Modified immune cells and uses thereof

Abstract

To provide modified immune cells including tumor infiltrating lymphocytes or B cells, compositions containing the immune cells, and methods for treating neoplasm or cancerous conditions comprising administration of the immune cells to the subject.SOLUTION: A modified immune cell that specifically binds to a neoantigen is disclosed, where the modified immune cell comprises: (a) a switch molecule comprising an extracellular domain (ECD) of a protein that, in unmodified immune cells, elicits an immune cell inactivation signal upon binding to its ligand, where the ECD is fused to an intracellular domain of a co-stimulatory molecule that mediates an immune cell activation signal, and where binding of the switch molecule to the ligand yields an immune cell activation signal instead of the immune cell inactivation signal in the modified immune cell, and (b) a chimeric antigen receptor comprising: (i) an antigen interacting domain capable of binding a B cell surface protein; (ii) a transmembrane domain; and (iii) an intracellular signaling domain.SELECTED DRAWING: Figure 10

Description

[Technical field]

[0001] [CROSS REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of and priority to Chinese Patent Application No. 201711101450.X, filed November 10, 2017, Chinese Patent Application No. 201810017770.5, filed January 9, 2018, Chinese Patent Application No. 201810037682.1, filed January 16, 2018, PCT International Application No. PCT / CN2018 / 090638, filed June 11, 2018, PCT International Application No. PCT / CN2018 / 094126, filed July 2, 2018, and PCT International Application No. PCT / CN2018 / 114897, filed November 9, 2018, which are incorporated by reference in their entireties herein. [Background technology]

[0002] Immunotherapy involves modifying a patient's own immune cells to redirect cytotoxicity to targeted cells, such as cancer cells. Modified immune cells, such as T cells, expressing chimeric antigen receptors (CARs) can exploit endogenous immune cell signaling for immune cytotoxicity.

[0003] Conventional immunotherapies suffer from a variety of deficiencies, including insufficient signaling from costimulatory receptors for sustained and / or adequate immune responses for therapeutic efficacy, insufficient specificity of engineered immune cells for diseased cells, such as cancer cells (e.g., on-target off-tumor effects and toxicity), and activation of immune suppressive mechanisms, all of which can minimize the effectiveness of the immune response. Summary of the Invention

[0004] In view of the above, there is a significant need for alternative systems and methods for performing immunotherapy. The compositions and methods of the present disclosure address this need and also provide additional advantages. In particular, various aspects of the present disclosure provide compositions and methods for inducing immune cell activation signals by signaling through binding to ligands that would normally induce immune cell inactivation signals. The compositions and methods can also induce immune cell activation signals through binding to B cell surface proteins.

[0005] In one aspect, the disclosure provides an engineered immune cell that specifically binds to a tumor antigen, wherein the engineered immune cell comprises a chimeric stimulating molecule, where the chimeric stimulating molecule comprises an extracellular domain (ECD) of a protein that induces an immune cell inactivation signal in a non-engineered immune cell upon binding to its ligand, where the ECD is fused to an intracellular domain (ICD) of a costimulatory molecule that mediates an immune cell activation signal, and where binding of the chimeric stimulating molecule to the ligand results in an immune cell activation signal instead of an immune cell inactivation signal in the engineered immune cell.

[0006] In some embodiments, the engineered immune cells are tumor infiltrating lymphocytes (TILs), where, optionally, the TILs express at least one of PD-1, CD137, and TIM-3.

[0007] In one aspect, the disclosure provides an engineered T cell that specifically binds to a neoantigen, wherein the engineered T cell comprises a switch molecule, wherein the switch molecule comprises an extracellular domain (ECD) of a protein that upon binding to its ligand induces an immune cell activation signal in a non-engineered T cell, wherein the ECD is fused to an intracellular domain (ICD) of a costimulatory molecule that mediates the immune cell activation signal, and wherein binding of the switch molecule to the ligand results in an immune cell activation signal instead of an immune cell deactivation signal in the engineered immune cell.

[0008] In some embodiments, the T cell may comprise a T cell receptor (TCR) complex that exhibits specific binding to a neoantigen. In some embodiments, the TCR complex may be an endogenous TCR complex. In some embodiments, the TCR complex may be an exogenous TCR complex.

