T cell immunotherapy). The compositions disclosed in this application (e.g., modified immune cells) include: TCR-like fusion molecules targeting CD70. The subject matter disclosed in this application also provides methods for producing such compositions, and 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 subject matter disclosed in this application is based at least in part on the following discovery: 70-HIT T cells can eradicate solid tumors (e.g., renal cell carcinoma, ovarian cancer, pancreatic cancer).

[0048] This specification and examples describe non-limiting embodiments of the subject matter disclosed in this application.

[0049] For clarity of the present disclosure and not by way of limitation, the detailed description is divided into the following subsections:

[0050] 1. Definition;

[0051] 2. Cells;

[0052] 3. Nucleic acid compositions and vectors;

[0053] 4. Formulation and administration;

[0054] 5. Treatment methods;

[0055] 6. Kit; and

[0056] 7. Exemplary embodiments.

[0057] 1. Definition

[0058] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The following references provide those of skill in the art with general definitions of many of the terms used in the subject matter disclosed in this application: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd edition 1994); The Cambridge Dictionary ofScience and Technology (edited by Walker, 1988); The Glossary of Genetics, 5th edition, R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The HarperCollins Dictionary of Biology (1991).

[0059] 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 will depend 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 according to the 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 for 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.

[0060] As used herein, "co-stimulatory molecules" refer to cell surface molecules other than antigen receptors or their ligands that can provide efficient responses of lymphocytes to antigens. In certain embodiments, co-stimulatory molecules can provide optimal lymphocyte activation.

[0061] As used herein, "costimulatory ligand" refers to a molecule that, when bound to its receptor (e.g., a co-stimulatory molecule), produces a co-stimulatory response, e.g., an intracellular response that affects the stimulation provided when an antigen recognition receptor (e.g., a chimeric antigen receptor (CAR)) binds to its target antigen.

[0062] "Immune response cell" refers to a cell or its ancestor or descendant that plays a role in an immune response. In certain embodiments, the immune response cell is a lymphocyte. Non-limiting examples of lymphocytes include T cells, natural killer (NK) cells, B cells, and stem cells from which lymphoid cells can be differentiated. In certain embodiments, the immune response cell is a myeloid cell.

[0063] "Activating immune response cells" means inducing changes in signal transduction or protein expression in cells, leading to the initiation of immune responses. For example, when CD3 chains aggregate in response to ligand binding and immune receptors based on tyrosine-based inhibitory motifs (ITAMs), a signal transduction cascade is generated. In certain embodiments, when endogenous TCR or exogenous CAR binds to an antigen, the formation of an immune synapse occurs, including many molecules that aggregate near the bound receptors (e.g., CD4 or CD8, CD3γ / δ / ε / ζ, etc.). This aggregation of membrane-bound signaling molecules causes the ITAM motif contained in the CD3 chain to be phosphorylated. This phosphorylation then activates the T cell activation pathway, ultimately activating transcription factors such as NF-κB and AP-1. These transcription factors induce the overall gene expression of T cells to increase the production of IL-2, which is used to regulate the proliferation and expression of T cell proteins, thereby initiating T cell-mediated immune responses.

[0064] "Stimulating immune response cells" means a signal that causes a robust and sustained immune response. In various embodiments, this occurs after immune cells (e.g., T cells) are activated or concomitantly mediated by receptors, including but not limited to CD28, CD137 (4-1BB), OX40, CD40, ICOS, DAP-10, CD27, NKG2D, CD2, CD150, CD226. Receiving a variety of stimulating signals may be important for establishing a robust and long-term T cell-mediated immune response. T cells may be quickly suppressed and have no responsiveness to antigens. Although the effects of these costimulatory signals may be different, they generally increase gene expression to produce long-term survival, proliferation, and anti-apoptotic T cells that produce a robust response to antigens to achieve complete and sustained eradication.

[0065] As used herein, the term "antigenic heterogeneity" refers to the differential expression of many antigens (eg, tumor antigens, eg, CD70, CD312), which results in variations in tumor cell phenotype and the distribution of tumor antigen-positive cells.

[0066] As used herein, the term "low antigen density" refers to a target molecule (e.g., antigen) having a cell surface density of less than about 5,000 molecules / cell. In certain embodiments, low antigen density is a cell surface density of less than about 4,000 molecules / cell, less than about 3,000 molecules / cell, less than about 2,000 molecules / cell, less than about 1,500 molecules / cell, less than about 1,000 molecules / cell, less than about 500 molecules / cell, less than about 200 molecules / cell, or less than about 100 molecules / cell. In certain embodiments, low antigen density is a cell surface density of less than about 2,000 molecules / cell. In certain embodiments, low antigen density is a cell surface density of less than about 1,500 molecules / cell. In certain embodiments, low antigen density is a cell surface density of less than about 1,000 molecules / cell. In certain embodiments, low antigen density is a cell surface density of between about 4,000 molecules / cell and about 2,000 molecules / cell, between about 2,000 molecules / cell and about 1,000 molecules / cell, between about 1,500 molecules / cell and about 1,000 molecules / cell, between about 2,000 molecules / cell and about 500 molecules / cell, between about 1,000 molecules / cell and about 200 molecules / cell, or between about 1,000 molecules / cell and about 100 molecules / cell.

[0067] As used herein, the term "low tumor cell frequency" refers to a target cell with a target cell frequency of less than about 50% / tumor. In certain embodiments, the low tumor cell frequency is less than about 40% / tumor, less than about 30% / tumor, less than about 20% / tumor, less than about 15% / tumor, less than about 10% / tumor, less than about 5% / tumor, less than about 2% / tumor or less than about 1% / tumor. In certain embodiments, the low tumor cell frequency is less than about 2% / tumor. In certain embodiments, the low tumor cell frequency is less than about 1.5% / tumor. In certain embodiments, the low tumor cell frequency is less than about 1% / tumor. In certain embodiments, the low tumor cell frequency is between about 40% / tumor and about 20% / tumor, between about 20% / tumor and about 10% / tumor, between about 15% / tumor and about 10% / tumor, between about 20% / tumor and about 5% / tumor, between about 10% / tumor and about 2% / tumor, or between about 10% / tumor and about 1% / tumor.

[0068] As used herein, the term "antigen recognition receptor" refers to a receptor capable of activating an immune or immune response cell (eg, a T cell) in response to its binding to an antigen.

[0069] As used herein, the term "antibody" refers not only to intact antibody molecules, but also to antibody molecule fragments that retain the ability to bind to an immunogen. Such fragments are also well known in the art and are commonly used in vitro and in vivo. Therefore, 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 Fab fragments lacking the Fc fragment of an intact antibody are cleared from the circulation more quickly and may have less nonspecific tissue binding of an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)). As used herein, antibodies include intact natural antibodies, bispecific antibodies; chimeric antibodies; Fab, Fab', single-chain variable fragments (scFv), fusion polypeptides, and unconventional antibodies. In certain embodiments, an "antibody" is a glycoprotein 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 (abbreviated herein as V H ) and heavy chain constant (C H ) region. The heavy chain constant region comprises three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as V L ) and light chain constant C L The light chain constant region consists of one domain, C L . V H and V L The V region can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). H and V L It is composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: 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 can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1 q) of the classical complement system.

[0070] As used herein, "CDR" is defined as the complementarity determining region amino acid sequence of an antibody, which is the hypervariable region of the immunoglobulin heavy and light chains. See, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 4 thUS Department of Health and Human Services, National Institutes of Health (1987). Typically, an antibody includes three heavy chain and three light chain CDRs or CDR regions in the variable region. CDRs provide most of the contact residues for the binding of an antibody to an antigen or epitope. In certain embodiments, the Kabat system is used to describe the CDR region (Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242). In certain embodiments, the PyIgClassify system is used to describe the CDR region (Adolf-Bryfogle et al., Nucleic acids research 43.D1 (2015): D432-D438).

[0071] As used herein, the term "linker" shall mean a functional group (e.g., a chemical substance or a polypeptide) that covalently attaches two or more polypeptides or nucleic acids to connect them to each other. As used herein, a "peptide linker" refers to a functional group (e.g., a chemical substance or a polypeptide) that is used to covalently attach two or more polypeptides or nucleic acids to connect them to each other. 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, which is provided below:

[0072] GGGGSGGGGSGGGGS [SEQ ID NO: 1]

[0073] In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 2, which is provided below:

[0074] GGGGSGGGGSGGGSGGGGS [SEQ ID NO: 2]

[0075] In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 3, which is provided below:

[0076] GGGGSGGGGSGGGGSGGGSGGGGS [SEQ ID NO: 3]

[0077] In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 4, which is provided below:

[0078] GGGGSGGGGSGGGGSGGGGSGGGSGGGGS [SEQ ID NO: 4]

[0079] In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 5, which is provided below:

[0080] GGGGS [SEQ ID NO: 5]

[0081] In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 6, which is provided below:

[0082] GGGGSGGGGS [SEQ ID NO: 6]

[0083] As used herein, the term "single-chain variable fragment" or "scFv" is a fragment of a polypeptide that is covalently linked to form a V H ::V L Heterodimer of immunoglobulin heavy chain (V H ) and light chain (V L ) fusion protein of the variable region. H and V L Directly joined or joined through a peptide-encoded linker (e.g., 10, 15, 20, 25 amino acids) that connects V H The N-terminus of L or connect the C-terminus of V H The C-terminus of L The linker is usually rich in glycine for flexibility and 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. The single-chain Fv polypeptide antibody can be composed of a V H and V LNucleic acid expression of coding sequences is described in Huston et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). See also U.S. Pat. Nos. 5,091,513, 5,132,405, and 4,956,778; and U.S. Pat. Nos. 20050196754 and 20050196754. Antagonistic scFvs with inhibitory activity have been described (see, e.g., Zhao et al., Hyrbidoma (Larchmt) 2008 27(6):455-51; Peter et al., J Cachexia Sarcopenia Muscle 2012 Aug 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). Agonistic 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).

[0084] As used herein, the term "affinity" means a measure of binding strength. Affinity may depend on the proximity of the stereochemical match between the antibody combining 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 the antigen-antibody bond after the formation of a reversible complex. Methods for calculating the affinity of an antibody for an antigen are known in the art, including but not limited to various antigen binding experiments, e.g., functional assays (e.g., flow cytometry assays).

[0085] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a molecule comprising an extracellular antigen binding domain and a transmembrane domain, which is fused to an intracellular signaling domain capable of activating or stimulating immune or immune response cells. In certain embodiments, the extracellular antigen binding domain of CAR comprises scFv. ScFv can be derived from fusing the variable heavy chain and light chain regions of an antibody. Alternatively or in addition, scFv can be derived from Fab' (rather than from an antibody, e.g., obtained from a Fab library). In certain embodiments, scFv is fused to a transmembrane domain and then to an intracellular signaling domain. In certain embodiments, CAR is selected to have a high binding affinity or avidity for an antigen.

[0086] As used herein, the term "substantially identical" or "substantially homologous" refers 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 sequences are 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 sequence or nucleic acid sequence used for comparison.

[0087] Sequence identity can be measured by using sequence analysis software (e.g., BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs, sequence analysis packages, Genetic Computer Group, 1710 University Avenue, University of Wisconsin, Madison, WI 53705). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions generally 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 method for determining the degree of identity, the BLAST program can be used, with a probability score between e-3 and e-100, indicating closely related sequences.

[0088] 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)), which has been 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. In addition, the percent homology between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossom 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. Additionally or alternatively, the amino acid sequences of the presently disclosed subject matter can be further used as a "query sequence" to search public databases to, for example, 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. BLAST protein searches can be performed using the XBLAST program with score = 50 and word length = 3 to obtain amino acid sequences homologous to the specific sequences disclosed herein (e.g., heavy and light chain variable region sequences). In order to obtain gapped alignments for comparison purposes, gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST programs and gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0089] As used herein, the term "conservative sequence modification" refers to an amino acid modification that does not significantly affect or change the binding properties of an antigen recognition receptor (e.g., an extracellular antigen binding domain of a CAR) comprising an amino acid sequence disclosed herein. Conservative modifications may include amino acid substitutions, additions, and deletions. Modifications may be introduced into the extracellular antigen binding domain of the currently disclosed CAR by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Amino acids may be classified into groups according to their physicochemical properties, such as charge and polarity. Conservative amino acid substitutions are substitutions in which amino acid residues are replaced by amino acids in the same group. For example, amino acids may be classified by charge: positively charged amino acids include lysine, arginine, and histidine; negatively charged amino acids include aspartic acid and glutamic acid; neutrally charged amino acids include alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In addition, amino acids can be classified by polarity: polar amino acids include arginine (basic polarity), asparagine, aspartic acid (acidic polarity), glutamic acid (acidic polarity), glutamine, histidine (basic polarity), lysine (basic polarity), serine, threonine and tyrosine; non-polar amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan and valine. Therefore, one or more amino acid residues in the CDR region can be replaced by other amino acid residues from the same group, and the retained function of the altered antibody (i.e., the function described in (c) to (l) above) can be tested 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, no more than five residues are changed in a specific sequence or CDR region.

[0090] "Disease" means any condition, illness or disorder that damages or interferes with the normal function of cells, tissues or organs (eg, neoplasms and infection with cellular pathogens).

[0091] "Effective amount" refers to an amount sufficient to have a therapeutic effect. In certain embodiments, an "effective amount" is an amount sufficient to prevent, reduce or inhibit the continued proliferation, growth or metastasis (eg, invasion or migration) of a neoplasm.

[0092] "Endogenous" refers to a nucleic acid molecule or polypeptide that is normally expressed in a cell or tissue.

[0093] "Exogenous" means a nucleic acid molecule or polypeptide that is not endogenously present in a cell. Thus, the term "exogenous" encompasses any recombinant nucleic acid molecule or polypeptide expressed in a cell, such as foreign, heterologous, and overexpressed nucleic acid molecules and polypeptides. "Exogenous" nucleic acid means a nucleic acid that is not present in a natural wild-type cell; for example, an exogenous nucleic acid may differ from an endogenous counterpart in sequence, in position / location, or in both. For clarity, an exogenous nucleic acid may have the same or different sequence relative to its natural endogenous counterpart; it may be introduced into the cell itself or its progenitors by genetic engineering, and may optionally be linked to an alternative control sequence, such as a non-natural promoter or secretory sequence.

[0094] "Increase" means a positive change of at least about 5%. The change can be about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, about 100% or more.

[0095] "Decrease" means a negative change of at least about 5%. The change can be about 5%, about 10%, about 25%, about 30%, about 50%, about 75% or even about 100%.

[0096] The terms "isolated", "purified" or "biologically pure" refer to materials that are free of normally accompanying components found in their natural state to varying degrees. "Isolated" means a degree of separation from the original source or surrounding environment. "Purification" means a higher degree of separation than separation. A "purified" or "biologically pure" protein is sufficiently free of other materials so that any impurities do not substantially affect the biological properties of the protein or cause other adverse consequences. That is, if the nucleic acid or peptide is substantially free of cellular material, viral material or culture medium when produced by recombinant DNA technology, or substantially free of chemical precursors or other chemicals when chemically synthesized, the nucleic acid or peptide is purified. Purity and uniformity are generally determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can mean that the nucleic acid or protein produces essentially one band in an electrophoretic gel. For proteins that can be modified (e.g., phosphorylated or glycosylated), different modifications can produce different isolated proteins, and the proteins can be purified separately.

[0097] An "isolated cell" refers to a cell that is separated from molecules and / or cellular components that naturally accompany the cell.

[0098] As used herein, the term "antigen binding domain" refers to a domain that is capable of specifically binding to a particular antigenic determinant or group of antigenic determinants present on a cell.

[0099] "Vegetation" or "malignant tumor" means a disease characterized by pathological proliferation of cells or tissues and subsequent migration to or invasion of other tissues or organs. Vegetation growth is usually uncontrolled and progressive, and occurs under conditions that will not cause normal cell reproduction or will cause normal cell reproduction to stop. Vegetation can affect a variety of cell types, tissues or organs, including but not limited to organs or their tissues or cell types selected from the following items: bladder, bone, brain, chest, cartilage, glial, esophagus, fallopian tube, gallbladder, heart, intestine, kidney, liver, lung, lymph node, nervous tissue, ovary, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testicle, thymus, thyroid, trachea, urogenital tract, ureter, urethra, uterus and vagina. Vegetation includes cancer, such as sarcoma, carcinoma or plasmacytoma (malignant tumor of plasma cells). In certain embodiments, the vegetation is cancer.

[0100] "Specific binding" means a polypeptide or fragment thereof that recognizes and binds to a biological molecule (eg, polypeptide) of interest but does not substantially recognize and bind to other molecules in a sample (eg, a biological sample that naturally contains the polypeptide disclosed herein).

[0101] As used herein, the term "tumor antigen" refers to an antigen (e.g., polypeptide) that is uniquely or differentially expressed on tumor cells compared to normal or non-neoplastic cells. In certain embodiments, tumor antigens include any polypeptide expressed by a tumor that can activate or induce an immune response via an antigen recognition receptor or can inhibit an immune response via receptor-ligand binding.

[0102] The terms "comprises," "comprising," are intended to have the broad meaning ascribed to them in U.S. patent law, and may mean "includes," "including," and the like.

[0103] As used herein, "treatment" refers to clinical intervention that attempts to alter the course of disease in the individual or cell being treated, and can be performed for prevention or during clinical pathology. The efficacy of treatment includes, but is not limited to, preventing the occurrence or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis. By preventing the progression of a disease or condition, treatment can prevent the affected or diagnosed subject or a subject suspected of having a condition from worsening due to the condition, and treatment can also prevent the occurrence of a condition or symptoms of a condition in a subject at risk for or suspected of having a condition.

[0104] An "individual" or "subject" herein is a vertebrate, such as a human or a non-human animal, such as a mammal. Mammals include, but are not limited to, humans, primates, farm animals, sports animals, rodents, and pets. Non-limiting examples of non-human animal subjects include: rodents, such as mice, rats, hamsters, and guinea pigs; rabbits; dogs; cats; sheep; pigs; goats; cattle; horses; and non-human primates, such as apes and monkeys. As used herein, the term "immunocompromised" refers to a subject who suffers from an immune deficiency. A subject is highly susceptible to opportunistic infections, which are infections caused by organisms that do not normally cause disease in people with healthy immune systems but can affect people with a weakened or suppressed immune system.

[0105] As used herein, a "functional fragment" of a molecule or polypeptide includes a molecule or polypeptide fragment 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.

[0106] Other aspects of the presently disclosed subject matter are described in the following disclosure and are within the scope of the presently disclosed subject matter.

[0107] 2. Cells

[0108] The subject matter disclosed in the present application provides cells comprising an antigen recognition receptor targeting CD70. In certain embodiments, the antigen recognition receptor is a TCR-like fusion molecule.

[0109] In certain embodiments, the cell is selected from the group consisting of lymphoid cells and myeloid cells. In certain embodiments, the cell is an immune response cell. In certain embodiments, the immune response cell is a lymphoid cell.

[0110] In certain embodiments, the cell is a cell of the lymphoid lineage. The cells of the lymphoid lineage can produce antibodies, regulate the cellular immune system, detect foreign agents in the blood, detect foreign cells in the host, etc. Non-limiting examples of cells of the lymphoid lineage 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 cell is a pluripotent stem cell (e.g., embryonic stem cell).

[0111] In certain embodiments, the cell is a T cell. T cells can be lymphocytes that mature in the thymus and are primarily responsible for cell-mediated immunity. T cells are involved in the adaptive immune system. The T cells of the subject matter disclosed in the present application can be any type of T cell, including but not limited to helper T cells, cytotoxic T cells, memory T cells (including central memory T cells), stem cell-like memory T cells (or stem cell-like memory T cells), and two types of effector memory T cells: for example, T EM Cells and T EMRAT cells, regulatory T cells (also known as suppressor T cells), tumor infiltrating lymphocytes (TILs), natural killer T cells, mucosa-associated invariant T cells, and gamma delta T cells. Cytotoxic T cells (CTLs or killer T cells) are a subset of T lymphocytes that are able to induce the death of infected somatic or tumor cells. A patient's own T cells can be genetically modified to target specific antigens by introducing antigen recognition receptors (e.g., 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 + T cells are independent of CD4. In certain embodiments, T cells are derived from induced pluripotent stem cells (iPSC). In certain embodiments, T cells are independent of CD4 CD8 + T cells, and CD8 + T cells are derived from iPSCs.

[0112] In certain embodiments, the cell is a NK cell. Natural killer (NK) cells can be lymphocytes that are part of cell-mediated immunity and function during the innate immune response. NK cells can produce cytotoxic effects on target cells without prior activation.

[0113] The types of human lymphocytes of the subject matter disclosed in the present application include, but are not limited to, peripheral donor lymphocytes, such as those disclosed in 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 recognition T cell receptor complex comprising 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 314:126-129 (disclosing peripheral donor lymphocytes genetically modified to express a full-length tumor antigen recognition T cell receptor complex comprising 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 the selective in vitro expansion of antigen-specific peripheral blood leukocytes using artificial antigen presenting cells (AAPCs) or pulsed dendritic cells).

[0114] In certain embodiments, the cell (e.g., T cell) is autologous. In certain embodiments, the cell (e.g., T cell) is non-autologous. In certain embodiments, the cell (e.g., T cell) is allogeneic. In certain embodiments, the cell (e.g., T cell) is derived in vitro from an engineered progenitor cell or stem cell.

[0115] In certain embodiments, the cell is a cell 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.

[0116] In certain embodiments, the stem cells are pluripotent stem cells (eg, embryonic stem cells or induced pluripotent stem cells).

[0117] 2.1. Antigen recognition receptors

[0118] The antigen recognition receptor (e.g., the first antigen recognition receptor) targets an antigen (e.g., the first antigen). The antigen (e.g., the first antigen) can be a tumor antigen or a pathogen antigen. In certain embodiments, the antigen recognition receptor (e.g., the first antigen recognition receptor) is a chimeric receptor. In certain embodiments, the chimeric receptor is a TCR-like fusion molecule.