[0009] In some embodiments, neoantigens may comprise peptide fragments of proteins encoded by mutated genes, where the genes are ABL1, ACOL 1997, ACVR2A, AFP, AKT1, ALK, ALPPL2, ANAPC1, APC, ARID1A, AR, AR-v7, ASCL2, β2M, BRAF, BTK, C15ORF40, CDH1, CLDN6, CNOT1, CT45A5, CTAG1B, DCT, DKK4, EEF1B2, EEF1DP3, EGFR, EIF2B3, env, EPHB2, ERBB3, ESR1, ESRP1, FAM11 IB, FGFR3, FRG1B, GAGE1, GAGE ​​10, GATA3, GBP3, HER2, IDH1, JAK1, KIT, KRAS, LMAN1, MABEB 16, MAGEA1, MAGEA10, MAGEA4, MAGEA8, MAGEB 17, MAGEB4, MAGEC1, MEK, MLANA, MLL2, MMP13, MSH3, MSH6, MYC, NDUFC2, NRAS, PAGE2, PAGE5, PDGFRa, PIK3CA, PMEL, pol protein, POLE, PTEN, RAC1, RBM27, RNF43, RPL22, RUNX1, SEC31A, SEC63, SF3B 1, SLC35F5, SLC45A2, SMAP1, SMAP1, SPOP, TFAM, TGFBR2, THAP5, TP53, TTK, TYR, UBR5, VHL, and XPOT. In some embodiments, the neoantigen may comprise a peptide fragment of a protein encoded by a mutated gene, where the gene is JAK2, KRAS, BRAF, TP53, PIK3CA, EGFR, IDH1, NRAS, CTNNB1, NPM1, CALR, FGFR3, CDKN2A, KIT, MYD88, APC, HRAS, MED12, DNMT3A, GNAS, IDH2, KCNJ5, PTEN, NOTCH1, SF3B1, FLT3, ASXL1, SRSF2, FOXL2, PTPN11, GNAQ, RET, HLA-A, MPL, IKZF1, KMT2C, TET2, PDGFRA, FBXW7, H3F3A, ALK, CEBPA, ESR1, AKT1, RUNX1, GNA11, VHL, WT1, U2AF1, ABL1, ERBB2, DICER1,NOTCH4, EZH2, HNF1A, SMARCB1, CXCR4, PLCG1, TSHR, PRKACA, RHOA STAT3, POLE, SETBP1, MET, AR, STK11, NF2, CBL, HLA-B, PRKCB, ATR PPP2R1A, CASC5, CD79B, PBRM1, PTK2B, GATA2, KMT2D, SULT1A1, F LNB, PRPF8, RNF43, MSH6, FGFR2, SMAD4, JAK3, USP8, DLC1, ESRP1 RP1B, MYH11, BRCA1, CARD11, HSP90AB1, MAP3K9, ADAMTSL3, PDGFR B RPTOR ROS1 NFKBIE AMER1 KLF4 RAC1 TERT MYOD1 ATP1A1C SF3R, NOTCH2, CCR4, PAX5, SPTAN1, MLH1, CUBN, RNF213, SMO, ABCC4 AXIN2, CSF1R, PER1, PKHD1, IL7R, RB1, ARID1A, ATM, FES, MTHFR TCH2, FANCI, CDH5, CIC, IL6ST, MYH9, NF1, TGFBR2, INSR, PTPN12. TNFAIP3, MEN1, NSD1, SLITRK6, SYT1, TNKS, CCND3, PSMD13, CYP2D6 HELQ, LPHN3, PRAME, STAT5B, BCL6, CCDC6, CCND1, FLCN, LMO2, MU C1, NFKBIZ, NRP2, CTCF, HIST1H3B, KEAP1, SLC22A2, ABCC2, EED, GA TA1, GLI3, IKZF3, PIK3CG, XPO1, CHRNA3, MAP2K1, SETD2, ZNF668. CCND2, FLT4, NT5C2, RECQL4, SSX1, ALOX12B, CDKN1B, ELF3, INPP4B MARVELD3, MLLT4, MLPH, NTRK3, SPOP, BCL2, EPHB1, ERCC4, ERCC6 ETNK1, JAK1, LRP2, MUTYH, NFKBIA, ARNT, BRCA2, and CDH2.

[0010] In some embodiments, neoantigens can be selected based on the genetic profile of a tumor sample from an individual. In some embodiments, neoantigens can be selected based on the somatic mutation profile of a tumor sample from an individual.

[0011] In some embodiments, the protein that induces an immune cell inactivation signal in unmodified TILs or unmodified T cells upon binding to its ligand may be a signaling receptor, hi some embodiments, the protein that induces an immune cell inactivation signal in unmodified TILs or unmodified T cells upon binding to its ligand may be a checkpoint receptor, a cytokine receptor, a chemokine receptor, a growth factor receptor, or a hormone receptor. In some embodiments, the protein that induces an immune cell inactivation signal in a non-modified TIL or non-modified T cell upon binding to its ligand can be selected from the group consisting of transforming growth factor-β receptor (TGF-β-R), programmed cell death 1 (PD-1), T cell co-stimulatory receptor 4 (CTLA-4), B- and T-lymphocyte attenuator (BTLA), killer cell immunoglobulin-like receptor (KIR), indoleamine-2,3-dioxygenase (IDO), lymphocyte activation gene-3 (LAG3), T cell immunoglobulin mucin-3 (TIM-3), and TIGIT.

[0012] In some embodiments, the costimulatory molecule may be interleukin-2 receptor (IL-2R), interleukin-12 receptor (IL-12R), CD2, CD3, CD4, CD7, CD8, CD27, CD28, CD30, CD40, 4-1BB / CD137, ICOS, lymphocyte function-associated antigen-1 (LFA-1), LIGHT, NKG2C, or OX40.

[0013] In some embodiments, the immune cell activity signal may be mediated by an activator. In some embodiments, the activator is a soluble cytokine, a soluble chemokine, or a growth factor. In some embodiments, the activator is a soluble cytokine, and wherein the soluble cytokine is IL-1, IL-2, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IL-21, TNF, TGF, IFN, or a functional fragment or variant thereof. In some embodiments, the immune cell activation signal can be clonal expansion of the modified TIL or modified T cell, cytokine release by the modified TIL or modified T cell, cytotoxicity of the modified TIL or modified T cell, proliferation of the modified TIL or modified T cell, differentiation, dedifferentiation, transdifferentiation of the modified TIL or modified T cell, migration and / or trafficking of the modified TIL or modified T cell, exhaustion and / or reactivation of the modified TIL or modified T cell, and release of other intercellular molecules, metabolites, compounds, or combinations thereof by the modified TIL or modified T cell.

[0014] In some embodiments, upon binding of the switch molecule to a ligand, the modified TIL or modified T cell may exhibit enhanced neoantigen binding compared to the unmodified TIL or unmodified T cell.

[0015] In some embodiments, the modified TIL or modified T cell may exhibit increased cytotoxicity against a target cell compared to an unmodified TIL or unmodified T cell when the switch molecule binds to a ligand and the modified TIL or modified T cell binds to a neoantigen present on the target cell.

[0016] In some embodiments, the engineered TIL or engineered T cell may exhibit increased cytokine secretion compared to unengineered TIL or unengineered T cell when the switch molecule binds to a ligand and the engineered TIL or engineered T cell binds to a neoantigen present on a target cell. In some embodiments, the cytokine may be IFN-γ or IL-2.

[0017] In one aspect, the present disclosure provides an engineered immune cell comprising a chimeric antigen receptor (CAR) and a T cell receptor (TCR) complex that exhibits specific binding to a neoantigen, wherein the CAR comprises (a) an antigen interacting domain capable of binding a B cell surface protein, (b) a transmembrane domain, and (c) an intracellular signaling domain.

[0018] In some embodiments, the immune cells may be tumor infiltrating lymphocytes (TILs). In some embodiments, the TILs may be triple positive T cells expressing PD-1, CD137 and TIM-3. In some embodiments, the TCR complex that exhibits specific binding to the neoantigen may be an endogenous TCR complex. In some embodiments, the TCR complex that exhibits specific binding to the neoantigen may be an exogenous TCR complex.

[0019] In some embodiments, the neoantigen comprises a peptide fragment of a protein encoded by a mutated gene, wherein the gene is ABL1, ACOL 1997, ACVR2A, AFP, AKT1, ALK, ALPPL2, ANAPC1, APC, ARID1A, AR, AR-v7, ASCL2, β2M, BRAF, BTK, C15ORF40, CDH1, CLDN6, CNOT1, CT45A5, CTAG1B, DCT, DKK4, EEF1B2, EEF1DP3, EGFR, EIF2B3, env, EPHB2, ERBB3, ESR1, ESRP1, FAM11 IB, FGFR3, FRG1B, GAGE1, GAGE ​​10, GATA3, GBP3, HER2, IDH1, JAK1, KIT, KRAS, LMAN1, MABEB 16, MAGEA1, MAGEA10, MAGEA4, MAGEA8, MAGEB 17, MAGEB4, MAGEC1, MEK, MLANA, MLL2, MMP13, MSH3, MSH6, MYC, NDUFC2, NRAS, PAGE2, PAGE5, PDGFRa, PIK3CA, PMEL, pol protein, POLE, PTEN, RAC1, RBM27, RNF43, RPL22, RUNX1, SEC31A, SEC63, SF3B 1, SLC35F5, SLC45A2, SMAP1, SMAP1, SPOP, TFAM, Tgfbr2, THAP5, TP53, TTK, TYR, UBR5, VHL, and XPOT.

[0020] In some embodiments, neoantigens can be selected based on the genetic profile of a tumor sample from an individual. In some embodiments, neoantigens can be selected based on the somatic mutation profile of a tumor sample from an individual.

[0021] In some embodiments, the B cell surface protein is selected from CD19, CD20 and CD22.

[0022] In some embodiments, the intracellular signaling domain may comprise an immunoreceptor tyrosine-based activation motif (ITAM). In some embodiments, the intracellular signaling domain may comprise an immunoreceptor tyrosine-based inhibitory motif. In some embodiments, the intracellular signaling domain may comprise an intracellular domain of a molecule selected from Fc gamma receptor (FcγR), Fcε receptor (FcεR), Fca receptor (FcαR), neonatal Fc receptor (FcRn), CD3, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD4, CD5, CD8, CD21, CD22, CD28, CD32, CD40L (CD154), CD45, CD66d, CD79a, CD79b, CD80, CD86, CD278 (also referred to as ICOS), CD247 zeta, CD247 eta, DAP10, DAP12, FYN, LAT, Lck, MAPK, MHC complex, NFAT, NF-κB, PLC-γ, iC3b, C3dg, C3d, and Zap70.

[0023] In some embodiments, the intracellular signaling domain may comprise the intracellular domain of CD3 zeta. In some embodiments, the CAR may further comprise a costimulatory domain. In some embodiments, the costimulatory domain may comprise a signaling domain of an MHC class I molecule, a TNF receptor protein, an immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocyte activation molecule (SLAM protein), an activating NK cell receptor, or a Toll ligand receptor.