[0119] 2.1.1. Antigens

[0120] In certain embodiments, the antigen is a tumor antigen. In certain embodiments, the tumor antigen is an antigen with a low antigen density. In certain embodiments, the tumor antigen is expressed on cells with a low tumor cell frequency.

[0121] Any tumor antigen (antigenic peptide) can be used in the tumor-related embodiments described herein. The source of the antigen includes, but is not limited to, oncoproteins. The antigen (e.g., a first antigen) can be expressed as a peptide or as a complete protein or a portion thereof. The complete protein or a portion thereof can be natural 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, CADM3, CAIX, CAPN3, CCDC155, CCR1, CD10, CD117, CD123, CD133, CD135 (FLT3), CD138, CD20, CD22, CD244.(2B4), CD25, CD26, CD276, CD30, CD300LF, CD312, CD32, CD321, CD33, CD34, CD36, CD38, CD41, CD44, CD44V6, C D47, CD49f, CD56, CD7, CD71, CD74, CD8, CD82, CD96, CD98, CD99, CDH13, CDHR1, CEA, CEACAM6, CHST3, CLEC12A, CLEC1A, CLL1, CNIH2, COL15A1, COLEC12, 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-B 4. 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, ICAM 1. 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, NKG2D ligand, NLGN3, NPAS2, NY-ESO-1, carcinoembryonic 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, SCIN, SCN11A, SCN2A, SCNN1D, SEC31B, SEMA4A, SH3PXD2A, SIGLEC11, SIRPB1, SLC16A6, SLC19A1, SLC22A5, SLC25A3 6. SLC25A41, SLC30A1, SLC34A3, SLC43A3, SLC44A1, SLC44A3, SLC45A3, SLC6A16, SLC6A6, SLC8A3, S LC9A1, SLCO2B1, SPAG17, STC1, STON2, SUN3, Survivin, 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. In certain embodiments, the antigen is CD70.

[0122] In certain embodiments, the antigen (eg, a first antigen) is a pathogen antigen. Non-limiting examples of viruses include: Retroviridae (e.g., human immunodeficiency viruses, such as HIV-1 (also known as HDTV-III, LAVE or HTLV-III / LAV, or HIV-III); and other isolates, such as 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); Flaviridae (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); Viruses); Paramyxoviridae (e.g., parainfluenza, mumps, measles, respiratory syncytial virus); Orthomyxoviridae (e.g., influenza); Bungaviridae (e.g., hantavirus, bunga, phlebovirus, and Naira virus); Arenaviridae (hemorrhagic fever viruses); Reoviridae (e.g., reoviruses, orbiviruses, 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, cytomegalovirus (CMV), herpesviruses); Poxviridae (smallpox virus, vaccinia virus, poxviruses); and Iridoviridae (e.g., African swine fever virus); as well as unclassified viruses (e.g., hepatitis D virus (thought to be a defective satellite of hepatitis B virus), non-A, non-B hepatitis viruses (category 1 = transmitted internally; category 2 = transmitted parenterally (i.e., hepatitis C)); Norwalk and related viruses, and astroviruses).

[0123] Non-limiting examples of bacteria include: Pasteurella, Staphylococci, Streptococcus, Escherichia coli, Pseudomonas species, and Salmonella species. Specific examples of infectious bacteria include, but are not limited to, Helicobacter pyloris, Borrelia burgdorferi, Legionella, Legionella pneumophilia, Mycobacteria sps (e.g., M. tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae, M. leprae), Staphylococcus aureus, Staphylococcus epidermidis, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes, pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcus pneumoniae, pathogenic Campylobacter sp., Campylobacter jejuni, Enterococcus sp.), Haemophilus influenzae, Bacillus antracis, corynebacterium diphtheriae, corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridium spp., Clostridium perfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Leptospira, Rickettsia and Actinomyces israelli, 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.

[0124] 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.

[0125] 2.1.2. TCR-like fusion molecules

[0126] In certain embodiments, the antigen recognition receptor is a TCR-like fusion molecule. Non-limiting examples of TCR fusion molecules include HLA-independent TCR-based chimeric antigen receptors (also referred to as "HITs," e.g., those disclosed in International Patent Application No. PCT / US19 / 017525, which are incorporated by reference in their entirety) and T cell receptor fusion constructs (TRuC) (e.g., Baeuerle et al., "Synthetic TRuC receptors engaging thecomplete T cell receptor for potent anti-tumor response," Nature Communications Vol. 10, Article No.: 2087 (2019), which are incorporated by reference in their entirety).

[0127] 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 its fragment, 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 antigen binding chain and the second antigen binding chain 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 a manner independent of HLA. Therefore, in certain embodiments, the recombinant TCR is an HLA-independent (or non-HLA-binding) TCR (referred to as "HIT").

[0128] 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 a light chain variable region (V L ). In certain embodiments, the first antigen binding chain comprises a V H The antigen-binding fragment of the present invention comprises a V L antigen-binding fragment.

[0129] In certain embodiments, the constant domain comprises a TCR constant region selected from the group consisting of a natural or modified TRAC polypeptide, a natural or modified TRBC polypeptide, a natural or modified TRDC polypeptide, a natural or modified TRGC polypeptide, and any variants or functional fragments thereof. In certain embodiments, the constant domain comprises a natural or modified TRAC polypeptide. In certain embodiments, the constant domain comprises a natural 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.

[0130] In certain embodiments, the first antigen-binding chain comprises the V H and a TRAC polypeptide, and the second antigen binding chain comprises an antibody V L and TRBC polypeptide.

[0131] In certain embodiments, the first antigen-binding chain comprises the V H and a TRBC polypeptide, and the second antigen binding chain comprises the V L and TRAC peptides.

[0132] 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.

[0133] In certain embodiments, the antigen binding chain can associate with the CD3 zeta polypeptide. In certain embodiments, the antigen binding chain can activate the CD3 zeta polypeptide associated with the antigen binding chain after binding with the antigen. In certain embodiments, the activation of the CD3 zeta polypeptide can activate immune response cells. In certain embodiments, the TCR-like fusion molecule can integrate with the CD3 complex and provide antigen recognition independent of HLA. In certain embodiments, the TCR-like fusion molecule replaces the endogenous TCR in the CD3 / TCR complex.

[0134] In certain embodiments, the first antigen-binding chain and the second antigen-binding chain are approximately 2 × 10 -7 M or less dissociation constant (K D ) binds to the antigen. In certain embodiments, the first antigen binding chain and the second antigen binding chain bind to the antigen with high binding affinity. In certain embodiments, K D About 2 × 10 -7 M or smaller, approximately 1 × 10 -7 M or smaller, about 9× 10 -8 M or smaller, approximately 1 × 10 -8 M or smaller, about 9 × 10 -9 M or smaller, about 5 × 10 -9 M or smaller, about 4× 10 -9 M or smaller, about 3 × 10 -9 or smaller, about 2 × 10 -9 M or less or about 1 × 10 -9 M or less. In certain embodiments, K D About 1 × 10 -8 M or less. In certain embodiments, K D About 3 × 10 -9 M or less. In certain embodiments, K D About 5 × 10 -9 M or less. In certain embodiments, K D About 1 × 10 -9 M to about 1 × 10 -8 M. In certain embodiments, K D About 1.5 × 10 -9 M to about 1 × 10 -8 M. In certain embodiments, K D About 5 × 10 -9 M to about 1 × 10 -8 M.

[0135] 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.

[0136] 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 shown in SEQ ID NO: 7 or a fragment thereof, and / or may optionally comprise at most one, or at most two, or at most three conservative amino acid substitutions. In certain embodiments, the TRAC polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 7. SEQ ID NO: 7 is provided below.

[0137] IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS [SEQID NO: 7]

[0138] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 7 is shown in SEQ ID NO: 8 provided below.

[0139] ATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTT GCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC [SEQ ID NO: 8]

[0140] 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 shown in SEQ ID NO: 9, 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 TRAC polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 9. SEQ ID NO: 9 is provided below.

[0141] IPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS[SEQ ID NO: 9]

[0142] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 9 is shown in SEQ ID NO: 10 provided below.

[0143] ATTCCCAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTG ACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC [SEQ ID NO: 10]

[0144] In certain embodiments, the 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 a gene of NCBI Genbank ID: 28755, NG_001332.3, range 925603 to 930229 (SEQ ID NO: 11), or a fragment thereof, and / or may optionally comprise at most one, or at most two, or at most three conservative amino acid substitutions. In certain embodiments, the TRAC polypeptide comprises or consists of an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 11. SEQ ID NO: 11 is provided below.

[0145]

[0146] In certain embodiments, the first antigen-binding chain or the second antigen-binding chain comprises a constant domain, and the constant domain comprises 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 shown in SEQ ID NO: 12 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 TRBC2 polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 12. SEQ ID NO: 12 is provided below.

[0147] DLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG [SEQ ID NO: 12]

[0148] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 12 is shown in SEQ ID NO: 13 provided below.

[0149] GATCTGAAAACGTGTTCCCTCCTGAAGTGGCTGTCTTTGAACCATCCGAGGCCGAGATTTCCCATACCCAGAAAGCAACTCTGGTCTGTCTGGCCACTGGATTCTACCCCGATCACGTGGAACTGTCTTGGT GGGTGAACGGCAAGGAAGTCCATTCCGGAGTCTCTACCGACCCTCAGCCCCTCAAGGAGCAGCCTGCTCTCAACGATTCTCGGTACTGCCTGTCATCTCGACTGAGAGTGTCTGCCACCTTCTGGCAGAACCCT AGAAAACCACTTTCGGTGTCAGGTCCAGTTTTACGGCCTGAGCGAGAACGATGAGTGGACACAGGATAGAGCCAAACCTGTGACACAGATTGTGAGCGCCGAGGCTTGGGGACGAGCCGATTGTGGCTTCACAT CCGAGTCTTACCAGCAGGGAGTGCTGTCTGCTACAATCCTCTACGAAATTCTCCTGGGGAAGGCCACCCTGTACGCTGTCCTCGTGTCTGCTCTGGTGCTCATGGCTATGGTCAAACGAAAGGACTCTAGAGGC [SEQ ID NO: 13]

[0150] 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 shown in SEQ ID NO: 14 or a fragment thereof, and / or may optionally comprise at most one, or at most two, or at most three conservative amino acid substitutions. In certain embodiments, the TRBC2 polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 14. SEQ ID NO: 14 is provided below.

[0151] LEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG [SEQ ID NO: 14]

[0152] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 14 is shown in SEQ ID NO: 15 provided below.

[0153] CTGGAGGATCTGAAAAACGTGTTCCCTCCTGAAGTGGCTGTCTTTGAACCATCCGAGGCCGAGATTTCCCATACCCAGAAAGCAACTCTGGTCTGTCTGGCCACTGGATTCTACCCCGATCACGTGGAACTGTCTTGGTGGGTGAACGGCAAGGAAGTCCATTCCGGAGTCTCTACCGACCCTCAGCCCCTCAAGGAGCAGCCTGCTCTCAACGATTCTCGGTACTGCCTGTCATCTCGACTGAGAGTGTCTGCCACCTTCTGGCAGAACCCTAGAAACCACTTTCGGTGTCAGGTCCAGTTTTACGGCCTGAGCGAGAACGATGAGTGGACACAGGATAGAGCCAAACCTGTGACACAGATTGTGAGCGCCGAGGCTTGGGGACGAGCCGATTGTGGCTTCACATCCGAGTCTTACCAGCAGGGAGTGCTGTCTGCTACAATCCTCTACGAAATTCTCCTGGGGAAGGCCACCCTGTACGCTGTCCTCGTGTCTGCTCTGGTGCTCATGGCTATGGTCAAACGAAAGGACTCTAGAGGC [SEQ ID NO: 15]

[0154] 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 shown in SEQ ID NO: 16 or a fragment thereof, and / or may optionally comprise at most one, or at most two, or at most three conservative amino acid substitutions. In certain embodiments, the TRBC1 polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 16. SEQ ID NO: 16 is provided below.

[0155] LNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF [SEQ ID NO: 16]

[0156] 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 shown in SEQ ID NO: 17, or a fragment thereof, and / or may optionally comprise at most one, or at most two, or at most three conservative amino acid substitutions. In certain embodiments, the TRBC1 polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 17. SEQ ID NO: 17 is provided below.

[0157] DLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF [SEQ ID NO: 17]

[0158] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 17 is shown in SEQ ID NO: 18 provided below.

[0159] GACCTGAACAAGGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTTCCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCCGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTTACCTCGGTGTCCTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAGGCCACCCTGTATGCTGTGCTGGTCAGCGCCCTTGTGTTGATGGCCATGGTCAAGAGAAAGGATTTC [SEQ ID NO: 18]

[0160] 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 an amino acid sequence encoded by a transcript expressed by a gene of NCBI Genbank ID: 28639, NG_001333.2, range 645749 to 647196 (TRBC1, SEQ ID NO: 19), NCBI Genbank ID: 28638, NG_001333.2, range 655095 to 656583 (TRBC2, SEQ ID NO: 20), or a fragment thereof, and / or may optionally comprise at most one, or at most two, or at most three conservative amino acid substitutions. In certain embodiments, the TRBC polypeptide comprises or consists of an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 19. In certain embodiments, the TRBC polypeptide comprises or consists of an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 20. SEQ ID NOs: 19 and 20 are provided below.

[0161]

[0162]

[0163] In certain embodiments, the first antigen binding chain or the second antigen binding chain comprises a constant domain comprising a natural or modified TRGC polypeptide. In certain embodiments, the TRGC polypeptide is a natural or modified TRGC1 polypeptide. In certain embodiments, the TRGC1 polypeptide comprises or consists of the following items: an amino acid sequence 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 shown in SEQ ID NO: 21 provided below. In certain embodiments, the TRGC1 polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 21.

[0164] DKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNTILGSQEGNTMKTNDTYMKFSWLTVPEKSLDKEHRCIVRHENNKNGVDQEIIFPPIKTDVITMDPKDNCSKDANDTLLLQLTNTSAYYMYLLLLLKSVVYFAIITCCLLRRTAFCCNGEKS [SEQ ID NO: 21]

[0165] In certain embodiments, the TRGC polypeptide is a natural 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 shown in SEQ ID NO: 22 provided below. In certain embodiments, the TRGC2 polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 22.

[0166] DKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDIIKIHWQEKKSNTILGSQEGNTMKTNDTYMKFSWLTVPESLDKEHRCIVRHENNKNGIDQEIIFPPIKTDVTTVDPKYNYSKDANDVITMDPKDNWSKDANDTLLLQLTNTSAYYTYLLLLLKSVVYFAIITCCLLRRTAFCCNGEKS [SEQ ID NO: 22]

[0167] In certain embodiments, the TRGC 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 an amino acid sequence encoded by a transcript expressed by a gene of NCBI Genbank ID: 6966, NG_001336.2, range 108270 to 113860 (TRGC1, SEQ ID NO: 23), NCBI Genbank ID: 6967, NG_001336.2, range 124376 to 133924 (TRGC2, SEQ ID NO: 24), or a fragment thereof, and / or may optionally comprise at most one, or at most two, or at most 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 an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 24. SEQ ID NOs: 23 and 24 are provided below.

[0168]

[0169]

[0170] [SEQ ID NO: 24]

[0171] 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 shown in SEQ ID NO: 25 provided below. In certain embodiments, the TRDC polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 25.

[0172] SQPHTKPSVFVMKNGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAVKLGKYEDSNSVTCSVQHDNKTVHSTDDFEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTEKVNMMSLTVLGLRMLFAKTVAVNFLLTAKLFFL [SEQ ID NO: 25]

[0173] In certain embodiments, the TCR-like fusion molecule comprises a hinge / spacer region connecting the first antigen binding chain to the constant domain. In certain embodiments, the TCR-like fusion molecule comprises a hinge / spacer region connecting the second antigen binding chain to the constant domain. The hinge / spacer region can be sufficiently flexible to allow the antigen binding chain to be oriented in different directions, thereby facilitating antigen recognition. In certain embodiments, the hinge / spacer region can be a hinge region from IgG1, a CH of an immunoglobulin, or a cytosine. 2 CH 3In certain embodiments, the hinge / spacer comprises a portion of a TCR alpha polypeptide, a portion of a TCR alpha polypeptide, a portion of a TCR β polypeptide, a portion of a CD28 polypeptide, a portion of a CD8 polypeptide, or a synthetic spacer sequence. In certain embodiments, the hinge / spacer comprises a portion of a TCR alpha polypeptide. In certain embodiments, the hinge / spacer comprises a portion of a variable region (TRAV), a portion of a diversity region (TRAD), a portion of a junction region (TRAJ), a portion of a constant region (TRAC), or a combination thereof. In certain embodiments, the hinge / spacer comprises a portion of a TRAJ region and a portion of a TRAC region of a TCR alpha polypeptide. In certain embodiments, the hinge / spacer comprises or consists of the amino acid sequence shown in SEQ ID NO: 26. In certain embodiments, the hinge / spacer comprises or consists of the following: amino acids 1 to 3 in the sequence shown in SEQ ID NO: 26. An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 26 is shown in SEQ ID NO: 27. SEQ ID NOs: 26 and 27 are provided below.

[0174] IPNIQNPDPA [SEQ ID NO: 26]

[0175] ATTCCCAATATCCAGAACCCTGACCCTGCC [SEQ ID NO: 27]

[0176] In certain embodiments, the hinge / spacer comprises a portion of a TCRβ polypeptide. In certain embodiments, the hinge / spacer comprises a portion of a variable region (TRBV), a portion of a diversity region (TRBD), a portion of a junction region (TRBJ), a portion of a constant region (TRBC), or a combination thereof. In certain embodiments, the hinge / spacer comprises a portion of a TRBJ region and a portion of a TRAC region (C) of a TCRβ polypeptide. In certain embodiments, the hinge / spacer comprises or consists of the amino acid sequence shown in SEQ ID NO: 28. In certain embodiments, the hinge / spacer comprises or consists of the following: amino acids 1 to 2 in the sequence shown in SEQ ID NO: 28. An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 28 is shown in SEQ ID NO: 29. SEQ ID NOs: 28 and 29 are provided below.

[0177] LEDLKNVFPPE [SEQ ID NO: 28]

[0178] CTGGAGGATCTGAAAAACGTGTTCCCTCCTGAA [SEQ ID NO: 29]

[0179] In certain embodiments, the antigen binding chain does not comprise an intracellular domain. In certain embodiments, the antigen binding chain is capable of associating with a CD3 zeta polypeptide. In certain embodiments, the antigen binding chain is associated with a CD3 zeta polypeptide via a constant domain. In certain embodiments, the CD3 zeta polypeptide is endogenous. In certain embodiments, the CD3 zeta polypeptide is exogenous. In certain embodiments, the binding of the antigen binding chain to the target antigen is capable of activating the CD3 zeta polypeptide associated with the antigen binding chain. In certain embodiments, the exogenous CD3 zeta polypeptide is fused or integrated with a co-stimulatory molecule disclosed herein.

[0180] 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 CD3 zeta polypeptide. In certain embodiments, the binding of the antigen binding chain to the antigen can activate the CD3 zeta polypeptide of the antigen binding chain.

[0181] In certain embodiments, the CD3 zeta 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 to the amino acid sequence shown in 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 CD3 zeta polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 53 of 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, CD3 zeta comprises or consists of an amino acid sequence of amino acids 1 to 164, 1 to 50, 50 to 100, 52 to 164, 100 to 150, or 150 to 164 of SEQ ID NO: 53. In certain embodiments, the CD3 zeta polypeptide comprises or consists of amino acids 52 to 164 of SEQ ID NO: 53.

[0182] In certain embodiments, the CD3 zeta 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 comprise at most one, or at most two, or at most three conservative amino acid substitutions. In certain embodiments, the CD3 zeta polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 54.

[0183] 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 CD3 zeta polypeptide. In certain embodiments, the intracellular domain comprises a costimulatory signaling region and does not comprise a CD3 zeta polypeptide. In certain embodiments, the costimulatory signaling region comprises at least the intracellular domain of a costimulatory molecule disclosed herein.

[0184] In certain embodiments, the TCR-like fusion molecule is capable of associating with a CD3 complex (also referred to as a "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 the natural and / or endogenous TCR in the CD3 / TCR complex. In certain embodiments, the CD3 complex comprises a CD3γ chain, a CD3δ chain, and two CD3ε chains.

[0185] 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 an amino acid sequence having NCBI Reference Number: NP_000064.1 (SEQ ID NO: 30), or a fragment thereof, and / or may optionally comprise at most one, or at most two, or at most three conservative amino acid substitutions. SEQ ID NO: 30 is provided below.

[0186] MEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRN [SEQ ID NO: 30]

[0187] 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 an amino acid sequence having NCBI Reference Number: NP_000723.1 (SEQ ID NO: 31) or a fragment thereof, or an amino acid sequence having NCBI Reference Number: NP_001035741.1 (SEQ ID NO: 32) or a fragment thereof, and / or may optionally comprise at most one, or at most two or at most three conservative amino acid substitutions. SEQ ID NOs: 31 and 32 are provided below.

[0188] MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALGVFCFAGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNK [SEQ ID NO: 31]

[0189] MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRTADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNK [SEQ ID NO: 32]

[0190] 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 an amino acid sequence having NCBI Reference Number: NP_000724.1 (SEQ ID NO: 33) or a fragment thereof, and / or may optionally comprise at most one, or at most two, or at most three conservative amino acid substitutions. SEQ ID NO: 33 is provided below.