[0024] In some embodiments, the costimulatory domain is selected from the group consisting of 2B4 / CD244 / SLAMF4, 4-1BB / TNFSF9 / CD137, B7-1 / CD80, B7-2 / CD86, B7-H1 / PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BAFF R / TNFRSF13C, BAFF / BLyS / TNFSF13B, BLAME / SLAMF8, BTLA / CD272, CD100 (SEMA4D), CD103, CD11a, CD11b, CD11c, CD11d, CD150, CD160 (BY55), CD18, CD19, CD2, CD200, CD229 / SLAMF3, CD27 Ligand / TNFSF7, CD27 / TNFRSF7, CD28, CD29, CD2F-10 / SLAMF9, CD30 Ligand / TNFSF8, CD30 / TNFRSF8, CD300a / LMIR1, CD4, CD40 Ligand / TNFSF5, CD40 / TNFRSF5, CD48 / SLAMF2, CD49a, CD49D, CD49f, CD53, CD58 / LFA-3, CD69, CD7, CD8 α, CD8 β, CD82 / Kai-1, CD84 / SLAMF5, CD90 / Thy1, CD96, CDS, CEACAM1, CRACC / SLAMF7, CRTAM, CTLA-4, DAP12, Dectin-1 / CLEC7A, DNAM1 (CD226), DPPIV / CD26, DR3 / TNFRSF25, EphB6, GADS, Gi24 / VISTA / B7-H5, GITR ligand / TNFSF18, GITR / TNFRSF18, HLA class I, HLA-DR, HVEM / TNFRSF14, IA4, ICAM-1, ICOS / CD278, Ikaros, IL2R β, IL2R γ, IL7R α, integrin α4 / CD49d, integrin α4β1, integrin α4β7 / LPAM-1, IPO-3, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIRDS2, LAG-3, LAT, LIGHT / TNFSF14, LTBR, ​​Ly108, Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1), lymphotoxin-α / TNF-β, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80(KLRF1), NTB-A / SLAMF6, OX40 Ligand / TNFSF4, OX40 / TNFRSF4, PAG / Cbp, PD-1, PDCD6, PD-L2 / B7-DC, PSGL1, RELT / TNFRSF19L, SELPLG (CD162), SLAM (SLAMF1), SLAM / CD150, SLAMF4 (CD244), SLAMF6 (NTB-A), SLAMF7, SLP-76, TACI / TNFRSF13B, TCL1A, TCL1B, TIM-1 / KIM-1 / HAVCR, TIM-4, TL1A / TNFSF15, TNF RII / TNFRSF1B, TNF-α, TRANCE / RANKL, TSLP, TSLP R, VLA1, and VLA-6.

[0025] In some embodiments, upon contacting an immune cell with a B cell surface protein, the immune cell may exhibit enhanced proliferation compared to an unmodified immune cell. In some embodiments, the enhanced proliferation may be confirmed in vitro. In some embodiments, the enhanced proliferation may be confirmed in vivo. In some embodiments, the immune cell may exhibit at least a two-fold increase in proliferation at least about 24, 48, or 96 hours after contact compared to an unmodified immune cell.

[0026] In one aspect, the disclosure provides an engineered tumor infiltrating lymphocyte (TIL) that specifically binds to a neoantigen, wherein the engineered TIL comprises (a) a switch molecule comprising an extracellular domain (ECD) of a protein that upon binding to its ligand induces an immune cell inactivation signal in a non-engineered TIL cell, where the ECD is fused to an intracellular domain (ICD) of a costimulatory molecule that mediates an immune cell activation signal, and where binding of the switch molecule to the ligand results in an immune cell activation signal instead of an immune cell inactivation signal in the engineered TIL, and (b) a chimeric antigen receptor comprising: (i) an antigen-interacting domain capable of binding to a B cell surface protein; (ii) a transmembrane domain; and (iii) an intracellular signaling domain.

[0027] In one aspect, the disclosure provides an engineered immune cell that specifically binds a neoantigen, wherein the engineered immune cell comprises (a) a switch molecule comprising an extracellular domain (ECD) of a protein that upon binding to its ligand induces an immune cell inactivation signal in a non-engineered immune cell, where the ECD is fused to an intracellular domain (ICD) of a costimulatory molecule that mediates an immune cell activation signal, and where binding of the switch molecule to the ligand results in an immune cell activation signal instead of an immune cell inactivation signal in the engineered immune cell, and (b) a chimeric antigen receptor comprising: (i) an antigen interacting domain capable of binding to a B cell surface protein; (ii) a transmembrane domain; and (iii) an intracellular signaling domain.

[0028] In some embodiments, the immune cell may comprise a T cell receptor (TCR) complex that exhibits specific binding to a neoantigen. In some embodiments, the TCR complex may be an endogenous TCR complex. In some embodiments, the TCR complex may be an exogenous TCR complex.

[0029] In some embodiments, the neoantigen comprises a peptide fragment of a protein encoded by a mutated gene, where the gene is ABL1, ACOL 1997, ACVR2A, AFP, AKT1, ALK, ALPPL2, ANAPC1, APC, ARID1A, AR, AR-v7, ASCL2, β2M, BRAF, BTK, C15ORF40, CDH1, CLDN6, CNOT1, CT45A5, CTAG1B, DCT, DKK4, EEF1B2, EEF1DP3, EGFR, EIF2B3, env, EPHB2, ERBB3, ESR1, ESRP1, FAM11 IB, FGFR3, FRG1B, GAGE1, GAGE ​​10, GATA3, GBP3, HER2, IDH1, JAK1, KIT, KRAS, LMAN1, MABEB 16, MAGEA1, MAGEA10, MAGEA4, MAGEA8, MAGEB 17, MAGEB4, MAGEC1, MEK, MLANA, MLL2, MMP13, MSH3, MSH6, MYC, NDUFC2, NRAS, PAGE2, PAGE5, PDGFRa, PIK3CA, PMEL, pol protein, POLE, PTEN, RAC1, RBM27, RNF43, RPL22, RUNX1, SEC31A, SEC63, SF3B 1, SLC35F5, SLC45A2, SMAP1, SMAP1, SPOP, TFAM, TGFBR2, THAP5, TP53, TTK, TYR, UBR5, VHL, and XPOT.

[0030] In some embodiments, neoantigens can be selected based on the genetic profile of a tumor sample from an individual. In some embodiments, neoantigens can be selected based on the somatic mutation profile of a tumor sample from an individual.

[0031] In some embodiments, the protein that induces an immune cell inactivation signal in unmodified TILs or unmodified immune cells upon binding to its ligand may be a signaling receptor, hi some embodiments, the protein that induces an immune cell inactivation signal in unmodified TILs or unmodified immune cells upon binding to its ligand may be a checkpoint receptor, a cytokine receptor, a chemokine receptor, a growth factor receptor, or a hormone receptor. In some embodiments, the protein that induces an immune cell inactivation signal in unmodified TIL or unmodified immune cells upon binding to its ligand can be selected from transforming growth factor-β receptor (TGF-β-R), programmed cell death 1 (PD-1), T cell co-stimulatory receptor 4 (CTLA-4), B- and T-lymphocyte attenuator (BTLA), killer cell immunoglobulin-like receptor (KIR), indoleamine-2,3-dioxygenase (IDO), lymphocyte activation gene-3 (LAG3), T cell immunoglobulin mucin-3 (TIM-3), and TIGIT.

[0032] In some embodiments, the costimulatory molecule may be interleukin-2 receptor (IL-2R), interleukin-12 receptor (IL-12R), CD2, CD3, CD4, CD7, CD8, CD27, CD28, CD30, CD40, 4-1BB / CD137, ICOS, lymphocyte function-associated antigen-1 (LFA-1), LIGHT, NKG2C, or OX40.

[0033] The immune cell activity signal can be mediated by an activator. In some embodiments, the activator can be a soluble cytokine, a soluble chemokine, or a growth factor. In some embodiments, the activator is a soluble cytokine, and the soluble cytokine can be IL-1, IL-2, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IL-21, TNF, TGF, IFN, or a functional fragment or variant thereof.

[0034] In some embodiments, immune cell activation signals can include clonal expansion of the modified TIL or modified immune cell, cytokine release by the modified TIL or modified immune cell, cytotoxicity of the modified TIL or modified immune cell, proliferation of the modified TIL or modified immune cell, differentiation, dedifferentiation, transdifferentiation of the modified TIL or modified immune cell, migration and / or trafficking of the modified TIL or modified immune cell, exhaustion and / or reactivation of the modified TIL or modified immune cell, and release of other intercellular molecules, metabolites, compounds, or combinations thereof by the modified TIL or modified immune cell.

[0035] In some embodiments, the B cell surface protein may be selected from CD19, CD20 and CD22.

[0036] In some embodiments, the intracellular signaling domain may comprise an immunoreceptor tyrosine-based activation motif (ITAM). In some embodiments, the intracellular signaling domain may comprise an immunoreceptor tyrosine-based inhibitory motif (ITIM). In some embodiments, the intracellular signaling domain may comprise an intracellular domain of a molecule selected from Fc gamma receptor (FcγR), Fcε receptor (FcεR), Fca receptor (FcαR), neonatal Fc receptor (FcRn), CD3, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD4, CD5, CD8, CD21, CD22, CD28, CD32, CD40L (CD154), CD45, CD66d, CD79a, CD79b, CD80, CD86, CD278 (also referred to as ICOS), CD247 zeta, CD247 eta, DAP10, DAP12, FYN, LAT, Lck, MAPK, MHC complex, NFAT, NF-κB, PLC-γ, iC3b, C3dg, C3d, and Zap70.

[0037] In some embodiments, the intracellular signaling domain may comprise an intracellular domain of CD3 zeta. In some embodiments, the intracellular domain of CD3 zeta may comprise an immunoreceptor tyrosine-based activation motif (ITAM). The CAR may further comprise a costimulatory domain. In some embodiments, the costimulatory domain may comprise an MHC class I molecule, a TNF receptor protein, an immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocyte activation molecule (SLAM protein), an activated NK cell receptor, or a Toll ligand receptor signaling domain.

[0038] In some embodiments, the costimulatory domain is selected from the group consisting of 2B4 / CD244 / SLAMF4, 4-1BB / TNFSF9 / CD137, B7-1 / CD80, B7-2 / CD86, B7-H1 / PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BAFF R / TNFRSF13C, BAFF / BLyS / TNFSF13B, BLAME / SLAMF8, BTLA / CD272, CD100 (SEMA4D), CD103, CD11a, CD11b, CD11c, CD11d, CD150, CD160 (BY55), CD18, CD19, CD2, CD200, CD229 / SLAMF3, CD27 Ligand / TNFSF7, CD27 / TNFRSF7, CD28, CD29, CD2F-10 / SLAMF9, CD30 Ligand / TNFSF8, CD30 / TNFRSF8, CD300a / LMIR1, CD4, CD40 Ligand / TNFSF5, CD40 / TNFRSF5, CD48 / SLAMF2, CD49a, CD49D, CD49f, CD53, CD58 / LFA-3, CD69, CD7, CD8 α, CD8 β, CD82 / Kai-1, CD84 / SLAMF5, CD90 / Thy1, CD96, CDS, CEACAM1, CRACC / SLAMF7, CRTAM, CTLA-4, DAP12, Dectin-1 / CLEC7A, DNAM1 (CD226), DPPIV / CD26, DR3 / TNFRSF25, EphB6, GADS, Gi24 / VISTA / B7-H5, GITR ligand / TNFSF18, GITR / TNFRSF18, HLA class I, HLA-DR, HVEM / TNFRSF14, IA4, ICAM-1, ICOS / CD278, Ikaros, IL2R β, IL2R γ, IL7R α, integrin α4 / CD49d, integrin α4β1, integrin α4β7 / LPAM-1, IPO-3, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIRDS2, LAG-3, LAT, LIGHT / TNFSF14, LTBR, ​​Ly108, Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1), lymphotoxin-α / TNF-β, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80(KLRF1), NTB-A / SLAMF6, OX40 Ligand / TNFSF4, OX40 / TNFRSF4, PAG / Cbp, PD-1, PDCD6, PD-L2 / B7-DC, PSGL1, RELT / TNFRSF19L, SELPLG (CD162), SLAM (SLAMF1), SLAM / CD150, SLAMF4 (CD244), SLAMF6 (NTB-A), SLAMF7, SLP-76, TACI / TNFRSF13B, TCL1A, TCL1B, TIM-1 / KIM-1 / HAVCR, TIM-4, TL1A / TNFSF15, TNF RII / TNFRSF1B, TNF-α, TRANCE / RANKL, TSLP, TSLP R, VLA1, and VLA-6.