[0191] MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKP EDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI[SEQ ID NO:33]

[0192] 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 combined with an antigen having a low antigen density on the surface of a tumor cell. In certain embodiments, cells comprising TCR-like fusion molecules can be used to treat subjects with tumor cells (e.g., recurrence of a disease) with low expression levels of surface antigens, wherein the subject has received treatment that causes residual tumor cells. In certain embodiments, tumor cells have a low antigen density of target molecules on the surface of 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 / cell, less than about 4,000 molecules / cell, less than about 3,000 molecules / cell, less than about 2,000 molecules / cell, less than about 1,500 molecules / cell, less than about 1,000 molecules / cell, less than about 500 molecules / cell, less than about 200 molecules / cell, or less than about 100 molecules / cell. In certain embodiments, the target molecule with low antigen density on the cell surface has a density of less than about 2,000 molecules / cell. In certain embodiments, the target molecule with low antigen density on the cell surface has a density of less than about 1,500 molecules / cell. In certain embodiments, the target molecule with low antigen density on the cell surface has a density of less than about 1,000 molecules / cell. In certain embodiments, the target molecule with low antigen density on the cell surface has a density of less than about 4,000 molecules / cell and about 2,000 molecules / cell, about 2,000 molecules / cell and about 1,000 molecules / cell, about 1,500 molecules / cell and about 1,000 molecules / cell, about 2,000 molecules / cell and about 500 molecules / cell, about 1,000 molecules / cell and about 200 molecules / cell or about 1,000 molecules / cell and about 100 molecules / cell.

[0193] In certain embodiments, the antigen recognition receptor is a TCR-like fusion molecule comprising: a first antigen binding chain comprising a V H and a constant domain comprising a TRBC polypeptide; and a second antigen binding chain comprising a V L and a constant domain comprising a TRAC polypeptide. In certain embodiments, the first antigen binding chain is designated as "V H In certain embodiments, the second antigen binding chain is designated as "V L -TRAC chain". In certain embodiments, the first antigen binding chain comprises V H 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 in V L 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.

[0194] In certain embodiments, the antigen recognition receptor is a TCR-like fusion molecule comprising: a first antigen binding chain comprising a V H and a constant domain comprising a TRAC polypeptide; and a second antigen binding chain comprising a V L and a constant domain comprising a TRBC polypeptide. In certain embodiments, the first antigen binding chain is designated as "V H In certain embodiments, the second antigen binding chain is designated as "V L -TRBC chain". In certain embodiments, the first antigen binding chain comprises V H In certain embodiments, the second antigen binding chain comprises a hinge region between V L and a hinge region between the TRBC polypeptide. In certain embodiments, the first antigen binding chain and the second antigen binding chain bind to an antigen (eg, human CD70).

[0195] In certain embodiments, the antigen recognition receptor is a TCR-like fusion molecule comprising: a first antigen binding chain comprising a V H and a constant domain comprising a TRBC polypeptide; and a second antigen binding chain comprising a V L and a constant domain comprising a TRAC polypeptide. In certain embodiments, the first antigen binding chain is designated as "VH In certain embodiments, the second antigen binding chain is designated as "V L -TRAC chain". In certain embodiments, the first antigen binding chain comprises V H In certain embodiments, the second antigen binding chain comprises a hinge region between V L and a hinge region between the TRBC polypeptide. In certain embodiments, the first antigen binding chain and the second antigen binding chain bind to an antigen (eg, human CD70).

[0196] In certain embodiments, the antigen recognition receptor is a TCR-like fusion molecule that binds to CD70 (e.g., human CD70) and comprises two antigen binding chains that can dimerize and bind to CD70, e.g., comprising V H and the first antigen binding chain of the TRBC polypeptide ("V H -TRBC chain") and contains V L and the second antigen binding chain of the TRBC polypeptide ("V L -TRAC chain"). In certain embodiments, V H The present invention comprises a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 34, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 35, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 36. In certain embodiments, V H Contains the amino acid sequence shown in SEQ ID NO: 40. In certain embodiments, V L The present invention comprises a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 37, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 38, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 39. In certain embodiments, V L In some embodiments, the TRAC polypeptide comprises the amino acid sequence shown in SEQ ID NO: 42. In some embodiments, the TRAC polypeptide comprises the amino acid sequence shown in SEQ ID NO: 7 or SEQ ID NO: 9. In some embodiments, the TRBC polypeptide is a TRBC2 polypeptide. In some embodiments, the TRBC2 polypeptide comprises the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 14. In some embodiments, the TCR-like fusion molecule is named "70-HIT" or "70H". SEQ ID NOs: 34 to 43 are provided in Table 1 below.

[0197] In certain embodiments, the Kabat system is used to describe the CDR regions / sequences disclosed herein (Swindells et al., J Mol Biol. 2017 Feb 3;429(3):356-364).

[0198] Table 1

[0199]

[0200] In certain embodiments, the antigen recognition receptor is a TCR-like fusion molecule that binds to CD70 (e.g., human CD70) and comprises two antigen binding chains that can dimerize and bind to CD70, e.g., comprising V H and the first antigen binding chain of the TRBC polypeptide ("V H -TRBC chain") and contains V L and the second antigen binding chain of the TRBC polypeptide ("V L -TRAC chain"). In certain embodiments, V H A V comprising an anti-CD70 antibody disclosed in International Patent Publication No. WO 2007 / 038637 H In certain embodiments, V H A V comprising the anti-CD70 antibody 2H5 disclosed in International Patent Publication No. WO 2007 / 038637 H In certain embodiments, V L A V comprising an anti-CD70 antibody disclosed in International Patent Publication No. WO 2007 / 038637 H In certain embodiments, V L A V comprising the anti-CD70 antibody 2H5 disclosed in International Patent Publication No. WO 2007 / 038637 H CDR1, CDR2 and CDR3 of the sequence.

[0201] Various TCR-like fusion molecules are disclosed in International Patent Application Publication No. WO2019 / 133969, which is hereby incorporated by reference in its entirety.

[0202] 2.1.3. Delivery of antigen recognition receptors

[0203] In certain embodiments, the antigen recognition receptor is delivered to the cell by 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, adeno-associated viral vectors, vaccinia virus, bovine papilloma virus, and herpes virus (e.g., such as Epstein-Barr virus).

[0204] In certain embodiments, antigen recognition receptors are delivered to cells by non-viral methods.Any targeted genome editing method can also be used to deliver the first antigen recognition receptor to cells.In certain embodiments, antigen recognition receptors are delivered to cells by methods including the following items: homologous recombination, zinc finger nucleases, meganucleases, transcription activator-like effector nucleases (TALEN), clustered regularly spaced short palindromic repeats (CRISPR) systems or combinations thereof.In certain embodiments, the CRISPR system is used to deliver the first antigen recognition receptor to cells.

[0205] 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: TRAC locus, TRBC locus, TRDC locus, and TRGC locus. In certain embodiments, the locus is a TRAC locus or a TRBC locus. In certain embodiments, the cell is a T cell, and the first antigen recognition receptor is integrated into the TRAC locus. The method of targeting a site within the genome of a T cell with CAR is disclosed in WO2017180989 and Eyquem et al., Nature. (March 2, 2017); 543(7643): 113–117, both of which are incorporated by reference in their entirety. In certain embodiments, the cell is a T cell, the first antigen recognition receptor is CAR, and the first antigen recognition receptor is integrated into the TRAC locus. In certain embodiments, the cell further includes a gene disruption to the TRBC locus. In certain embodiments, the gene disruption to the TRBC locus causes a knockout of the TRBC locus.

[0206] 2.1.4. Second antigen recognition receptor

[0207] In certain embodiments, the cells disclosed herein that contain an antigen recognition receptor (e.g., a first antigen recognition receptor) further contain a second antigen recognition receptor that targets a second antigen. In certain embodiments, the second antigen recognition 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).

[0208] 2.1.4.1. Second antigen

[0209] In certain embodiments, the second antigen is a tumor antigen, e.g., a tumor antigen disclosed in Section 2.1.1. In certain embodiments, the tumor antigen is an antigen with a low antigen density. In certain embodiments, the tumor antigen is expressed on cells with a low tumor cell frequency.

[0210] 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 cytomegalovirus (CMV) infected cells (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 (FLT3), CD138, CD20, CD22, CD244. (2B4), CD25, CD26, CD276, CD30, CD300LF, CD312, CD32, CD321, CD33, CD34, CD36, CD38, CD 41. 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, CX3CR1, CXCR4, CYP4F11, DAGLB, DARC, DFNB31, DGKI, EGF1R, EGFR-VIII, EGP-2, EGP-40, E LOVL6, 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, NKG2D ligand, NLGN3, NPAS2, NY-ESO-1, carcinoembryonic 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, SCIN, SCN11A, SCN2A, SCNN1D, SEC31B, SEMA4A, SH3PXD2A, SIGLEC11, SIRPB1, SLC16A6, SLC19A1, SLC22A5, SLC25A3 6. SLC25A41, SLC30A1, SLC34A3, SLC43A3, SLC44A1, SLC44A3, SLC45A3, SLC6A16, SLC6A6, SLC8A3, SLC9A1, SLCO2B1, SPAG17, STC1, STON2, SUN3, survivin, 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.

[0211] In certain embodiments, the second antigen is a pathogen antigen, eg, a pathogen antigen disclosed in Section 2.1.1.

[0212] 2.1.4.2. Chimeric Antigen Receptor (CAR)

[0213] CAR is an engineered receptor that transfers or imparts the specificity of interest to immune effector cells. CAR can be used to transfer the specificity of a monoclonal antibody to a T cell; wherein the transfer of its coding sequence is facilitated by a retroviral vector.

[0214] There are three generations of CARs. A "first generation" CAR is 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. A "first generation" CAR can provide de novo antigen recognition and elicit CD4 Tg expression independently of HLA-mediated antigen presentation through its CD3 zeta chain signaling domain in a single fusion molecule. + and CD8 + T cell activation. "Second generation" CARs include costimulatory molecules (e.g., CD28, 4-1BB, ICOS, OX40, CD27, CD40, NKG2D, DAP-10, CD2, CD150, CD226) to the signaling domain of the intracellular signaling domain of the CAR for providing a costimulatory signal to a cell (e.g., T cell or NK cell). "Second generation" CARs include those that provide both costimulation (e.g., CD28 or 4-1BB) and activation (CD3ζ). "Third generation" CARs include those that provide multiple costimulations (e.g., CD28 and 4-1BB) and activation (CD3ζ).

[0215] In certain embodiments, the second antigen recognition receptor is a CAR comprising an extracellular antigen binding domain and an intracellular signaling domain that binds to a second antigen. In certain embodiments, CAR further comprises a transmembrane domain. In certain embodiments, CAR further comprises a hinge / spacer.

[0216] In certain embodiments, the extracellular antigen binding domain of the CAR (e.g., scFv) is present at about 5 × 10 -7 M or smaller, approximately 1 × 10 -7 M or smaller, about 5 × 10 -8 M or smaller, approximately 1 × 10 -8M or smaller, about 5 × 10 -9 M or smaller, or about 1 × 10 -9 M or less or about 1 × 10 -10 M or less dissociation constant (K D ) binds to the first antigen. In certain embodiments, the extracellular antigen binding domain of the CAR (e.g., scFv) is approximately 1 × 10 -8 M or smaller K D Binds to the first antigen.

[0217] The binding of the extracellular antigen binding domain (e.g., in scFv) can be confirmed by, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition) or Western blot assay. Each of these assays is usually determined by using a labeled reagent (e.g., antibody or scFv) specific for the complex of interest to detect the presence of a protein-antibody complex of particular interest. For example, scFv can be radiolabeled and used in radioimmunoassay (RIA) (see, e.g., Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March 1986, which is incorporated herein by reference). Radioisotopes can be detected by means of a gamma counter or a scintillation counter or by autoradiography. In certain embodiments, the extracellular antigen binding domain of CAR is labeled with a fluorescent marker. Non-limiting examples of fluorescent markers include: green fluorescent protein (GFP), blue fluorescent proteins (e.g., EBFP, EBFP2, Azurite, and mKalama1), cyan fluorescent proteins (e.g., ECFP, Azure, and CyPet), and yellow fluorescent proteins (e.g., YFP, Citrine, Venus, and YPet).

[0218] The extracellular antigen binding domain may comprise or may be a scFv, Fab (which is optionally cross-linked) or F(ab) 2 In certain embodiments, any of the foregoing molecules may be included in a fusion protein having a heterologous sequence for forming an extracellular antigen binding domain. In certain embodiments, the extracellular antigen binding domain comprises or is a 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.

[0219] In addition, the extracellular antigen binding domain of CAR may include a leader peptide or signal peptide that guides the nascent protein into the endoplasmic reticulum. CAR is glycosylated and anchored in the cell membrane, then the signal peptide or leader peptide may be essential. The signal sequence or leader sequence may be a peptide sequence (e.g., about 5, about 10, about 15, about 20, about 25, or about 30 amino acids) present at the N-terminus of the newly synthesized protein, which guides the protein into the secretory pathway. In certain embodiments, the signal peptide is covalently bonded to the 5' end (N-terminus) of the extracellular antigen binding domain of 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 shown 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 shown in SEQ ID NO: 45); a human κ leader sequence (e.g., a human κ leader sequence comprising or consisting of the amino acid sequence shown in SEQ ID NO: 46), a mouse κ leader sequence (e.g., a mouse κ leader sequence comprising or consisting of the amino acid sequence shown 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 shown 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 shown in SEQ ID NO: 49); a human albumin signal sequence (e.g., a human CD8 leader sequence comprising or consisting of the amino acid sequence shown in SEQ ID NO: 41). 50 or a human albumin signal sequence consisting of the amino acid sequence shown in SEQ ID NO: 50); and a human prolactin signal sequence (e.g., a human prolactin signal sequence comprising the amino acid sequence shown in SEQ ID NO: 51 or a human prolactin signal sequence consisting of the amino acid sequence shown in SEQ ID NO: 52). SEQ ID NOs: 44 to 51 are provided below.

[0220] MYRMQLLSCIALSLALVTNS [SEQ ID NO: 44]

[0221] MYSMQLASCVTLTLVLLVNS [SEQ ID NO: 45]

[0222] METPAQLLFLLLLLWLPDTTG [SEQ ID NO: 46]

[0223] METDTLLLWVLLLWVPGSTG [SEQ ID NO: 47]

[0224] MALPVTALLLPLALLLHAARP [SEQ ID NO: 48]

[0225] MALPVTALLLPLALLLHA [SEQ ID NO: 49]

[0226] MKWVTFISLLFSSAYS [SEQ ID NO: 50]

[0227] MDSKGSSQKGSRLLLLLVVSSNLLLCQGVVS [SEQ ID NO: 51]

[0228] In certain embodiments, the signal peptide includes a CD8 polypeptide, for example, CAR includes a truncated CD8 signal peptide. In certain embodiments, the signal peptide includes the amino acid sequence shown in SEQ ID NO: 49 or consists of the amino acid sequence.

[0229] In certain embodiments, the second antigen recognition receptor is a CAR comprising a transmembrane domain. Different transmembrane domains produce different receptor stabilities. After antigen recognition, the receptors aggregate and the signal is transmitted to the cell. According to the subject matter disclosed in the present application, the transmembrane domain of the first antigen recognition receptor may include a natural or modified transmembrane domain of the following items: CD8 polypeptide, CD28 polypeptide, CD3ζ polypeptide, CD40 polypeptide, 4-1BB polypeptide, OX40 polypeptide, CD84 polypeptide, CD166 polypeptide, CD8a polypeptide, CD8b polypeptide, ICOS polypeptide, ICAM-1 polypeptide, CTLA-4 polypeptide, CD27 polypeptide, CD40 polypeptide, NKG2D polypeptide, synthetic polypeptide (not based on proteins associated with immune response) or a combination thereof.

[0230] In certain embodiments, the transmembrane domain of CAR comprises a CD28 polypeptide (e.g., a transmembrane domain of CD28 or a portion thereof). In certain embodiments, the transmembrane domain of CAR comprises a transmembrane domain of human CD28 or a portion thereof. In certain embodiments, the CD28 polypeptide comprises or consists of the following: an amino acid sequence having a continuous portion of an amino acid sequence with NCBI reference number: NP_006130 (SEQ ID NO: 52) of at least about 20, or at least about 25, or at least about 30 and / or at most about 220 amino acids in length. In certain embodiments, the CD28 polypeptide comprises or consists of the following: an amino acid sequence of amino acids 1 to 220, 1 to 50, 50 to 100, 100 to 150, 114 to 220, 150 to 200, 153 to 179 or 200 to 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 to 179 of SEQ ID NO: 52. SEQ ID NO: 52 is provided below.

[0231] MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFC KIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS[SEQ ID NO: 52]

[0232] In certain embodiments, the second antigen recognition receptor is a CAR further comprising a hinge / spacer region connecting the extracellular antigen binding domain to the transmembrane domain. The hinge / spacer region can be sufficiently flexible to allow the antigen binding domain to be oriented in different directions, thereby facilitating antigen recognition. In certain embodiments, the hinge / spacer region of CAR may include a natural or modified hinge region of the following items: CD8 polypeptide, CD28 polypeptide, CD3ζ polypeptide, CD40 polypeptide, 4-1BB polypeptide, OX40 polypeptide, CD84 polypeptide, CD166 polypeptide, CD8a polypeptide, CD8b polypeptide, ICOS polypeptide, ICAM-1 polypeptide, CTLA-4 polypeptide, CD27 polypeptide, CD40 polypeptide, NKG2D polypeptide, synthetic polypeptide (not based on proteins associated with immune response) or a combination thereof. The hinge / spacer region can be a hinge region from IgG1 or a CH of an immunoglobulin.2 CH 3 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.

[0233] In certain embodiments, the second antigen recognition receptor is a CAR further comprising a hinge / spacer region of a native or modified hinge region comprising a CD28 polypeptide. In certain embodiments, the hinge / spacer region of the first antigen recognition receptor (e.g., CAR) comprises a CD28 polypeptide comprising or consisting of: amino acids 114 to 152 of SEQ ID NO: 52.

[0234] In certain embodiments, hinge / spacer is positioned between extracellular antigen binding domain and transmembrane domain.In certain embodiments, hinge / spacer includes the following items: CD8 polypeptide, CD28 polypeptide, CD3 ζ polypeptide, CD4 polypeptide, 4-1BB polypeptide, OX40 polypeptide, CD166 polypeptide, CD8a polypeptide, CD8b polypeptide, ICOS polypeptide, ICAM-1 polypeptide, CTLA-4 polypeptide, CD27 polypeptide, CD40 polypeptide, NKG2D polypeptide, synthetic polypeptide (not based on protein associated with immune response) or their combination.In certain embodiments, transmembrane domain includes the following items: CD8 polypeptide, CD28 polypeptide, CD3 ζ polypeptide, CD4 polypeptide, 4-1BB polypeptide, OX40 polypeptide, CD166 polypeptide, CD8a polypeptide, CD8b polypeptide, ICOS polypeptide, ICAM-1 polypeptide, CTLA-4 polypeptide, CD27 polypeptide, CD40 polypeptide, NKG2D polypeptide, synthetic polypeptide (not based on protein associated with immune response) or their combination.

[0235] In certain embodiments, the membrane spaning domain and hinge / spacer are derived from the same molecule. In certain embodiments, the membrane spaning domain and hinge / spacer are derived from different molecules. In certain embodiments, hinge / spacer comprises CD28 polypeptide and membrane spaning domain comprises CD28 polypeptide. In certain embodiments, hinge / spacer comprises CD28 polypeptide and membrane spaning domain comprises CD28 polypeptide. In certain embodiments, hinge / spacer comprises CD84 polypeptide and membrane spaning domain comprises CD84 polypeptide. In certain embodiments, hinge / spacer comprises CD166 polypeptide and membrane spaning domain comprises CD166 polypeptide. In certain embodiments, hinge / spacer comprises CD8a polypeptide and membrane spaning domain comprises CD8a polypeptide. In certain embodiments, hinge / spacer comprises CD8b polypeptide and membrane spaning domain comprises CD8b polypeptide. In certain embodiments, hinge / spacer comprises CD28 polypeptide and membrane spaning domain comprises ICOS polypeptide.

[0236] In certain embodiments, the second antigen recognition 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., lymphocytes, e.g., T cells). Wild-type ("natural") CD3ζ includes three functional immunoreceptor tyrosine-based activation motifs (ITAMs), three functional base-rich stretches (BRS) regions (BRS1, BRS2, ​​and BRS3). After the antigen is bound, CD3ζ transmits an activation signal to the cell (e.g., lymphocytes, e.g., T cells). The intracellular signaling domain of the CD3ζ chain is the main transmitter of the signal of the endogenous TCR.

[0237] In certain embodiments, the intracellular signaling domain of the CAR comprises a native CD3 zeta. In certain embodiments, the native CD3 zeta 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 an amino acid sequence or a fragment thereof with NCBI reference number: NP_932170 (SEQ ID NO: 53), and / or may optionally comprise at most one, or at most two, or at most three conservative amino acid substitutions. In certain embodiments, the CD3 zeta 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 at most about 164 amino acids in length as a continuous portion of SEQ ID NO: 53. In certain embodiments, the native CD3 zeta comprises or consists of an amino acid sequence of amino acids 1 to 164, 1 to 50, 50 to 100, 52 to 164, 100 to 150, or 150 to 164 of SEQ ID NO: 53. In certain embodiments, the intracellular signaling domain of the CAR contains a native CD3 zeta comprising or consisting of an amino acid sequence of amino acids 52 to 164 of SEQ ID NO: 53. SEQ ID NO: 53 is provided below:

[0238] MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR [SEQ ID NO: 53]

[0239] 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 shown in SEQ ID NO: 54. SEQ ID NO: 54 is provided below:

[0240] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR [SEQ ID NO: 54]

[0241] In certain embodiments, the intracellular signaling domain of the CAR includes a modified CD3 zeta polypeptide. In certain embodiments, the modified CD3 zeta polypeptide comprises one, two or three ITAMs. In certain embodiments, the modified CD3 zeta polypeptide includes a native ITAM1. In certain embodiments, the native ITAM1 includes or consists of the amino acid sequence shown in SEQ ID NO: 55.

[0242] QNQLYNELNLGRREEYDVLDKR [SEQ ID NO: 55]

[0243] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 55 is set forth in SEQ ID NO: 56 provided below.

[0244] CAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGA[SEQ ID NO: 56]

[0245] In certain embodiments, the modified CD3ζ polypeptide contains 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 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 shown in SEQ ID NO: 57, which is provided below.

[0246] QNQLFNELNLGRREEFDVLDKR [SEQ ID NO: 57]

[0247] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 57 is set forth in SEQ ID NO: 58 provided below.

[0248] CAGAACCAGCTCTTTAACGAGCTCAATCTAGGACGAAGAGAGGAGTTCGATGTTTTGGACAAGAGA[SEQ ID NO: 58]

[0249] In certain embodiments, the modified CD3 zeta polypeptide comprises native ITAM 2. In certain embodiments, native ITAM 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 59, which is provided below.

[0250] QEGLYNELQKDKMAEAYSEIGMK [SEQ ID NO: 59]

[0251] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 59 is shown in SEQ ID NO: 60 provided below.

[0252] CAGGAAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAA[SEQ ID NO: 60]

[0253] 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 shown in SEQ ID NO: 61, which is provided below.

[0254] QEGLFNELQKDKMAEAFSEIGMK [SEQ ID NO: 61]

[0255] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 61 is shown in SEQ ID NO: 62 provided below.

[0256] CAGGAAGGCCTGTTCAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTTCAGTGAGATTGGGATGAAA[SEQ ID NO: 62]

[0257] In certain embodiments, the modified CD3 zeta polypeptide comprises native ITAM 3. In certain embodiments, native ITAM 3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 63, which is provided below.

[0258] HDGLYQGLSTATKDTYDALHMQ [SEQ ID NO: 63]

[0259] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 63 is shown in SEQ ID NO: 64 provided below.

[0260] CACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAG[SEQ ID NO: 64]

[0261] In certain embodiments, the modified CD3 zeta 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 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 shown in SEQ ID NO: 65, which is provided below.

[0262] HDGLFQGLSTATKDTFDALHMQ [SEQ ID NO: 65]

[0263] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 65 is shown in SEQ ID NO: 66 provided below.

[0264] CACGATGGCCTTTTCCAGGGGCTCAGTACAGCCACCAAGGACACCTTCGACGCCCTTCACATGCAG[SEQ ID NO: 66]

[0265] Various modified CD3ζ polypeptides and CARs comprising modified CD3ζ polypeptides are disclosed in International Patent Application Publication No. WO2019 / 133969, which is hereby incorporated by reference in its entirety.

[0266] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3 zeta polypeptide comprising a native ITAM1, an ITAM2 variant comprising one or more (e.g., two) loss-of-function mutations or consisting thereof, and an ITAM3 variant comprising one or more (e.g., two) loss-of-function mutations or consisting thereof. In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3 zeta 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 the CAR comprises a modified CD3 zeta polypeptide comprising: a native ITAM1 consisting of the amino acid sequence shown in SEQ ID NO: 55, an ITAM2 variant consisting of the amino acid sequence shown in SEQ ID NO: 59, and an ITAM3 variant consisting of the amino acid sequence shown in SEQ ID NO: 63. In certain embodiments, the CAR is designated as "1XX". In certain embodiments, the modified CD3 zeta polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 67. SEQ ID NO: 67 is provided below:

[0267] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR [SEQ ID NO: 67]

[0268] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3 zeta polypeptide comprising or consisting 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% identical to SEQ ID NO: 67 or a fragment thereof, and / or may optionally comprise up to one, up to two, or up to three conservative amino acid substitutions.

[0269] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 67 is shown in SEQ ID NO: 68 provided below.

[0270] AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAA GGCCTGTTCAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTTCAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTCAGGGGCTCAGTACAGCCACCAAGGACACCTTCGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC [SEQ ID NO: 68]

[0271] In certain embodiments, the intracellular signaling domain of 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 one 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.

[0272] In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising a CD28 polypeptide, e.g., an intracellular domain of CD28 or a portion thereof. In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising an intracellular domain of human CD28 or a portion thereof.

[0273] In certain embodiments, the CD28 polypeptide contained in the costimulatory signaling region of the second antigen recognition 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%, at least about 100% identical or homologous to the amino acid sequence shown in SEQ ID NO: 52 or a fragment thereof, and / or may optionally comprise at most one, or at most two, or at most three conservative amino acid substitutions. In certain embodiments, the CD28 polypeptide contained in the costimulatory signaling region of the CAR comprises or consists of an amino acid sequence that is a continuous portion of SEQ ID NO: 52 of at least about 20, or at least about 30, or at least about 40, or at least about 50 and at most about 220 amino acids in length. Alternatively or additionally, in certain embodiments, the CD28 polypeptide contained in the costimulatory signaling region of the CAR comprises or consists of amino acids 1 to 220, 1 to 50, 50 to 100, 100 to 150, 114 to 220, 150 to 200, 180 to 220, or 200 to 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 to 220 of SEQ ID NO: 52.

[0274] An exemplary nucleic acid sequence encoding the amino acid sequence of amino acids 180 to 220 of SEQ ID NO: 52 is shown in SEQ ID NO: 69 provided below.

[0275] AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCCACCACGCGACTTCGCAGCCTATCGCTCC [SEQ ID NO: 69]

[0276] In certain embodiments, the intracellular signaling domain of the second antigen recognition receptor comprises a co-stimulatory signaling region, the co-stimulatory signaling region comprising the intracellular domain of mouse CD28 or a portion thereof. In certain embodiments, the CD28 polypeptide contained in the co-stimulatory 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%, at least about 100% identical or homologous to an amino acid sequence having NCBI reference number: NP_031668.3 (or SEQ ID NO: 70) or a fragment thereof, and / or may optionally comprise at most one, or at most two, or at most three conservative amino acid substitutions. In certain embodiments, the CD28 polypeptide contained in the costimulatory signaling region of the CAR comprises or consists of an amino acid sequence of 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 as a contiguous portion of SEQ ID NO: 70. In certain embodiments, the CD28 polypeptide contained in the costimulatory signaling region of the CAR comprises or consists of an amino acid sequence of amino acids 1 to 218, 1 to 50, 50 to 100, 100 to 150, 150 to 218, 178 to 218, or 200 to 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 to 218 of SEQ ID NO: 70. SEQ ID NO: 70 is provided below.

[0277] MTLRLLFLAL NFFSVQVTEN KILVKQSPLL VVDSNEVSLS CRYSYNLLAK EFRASLYKGV

[0278] NSDVEVCVGN GNFTYQPQFR SNAEFNCDGD FDNETVTFRL WNLHVNHTDI YFCKIEFMYP

[0279] PPYLDNERSN GTIIHIKEKH LCHTQSSPKL FWALVVVAGV LFCYGLLVTV ALCVIWTNSR

[0280] RNRLLQSDYM NMTPRRPGLT RKPYQPYAPA RDFAAYRP [SEQ ID NO: 70]

[0281] In certain embodiments, the intracellular signaling domain of CAR includes a costimulatory signaling region, and the costimulatory signaling region includes a 4-1BB polypeptide, for example, an intracellular domain of 4-1BB or a portion thereof. In certain embodiments, the costimulatory signaling region includes an intracellular domain of human 4-1BB or a portion thereof. In certain embodiments, the 4-1BB included in the costimulatory signaling region of CAR includes or consists of the following items: with NCBI reference number: NP_001552 (SEQ ID NO: 71) sequence or its fragment 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 or homologous amino acid sequence, and / or may optionally include at most one, or at most two or at most three conservative amino acid substitutions. In certain embodiments, the 4-1BB contained in the costimulatory signaling region of the CAR comprises or consists of an amino acid sequence of at least about 20, or at least about 30, or at least about 40, or at least about 50, and / or at most about 50, at most about 60, at most about 70, at most about 80, at most about 90, at most about 100, at most about 200, or at most about 255 amino acids in length as a continuous portion of SEQ ID NO: 71. In certain embodiments, the costimulatory signaling region of the CAR comprises a 4-1BB polypeptide comprising or consisting of an amino acid sequence of amino acids 1 to 255, 1 to 50, 50 to 100, 100 to 150, 150 to 200, or 200 to 255 of SEQ ID NO: 71. In certain embodiments, the co-stimulatory signaling region of the CAR comprises a 4-1BB polypeptide comprising or consisting of an amino acid sequence of amino acids 214 to 255 of SEQ ID NO: 71. SEQ ID NO: 71 is provided below.

[0282] MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGTFND QKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL [SEQ ID NO: 71]

[0283] In certain embodiments, the intracellular signaling domain of CAR comprises two costimulatory signaling regions, wherein the first costimulatory signaling region comprises the intracellular domain of the first costimulatory molecule or a portion thereof, and the second costimulatory signaling region comprises the intracellular domain of the second costimulatory molecule or a portion thereof. The first costimulatory molecule and the second costimulatory molecule are independently selected from the group consisting of the following items: 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 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.

[0284] 2.1.4.3. Chimeric ligand receptors

[0285] In certain embodiments, the second antigen recognition receptor is a chimeric ligand receptor, which comprises a ligand or a portion thereof that binds to the first antigen. In certain embodiments, the chimeric ligand receptor further comprises a transmembrane domain and an intracellular signaling domain.

[0286] In certain embodiments, the transmembrane domain is fused to the ligand or a portion thereof. In certain embodiments, the transmembrane domain is fused to the intracellular signaling domain. In certain embodiments, the transmembrane domain is positioned between the ligand or a 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 CD3 zeta polypeptide (e.g., as disclosed in Section 2.1.5.2).

[0287] Additional 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.

[0288] 2.1.4.4.CCR

[0289] In certain embodiments, the cell comprising the first antigen recognition receptor disclosed in the present application further comprises a CCR. The term "chimeric co-stimulatory receptor" or "CCR" refers to a chimeric receptor that binds to an antigen and provides a co-stimulatory signal but does not provide a T cell activation signal to a cell comprising a CCR. Various CCRs are described in US20020018783, the contents of which are incorporated by reference in their entirety. CCR mimics co-stimulatory signals, but unlike CAR, does not provide T cell activation signals. In certain embodiments, CCR lacks a CD3 ζ polypeptide.

[0290] In the absence of natural co-stimulatory ligands on antigen presenting cells, CCR provides co-stimulatory signals (e.g., CD28-like signals or 4-1BB-like signals). Combination antigen recognition (i.e., CCR used in combination with CAR) can enhance T cell reactivity against T cells expressing dual antigens, thereby improving selective tumor targeting. Kloss et al. describe a strategy that integrates combined antigen recognition, split signaling, and balanced intensity of key T cell activation and co-stimulation to generate T cells that eliminate target cells expressing antigen combinations while retaining 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). Using this method, T cell activation requires CAR-mediated recognition of one antigen, but co-stimulation is independently mediated by CCRs that are specific for the second antigen. In order to achieve tumor selectivity, the combined antigen recognition method reduces the efficiency of T cell activation to a level where it is ineffective without the rescue provided by simultaneous CCR recognition of the second antigen.

[0291] In certain embodiments, CCR includes an extracellular antigen binding domain that is combined with a third antigen, and an intracellular domain that can deliver a co-stimulatory signal to a cell but does not deliver an activation signal to the cell alone. In certain embodiments, CCR further includes a transmembrane domain. In certain embodiments, the intracellular domain of CCR includes at least an intracellular domain of a co-stimulatory molecule or a portion thereof. In certain embodiments, co-stimulatory molecules are selected from the group consisting of the following items: CD28, 4-1BB, OX40, CD27, CD40, CD154, CD97, CD11a / CD18, ICOS, DAP-10, CD2, CD150, CD226 and NKG2D.

[0292] In certain embodiments, CCR includes the intracellular domain of CD28 or a portion thereof. In certain embodiments, CCR includes the intracellular domain of 4-1BB or a portion thereof. In certain embodiments, CCR includes the intracellular domain of CD28 or a portion thereof and the intracellular domain of 4-1BB or a portion thereof.

[0293] In some embodiments, a second antigen is selected such that the expression of both the first antigen and the second antigen is restricted to the target cells (e.g., cancer tissue or cancer cells). Similar to CARs, the extracellular antigen-binding domain can be an scFv, Fab, F(ab) 2 or a fusion protein having a heterologous sequence for forming an extracellular antigen-binding domain.

[0294] In some embodiments, cells comprising a first antigen recognition receptor and a CCR exhibit a greater degree of cytolytic activity against cells that are positive for both the first / second antigen and a third antigen, compared to cells that are singly positive for the first / second antigen. In some embodiments, cells comprising a first antigen recognition receptor and a CCR exhibit substantially no or negligible cytolytic activity against cells that are singly positive for the first / second antigen.

[0295] In some embodiments, the first antigen recognition receptor (e.g., the receptor disclosed in Section 2.1.2) binds to the antigen with a low binding affinity (e.g., about 1 × 10 -8 M or greater, about 5 × 10 -8 M or greater, about 1 × 10 -7 M or greater, about 5 × 10 -7 M or greater, or about 1 × 10 -6 M or greater or about 1 × 10 -8 M to about 1 × 10 -6 M dissociation constant (K D ). In some embodiments, the antigen recognition receptor (e.g., a CAR, TCR, or TCR-like fusion molecule) binds to the antigen with a low binding affinity. In some embodiments, the antigen recognition receptor (e.g., a TCR-like fusion molecule) binds to the antigen at an epitope with low accessibility. In some embodiments, the antigen recognition receptor (e.g., a TCR-like fusion molecule) binds to the antigen with a binding affinity that is lower than the binding affinity with which a second antigen recognition receptor (e.g., a CCR) binds to the second antigen. In some embodiments, the CCR binds to the second antigen with a binding affinity of about 1 × 10 -9 M to about 1 × 10 -7 M, e.g., about 1 × 10 -7 M or less, about 1 × 10 -8 M or less or about 1 × 10 -9 M or less binding affinity K D for binding to the second antigen.

[0296] 2.1.5.5. T cell receptor (TCR)

[0297] In certain embodiments, the cell comprising the first antigen recognition receptor disclosed in the present application further comprises a TCR. TCR is a disulfide-linked heterodimeric protein composed of two variable chains expressed as part of a complex with an invariant CD3 chain molecule. TCR is found on the surface of T cells and is responsible for identifying antigens as peptides bound to major histocompatibility complex (MHC) molecules. In certain embodiments, TCR includes an alpha chain and a beta chain (encoded by TRA and TRB, respectively). In certain embodiments, TCR includes a gamma chain and a delta chain (encoded by TRG and TRD, respectively).

[0298] Each chain of the TCR consists of two extracellular domains: a variable (V) region and a constant (C) region. The constant region is close to the cell membrane, followed by a transmembrane region and a short cytoplasmic tail. The variable region binds to the peptide / MHC complex. The variable domains of the two chains each have three complementarity determining regions (CDRs).

[0299] In certain embodiments, the TCR can form a receptor complex with three dimeric signaling modules CD3δ / ε, CD3γ / ε, and CD247ζ / ζ or ζ / η. When the TCR complex engages with its antigen and MHC (peptide / MHC), the T cell expressing the TCR complex is activated.

[0300] In certain embodiments, the TCR is an endogenous TCR. In certain embodiments, the TCR is a naturally occurring TCR.

[0301] In certain embodiments, TCR is exogenous TCR. In certain embodiments, TCR is recombinant TCR. In certain embodiments, TCR is non-natural TCR. In certain embodiments, non-natural TCR differs from any naturally occurring TCR by at least one amino acid residue. In certain embodiments, non-natural 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, non-natural TCR is modified by at least one amino acid residue of naturally occurring TCR. In certain embodiments, the non-naturally occurring TCR is modified from a 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.

[0302] 2.1.5.6. Delivery of Second Antigen Recognition Receptor

[0303] In certain embodiments, the second antigen recognition receptor is delivered to the cell by 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, adeno-associated viral vectors, vaccinia virus, bovine papilloma virus, and herpes virus (e.g., such as Epstein-Barr virus).

[0304] In certain embodiments, the second antigen recognition receptor is delivered to cells by non-viral methods.Any targeted genome editing method can also be used to deliver the second antigen recognition receptor to cells.In certain embodiments, the second antigen recognition receptor is delivered to cells by a method including the following items: homologous recombination, zinc finger nuclease, meganuclease, transcription activator-like effector nuclease (TALEN), clustered regularly spaced short palindromic repeats (CRISPR) system or a combination thereof.In certain embodiments, the CRISPR system is used to deliver the second antigen recognition receptor to cells.

[0305] In certain embodiments, the cell is a T cell, and the second antigen recognition receptor is integrated into a locus within the genome of the T cell. Non-limiting examples of loci include: TRAC locus, TRBC locus, TRDC locus, and TRGC locus. In certain embodiments, the locus is a TRAC locus or a TRBC locus. In certain embodiments, the cell is a T cell, and the second antigen recognition receptor is integrated into the TRAC locus.

[0306] 2.2. Co-stimulatory ligands

[0307] In certain embodiments, the cells disclosed herein that contain an antigen recognition receptor (e.g., a first antigen recognition receptor, e.g., a receptor disclosed in Section 2.1.2) further contain at least one recombinant or exogenous co-stimulatory ligand. For example, the cells disclosed herein may be further transduced with at least one co-stimulatory ligand so that the cells express or are induced to express the first antigen recognition receptor, the second antigen recognition receptor, and the at least one co-stimulatory ligand. The at least one co-stimulatory ligand provides a co-stimulatory signal to the cell.

[0308] 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 that participates in systemic inflammation and stimulates acute phase reactions. Its main function is to regulate immune cells. Members of the TNF superfamily have many common features. Most of the TNF superfamily members are synthesized as type II transmembrane proteins (extracellular C-terminals), which contain short cytoplasmic portions and relatively long extracellular regions. 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 β (TNFβ) / lymphotoxin-α (LTα), lymphotoxin-β (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 class of cell surface and soluble proteins involved in the recognition, binding or adhesion of cells. These proteins have the same structural features as immunoglobulins - they have immunoglobulin domains (folds). Non-limiting examples of immunoglobulin superfamily ligands include: CD80, CD86 and ICOSLG. In certain embodiments, the at least one co-stimulatory ligand is selected from the group consisting of 4-1BBL, CD80, CD86, CD70, GITRL, CD40L, OX40L, CD30L, TNFRSF14, ICOSLG, TRAIL, and combinations thereof.

[0309] In certain embodiments, cell further comprises a kind of exogenous costimulatory ligand, it is 4-1BBL.In certain embodiments, costimulatory ligand is mankind 4-1BBL.In certain embodiments, 4-1BBL includes the following items or is made up of the following items: with Uniprot reference number: P41273-1 (SEQ ID NO: 72) amino acid sequence or its fragment 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% homology or identical amino acid sequence, and / or can optionally include up to one or up to two or up to three conservative amino acid substitutions.In certain embodiments, 4-1BBL includes the following items or is made up of the following items: as SEQ IDNO: The amino acid sequence of the continuous part of 72 amino acid sequence.SEQ ID NO: 72 is provided below.

[0310] MEYASDASLDPEAPWPPARARACRVLPWALVAGLLLLLLLAAACAVFLACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYK EDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPPSPRSE [SEQ ID NO: 72]

[0311] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 72 is shown in SEQ ID NO: 73.

[0312] ATGGAATACGCCTCTGACGCTTCACTGGACCCCGAAGCCCCGTGGCCTCCCGCGCCCCGCGCTCGCGCCTGCCGCGTACTGCCTTGGGCCCTGGTCGCGGGGCTGCTGCTGCTGCTGCTGCTCGCTGCCGCCTGCGCCGTCTTCCTCGCCTGCCCCTGGGCCGTGTCCGGGGCTCGCGCCTCGCCCGGCTCCGCGGCCAGCCCGAGACTCCGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA [SEQ ID NO: 73]

[0313] In certain embodiments, the cell further comprises an exogenous co-stimulatory ligand which is CD80. In certain embodiments, the co-stimulatory ligand is human CD80. In certain embodiments, 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 an amino acid sequence or fragment thereof having NCBI reference number: NP_005182 (SEQ ID NO: 74), and / or may optionally comprise at most one, or at most two, or at most three conservative amino acid substitutions. In certain embodiments, 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.

[0314] MGHTRRQGTSPSKCPYLNFFQLLVLAGLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPT PSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV [SEQ IDNO: 74]

[0315] 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.

[0316] ATGGGCCACACACGGAGGCAGGGAACATCACCATCCAAGTGTCCATACCTCAATTTCTTTCAGCTCTTGGTGCTGGCTGGTCTTTCTCACTTCTGTTCAGGTGTTATCCACGTGACCAAGGAAGTGAAAGAAGTGGCAACGCTGTCCTGTGGTCACAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCTACTGGCAAAAGGAGAAGAAAATG GTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATCCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTGTTCTGAAGTATGAAAAGACGCTTTCAAGCGGGAACACCTGGCTGAAGTGACGTTATCAGTCAAAGCTGACTTCCCTACA CCTAGTATATCTGACTTTGAAATTCCAACTTCTAATATTAGAAGGATAATTTGCTCAACCTCTGGAGGTTTTCCAGAGCCTCACCTCTCCTGGTTGGAAAATGGAGAAGAATTAAATGCCATCAACACAACAGTTTCCCAAGATCCTGAAACTGAGCTCTATGCTGTTAGCAGCAAACTGGATTTCAATATGACAACCAACCACAGCTTCATGTGTCTCATCAAGTATGGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACAACCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCTTAAATCTCAGTAAATGGAATTTTTGTGATATGCTGCCTGACCTACTGCTTTGCCCCAAGATGCAGAGAGAGAAGGAGGAATGAGAGATTGAGAAGGGAAAGTGTACGCCCTGTA [SEQ ID NO: 75]

[0317] In certain embodiments, the cell further comprises two exogenous costimulatory ligands, which are 4-1BBL and CD80. In certain embodiments, the cell further comprises two exogenous costimulatory ligands, which are 4-1BBL and CD80, wherein 4-1BBL comprises or consists of the amino acid sequence shown in SEQ ID NO: 72, and CD80 comprises or consists of the amino acid sequence shown in SEQ ID NO: 74.