[0039] In some embodiments, upon binding of the switch molecule to a ligand, the modified TIL or modified immune cell may exhibit enhanced neoantigen binding compared to the unmodified TIL or unmodified immune cell.

[0040] In some embodiments, the modified TIL or modified T cell may exhibit increased cytotoxicity against a target cell compared to an unmodified TIL or unmodified T cell when the switch molecule binds to a ligand and the modified TIL or modified immune cell binds to a neoantigen present on the target cell.

[0041] In some embodiments, the engineered TIL or engineered immune cell may exhibit increased cytokine secretion compared to unengineered TIL or unengineered immune cell when the switch molecule binds to a ligand and the engineered TIL or engineered immune cell binds to a neoantigen present on a target cell. In some embodiments, the cytokine may be IFN-γ or IL-2.

[0042] In one aspect, the disclosure provides a method of treating cancer in a subject, comprising: (a) administering to the subject a modified TIL, modified T cell, or modified immune cell as described in any one of the claims; and (b) contacting a cancer target cell that expresses a neoantigen with the modified TIL, modified T cell, or modified immune cell under conditions that induce cytotoxicity of the modified TIL, modified T cell, or modified immune cell against the cancer target cell, thereby inducing death of the cancer target cell.

[0043] In one aspect, the disclosure provides a method for expanding a T cell population, the method comprising: (a) providing a T cell population comprising at least one modified immune cell of any one of claims 21-40; and (b) exposing the population of T cells to a B cell surface protein to effect expansion of the population of T cells. In some embodiments, the population of T cells may be exposed to B cells comprising the B cell surface protein.

[0044] In one aspect, the disclosure provides a method for expanding a T cell population comprising: (a) introducing into a T cell population a nucleic acid encoding a chimeric antigen receptor (CAR), thereby creating a first CAR-expressing cell population, wherein the CAR comprises (i) an antigen interacting domain capable of binding to a B cell surface protein; (ii) a transmembrane domain; and (iii) an intracellular signaling domain; and (b) contacting the first CAR-expressing cell population with the B cell surface protein, thereby creating an expanded and / or activated immune cell population.

[0045] In one aspect, the disclosure provides a composition comprising: (a) a switch molecule comprising an extracellular domain (ECD) of a protein that, upon binding to its ligand, induces an immune inactivation signal in an unmodified immune cell, where the ECD is fused to an intracellular domain (ICD) of a costimulatory molecule that mediates an immune cell activation signal; and (b) one or more polynucleotides encoding one or more of an antigen-specific T cell receptor complex, or one or more components thereof.

[0046] Another aspect of the present disclosure provides a composition comprising one or more polynucleotides encoding one or more of: (a) an antigen-specific T cell receptor complex, or one or more components thereof; and (b) a chimeric antigen receptor comprising: (i) an antigen interacting domain capable of binding to a B cell surface protein; (ii) a transmembrane domain; and (iii) an intracellular signaling domain.

[0047] In some embodiments, neoantigens may comprise peptide fragments of proteins encoded by mutated genes, where the genes are ABL1, ACOL 1997, ACVR2A, AFP, AKT1, ALK, ALPPL2, ANAPC1, APC, ARID1A, AR, AR-v7, ASCL2, β2M, BRAF, BTK, C15ORF40, CDH1, CLDN6, CNOT1, CT45A5, CTAG1B, DCT, DKK4, EEF1B2, EEF1DP3, EGFR, EIF2B3, env, EPHB2, ERBB3, ESR1, ESRP1, FAM11 IB, FGFR3, FRG1B, GAGE1, GAGE ​​10, GATA3, GBP3, HER2, IDH1, JAK1, KIT, KRAS, LMAN1, MABEB 16, MAGEA1, MAGEA10, MAGEA4, MAGEA8, MAGEB 17, MAGEB4, MAGEC1, MEK, MLANA, MLL2, MMP13, MSH3, MSH6, MYC, NDUFC2, NRAS, PAGE2, PAGE5, PDGFRa, PIK3CA, PMEL, pol protein, POLE, PTEN, RAC1, RBM27, RNF43, RPL22, RUNX1, SEC31A, SEC63, SF3B 1, SLC35F5, SLC45A2, SMAP1, SMAP1, SPOP, TFAM, TGFBR2, THAP5, TP53, TTK, TYR, UBR5, VHL, and XPOT.

[0048] In some embodiments, the protein that induces immune cell inactivation signals in unmodified TIL or unmodified T cell upon binding to its ligand may be a signaling receptor. In some embodiments, the protein may be a checkpoint receptor, a cytokine receptor, a chemokine receptor, a growth factor receptor, or a hormone receptor. In some embodiments, the protein may be selected from the group consisting of transforming growth factor-β receptor (TGF-β-R), programmed cell death 1 (PD-1), T cell co-stimulatory receptor 4 (CTLA-4), B and T lymphocyte attenuator (BTLA), killer cell immunoglobulin-like receptor (KIR), indoleamine-2,3-dioxygenase (IDO), lymphocyte activation gene-3 (LAG3), T cell immunoglobulin mucin-3 (TIM-3) and TIGIT.

[0049] In some embodiments, a costimulatory molecule that mediates an immune cell activation signal may be interleukin-2 receptor (IL-2R), interleukin-12 receptor (IL-12R), CD2, CD3, CD4, CD7, CD8, CD27, CD28, CD30, CD40, 4-1BB / CD137, ICOS, lymphocyte function-associated antigen-1 (LFA-1), LIGHT, NKG2C, or OX40.

[0050] In some embodiments, the B cell surface protein may be selected from CD19, CD20 and CD22. In some embodiments, the intracellular signaling domain may comprise an immunoreceptor tyrosine-based inhibitory motif. In some embodiments, the intracellular signaling domain may comprise an intracellular domain of a molecule selected from Fc gamma receptor (FcγR), Fcε receptor (FcεR), Fca receptor (FcαR), neonatal Fc receptor (FcRn), CD3, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD4, CD5, CD8, CD21, CD22, CD28, CD32, CD40L (CD154), CD45, CD66d, CD79a, CD79b, CD80, CD86, CD278 (also referred to as ICOS), CD247 zeta, CD247 eta, DAP10, DAP12, FYN, LAT, Lck, MAPK, MHC complex, NFAT, NF-κB, PLC-γ, iC3b, C3dg, C3d, and Zap70.

[0051] In some embodiments, the chimeric antigen receptor may further comprise a costimulatory domain, such as 2B4 / CD244 / SLAMF4, 4-1BB / TNFSF9 / CD137, B7-1 / CD80, B7-2 / CD86, B7-H1 / PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BAFF R / TNFRSF13C, BAFF / BLyS / TNFSF13B, BLAME / SLAMF8, BTLA / CD272, CD100 (SEMA4D), CD103, CD11a, CD11b, CD11c, CD11d, CD150, CD160 (BY55), CD18, CD19, CD2, CD200, CD229 / SLAMF3, CD27 Ligand / TNFSF7, CD27 / TNFRSF7, CD28, CD29, CD2F-10 / SLAMF9, CD30 Ligand / TNFSF8, CD30 / TNFRSF8, CD300a / LMIR1, CD4, CD40 Ligand / TNFSF5, CD40 / TNFRSF5, CD48 / SLAMF2, CD49a, CD49D, CD49f, CD53, CD58 / LFA-3, CD69, CD7, CD8 α, CD8 β, CD82 / Kai-1, CD84 / SLAMF5, CD90 / Thy1, CD96, CDS, CEACAM1, CRACC / SLAMF7, CRTAM, CTLA-4, DAP12, Dectin-1 / CLEC7A, DNAM1 (CD226), DPPIV / CD26, DR3 / TNFRSF25, EphB6, GADS, Gi24 / VISTA / B7-H5, GITR ligand / TNFSF18, GITR / TNFRSF18, HLA class I, HLA-DR, HVEM / TNFRSF14, IA4, ICAM-1, ICOS / CD278, Ikaros, IL2R β, IL2R γ, IL7R α, integrin α4 / CD49d, integrin α4β1, integrin α4β7 / LPAM-1, IPO-3, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIRDS2, LAG-3, LAT, LIGHT / TNFSF14, LTBR, ​​Ly108, Ly9(CD229), lymphocyte function-associated antigen-1 (LFA-1), lymphotoxin-α / TNF-β, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), NTB-A / SLAMF6, OX40 ligand / TNFSF4, OX40 / TNFRSF4, PAG / Cbp, PD-1, PDCD6, PD-L2 / B7-DC, PSGL1, RELT / TNFRSF19L, SELPLG (CD162), SLAM (SLAMF1), SLAM / CD150, SLAMF4 (CD244), SLAMF6 (NTB-A), SLAMF7, SLP-76, TACI / TNFRSF13B, TCL1A, TCL1B, TIM-1 / KIM-1 / HAVCR, TIM-4, TL1A / TNFSF15, TNF RII / TNFRSF1B, TNF-α, TRANCE / RANKL, TSLP, TSLP R, VLA1, and VLA-6.

[0052] In one aspect, the disclosure provides a composition comprising: (a) a switch molecule, the switch molecule comprising an extracellular domain (ECD) of a protein that, upon binding to its ligand, induces an immune inactivation signal in an unmodified immune cell, wherein the ECD is fused to an intracellular domain (ICD) of a costimulatory protein that mediates an immune cell activation signal; (b) an antigen-specific T cell receptor complex, or one or more components thereof; and (c) one or more polynucleotides encoding one or more of a chimeric antigen receptor comprising: (i) an antigen interacting domain capable of binding to a B cell surface protein; (ii) a transmembrane domain; and (iii) an intracellular signaling domain.

[0053] In one aspect, the disclosure provides an engineered tumor infiltrating lymphocyte (TIL) that specifically binds to a neoantigen, the engineered TIL comprising a chimeric stimulating molecule, wherein the chimeric stimulating molecule comprises a polypeptide extracellular domain (PED) that binds to a neoantigen, wherein the PED is fused to an intracellular domain (ICD) of a costimulatory molecule that mediates an immune cell activation signal, and wherein binding of the chimeric stimulating molecule to the neoantigen results in an immune cell activation signal in the engineered TIL.