[0318] Cells comprising a receptor and comprising at least one exogenous costimulatory ligand are described in U.S. Patent No. 8,389,282, which is incorporated herein by reference in its entirety.

[0319] 2.3. Fusion polypeptide

[0320] In certain embodiments, the cells disclosed in the present application that comprise an antigen recognition receptor (e.g., a first antigen recognition receptor, e.g., the receptor disclosed in Section 2.1.2) further comprise a fusion polypeptide. For example, the cells disclosed in the present application can be further transduced with a fusion polypeptide such that the cells express or are induced to express a first antigen recognition receptor, a second antigen recognition receptor, and the fusion polypeptide. The fusion polypeptide provides a costimulatory signal to the cell. The fusion polypeptide is capable of enhancing the activity and / or efficacy of cells comprising a first antigen recognition receptor (e.g., a CAR or TCR-like fusion molecule). In certain embodiments, the fusion polypeptide comprises: a) an extracellular domain and a transmembrane domain of a costimulatory ligand, and b) an intracellular domain of a first costimulatory molecule.

[0321] 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.

[0322] In certain embodiments, the fusion polypeptide comprises an extracellular domain and a transmembrane domain that is a co-stimulatory ligand of CD80. In certain embodiments, the co-stimulatory ligand is human CD80. In certain embodiments, 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 shown in SEQ ID NO: 69 or a fragment thereof, and / or may optionally comprise at most one, or at most two, or at most three conservative amino acid substitutions. In certain embodiments, CD80 comprises or consists of an amino acid sequence that is a continuous portion of the amino acid sequence of SEQ ID NO: 74.

[0323] 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 to 242 of SEQ ID NO: 74. In certain embodiments, the extracellular domain of CD80 comprises or consists of amino acids 1 to 242 of SEQ ID NO: 74, or a functional fragment thereof. The functional fragment can be a continuous portion of amino acids 1 to 242 of SEQ ID NO: 74 with a length of 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 certain embodiments, the 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 main function of the extracellular domain of CD80. Non-limiting examples of the main functions of the extracellular domain of CD80 include: binding / interacting with CD28, binding / interacting with CTLA-4, binding / interacting with PD-L1, and promoting CD80 homodimerization. In certain embodiments, the extracellular domain of CD80 comprises or consists of amino acids 1 to 242 of SEQ ID NO: 74.

[0324] 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 to 263 of SEQ ID NO: 74. In certain embodiments, the transmembrane domain of CD80 comprises or consists of amino acids 243 to 263 of SEQ ID NO: 74, or a fragment thereof. Such fragments may 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.

[0325] Non-limiting examples of co-stimulatory molecules include: CD28, 4-1BB, OX40, ICOS, DAP-10, CD27, CD40, NKG2D, CD2, and combinations thereof.

[0326] In certain embodiments, the fusion polypeptide includes the extracellular domain and the transmembrane domain of the costimulatory molecule of 4-1BB.In certain embodiments, the costimulatory molecule is human 4-1BB.In certain embodiments, 4-1BB includes the following items or consists of the following items: with SEQ ID NO: The amino acid sequence shown in 71 or its fragment 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 amino acid sequence, and / or it can optionally include up to one, or up to two or up to three conservative amino acid substitutions.In certain embodiments, 4-1BB includes the following items or consists of the following items: as SEQ ID NO: The amino acid sequence of the continuous part of the amino acid sequence of 71. In certain embodiments, the intracellular domain of 4-1BB comprises or consists of the following: 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 amino acids 214 to 255 of SEQ ID NO: 71 or a fragment thereof, and / or may optionally comprise at most one, or at most two, or at most three conservative amino acid substitutions. In certain embodiments, the intracellular domain of 4-1BB comprises or consists of the following: amino acids 214 to 255 of SEQ ID NO: 71 or a functional fragment thereof. Such a functional fragment may be a continuous portion of amino acids 214 to 255 of SEQ ID NO: 71 having a length of at least about 20, at least about 25, at least about 30, at least about 35, or at least about 40 amino acids. In certain embodiments, the functional fragment of amino acids 214 to 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 main function of the intracellular domain of 4-1BB. Non-limiting examples of the main functions of the intracellular domain of 4-1BB include: providing costimulatory signaling for activation and proliferation of immune response cells (e.g., T cells), and interacting with and activating downstream adapters (e.g., TRAFs). In certain embodiments, the intracellular domain of 4-1BB comprises or consists of the following items: amino acids 214 to 255 of SEQ ID NO: 71.

[0327] 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%, at least about 100% homologous or identical to the amino acid sequence shown in SEQ ID NO: 52 or a fragment thereof, and / or may optionally comprise at most one, or at most two, or at most three conservative amino acid substitutions. In certain embodiments, CD28 comprises or consists of an amino acid sequence that is a continuous 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%, at least about 100% homologous or identical to amino acids 180 to 219 of SEQ ID NO: 52 or a fragment thereof, and / or may optionally comprise at most one, or at most two, or at most three conservative amino acid substitutions. In certain embodiments, the intracellular domain of CD28 comprises or consists of amino acids 180 to 219 of SEQ ID NO: 52 or a functional fragment thereof. A functional fragment of amino acids 180 to 219 of SEQ ID NO: 52 may be a continuous portion of amino acids 180 to 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 the primary functions of the intracellular domain of CD28 include: providing co-stimulatory signaling for activation and proliferation of immune response 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.

[0328] 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 costimulatory molecule, the second costimulatory molecule, the third costimulatory molecule, the fourth costimulatory molecule, and the fifth costimulatory molecule may be the same or different from each other.

[0329] In certain embodiments, the fusion polypeptide comprises an extracellular domain and a transmembrane domain of a costimulatory ligand for CD80, and an intracellular domain of a costimulatory molecule for 4-1BB. In certain embodiments, the fusion polypeptide comprises or consists of the following: an amino acid sequence 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 shown in SEQ ID NO: 76. In certain embodiments, the fusion polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 76. SEQ ID NO: 76 is provided below.

[0330] MGHTRRQGTSPSKCPYLNFFQLLVLAGLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPT SNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL [SEQ ID NO: 76]

[0331] In certain embodiments, the fusion polypeptide comprises the extracellular domain and transmembrane domain of a co-stimulatory ligand that is CD80, the intracellular domain of a first co-stimulatory molecule that is 4-1BB, and the intracellular domain of a second co-stimulatory molecule that is CD28.

[0332] 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 shown in SEQ ID NO: 77. In certain embodiments, the fusion polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 77. SEQ ID NO: 77 is provided below.

[0333] MGHTRRQGTSPSKCPYLNFFQLLVLAGLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLS WLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL [SEQ ID NO: 77]

[0334] Various modified fusion polypeptides are disclosed in International Patent Application No. PCT / US20 / 42753, which is hereby incorporated by reference in its entirety.

[0335] 2.4. Gene disruption and gene modification

[0336] In certain embodiments, the cells disclosed herein that contain an antigen recognition receptor (e.g., a first antigen recognition receptor, e.g., a receptor disclosed in Section 2.1.2) further comprise a gene disruption of the CD70 locus. The gene disruption of the CD70 locus can cause a non-functional CD70 protein or a knockout of CD70 gene expression. In certain embodiments, the gene disruption of the CD70 locus causes a knockout of CD70 gene expression.

[0337] The limiting examples of gene disruption include substitution, deletion, insertion or a combination thereof. In certain embodiments, mutation includes missense mutation, nonsense mutation or a combination thereof. In certain embodiments, deletion includes non-frameshift deletion, frameshift deletion or a combination thereof. In certain embodiments, insertion includes non-frameshift insertion, frameshift insertion or a combination thereof.

[0338] In certain embodiments, the CD70 locus is a human CD70 locus. Gene disruption of the CD70 locus can be generated by any suitable gene editing method. In certain embodiments, a viral method is used to generate a genetic disruption of the CD70 locus (e.g., a knockout of the CD70 locus). 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, adeno-associated viral vectors, vaccinia virus, bovine papilloma virus, and herpes virus (e.g., such as Epstein-Barr virus).

[0339] In certain embodiments, non-viral methods are used to generate genetic disruption of the CD70 locus (eg, knockout of the CD70 locus). Non-viral methods can also be used for genetic modification of cells. For example, nucleic acid molecules can be introduced into cells by administering the nucleic acid in the presence of 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), by asialomucoid-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 have potential benefits for delivering DNA into cells. Transplanting normal genes into affected tissues of a subject can also be accomplished by transferring normal nucleic acids ex vivo into culturable cell types (e.g., autologous or allogeneic primary cells or their progeny), followed by injection of the cells (or their progeny) into targeted tissues or systemic injection. Recombinant receptors can also be derived or obtained using transposases or targeted nucleases (e.g., zinc finger nucleases, large-range nucleases or TALE nucleases, CRISPR). Transient expression can be obtained by RNA electroporation.

[0340] Any targeted genome editing method can also be used to generate gene disruption to the CD70 locus. In certain embodiments, gene disruption to the CD70 locus is generated by a method comprising homologous recombination, zinc finger nucleases, meganucleases, transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR) system, or a combination thereof.

[0341] In certain embodiments, a CRISPR system is used to generate a genetic disruption of the CD70 locus.

[0342] The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system is a genome editing tool found in prokaryotic cells. When used for genome editing, the system contains Cas9 (a protein that can modify DNA using crRNA as its guide), CRISPR RNA (crRNA, containing RNA used by Cas9 to guide it to the correct section of host DNA, and a region that binds to tracrRNA (usually in the form of a hairpin loop) to form an active complex with Cas9), trans-activating crRNA (tracrRNA, which binds to crRNA and forms an active complex with Cas9), and an optional section of DNA repair template (DNA that guides the cell's repair process, allowing for the insertion of a specific DNA sequence). CRISPR / Cas9 is usually transfected into target cells using a plasmid. The crRNA needs to be designed for each application, as this is the sequence that Cas9 uses to identify and directly bind to the target DNA in the cell. The repair template carrying the CAR expression cassette also needs to be designed for each application, as it must overlap with the sequences on either side 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 joined to the Cas9 gene and made into a plasmid for transfection into a cell. 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 guide editor. In certain embodiments, the CRISPR system comprises an epigenetic regulator. In certain embodiments, the CRISPR system comprises a CRISPRoff system. Additional details of the CRISPR system of the subject matter disclosed in this application can be found in Anzalone et al., Nature biotechnology 38.7 (2020): 824-844 and Nuñez et al., Cell 184.9 (2021): 2503-2519, the contents of each of these references being incorporated by reference in their entirety.

[0343] In certain embodiments, a gRNA molecule is used to disrupt the CD70 locus to knock out the expression of CD70. The gRNA molecule can target the coding sequence of a CD70 gene (e.g., a human CD70 gene) or the non-coding sequence of a CD70 gene (e.g., a human CD70 gene). In certain embodiments, the gRNA molecule targets the 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.

[0344] In certain embodiments, zinc finger nucleases are used to generate gene disruption at the CD70 locus. Zinc finger nucleases (ZFNs) are artificial restriction enzymes that are generated by combining zinc finger DNA-binding domains with DNA cleavage domains. Zinc finger domains can be engineered to target specific DNA sequences, which allows ZFNs to target desired sequences within the genome. The DNA-binding domain of a single ZFN typically contains multiple individual zinc finger repeats, and each can recognize multiple base pairs. The most common method for generating new zinc finger domains is to combine smaller zinc finger "modules" with known specificities. The most common cleavage domain in ZFNs is the non-specific cleavage domain from the type IIs restriction endonuclease FokI. Using the endogenous homologous recombination (HR) mechanism and a homologous DNA template carrying a CAR expression cassette, ZFNs can be used to insert the CAR expression cassette into the genome. When the target sequence is cleaved by a ZFN, the HR mechanism searches for homology between the damaged chromosome and the homologous DNA template, and then replicates the sequence of the template between the two broken ends of the chromosome, whereby the homologous DNA template is integrated into the genome.

[0345] In certain embodiments, the TALEN system is used to generate gene disruption at the CD70 locus. Transcription activator-like effector nucleases (TALENs) are restriction enzymes that can be engineered to cleave specific DNA sequences. The operating principle of the TALEN system is almost the same as that of ZFNs. They are generated by combining transcription activator-like effector DNA-binding domains with DNA cleavage domains. Transcription activator-like effectors (TALEs) are composed of 33-34 amino acid repeat motifs with two variable positions that have a strong recognition ability for specific nucleotides. By assembling arrays of these TALEs, the TALE DNA-binding domain can be engineered to bind the desired DNA sequence and thereby direct the nuclease to cleave at a specific location in the genome. cDNA expression for polynucleotide therapy methods can be directed by 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 construct). For example, if desired, enhancers known to preferentially direct gene expression in specific cell types can be used to direct the expression of the nucleic acid. The enhancers used can include, but are not limited to, enhancers characterized as tissue- or cell-specific enhancers. Alternatively, if a genomic clone is used as the therapeutic construct, regulation can be mediated by homologous regulatory sequences or (if desired) by regulatory sequences derived from a heterologous source containing any of the promoters or regulatory elements described above.

[0346] Methods for delivering genome editing agents / systems can vary as needed. In certain embodiments, the components of the selected genome editing method are delivered as DNA constructs in one or more plasmids. In certain embodiments, these components are delivered via viral vectors. Common delivery methods include, but are not limited to, electroporation, microinjection, gene gun, puncture, hydrodynamic pressure, continuous infusion, sonication, magnetofection, adeno-associated virus, pseudotyping of viral vectors with envelope proteins, cis- and trans-acting elements of replicative vectors, herpes simplex virus, and chemical agents (e.g., oligonucleotides, lipoplexes, polymersomes, polyplexes, dendrimers, inorganic nanoparticles, and cell-penetrating peptides).

[0347] In certain embodiments, gene disruption of the CD70 locus can be disruption of the coding region of the CD70 locus and / or disruption of the non-coding region of the CD70 locus. In certain embodiments, gene disruption of the CD70 locus includes disruption of the coding region of the CD70 locus. In certain embodiments, gene disruption of the CD70 locus includes an insertion at the coding region of the CD70 locus. The human CD70 protein contains three exons: exon 1, exon 2, and exon 3. In certain embodiments, gene disruption of the CD70 locus includes disruption at one or more of exon 1, exon 2, and exon 3 of the CD70 locus. In certain embodiments, gene disruption of the CD70 locus includes disruption at exon 1 of the CD70 locus. In certain embodiments, gene disruption of the CD70 locus includes an insertion at exon 1 of the CD70 locus.

[0348] In certain embodiments, the cells disclosed in this application that contain an antigen recognition receptor (e.g., a first antigen recognition receptor, e.g., the receptor disclosed in Section 2.1.2) further include gene disruption of the TRAC locus. In certain embodiments, gene disruption of the TRAC locus results in a non-functional TCR. In certain embodiments, gene disruption of the TRAC locus results in knockout of TCR gene expression.

[0349] Any method for generating gene disruption of the CD70 locus as disclosed above can be used to generate gene disruption of the TRAC locus. In certain embodiments, gene disruption of the TRAC locus is generated by a method that includes a gene editing method, the gene editing method including: homologous recombination, zinc finger nucleases, meganucleases, transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR) systems, or combinations thereof.

[0350] In certain embodiments, the cells disclosed herein further include genetic modification of the CD70 gene. Genetic modification of the CD70 gene may cause non-functional CD70 protein or knockdown of CD70 gene expression. In certain embodiments, genetic modification of the CD70 gene causes knockout of CD70 gene expression.

[0351] In certain embodiments, modification of the CD70 gene includes the use of RNAi agents (including but not limited to shRNA, siRNA, LNA, dsRNA and miRNA). In certain embodiments, the RNAi agent includes shRNA. In certain embodiments, the RNAi agent (e.g., shRNA) targets one or more isoforms of the CD70 gene, and thereby reduces or eliminates the expression of the CD70 gene or CD70 protein. In certain embodiments, the RNAi agent (e.g., shRNA) is expressed by the same construct expressing the first antigen recognition receptor and / or the second antigen recognition receptor disclosed herein. In certain embodiments, the expression of the RNAi agent (e.g., shRNA), the first antigen recognition receptor, and the second antigen recognition receptor is driven by the same promoter (e.g., the same promoter). In certain embodiments, the expression of the shRNA, the first antigen recognition receptor, and the second antigen recognition receptor disclosed herein is driven by different promoters.

[0352] In certain embodiments, the 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, the RNAi agent (e.g., shRNA) comprises a nucleotide sequence of at least about 10 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides that is complementary to a CD70 gene. In certain embodiments, the RNAi agent (e.g., shRNA) comprises a nucleotide sequence of up to 15 nucleotides, up to 20 nucleotides, up to 25 nucleotides, up to 30 nucleotides, up to 35 nucleotides, up to 40 nucleotides, up to 55 nucleotides, up to 60 nucleotides, up to 65 nucleotides, up to 70 nucleotides, up to 75 nucleotides, up to 80 nucleotides, up to 85 nucleotides, up to 90 nucleotides, up to 95 nucleotides, or up to 100 nucleotides in length. In certain embodiments, the RNAi agent comprises DNA or atypical or non-naturally occurring residues, such as, but not limited to, phosphorothioate residues.

[0353] In certain embodiments, the RNAi agent reduces the 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 thereof.

[0354] In certain embodiments, the gene disruption to the TRAC locus can be the disruption of the coding region of the TRAC locus and / or the disruption of the non-coding region of the TRAC locus. In certain embodiments, the gene disruption to the TRAC locus includes the disruption of the coding region of the TRAC locus. In certain embodiments, the gene disruption to the TRAC locus includes the insertion at the coding region of the TRAC locus. Human TRAC protein comprises 4 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 to the TRAC locus includes the disruption of one or more of exon 1 to exon 4 of the TRAC locus. In certain embodiments, the gene disruption to the TRAC locus includes the disruption at exon 1 of the TRAC locus. In certain embodiments, the gene disruption to the TRAC locus includes the insertion at exon 1 of the TRAC locus.

[0355] The cells disclosed herein can be isolated and activated by using CD3 / CD28 antibodies before the gene disruption is generated. In certain embodiments, the cells disclosed herein comprising an antigen recognition receptor (e.g., a first antigen recognition receptor, e.g., a receptor disclosed in Section 2.1.2) further comprise gene disruptions to the TRAC locus and the CD70 locus.

[0356] In certain embodiments, the gene disruption to the TRAC locus and the gene disruption to the CD70 locus are generated after isolation and activation of cells (e.g., T cells). In certain embodiments, the gene disruption to the TRAC locus and the gene disruption to the CD70 locus are generated prior to isolation and activation of cells (e.g., T cells).

[0357] In certain embodiments, the gene disruption to the TRAC locus is generated prior to isolation and activation of the cells (e.g., T cells), and the gene disruption to the CD70 locus is generated after isolation and activation of the cells (e.g., T cells). In certain embodiments, the gene disruption to the CD70 locus is generated prior to isolation and activation of the cells (e.g., T cells), and the gene disruption to the TRAC locus is generated after isolation and activation of the cells (e.g., T cells).

[0358] 3. Nucleic acid compositions and vectors

[0359] The subject matter disclosed in the present application provides a nucleic acid composition comprising a first polynucleotide encoding an antigen recognition receptor disclosed herein (e.g., disclosed in Section 2.1). A cell comprising such a nucleic acid composition is also provided. In certain embodiments, the nucleic acid composition further comprises a promoter operably connected to the antigen recognition receptor. In certain embodiments, the nucleic acid composition further comprises a second promoter operably connected to a second antigen recognition receptor (e.g., disclosed in Section 2.1).

[0360] In addition, the subject matter disclosed in the present application provides a nucleic acid composition comprising a polynucleotide encoding an antigen recognition receptor disclosed herein (e.g., disclosed in Section 2.1). A cell comprising such a nucleic acid composition is also provided. In certain embodiments, the nucleic acid composition further comprises a second promoter operably connected to a second antigen recognition receptor.

[0361] In certain embodiments, the subject matter disclosed in the present application provides a nucleic acid composition comprising a polynucleotide encoding an antigen recognition receptor disclosed herein (e.g., disclosed in Section 2.1) and a co-stimulatory ligand (e.g., disclosed in Section 2.2). In certain embodiments, the nucleic acid composition further comprises a promoter operably linked to the antigen recognition receptor. In certain embodiments, the nucleic acid composition further comprises a second promoter operably linked to the co-stimulatory ligand (e.g., disclosed in Section 2.2). In certain embodiments, the nucleic acid composition further comprises a third promoter operably linked to the second co-stimulatory ligand (e.g., disclosed in Section 2.2).

[0362] Additionally or alternatively, the subject matter disclosed in the present application provides a nucleic acid composition comprising a polynucleotide encoding an antigen recognition receptor disclosed herein (e.g., disclosed in Section 2.1) and a fusion polypeptide (e.g., disclosed in Section 2.3). In certain embodiments, the nucleic acid composition further comprises a first promoter operably linked to the antigen recognition receptor. In certain embodiments, the nucleic acid composition further comprises a second promoter operably linked to the fusion polypeptide.

[0363] In certain embodiments, one or both of the first promoter and the second promoter are endogenous or exogenous.

[0364] In certain embodiments, the exogenous promoter is selected from the group consisting of elongation factor (EF) -1 promoter, CMV promoter, SV40 promoter, PGK promoter, and metallothionein promoter. In certain embodiments, one or both of the first promoter and the second promoter are inducible promoters. In certain embodiments, the inducible promoter is selected from the group consisting of NFAT transcriptional response element (TRE) promoter, CD69 promoter, CD25 promoter, and IL-2 promoter.

[0365] In certain embodiments, the first antigen recognition receptor and / or the second antigen recognition receptor are integrated into a locus (e.g., a TRAC locus, a TRBC locus, a TRDC locus, or a TRGC locus) within the genome of the T cell. In certain embodiments, the locus is a TRAC locus. In certain embodiments, the expression of the first antigen recognition receptor and / or the second antigen recognition receptor is under the control of an endogenous promoter. Non-limiting examples of endogenous promoters include: an endogenous TRAC promoter, an endogenous TRBC promoter, an endogenous TRDC promoter, and an endogenous TRGC promoter. In certain embodiments, the endogenous promoter is an endogenous TRAC promoter.

[0366] In certain embodiments, the nucleic acid composition is a vector. In certain embodiments, the vector is a retroviral vector (e.g., a gamma-retroviral vector or a lentiviral vector). In certain embodiments, the vector is a viral vector selected from the group consisting of an adenoviral vector, an adeno-associated viral vector, a vaccinia virus, a bovine papilloma virus, and a herpes virus (e.g., such as Epstein-Barr virus).

[0367] In addition, nucleic acid compositions can be applied to subjects or / and 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 accomplished by transducing substantially homogeneous cell compositions with recombinant DNA constructs. In certain embodiments, retroviral vectors (gamma retroviruses or slow viruses) are used to introduce nucleic acid compositions into cells. For example, the first polynucleotide and the second polynucleotide can be cloned into a retroviral vector, and expression can be driven from its endogenous promoter, from a retroviral long terminal repeat sequence, or from a promoter specific to the target cell type of interest. Non-viral vectors can also be used.

[0368] The first polynucleotide and the second polynucleotide can be constructed in a single polycistronic expression cassette, in multiple expression cassettes of a single vector, or in multiple vectors. Examples of elements that generate polycistronic expression cassettes include, but are not limited to, various viral and non-viral internal ribosome entry sites (IRES, e.g., FGF-1 IRES, FGF-2 IRES, VEGF IRES, IGF-IIIRES, NF-κB IRES, RUNX1 IRES, p53 IRES, hepatitis A IRES, hepatitis C IRES, pestivirus IRES, aphthous virus IRES, picornavirus IRES, poliovirus IRES, and encephalomyocarditis virus IRES) and cleavable linkers (e.g., 2A peptides, e.g., P2A peptides, T2A peptides, E2A peptides, and F2A peptides). A combination of a retroviral vector and an appropriate packaging system is also suitable, in which the capsid protein will have the function of infecting human cells. Various cell lines that produce amphotropic viruses 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 are also suitable, for example, particles pseudotyped with VSVG, RD114 or GALV envelopes and any other known in the art.

[0369] Possible transduction methods also include direct co-cultivation of cells with producer cells, for example, by the method of Bregni, et al. (1992) Blood 80:1418-1422, or culturing with viral supernatant alone or concentrated vector stock with or without appropriate growth factors and polycations, for example, by the method of Xu, et al. (1994) Exp. Hemat. 22:223-230; and Hughes, et al. (1992) J. Clin. Invest. 89:1817.

[0370] Other transduction 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. U.S.A. 94:10319, 1997). Other viral vectors that can be used include, for example, adenovirus, lentivirus, and adeno-associated virus vectors, vaccinia virus, bovine papillomavirus, 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; LeGalLa Salle et al., Science 259:988-990, 1993; and Johnson, Chest 107:77S-83S, 1995 for vectors). Retroviral vectors are particularly well-established and have been used in the clinical setting (Rosenberg et al., N. Engl. J. Med 323:370, 1990; Anderson et al., U.S. Patent No. 5,399,346).

[0371] Non-viral methods can also be used for genetic modification of cells. For example, nucleic acid molecules can be introduced into cells by administering nucleic acids in the presence of 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), by asialomucoid-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 have potential benefits for delivering DNA into cells. Transplanting normal genes into the affected tissues of a subject may also be accomplished by transferring normal nucleic acids in vitro into culturable cell types (e.g., autologous or allogeneic primary cells or their progeny), followed by injection of cells (or their progeny) into targeted tissues or systemic injection. Transient expression can be obtained by RNA electroporation.

[0372] The method for delivering genome editing agents / systems can be varied as needed. In certain embodiments, the components of the selected genome editing method are delivered as DNA constructs in one or more plasmids. In certain embodiments, these components are delivered by viral vectors. Common delivery methods include but are not limited to electroporation, microinjection, gene guns, puncture, hydrostatic pressure, continuous infusion, ultrasonic treatment, magnetic infection, adeno-associated viruses, envelope protein pseudotypes of viral vectors, replicative vector cis- and trans-acting elements, herpes simplex viruses, and chemical mediators (e.g., oligonucleotides, lipoplexes, polymer vesicles, polycomplexes, dendrimers, inorganic nanoparticles, and cell penetrating peptides).

[0373] 3.1. Delivery method

[0374] The method for delivering genome editing agents / systems can be varied as needed. In certain embodiments, the components of the selected genome editing method are delivered as DNA constructs in one or more plasmids. In certain embodiments, these components are delivered by viral vectors. Common delivery methods include but are not limited to electroporation, microinjection, gene guns, puncture, hydrostatic pressure, continuous infusion, ultrasonic treatment, magnetic infection, adeno-associated viruses, envelope protein pseudotypes of viral vectors, replicative vector cis- and trans-acting elements, herpes simplex viruses, and chemical mediators (e.g., oligonucleotides, lipoplexes, polymer vesicles, polycomplexes, dendrimers, inorganic nanoparticles, and cell penetrating peptides).

[0375] In certain embodiments, the delivery method includes the use of a colloid. As used herein, the term "colloid" refers to a system in which two or more phases are present, wherein one phase (e.g., a dispersed phase) is distributed in another phase (e.g., a continuous phase). In addition, at least one of the phases has a small size (on the order of about 10 −9 to about 10 −6 m). Non-limiting examples of colloids encompassed by the presently disclosed subject matter include macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems (e.g., micelles, liposomes, and lipid nanoparticles).

[0376] In certain embodiments, the delivery method includes the use of liposomes. As used herein, the term "liposome" refers to a single or multilayer spherical lipid bilayer structure produced by lipids dissolved in an organic solvent and then dispersed in an aqueous medium. It is used experimentally and therapeutically to deliver active pharmaceutical ingredients (e.g., nucleic acid compositions disclosed herein) to cells, where the liposomes fuse with the cell membrane so that the contents are transferred to the cytoplasm.

[0377] In certain embodiments, the delivery method includes the use of lipid nanoparticles. As used herein, the term "lipid nanoparticle" refers to a particle having at least one nanometer-scale (e.g., about 1 nm to about 1,000 nm) size and including at least one lipid. In certain embodiments, the lipid nanoparticle may include an active pharmaceutical ingredient (e.g., a nucleic acid composition disclosed herein) for delivery to a cell. The morphology of the lipid nanoparticle may be different from a liposome. 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 reverse micelles surrounding an active pharmaceutical ingredient (e.g., a nucleic acid composition disclosed herein). Additional 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.

[0378] 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 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.

[0379] In certain embodiments, lipid nanoparticles may include cationic lipids or ionizable lipids. The term "cationic lipid" refers to a lipid comprising a head group with a permanent positive charge. Non-limiting examples of cationic lipids encompassed by the current disclosed subject matter include 1,2-di-O-octadecenyl-3-trimethylammonium-propane (DOTMA), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 2,3-dioleoyloxy-N-[2-(sperminecarboxamido)ethyl]-N, N-dimethyl-1-propylammonium trifluoroacetate (DOSPA) and ethylphosphatidylcholine (ePC).

[0380] As used herein, the term "ionizable lipid" refers to a lipid that is protonated at low pH and neutral at physiological pH. The pH sensitivity of ionizable lipids is particularly advantageous for in vivo delivery (e.g., delivery of the nucleic acid compositions disclosed herein) because neutral lipids interact less with the anionic membranes of blood cells and thus improve the biocompatibility of lipid nanoparticles. Once trapped in the endosome, the ionizable lipids are protonated and promote membrane instability, thereby allowing the nanoparticles to escape the endosome. Non-limiting examples of ionizable lipids encompassed by the subject matter disclosed herein include: 3,3',3'',3'''-(((methylazadiyl)bis(propane-3,1-diyl))bis(azatriyl))tetrapropionic acid tetra(8-methylnonyl) ester; (2-(dioctylammonio)ethyl)decyl phosphate; ((4-hydroxybutyl)azadiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate); 3,3'-((3- 9-oxo-10-oxa-13,14-dithia-3,6-diazacosanoyl)azadiyl)dipropionic acid bis(2-(dodecyldisulfanyl)ethyl) ester; 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azadiyl)bis(dodecan-2-ol); cKK-E12, 3,6-Bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione; (6Z,9Z,28Z,31Z)-heptahexaenoic acid-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate; Hexa(octan-3-yl) 9,9',9'',9''',9'''',9''''-((((benzene-1,3,5-tricarbonyl)tri(azadiyl))tri(propane-3,1-diyl))tri(azatri 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate; and (((3,6-dioxopiperazine-2,5-diyl)bis(butane-4,1-diyl))bis(azatriyl))tetra(ethane-2,1-diyl)(9Z,9'Z,9''Z,9'''Z,12Z,12'Z,12''Z,12'''Z)-tetra(octadec-9,12-dienoate).

[0381] In addition, in certain embodiments, the lipid nanoparticles may include other lipids. For example, but not limited to, the lipid nanoparticles of the currently disclosed subject matter may include 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 regulating integrity and rigidity. Non-limiting examples of other lipids present in 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 (POP E) and 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-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), and l,2-ditransoleoyl-sn-glycero-3-phosphoethanolamine (trans-DOPE).

[0382] In certain embodiments, the lipid nanoparticle may include a targeting portion that binds to a ligand. The use of a targeting portion allows the active pharmaceutical ingredient (e.g., a nucleic acid composition disclosed herein) to be selectively delivered to a target cell (e.g., a T cell) that expresses a ligand. In certain embodiments, the targeting portion 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 is an antibody or antigen binding fragment thereof that binds to a receptor (e.g., CD3, CD4, CD8, CD16, CD40L, CD95, FasL, CTLA-4, OX40, GITR, LAG3, ICOS, and PD-1) expressed on the surface of a T cell.

[0383] In certain embodiments, the delivery method is an in vivo delivery method. In certain embodiments, the delivery method is an ex vivo delivery method.

[0384] 4. Formulation and Administration

[0385] The presently disclosed subject matter provides compositions comprising cells disclosed herein (eg, disclosed in Section 2). In certain embodiments, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable excipient.

[0386] Compositions comprising cells disclosed herein can be conveniently provided in the form of sterile liquid preparations, for example, isotonic aqueous solutions, suspensions, emulsions, dispersions or viscous compositions, which can be buffered to selected pH. Liquid preparations are generally easier to prepare than gels, other viscous compositions and solid compositions. Additionally, it is slightly more convenient to use liquid compositions (especially by injection). On the other hand, viscous compositions can be formulated in a suitable viscosity range to provide a longer contact period with a particular tissue. 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 (for example, glycerol, propylene glycol, liquid polyethylene glycol, etc.) and a mixture suitable therefor.

[0387] Compositions comprising cells disclosed herein can be provided to subjects systemically or directly for inducing and / or enhancing immune responses to antigens and / or treating and / or preventing vegetation. In certain embodiments, cells disclosed herein or compositions comprising them are directly injected into organs of interest (e.g., organs affected by vegetation). Alternatively, cells disclosed herein or compositions comprising them are indirectly provided to organs of interest, e.g., by administration to the circulatory system (e.g., tumor vasculature). Amplifiers and differentiation agents can be provided before, during, or after administration of cells or compositions to increase in vitro or in vivo production of cells.

[0388] The number of cells to be administered can vary depending on the subject being treated. In certain embodiments, about 10 4 About 10 10 Between 10 4 About 10 7 Between 10 5 About 10 7 Between 10 5 About 10 9 Between or about 10 6 About 10 8 In certain embodiments, about 10 5 About 10 7 The cells disclosed herein are administered to a subject in an amount of at least about 1 × 10 cells. More potent cells can be administered in even smaller amounts. Typically, at least about 1 × 10 cells are administered. 5 cells, eventually reaching about l × 10 10 In certain embodiments, at least about 1 × 10 5 pcs, about 5 × 10 5 pcs, about 1 × 10 6 pcs, about 5 × 10 6pcs, about 1 × 10 7 pcs, about 5× 10 7 pcs, about 1 × 10 8 or about 5 × 10 8 In certain embodiments, about 1 × 10 cells disclosed herein are administered to a subject. 5 In certain embodiments, about 5 × 10 5 In certain embodiments, about 1 × 10 6 The presently disclosed cells. The exact determination of the dosage that will be considered an effective dose can be based on individual factors for each subject, including their size, age, sex, weight, and the condition of the particular subject. The dosage can be easily determined by those skilled in the art from this disclosure and knowledge in the art.

[0389] 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 the subject. Cells disclosed herein can be administered with any physiologically acceptable vehicle (usually intravascular), although it can also be introduced into other convenient positions (e.g., thymus) where the cells can find the appropriate position for regeneration and differentiation. Cells can be introduced by injection, catheter, etc.

[0390] Compositions comprising cells disclosed herein can be provided to subjects systemically or directly for inducing and / or enhancing immune responses to antigens and / or treating and / or preventing vegetation (e.g., cancer), pathogen infection or infectious diseases. In certain embodiments, cells, compositions or nucleic acid compositions disclosed herein are directly injected into organs of interest (e.g., organs affected by vegetation). Alternatively, cells, compositions or nucleic acid compositions disclosed herein are provided indirectly to organs of interest, for example, by administration to the circulatory system (e.g., tumor vasculature). Amplifiers and differentiation agents can be provided before, during or after administration of cells, compositions or nucleic acid compositions to increase in vitro or in vivo production of cells (e.g., T cells (e.g., CTL cells) or NK cells).

[0391] Compositions disclosed in the present application can be pharmaceutical compositions comprising cells disclosed in the present application or their progenitor cells and a pharmaceutically acceptable carrier. Administration can be autologous or allogeneic. For example, cells or progenitor cells can be obtained from a subject and applied to the same subject or different compatible subjects. Cells derived from peripheral blood or their progeny (e.g., in vivo, ex vivo or in vitro derived) can be administered via local injection, including catheter administration, systemic injection, local injection, intravenous injection or parenteral administration. When the therapeutic composition of the subject disclosed in the present application (e.g., a pharmaceutical composition comprising cells disclosed in the present application) is administered, it can be formulated into a unit dose injectable form (solution, suspension, emulsion).

[0392] 5. Treatment

[0393] The subject matter disclosed in this application provides various methods of using the cells disclosed in this application or compositions comprising them. The cells disclosed in this application and compositions comprising them can be used in therapy or medicine. For example, the subject matter disclosed in this application provides methods for inducing and / or increasing immune responses in subjects in need thereof. The cells disclosed in this application and compositions comprising them can be used to reduce tumor burden in subjects. The cells disclosed in this application and compositions comprising them can reduce the number of tumor cells, reduce tumor size and / or eradicate tumors in subjects. The cells disclosed in this application and compositions comprising them can be used to treat and / or prevent tumors (or neoplasms) in subjects. The cells disclosed in this application and compositions comprising them can be used to extend the survival of subjects with tumors. In certain embodiments, the tumor is cancer. The cells, compositions and nucleic acid compositions disclosed in this application can also be used to treat and / or prevent pathogen infection or other infectious diseases in subjects (such as immunocompromised human subjects). The cells, compositions and nucleic acid compositions disclosed in this application can also be used to treat and / or prevent autoimmune diseases in subjects. In certain embodiments, each of the above methods comprises: administering the cells disclosed herein or a composition (e.g., a pharmaceutical composition) comprising the cells to achieve a desired effect, e.g., to alleviate an existing condition or prevent recurrence. For treatment, the amount administered is an amount that is effective to produce the desired effect. An effective amount may be provided in a single administration or in a series of administrations. An effective amount may be provided in a bolus or by continuous perfusion.

[0394] Non-limiting examples of tumors (or neoplasms) include: blood cancers (e.g., leukemias, lymphomas, and myelomas), 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, laryngeal cancer, melanoma, neuroblastoma, adenocarcinoma, glioma, soft tissue sarcomas, and various carcinomas (including prostate cancer and small cell lung cancer). Suitable cancers further include any cancer known in the art of oncology, including, but not limited to, 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, endotheliosarcoma, squamous cell carcinoma, bronchoalveolar carcinoma, epithelial adenocarcinoma and its liver metastasis, lymphangiosarcoma, lymphangioendotheliosarcoma, hepatoma, bile duct carcinoma, synovioma, 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, Wilm's 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, cervical squamous and adenocarcinoma, uterine and ovarian epithelial carcinoma, prostate adenocarcinoma, bladder transitional squamous cell carcinoma, B cell 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 a cancer.

[0395] 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-related nasopharyngeal carcinoma, and ovarian cancer.

[0396] 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.

[0397] In certain embodiments, the tumor and / or neoplasm comprises tumor cells having a low antigen density of CD70. In certain embodiments, cells having a low antigen density of CD70 comprise a CD70 cell surface density of less than about 5,000 molecules / cell, less than about 4,000 molecules / cell, less than about 3,000 molecules / cell, less than about 2,000 molecules / cell, less than about 1,500 molecules / cell, less than about 1,000 molecules / cell, less than about 500 molecules / cell, less than about 200 molecules / cell, or less than about 100 molecules / cell.

[0398] In certain embodiments, the tumor and / or neoplasm comprises CD70 with a low frequency of tumor cells. + In certain embodiments, CD70 + Tumor cells have a frequency of less than about 50% / tumor, less than about 40% / tumor, less than about 30% / tumor, less than about 20% / tumor, less than about 15% / tumor, less than about 10% / tumor, less than about 5% / tumor, less than about 2% / tumor, or less than about 1% / tumor.

[0399] like Fig. 20A As shown, low antigen density and / or low tumor cell frequency can render CD70 polypeptide levels undetectable 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 blot, conjugate-ligand assay, immunohistochemical techniques, agglutination, complement assay, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), superdiffusion 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 generate false negative results, which may result in a lack of treatment for certain patients.

[0400] In certain embodiments, the tumor and / or neoplasm comprises a CD70 polypeptide that is not detectable by immunodiffusion, immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, Western blot, conjugate-ligand assay, immunohistochemical techniques, agglutination, complement assay, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), superdiffusion chromatography, or a combination thereof. In certain embodiments, the tumor and / or neoplasm comprises a CD70 polypeptide that is not detectable by immunohistochemistry (IHC).

[0401] further, Fig. 20A It is shown that CD70 polynucleotide levels allow detection of low antigen density and / or low tumor cell frequency. For example, but not limited to, CD70 polynucleotides can be detected by RNA-seq, single cell RNA-seq, quantitative RT-PCR, single cell qPCR, fluorescence 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 contain CD70 polynucleotides that can be detected by RNA-seq, single cell RNA-seq, quantitative RT-PCR, single cell qPCR, fluorescence 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 contain CD70 polynucleotides that can be detected by fluorescence in situ hybridization (FISH).

[0402] Additionally or alternatively, the subject matter disclosed herein provides methods for inducing and / or increasing an immune response, reducing tumor burden, treating and / or preventing tumors (or neoplasms), and / or prolonging survival in a subject having a tumor and / or neoplasm with undetectable levels of CD70 polypeptide. In certain embodiments, the method comprises: obtaining a sample from the subject. In certain embodiments, the sample is a tumor sample.

[0403] In certain embodiments, the sample comprises a CD70 polynucleotide that can be detected by FISH. In certain embodiments, when a CD70 polynucleotide is detected in a sample by FISH, the method comprises administering a cell disclosed herein or a composition (e.g., a pharmaceutical composition) comprising the cell to achieve a desired effect, for example, alleviating an existing condition or preventing recurrence.

[0404] like Fig. 17C and Fig.17DAs shown, Ezh2 inhibitors can increase CD70 polypeptide levels and make these detectable. Therefore, 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: tazerestat, 3-deazaadenine A (DZNep), EPZ005687, EI1, GSK126, and UNC1999. In certain embodiments, the Ezh2 inhibitor is tazerestat. 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 12 days, up to about 13 days, or up to about 14 days. In certain embodiments, contacting the sample with the Ezh2 inhibitor increases the level of CD70 polypeptide (e.g., detectable by IHC, immunodiffusion, immunoelectrophoresis, RIA, ELISA, immunofluorescence assay, Western blot, conjugate-ligand assay, immunohistochemical technique, agglutination, complement assay, HPLC, TLC, superdiffusion chromatography, or a combination thereof). In certain embodiments, when the Ezh2 inhibitor increases the level of CD70 polypeptide in the sample, the method includes 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.

[0405] In certain embodiments, the methods disclosed herein include: a) obtaining a sample having undetectable levels of CD70 polypeptide from a subject; b) detecting CD70 polynucleotides by FISH; and c) if CD70 polynucleotides are detected (e.g., detected by FISH), administering a cell disclosed herein or a composition comprising the same (e.g., a pharmaceutical composition).

[0406] In certain embodiments, the methods disclosed herein include: a) obtaining a sample having undetectable levels of CD70 polypeptide from a subject; b) contacting the sample with an Ezh2 inhibitor; and c) if the level of CD70 polypeptide is increased, administering a cell disclosed herein or a composition (e.g., a pharmaceutical composition) comprising the same.

[0407] In certain embodiments, the methods disclosed herein include: a) obtaining a sample having undetectable levels of CD70 polypeptide from a subject; b) contacting the sample with an Ezh2 inhibitor; and c) if CD70 polypeptide is detected (e.g., detected by IHC), administering a cell disclosed herein or a composition (e.g., a pharmaceutical composition) comprising the same.

[0408] The subject disclosed in the present application provides a method for treating and / or preventing viral infection in a subject. The method may include: applying an effective amount of the cell disclosed in the present application, the composition disclosed in the present application, or the nucleic acid composition disclosed in the present application to a subject suffering from viral infection. Non-limiting examples of viral infection include viral infection caused by the following items: cytomegalovirus (CMV), Epstein-Barr virus (EBV), hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, hepatitis F or hepatitis G, human immunodeficiency virus (HIV), adenovirus, BK polyomavirus, coronavirus, coxsackievirus, poliovirus, herpes simplex virus type 1, herpes simplex virus type 2, human cytomegalovirus, human herpes virus type 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., pneumovirus, measles virus, metapneumovirus, respiratory virus, or mumps virus), 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., hepatitis virus or flavivirus), Viruses), Herpesviridae (e.g., herpes simplex virus, varicella virus, cytomegalovirus, roseola virus, or lymphocytic cryptovirus), Orthomyxoviridae (e.g., influenza virus or togovirus), Parvoviridae (e.g., parvovirus), Picomaviridae (e.g., enterovirus or hepatovirus), Poxviridae (e.g., orthopoxvirus, fowlpox virus, or leporpoxvirus), Retroviridae (e.g., lentivirus or foamy virus), Reoviridae (e.g., rotavirus), Rhabdoviridae (e.g., rabies virus, exorbiculare, or vesiculovirus), and Togaviridae (e.g., alphavirus or rubella virus).In certain embodiments, viral infections include: human respiratory coronavirus, influenza virus types A to C, hepatitis virus types A to G, and herpes simplex virus 1 to 9. In certain embodiments, the subject suffers from an immunodeficiency.

[0409] The subject matter disclosed in this application provides a method for treating and / or preventing bacterial infection in a subject. The method may include administering an effective amount of the cell disclosed in this application, the composition disclosed in this application, or the nucleic acid composition disclosed in this application to a subject suffering from a bacterial infection. Bacterial infections include, but are not limited to, Mycobacteria, Rickettsia, Mycoplasma, Neisseria meningitides, Neisseria gonorrheoeae, Legionella, Vibrio cholerae, Streptococci, Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Corynobacteria diphtheriae, Clostridium spp., enterotoxigenic Eschericia coli, Bacillus anthracis, 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.

[0410] The subject matter disclosed in this application provides a method for treating and / or preventing an autoimmune disease in a subject. The method may include administering an effective amount of a cell disclosed in this application, a composition disclosed in this application, or a nucleic acid composition disclosed in this application to a subject suffering from an autoimmune disease.

[0411] The subject matter disclosed in the present application provides a method for treating and / or preventing an infectious disease in a subject. The method may include administering an effective amount of a cell disclosed in the present application, a composition disclosed in the present application, or a nucleic acid composition disclosed in the present application to a subject suffering from an infectious disease.

[0412] Non-limiting examples of autoimmune and inflammatory diseases or conditions thereof include: arthritis (e.g., rheumatoid arthritis (RA)), type I diabetes, systemic lupus erythematosus (SLE), inflammatory bowel disease, ulcerative colitis, psoriasis, psoriatic arthritis, scleroderma, autoimmune thyroid disease, Grave's disease, Crohn's disease, multiple sclerosis, systemic sclerosis, asthma, organ transplant rejection, diseases or conditions associated with transplantation, Takayasu's arteritis, giant cell arteritis, Kawasaki disease, polyarteritis nodosa, Behcet's syndrome, Wegener's granulomatosis, ANCA vasculitis, Chargé-Strauss syndrome, microscopic polyangiitis, connective tissue disease vasculitis, Hennoch-Schonlein purpura, cryoglobulinemia vasculitis, cutaneous leukocytoclastic vasculitis, sarcoidosis, Cogan's syndrome, Wiskott-Aldrich syndrome, primary CNS vasculitis, thromboembolic vasculitis, Thrombotic vasculitis, paraneoplastic arteritis, myelodysplastic syndrome, erythema acuminatum, amyloidosis, autoimmune myositis, Guillain-Barré syndrome, histiocytosis, atopic dermatitis, pulmonary fibrosis, glomerulonephritis, Whipple's disease, Still's disease, Sjogren's syndrome, myelofibrosis, chronic inflammatory demyelinating polyneuropathy, Kimura's disease, systemic sclerosis, chronic periaortitis, chronic prostatitis, idiopathic pulmonary fibrosis , chronic granulomatous disease, idiopathic bleomycin-induced lung inflammation, cytarabine-induced lung inflammation, 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. According to the subject matter disclosed in the present application, the above various methods may include: administering a checkpoint immune blocker to the subject.

[0413] The subject may have advanced disease, in which case the goal of treatment may include slowing or reversing disease progression, and / or ameliorating side effects. The subject may have a history of having been treated previously, in which case the goal of treatment will generally include reducing or delaying the risk of relapse.

[0414] Further modifications may be introduced into the cells disclosed herein to avoid or minimize the risk of immunological complications (referred to as "malignant T cell transformation"), for example, graft-versus-host disease (GvHD), or to cause results similar to GvHD when healthy tissue expresses the same target antigen as tumor cells. A potential solution to this problem is to engineer suicide genes 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) polypeptides. In certain embodiments, the suicide gene is an EGFRt polypeptide. The EGFRt polypeptide can be eliminated by administering an anti-EGFR monoclonal antibody (e.g., cetuximab) to achieve T cell elimination. EGFRt can be covalently linked to the upstream of an antigen recognition receptor. The suicide gene may be contained in a vector comprising a nucleic acid encoding an antigen recognition receptor disclosed herein. In this way, administration of a prodrug designed to activate a suicide gene (e.g., a prodrug (e.g., AP1903 that can activate iCasp-9)) during malignant T cell transformation (e.g., GVHD) triggers apoptosis in suicide gene-activated cells expressing the antigen recognition receptor disclosed herein. Integrating the suicide gene into the antigen recognition receptor disclosed herein confers an increased level of safety in the case of being able to eliminate the majority of the receptor-expressing cells in a very short period of time. The cells disclosed herein that incorporate the suicide gene can be pre-eliminated at a given time point after cell infusion, or eradicated at the earliest sign of toxicity.

[0415] 6. Kit

[0416] The subject matter disclosed in the present application provides a kit for inducing and / or enhancing an immune response and / or treating and / or preventing a neoplasm or pathogen infection (e.g., an autoimmune disease or an infectious disease) in a subject. In certain embodiments, the kit comprises an effective amount of a cell disclosed in the present application, a composition disclosed in the present application, or a nucleic acid composition disclosed in the present application. In certain embodiments, the kit comprises a sterile container; such a container may be a box, an ampoule, a bottle, a vial, a tube, a bag, a pouch, a blister pack, or other suitable container forms known in the art. Such containers may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for containing drugs. 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) for an antigen of interest in an expressible form, which may optionally be contained in the same or different carriers.

[0417] If desired, the cell, composition or nucleic acid composition is provided with instructions for administering the cell, composition or nucleic acid composition to a subject with a tumor (e.g., cancer) or pathogen infection (e.g., infectious disease) or immune disorder (e.g., autoimmune disease) or a risk of developing a tumor, pathogen infection or immune disorder. The instructions generally contain information about the use of the cell, composition or nucleic acid composition for treating and / or preventing a neoplasm or pathogen infection (e.g., infectious disease) or immune disorder (e.g., autoimmune disease). In certain embodiments, the instructions contain at least one of the following: a description of the therapeutic agent; dosage regimens and administration for treating or preventing a neoplasm, pathogen infection (e.g., infectious disease) or immune disorder (e.g., autoimmune disease) or symptoms thereof; precautions; warnings; indications; contraindications; overdose information; adverse reactions; animal pharmacology; clinical studies; and / or references. These instructions may be printed directly on the container (if present), or as a label applied to the container or as a separate sheet, booklet, card or folder provided with or in the container.

[0418] 7. Exemplary Implementation

[0419] Example 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 targeting CD70.

[0420] Embodiment 2. The method according to embodiment 2, wherein the method reduces the number of tumor cells, reduces the size of the tumor and / or eradicates the tumor in the subject.

[0421] Example 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 targeting CD70, wherein the tumor is renal cell carcinoma, pancreatic cancer, or ovarian cancer.

[0422] Embodiment 4. The method according to embodiment 3, wherein said method reduces the number of tumor cells, reduces the size of a tumor and / or eradicates a tumor in said subject.

[0423] Example 5. A method for preventing and / or treating a tumor in a subject having renal cell carcinoma neoplasms, 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 targeting CD70.

[0424] Example 6. 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 targeting CD70, wherein the tumor is a renal cell carcinoma neoplasm, renal cell carcinoma, pancreatic cancer, or ovarian cancer.

[0425] Example 7. A method for preventing and / or treating a tumor in a subject in need thereof, the method comprising: a) obtaining a tumor sample having undetectable levels of CD70 polypeptide from the subject; b) detecting CD70 polynucleotides by FISH; and c) if the CD70 polynucleotides are detected, administering to the subject an effective amount of cells comprising a TCR-like fusion molecule targeting CD70.

[0426] Example 8. A method for preventing and / or treating a tumor in a subject in need thereof, the method comprising: a) obtaining a tumor sample having undetectable levels of CD70 polypeptide from the subject; b) contacting the sample with an Ezh2 inhibitor; and c) if the CD70 polypeptide is detected in the sample, administering to the subject an effective amount of cells comprising a TCR-like fusion molecule targeting CD70.

[0427] Embodiment 9. A method according to any one of embodiments 1 to 8, wherein the TCR-like fusion molecule comprises: i) a first antigen binding chain, which comprises an antigen binding fragment of the heavy chain variable region (VH) of an antibody; and ii) a second antigen binding chain, which comprises an antigen binding fragment of the light chain variable region (VL) of the antibody; wherein the first antigen binding chain and the second antigen binding chain a) each comprise a TRAC polypeptide or a TRBC polypeptide, and b) bind to a second antigen, wherein the TCR-like fusion molecule binds to the second antigen in an HLA-independent manner.

[0428] Embodiment 10. The method of embodiment 9, wherein at least one of the TRAC polypeptide and the TRBC polypeptide is endogenous.

[0429] Embodiment 11. The method according to embodiment 9 or 10, wherein the first antigen binding chain and the second antigen binding chain are about 1 × 10 -8 The present invention binds to the second antigen with a dissociation constant (KD) of M or less.

[0430] Embodiment 12. The method according to any one of embodiments 9 to 11, wherein the first antigen binding chain and the second antigen binding chain are about 5×10 -9 The present invention binds to the second antigen with a dissociation constant (KD) of M or less.

[0431] Embodiment 13. A method according to any one of embodiments 9 to 12, wherein the first antigen binding chain comprises an antigen binding fragment of the VH of the antibody and a TRBC polypeptide, and the second antigen binding chain comprises an antigen binding fragment of the VL of the antibody and a TRAC polypeptide.

[0432] Embodiment 14. A method according to any one of embodiments 9 to 13, wherein the first antigen binding chain comprises an antigen binding fragment of the VH of the antibody and a TRAC polypeptide, and the second antigen binding chain comprises an antigen binding fragment of the VL of the antibody and a TRBC polypeptide.

[0433] 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 shown in SEQ ID NO: 34, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 35, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 36, and the second antigen-binding chain comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 37, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 38, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 39; or ii) the first antigen-binding chain comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 37, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 38, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 39, and the second antigen-binding chain comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 34, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 35, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: CDR3 with the amino acid sequence shown in 36.

[0434] Embodiment 16. A 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 shown in SEQ ID NO: 40, and the second antigen binding chain comprises CDR1, CDR2 and CDR3 of the light chain variable region shown in SEQ ID NO: 42; or ii) the first antigen binding chain comprises CDR1, CDR2 and CDR3 of the light chain variable region shown in SEQ ID NO: 42, and the second antigen binding chain comprises CDR1, CDR2 and CDR3 of the heavy chain variable region shown in SEQ ID NO: 40.

[0435] Embodiment 17. A method according to any one of Embodiments 9 to 15, wherein i) the first antigen-binding chain comprises the heavy chain variable region shown in SEQ ID NO: 40, and the second antigen-binding chain comprises CDR1, CDR2 and CDR3 of the light chain variable region shown in SEQ ID NO: 42; or ii) the first antigen-binding chain comprises the light chain variable region shown in SEQ ID NO: 42; and the second antigen-binding chain comprises the heavy chain variable region shown in SEQ ID NO: 40.

[0436] Embodiment 18. The method of any one of embodiments 9 to 17, wherein the first antigen binding chain and the second antigen binding chain are capable of associating with a CD3 zeta polypeptide.

[0437] Embodiment 19. A method according to embodiment 18, wherein the first antigen binding chain and the second antigen binding chain are capable of activating the CD3ζ polypeptide when bound to the second antigen.

[0438] Embodiment 20. The method according to embodiment 19, wherein said activation of said CD3ζ polypeptide is capable of activating said cells.

[0439] Embodiment 21. The method of any one of embodiments 9 to 20, wherein the cell further comprises a genetic disruption to the TRAC locus.

[0440] Embodiment 22. The method of any one of embodiments 9 to 20, wherein the cell further comprises a genetic disruption of the CD70 locus.

[0441] Embodiment 23. The method of any one of embodiments 9 to 20, wherein the cell further comprises genetic disruptions to the TRAC locus and CD70.

[0442] Embodiment 24. The method of any one of embodiments 1 to 23, wherein the tumor comprises tumor cells having a low CD70 antigen density.

[0443] Embodiment 25. The method of any one of embodiments 1 to 24, wherein the tumor has a low tumor cell frequency of CD70+ tumor cells.

[0444] 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 blot, conjugate-ligand assay, immunohistochemical technique, agglutination, complement assay, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), superdiffusion chromatography, or a combination thereof. In certain embodiments, the tumor and / or neoplasm comprises a CD70 polypeptide that is not detectable by immunohistochemistry (IHC).

[0445] Embodiment 27. The method of any one of Embodiments 1 to 26, wherein the tumor comprises a CD70 polypeptide by immunohistochemistry (IHC).

[0446] Embodiment 28. A method according to any one of embodiments 1 to 27, wherein the cell is a cell of the lymphoid lineage or a cell of the myeloid lineage.

[0447] Embodiment 29. The method according to 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.

[0448] Embodiment 30. A method according to any one of embodiments 1 to 29, wherein the cell is a T cell.

[0449] Embodiment 31. A method according to embodiment 30, wherein the T cells are derived from induced pluripotent stem cells.

[0450] Embodiment 32. The method according to embodiment 30 or 31, wherein the T cells are CD8+ T cells.

[0451] Embodiment 33. The method according to embodiment 32, wherein the CD8+ T cells are independent of CD4.

[0452] Embodiment 34. A method according to any one of embodiments 30 to 33, wherein 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.

[0453] Embodiment 35. A method according to any one of Embodiments 1 to 34, wherein the cell further comprises a chimeric antigen receptor (CAR) targeting a second antigen.

[0454] Example 36. A method according to Example 35, wherein the CAR comprises an extracellular antigen binding domain that binds to a first antigen, and an intracellular signaling domain capable of delivering an activation signal to the cell.

[0455] Example 37. A method according to Example 36, wherein the intracellular signaling domain of the CAR comprises a CD3ζ polypeptide.

[0456] Embodiment 38. The method according to embodiment 37, wherein the CD3ζ polypeptide is a natural CD3ζ polypeptide or a modified CD3ζ polypeptide.

[0457] Embodiment 39. The method according to embodiment 38, wherein the modified CD3ζ polypeptide comprises native ITAM1, an ITAM2 variant consisting of two loss-of-function mutations, and an ITAM3 variant consisting of two loss-of-function mutations.

[0458] Embodiment 40. A method according to any one of Embodiments 36 to 39, wherein the intracellular signaling domain of the CAR further comprises at least one co-stimulatory signaling region.

[0459] Embodiment 41. The method of embodiment 40, wherein the at least one co-stimulatory signaling region comprises at least the intracellular domain of a co-stimulatory molecule or a portion thereof.

[0460] Embodiment 42. A method according to embodiment 41, wherein the co-stimulatory 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.

[0461] Embodiment 43. A method according to any one of Embodiments 36 to 42, wherein the CAR comprises a transmembrane domain.

[0462] Embodiment 44. The method of any one of Embodiments 1 to 43, wherein said cell further comprises a chimeric co-stimulatory receptor (CCR).

[0463] Embodiment 45. A method according to embodiment 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 co-stimulatory signal to the cell but not delivering an activation signal to the cell alone.

[0464] Embodiment 46. A method according to Embodiment 45, wherein the intracellular domain of the CCR comprises at least the intracellular domain of a co-stimulatory molecule or a portion thereof.

[0465] Embodiment 47. The method according to embodiment 46, wherein the co-stimulatory 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.

[0466] Embodiment 48. The method of any one of Embodiments 35 to 47, wherein the second antigen is a tumor antigen or a pathogen antigen.

[0467] Embodiment 49. The method according to embodiment 48, wherein the tumor antigen is selected from the group consisting of: CD19, 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, CADM3, CAIX, CAPN3, CCDC155, CCR1, CD10, CD117, CD123, CD133, CD135 (FLT3), CD138, CD20, CD22, CD244(2B4), CD25, CD26, CD30, CD300LF, CD312, CD32, CD321, CD33, CD34, CD36, CD38, CD41, CD44, CD44V6, CD47, CD 49f, CD56, CD7, CD71, CD74, CD8, CD82, CD96, CD98, CD99, CDH13, CDHR1, CEA, CEACAM6, CHST3, CLEC12A, CLEC1 A. CLL1, CNIH2, COL15A1, COLEC12, CPM, CR1, CX3CR1, CXCR4, CYP4F11, DAGLB, DARC, DFNB31, DGKI, EGF1R, EGF R-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 ligand, NLGN3, NPAS2, NY-ESO-1, carcinoembryonic 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, SCIN, SCN11A, SCN2A, SCNN1D, SEC31B, SEMA4A, SH3PXD2A, SIGLEC11, SIRPB1, SLC16A6, SLC19A1, SLC22A5, SLC25A3 6. SLC25A41, SLC30A1, SLC34A3, SLC43A3, SLC44A1, SLC44A3, SLC45A3, SLC6A16, SLC6A6, SLC8A3, S LC9A1, SLCO2B1, SPAG17, STC1, STON2, SUN3, Survivin, 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.

[0468] Embodiment 50. The method of any one of Embodiments 1 to 49, wherein said cell further comprises at least one exogenous co-stimulatory ligand.

[0469] Embodiment 51. The method of embodiment 50, wherein the at least one exogenous co-stimulatory ligand is selected from the group consisting of: a tumor necrosis factor (TNF) family member, an immunoglobulin (Ig) superfamily member, and a combination thereof.

[0470] Embodiment 52. The method according to 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.

[0471] Embodiment 53. The method according to embodiment 51 or 52, wherein the Ig superfamily member is selected from the group consisting of: CD80, CD86, ICOSLG and combinations thereof.

[0472] Embodiment 54. The method of any one of Embodiments 50 to 53, wherein the at least one exogenous co-stimulatory ligand comprises CD80.

[0473] Embodiment 55. The method of any one of Embodiments 50 to 53, wherein the at least one exogenous co-stimulatory ligand comprises 4-1BBL.

[0474] Embodiment 56. The method of any one of Embodiments 50 to 53, wherein said cells comprise two exogenous co-stimulatory ligands.

[0475] Embodiment 57. The method of embodiment 56, wherein the at least two exogenous co-stimulatory ligands include CD80 and 4-1BBL.

[0476] Embodiment 58. A method according to any one of Embodiments 1 to 57, wherein the cell further comprises a fusion polypeptide comprising: a) an extracellular domain and a transmembrane domain of a co-stimulatory ligand, and b) an intracellular domain of a first co-stimulatory molecule.

[0477] Embodiment 59. The method of embodiment 58, wherein the co-stimulatory ligand is selected from the group consisting of: a tumor necrosis factor (TNF) family member, an immunoglobulin (Ig) superfamily member, and a combination thereof.

[0478] 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.

[0479] Embodiment 61. The method according to embodiment 59 or 60, wherein the Ig superfamily member is selected from the group consisting of: CD80, CD86, ICOSLG and combinations thereof.

[0480] Embodiment 62. The method of any one of Embodiments 58 to 61, wherein the co-stimulatory ligand is CD80.

[0481] Embodiment 63. A method according to any one of Embodiments 58 to 62, wherein the first co-stimulatory molecule is selected from the group consisting of: CD28, 4-1BB, OX40, ICOS, DAP-10, CD27, CD40, NKG2D, CD2 and combinations thereof.

[0482] Embodiment 64. The method of embodiment 63, wherein the first co-stimulatory molecule is 4-1BB.

[0483] Embodiment 65. The method of any one of Embodiments 58 to 64, wherein said co-stimulatory ligand is CD80 and said first co-stimulatory molecule is 4-1BB.

[0484] Embodiment 66. The method of any one of embodiments 58 to 65, wherein said fusion polypeptide further comprises an intracellular domain of a second costimulatory molecule.

[0485] Embodiment 67. The method of embodiment 66, wherein the second co-stimulatory molecule is selected from the group consisting of: CD28, 4-1BB, OX40, ICOS, DAP-10, CD27, CD40, NKG2D, CD2, and combinations thereof.

[0486] Embodiment 68. The method according to embodiment 66 or 67, wherein the second co-stimulatory molecule is CD28.

[0487] Embodiment 69. The method of any one of Embodiments 61 to 68, wherein the co-stimulatory ligand is CD80, the first co-stimulatory molecule is 4-1BB, and the second co-stimulatory molecule is CD28.

[0488] Embodiment 70. The method of any one of Embodiments 1 to 69, wherein said cells are autologous.

[0489] Embodiment 71. A method according to any one of Embodiments 1 to 69, wherein the cells are allogeneic.

[0490] Examples

[0491] Unless otherwise indicated, the practice of the present disclosure employs conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology within the skill of the art. Such techniques are fully explained in the following literature, such 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); "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of polynucleotides and polypeptides disclosed herein, and therefore can be considered in making and practicing the presently disclosed subject matter.Techniques particularly useful for specific embodiments are discussed in the following sections.

[0492] 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 their invention.

[0493] Example 1

[0494] To date, CAR-T cells have not been effective in eradicating solid tumors. Early 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 see good tumor responses. The main reason for the failure of CAR-T cells is due to tumor antigen heterogeneity. See Figure 1It is challenging to find a tumor target that is expressed on every tumor cell and is not also expressed on normal tissue. In RCC, CD70 is expressed on tumors. So far, approaches that include CD70 CAR or CAIX CAR or a combination of these have had limited efficacy.

[0495] To study renal cell carcinoma (RCC), the presently disclosed subject matter developed two patient-derived xenograft models (K5 and K7) from clear cell RCC patients.

[0496] like Figure 2B As shown, CD70 expression is highly maintained in an in vitro setting. Furthermore, T cells expressing anti-CD70 CAR can activate cytotoxicity in an in vitro model. Figure 3A Next, we demonstrated that anti-CD70 CAR T cells were able to induce cytotoxicity in an RCC model established by tail vein administration but not by orthotopic renal administration. 4A to 4D .

[0497] Next, we determined whether antigen downregulation was a possible mechanism for the observed resistance. Figure 6 As shown, CD70 was almost 100% positive in the lung, but only a portion of the renal tumor was CD70 positive. Next, it was determined that in vitro culture of untreated K5 and K7 cells with reduced CD70 expression restored the expression level of the antigen. Fig. 8A and Figure 8B Therefore, we explored whether expression of CD70 fusion proteins would improve tumor clearance in in vivo models. FIG. 10A to FIG. 10E As shown, overexpression of CD70 is sufficient to cause tumor clearance in the in vivo model. Collectively, these data indicate that: i) CD70 is downregulated in the kidney but not in the lung in untreated mice, ii) in vivo CD70 downregulation is reversed by culturing “CD70 low antigen” cells in vitro, and iii) lentiviral overexpression of CD70 causes clearance of renal tumors in K5 and K7 by SFG CD70 28z1xx but soon relapses in K7.

[0498] Because carbonic anhydrase IX (CAIX) is expressed in clear cell renal cell carcinoma and in sarcomatoid renal cell carcinoma, we determined whether this antigen is regulated and heterogeneously expressed as CD70. FIG. 11A to FIG. 11C It was shown that CAIX is heterogeneously expressed in K5 and K7 RCC cells and that it is activated by hypoxia (2% O 2) and is upregulated. Transcriptomic and proteomic analyses of sorted CD70 / CAIX double-negative, double-positive, and single-positive untreated renal tumors showed very low CD70 expression detected in the CD70 / CAIX double-negative population. Thus, CAIX and CD70 are regulated target antigens in RCC.

[0499] Next, a dual-targeting approach (pooling two CAR-T cells or dual transduction approach) using SFG-CD70 28z1xx CAR and SFG CAIX 28z1xx CAR was used. Anti-CAIX CAR T cells as well as the dual-targeting approach including anti-CD70 CAR T cells were able to kill in vitro. See Fig. 12A and Fig. 12B . However, in vivo studies showed that CD70 and CAIX dual-targeting did not result in tumor clearance due to residual low-density antigen populations. See FIG. 12C to FIG. 12I . In summary, CD70 / CAIX dual-targeting improved tumor control in K5 cells but not in K7 cells. Further, a significant amount of cells with low-density antigen was observed, and a slight improvement in tumor control was observed, but the "CD70 / CAIX double-negative" population still remained.

[0500] The subject matter disclosed in this application found that RCC contains such tumor cells that include a population of "low-density antigen" cells with low CD70 density (between 500 molecules / cell and 2,000 molecules / cell) that can be targeted by HIT CD70. RNA sequencing showed that this "CD70 lo" tumor cell population may be a tumor fraction with greater aggressiveness and enriched cancer stem cell pathways. Additionally, in vivo dynamic regulation of CD70 expression in the case where the low population can differentiate into the high population. See Fig.13E .

[0501] Thus, it is speculated that 70-HIT can completely eradicate tumors because it targets the more aggressive tumor population / cancer stem cell-like population. In fact, as FIG. 14A to FIG. 14E shown, 70-HIT cells co-expressing co-stimulatory ligands (e.g., including CD80 polypeptide and 4-1BBL, disclosed in Section 2.2) are able to address broad-spectrum expression (e.g., low-density antigen). This method successfully overcomes the antigen heterogeneity observed in RCC.

[0502] The 70-HIT disclosed in this application can be used to treat other solid tumors that express CD70 and have heterogeneous CD70 expression (e.g., glioblastoma, ovarian cancer, and pancreatic cancer).

[0503] Here, the subject matter disclosed in this application shows that HIT is a superior strategy to CAR for targeting heterogeneous targets in solid tumors. It is now possible to target other tumor targets (such as PMSA, mesothelin, CEA) knowing that expression is no longer positive vs. negative but a range of expression.

[0504] In the context of RCC and other solid tumors, HIT + SFG 80 / 41BBL is effective against K5 and K7 tumors. 70-HIT co-expressing co-stimulatory ligands (e.g., including CD80 polypeptide and 4-1BBL) disclosed in the present application represents a safe and effective therapy for RCC as well as for other solid tumors.

[0505] Example 2

[0506] CD70 is a cancer antigen that is expressed on the cell surface membrane of clear cell RCC (ccRCC) tumors but not on normal kidneys (Jilaveanu et al., Hum. Path. 2012). Importantly, its expression is retained in metastatic tissues. However, CD70 expression is heterogeneous. IHC ccRCC tumor microarrays identified 22% of ccRCC cases with ≥50% tumor cells positive for CD70 expression (Ye et al. J. Clinical Onc. 2022). Therefore, the subject matter disclosed in the present application determines whether CD70 represents an immunotherapy target in RCC.

[0507] To study renal cell carcinoma (RCC), two patient-derived xenograft models (K5 and K7) from clear cell RCC patients were developed. Details of the K5 and K7 cell lines are described in the table below and Figure 2A , while the expression level of CD70 is depicted in Figure 2B middle.

[0508]

[0509] Injection of both cell lines into the tail vein resulted in engraftment in the lungs and subsequently in the liver ( Figure 3B and Figure 3C ).

[0510] Next, the ability of T cells expressing anti-CD70 CARs to kill K5 and K7 cells in vitro was determined. Briefly, T cells were engineered to overexpress a CAR targeting CD70 and containing a 1XX domain (for details, see Figure 2C and Section 2.1). Figure 3BAs shown in Figure 2, anti-CD70 CAR T cells were able to induce killing of K5 and K7 cells in vitro. However, although in vivo killing by anti-CD70 CAR T cells was observed in tumors established in the lung, no effect was observed in the primary orthotopic RCC model ( 4A to 4D ). Figure 5 This shows that the effect is not due to a different ability to reach the tumor site. Therefore, differential CD70 expression leads to differential killing ability between tumor sites.

[0511] Next, the presently disclosed subject matter identified the mechanism behind the observed resistance. 4A to 4D As shown, tumors established at the orthotopic site are not inherently resistant to CAR therapy. Therefore, it is speculated that CD70 may play a role in the observed resistance. FACS analysis showed differential CD70 expression between untreated lung tumors and kidney tumors established by K5 and K7 cell lines ( Figure 6 Because these tumors are enriched for low levels of CD70 (CD70 lo - ) of cells ( Figure 7 ), so these cells were cultured in vitro to study their expression profile. Of note, CD70 lo - Cells regained CD70 expression over time ( Fig. 8A and Figure 8B Epigenetic analysis showed that the CD70 locus, but not the CAIX promoter locus, was epigenetically regulated ( Fig.9A and Fig. 9B ), and Ezh2 protein is able to approach the CD70 promoter ( Fig. 9C Therefore, we determined whether inhibition of Ezh2 could regulate CD70 expression. Fig.9D As shown, treatment with tazemetostat restored CD70 lo - Thus, the presently disclosed subject matter shows that CD70 expression is epigenetically regulated at different organ sites (e.g., primary vs. metastatic) in the same mouse via Ezh2-mediated repression of H3K27me3, and that in vitro inhibition of Ezh2 results in restoration of CD70 in CD70 lo renal tumors.

[0512] To further confirm the observed data, K5 and K7 cells were engineered to overexpress CD70 and implanted to establish tumors. Fig. 10D and Fig. 10EAs shown, overexpression of CD70 in an orthotopic model caused tumor clearance. Collectively, these data suggest that: i) differential CD70 expression causes differential killing between sites; ii) CD70 expression is epigenetically regulated at different organ sites (primary vs. metastatic) in the same mouse via Ezh2-mediated H3K27me3 repression; iii) Ezh2 inhibition in vitro causes CD70 restoration in CD70 lo renal tumors; iv) CD70 CAR T cells fail to clear orthotopic sites because CD70 is downregulated in vivo below the detection threshold of CAR T cells; and v) exogenous CD70 overexpression achieves CAR killing. Carbonic anhydrase 9 (CAIX) is a tumor antigen expressed in several solid tumors, including, for example, ccRCC, GBM, ovarian cancer, and colorectal cancer (Campos, NSPd et al. Cancers 2022). CAIX is induced by hypoxia, and its expression in normal tissues is observed in intrahepatic bile ducts, gastric mucosa, and duodenum. The first generation of anti-CAIX CAR T-cell therapy for metastatic ccRCC showed no clinical response and toxicity (Lamers et al. 2016). Given these characteristics, the role of CAIX and CD70 was further investigated.

[0513] In view of these findings, the inventors of the subject matter disclosed in this application wanted to determine whether alternative antigen recognition receptors and fusion polypeptides that can provide co-stimulatory signals to cells can restore the resistance of K5 and K7 derived renal orthotopic tumors. T cells were engineered to express a CAR targeting CD70 and comprising an 1XX domain or a HIT receptor targeting CD70 using the same antigen binding fragment as the CAR; in addition, some T cells were further engineered to contain a CD80 polypeptide and 4-1BBL, as disclosed in Section 2.2. As FIG. 14A to FIG. 14E and Fig.15 As shown, T cells expressing the HIT receptor and fusion protein targeting CD70 (e.g., a fusion protein having SEQ ID NO: 76) were able to induce a complete response in K5 and K7 derived renal orthotopic tumors. This effect was due to the exhaustion of CAR T cells, as evidenced by the higher expression profile of PD1, TIM3, and LAG3 observed in CAR T cells compared to HIT cells ( Fig.15 ).

[0514] Further, the presently disclosed subject matter establishes that HIT CD70 T cell efficacy is not due to bystander killing of CD70 negative tumors ( FIG. 16A to FIG. 16C), because knocking out CD70 on K5 or K7 PDX lines renders tumors resistant to HIT CD70 killing. Overall, the data disclosed in this application show that: i) CD70 expression is not binary, but rather a spectrum of expression ranging from high to very low; ii) highly sensitive CD70 HIT T cells can effectively target this very low CD70 population; iii) CAR T cells were up to 40% to 50% positive for the three exhaustion markers at days 7 and 14, compared to less than 15% for HIT T cells; and iv) HIT CD70 T cell efficacy was not due to bystander killing of CD70-negative tumors.

[0515] Recently, adoptive cell therapy involving an anti-CD70 CAR has been developed in which responses were observed only in tumors expressing high levels of CD70 (Srour et al., Cancer Research 83.8_Suppl (2023): CT011-CT011). Therefore, the subject matter disclosed in this application investigates whether CD70 lo responses are observed in other tumors. - SK-OV3 is a cell line that can develop ovarian carcinoma in situ and intraperitoneal carcinoma expressing CD70 ( Fig.19A and Fig.19B Importantly, administration of cells expressing the HIT receptor and fusion protein targeting CD70 (e.g., a fusion protein having SEQ ID NO: 76) was able to induce a complete and durable response and overcome tumor ( FIG. 19C to FIG. 19E ).

[0516] Next, it was determined whether cells expressing the HIT receptor and fusion protein targeting CD70 (e.g., a fusion protein having SEQ ID NO: 76) could rescue the effects of resistant pancreatic cancer. The PANC-1 cell line was analyzed for its CD70 expression profile and ability to establish orthotopic cancer ( Fig.18A Notably, cells expressing the HIT receptor and fusion protein targeting CD70 (e.g., a fusion protein having SEQ ID NO: 76) were able to induce a complete response and overcome tumor challenge in pancreatic cancer, due to the CD70 lo - The presence of cells ( Fig.18B ). Further, pancreatic ductal adenocarcinoma PDX (PDAC2) was analyzed for CD70 expression. Notably, this invasive pancreatic PDX in vitro was heterogeneous for CD70, with only approximately 23% positive CD70 expression in vitro and 30% positive CD70 expression in vivo at the orthotopic site in the pancreas ( FIG. 17A to FIG. 17BInterestingly, cells expressing the HIT receptor and fusion protein targeting CD70 (e.g., a fusion protein having SEQ ID NO: 76) were able to induce a complete response in an orthotopic model of pancreatic PDAC2, and this effect was specific for CD70, as knockout of CD70 on PDAC2 tumors rendered the tumors resistant to HIT CD70 T cell killing ( Fig.17E ). The CD70 locus was confirmed to be expressed in PDAC2 (an alternative pancreatic cancer cell type; see Fig. 17C and Fig.17D These data demonstrate the heterogeneity of CD70 and its epigenetic regulation in pancreatic, ovarian, and renal cancers.

[0517] Finally, it was determined whether conventional diagnostic methods (e.g., IHC) could identify these cells with CD70 lo - Cell tumors. Fig. 20A As shown, CD70 lo - The tumor-causing cells were negative for IHC testing, highlighting the limitations of conventional diagnostic methods.

[0518] In summary, the subject matter disclosed in this application shows that: i) HIT outperforms CAR in RCC, pancreatic cancer, and ovarian cancer with in vivo heterogeneous CD70 expression; ii) the mechanism of CD70 downregulation by Ezh2 inhibition may be conserved across CD70 heterogeneous tumors; iii) in CD70 heterogeneous PDAC2 tumors, in vitro Ezh2 inhibition can restore CD70 expression, indicating that partial CD70-positive tumors may mask tumors with very low CD70 expression, which can be revealed by Ezh2 inhibition; iv) conventional IHC cannot capture very low CD70 expression, and RNA FISH methods can be applied as a clinical diagnostic tool; v) it may be necessary to re-stratify patients who may now benefit from treatment, who would have been overlooked based on partial / negative CD70 expression; and vi) the identification that CD70-negative tumors may have very low CD70 expression opens a way forward for single targeted approaches or HITCD70 as the basis in dual targeted approaches.

[0519] Embodiments of the Presently Disclosed Subject Matter

[0520] From the foregoing description, it is obvious that the presently disclosed subject matter can be changed and modified to adapt it to various usages and conditions.Such embodiments are also within the scope of the following claims.

[0521] The recitation of a list of elements in any definition of a variable herein includes defining the 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.

[0522] All patents and publications mentioned in this specification are herein incorporated by reference as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.

Claims

1. A method of reducing tumor burden in a subject suffering from renal cell carcinoma, pancreatic cancer or ovarian cancer, the method comprising: include: An effective amount of cells comprising a TCR-like fusion molecule targeting CD70 is administered to the subject.

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

3. A method for reducing tumor burden in a subject, the method comprising: include: An effective amount of cells comprising a TCR-like fusion molecule targeting CD70 is administered to the subject, wherein the tumor is renal cell carcinoma, pancreatic cancer, or ovarian cancer.

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

5. A method for preventing and / or treating a tumor in a subject having renal cell carcinoma neoplasms, renal cell carcinoma, pancreatic cancer or ovarian cancer, the method comprising: include: An effective amount of cells comprising a TCR-like fusion molecule targeting CD70 is administered to the subject.

6. A method for preventing and / or treating a tumor in a subject, the method comprising: include: An effective amount of cells comprising a TCR-like fusion molecule targeting CD70 is administered to the subject, wherein the tumor is a renal cell carcinoma neoplasm, renal cell carcinoma, pancreatic cancer, or ovarian cancer.

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

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

9. A method according to any one of claims 1 to 8, wherein the TCR-like fusion molecule comprises: i) a first antigen binding chain, which comprises an antigen binding fragment of the heavy chain variable region (VH) of an antibody; and ii) a second antigen binding chain, which comprises an antigen binding fragment of the light chain variable region (VL) of the antibody; wherein the first antigen binding chain and the second antigen binding chain a) each comprise a TRAC polypeptide or a TRBC polypeptide, and b) bind to a second antigen, wherein the TCR-like fusion molecule binds to the second antigen in an HLA-independent manner.

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

11. The method of claim 9, wherein the first antigen-binding chain and the second antigen-binding chain are approximately 1 × 10 -8 The present invention binds to the second antigen with a dissociation constant (KD) of M or less.

12. The method of claim 9, wherein the first antigen-binding chain and the second antigen-binding chain are approximately 5 × 10 -9 The present invention binds to the second antigen with a dissociation constant (KD) of M or less.

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

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

15. The method according to claim 9, wherein i) the first antigen-binding chain comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 34, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 35, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 36, and the second antigen-binding chain comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 37, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 38, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 39; or ii) the first antigen-binding chain comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 37, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 38, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 39, and the second antigen-binding chain comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 34, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 35, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO:

36.

16. The method according to claim 9, wherein i) the first antigen-binding chain comprises CDR1, CDR2 and CDR3 of the heavy chain variable region shown in SEQ ID NO: 40, and the second antigen-binding chain comprises CDR1, CDR2 and CDR3 of the light chain variable region shown in SEQ ID NO: 42; or ii) the first antigen-binding chain comprises CDR1, CDR2 and CDR3 of the light chain variable region shown in SEQ ID NO: 42, and the second antigen-binding chain comprises CDR1, CDR2 and CDR3 of the heavy chain variable region shown in SEQ ID NO:

40.

17. The method according to claim 9, wherein i) the first antigen-binding chain comprises the heavy chain variable region shown in SEQ ID NO: 40, and the second antigen-binding chain comprises CDR1, CDR2 and CDR3 of the light chain variable region shown in SEQ ID NO: 42; or ii) the light chain variable region shown in SEQ ID NO: 42 of the first antigen-binding chain; and the second antigen-binding chain comprises the heavy chain variable region shown in SEQ ID NO:

40.

18. The method of claim 9, wherein the first antigen binding chain and the second antigen binding chain are capable of associating with a CD3 zeta polypeptide.

19. The method of claim 18, wherein the first antigen binding chain and the second antigen binding chain are capable of activating the CD3 zeta polypeptide when bound to the second antigen.

20. The method of claim 19, wherein said activation of said CD3zeta polypeptide is capable of activating said cell.

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

22. The method of claim 9, wherein the cells further comprise a genetic disruption of the CD70 locus.

23. The method of claim 9, wherein the cells further comprise genetic disruptions to the TRAC locus and CD70.

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

25. The method of claim 9, wherein the tumor contains a low frequency of CD70 + Tumor cells.

26. The method of claim 9, wherein the tumor comprises a CD70 polypeptide that is not detectable by immunodiffusion, immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, Western blot, conjugate-ligand assay, immunohistochemical techniques, agglutination, complement assay, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), superdiffusion chromatography, or a combination thereof.

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

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

32. The method according to claim 30 or 31, wherein the T cells are CD8 + T cells.

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

34. The method of any one of claims 30 to 33, wherein the T cell is 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.

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

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

37. The method of claim 36, wherein the intracellular signaling domain of the CAR comprises a CD3 zeta polypeptide.

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

39. The method of claim 38, wherein the modified CD3zeta polypeptide comprises native ITAM1, an ITAM2 variant consisting of two loss-of-function mutations, and an ITAM3 variant consisting of two loss-of-function mutations.

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

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

42. The method of claim 41, wherein the co-stimulatory 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. The method of any one of claims 1 to 43, wherein the cell further comprises a chimeric co-stimulatory receptor (CCR).

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 co-stimulatory signal to the cell but not delivering an activation signal to the cell alone.

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

47. The method of claim 46, wherein the co-stimulatory 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. The method of any one of claims 35 to 47, wherein the second antigen is a tumor antigen or a pathogen antigen.

49. The method of claim 48, wherein 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 cytomegalovirus (CMV) infected cells (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 (FLT3), CD138, CD20, CD22, CD244 (2B4), CD25, CD26, CD30, CD300LF, CD312, CD32, CD321, CD33, CD34, CD36, CD38, CD41, CD 44. CD44V6, CD47, CD49f, CD56, CD7, CD71, CD74, CD8, CD82, CD96, CD98, CD99, CDH13, CDHR 1. CEA, CEACAM6, CHST3, CLEC12A, CLEC1A, CLL1, CNIH2, COL15A1, COLEC12, CPM, CR1, CX3CR1, CXCR4, CYP4F11, DAGLB, DARC, DFNB31, DGKI, EGF1R, EGFR-VIII, EGP-2, EGP-40, ELOV L6, 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, 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, NKG2D ligand, NLGN3, NPAS2, NY-ESO-1, carcinoembryonic 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, SCIN, SCN11A, SCN2A, SCNN1D, SEC31B, SEMA4A, SH3PXD2A, SIGLEC11, SIRPB1, SLC16A6, SLC19A1, SLC22A5, SLC25A3 6. SLC25A41, SLC30A1, SLC34A3, SLC43A3, SLC44A1, SLC44A3, SLC45A3, SLC6A16, SLC6A6, SLC8A3, SLC9A1, SLCO2B1, SPAG17, STC1, STON2, SUN3, survivin, 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.

50. The method of any one of claims 1 to 49, wherein the cell further comprises at least one exogenous co-stimulatory ligand.

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

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), CD40L.

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. The method of any one of claims 50 to 53, wherein the at least one exogenous co-stimulatory ligand comprises CD80.

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

56. The method of any one of claims 50 to 53, wherein the cells comprise two exogenous co-stimulatory ligands.

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

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

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

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. 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. The method of any one of claims 58 to 61, wherein the co-stimulatory ligand is CD80.

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

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

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

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

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

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

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

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

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

Images