[0054] In one aspect, the disclosure provides an engineered immune cell comprising: (a) a switch molecule comprising the extracellular domain (ECD) of a protein that upon binding to its ligand induces an immune inactivation signal in a non-modified immune cell, wherein the ECD is fused to the intracellular domain (ICD) of a costimulatory molecule that mediates an immune cell activation signal, such that binding of the switch molecule to the ligand results in an immune cell activation signal instead of an immune cell inactivation signal in the engineered immune cell; and (b) a chimeric antigen receptor (CAR) comprising: (i) an antigen interacting domain capable of binding to a B cell surface protein; (ii) a transmembrane domain; and (iii) an intracellular signaling domain.

[0055] In some embodiments, the modified immune cells may express at least one of PD1, CD137, and TIM-3. In some embodiments, the immune cells may be obtained from a tumor. In some embodiments, the immune cells may be obtained from peripheral blood of mononuclear cells. In some embodiments, the immune cells may include an exogenous TCR complex. In some embodiments, the TCR complex may bind to a tumor cell. In some embodiments, the TCR complex may bind to a neoantigen. In some embodiments, the protein that induces an immune cell inactivation signal in a non-modified immune cell upon binding to its ligand may be a signaling receptor. In some embodiments, the protein that induces an immune cell inactivation signal in a non-modified immune cell upon binding to its ligand may be a checkpoint receptor, a cytokine receptor, a chemokine receptor, a growth factor receptor, or a hormone receptor. In some embodiments, the protein that induces an immune cell inactivation signal in unmodified immune cells upon binding to its ligand may be selected from the group consisting of transforming growth factor-β receptor (TGF-β-R), programmed cell death 1 (PD-1), T cell co-stimulatory receptor 4 (CTLA-4), B- and T-lymphocyte attenuator (BTLA), killer cell immunoglobulin-like receptor (KIR), indoleamine-2,3-dioxygenase (IDO), lymphocyte activation gene-3 (LAG3), T cell immunoglobulin mucin-3 (TIM-3), and TIGIT. In some embodiments, the costimulatory molecule may be interleukin-2 receptor (IL-2R), interleukin-12 receptor (IL-12R), CD2, CD3, CD4, CD7, CD8, CD27, CD28, CD30, CD40, 4-1BB / CD137, ICOS, lymphocyte function-associated antigen-1 (LFA-1), LIGHT, NKG2C, or OX40. In some embodiments, the immune cell activity signal may be mediated by an activator. In some embodiments, the activator may be a soluble cytokine, a soluble chemokine, or a growth factor. In some embodiments,The activator is a soluble cytokine, and wherein the soluble cytokine can be IL-1, IL-2, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IL-21, TNF, TGF, IFN, or a functional fragment or variant thereof. In some embodiments, the immune cell activation signal can include clonal expansion of the modified immune cell, cytokine release by the modified immune cell, cytotoxicity of the modified immune cell, proliferation of the modified immune cell, differentiation, dedifferentiation, transdifferentiation of the modified immune cell, migration and / or trafficking of the modified immune cell, exhaustion and / or reactivation of the modified immune cell, and release of other intercellular molecules, metabolites, compounds, or combinations thereof by the modified immune cell. In some embodiments, the B cell surface protein can be selected from CD19, CD20, and CD22. In some embodiments, the intracellular signaling domain can include an immunoreceptor tyrosine-based activation motif (ITAM). In some embodiments, the intracellular signaling domain may comprise an immunoreceptor tyrosine-based inhibitory motif (ITIM). In some embodiments, the intracellular signaling domain may comprise an Fcγ receptor (FcγR), Fcε receptor (FcεR), Fcα receptor (FcαR), neonatal Fc receptor (FcRn), CD3, CD3 ζ, CD3 γ, CD3 δ, CD3 ε, CD4, CD5, CD8, CD21, CD22, CD28, CD32, CD40L (CD154), CD45, CD66d, CD79a, CD79b, CD80, CD86, CD278 (also referred to as ICOS), CD247 ζ, CD247 In some embodiments, the intracellular signaling domain may comprise an intracellular domain of a molecule selected from η, DAP10, DAP12, FYN, LAT, Lck, MAPK, MHC complex, NFAT, NF-κB, PLC-γ, iC3b, C3dg, C3d, and Zap70. In some embodiments, the intracellular signaling domain may comprise an intracellular domain of CD3 ζ. In some embodiments, the intracellular domain of CD3 ζ may comprise an immunoreceptor tyrosine-based activation motif (ITAM). The CAR may further comprise:In some embodiments, the costimulatory domain may include a signaling domain of an MHC class I molecule, a TNF receptor protein, an immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocyte activation molecule (SLAM protein), an activating NK cell receptor, or a Toll ligand receptor. In some embodiments, the costimulatory domain is selected from the group consisting of 2B4 / CD244 / SLAMF4, 4-1BB / TNFSF9 / CD137, B7-1 / CD80, B7-2 / CD86, B7-H1 / PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BAFF R / TNFRSF13C, BAFF / BLyS / TNFSF13B, BLAME / SLAMF8, BTLA / CD272, CD100 (SEMA4D), CD103, CD11a, CD11b, CD11c, CD11d, CD150, CD160 (BY55), CD18, CD19, CD2, CD200, CD229 / SLAMF3, CD27 Ligand / TNFSF7, CD27 / TNFRSF7, CD28, CD29, CD2F-10 / SLAMF9, CD30 Ligand / TNFSF8, CD30 / TNFRSF8, CD300a / LMIR1, CD4, CD40 Ligand / TNFSF5, CD40 / TNFRSF5, CD48 / SLAMF2, CD49a, CD49D, CD49f, CD53, CD58 / LFA-3, CD69, CD7, CD8 α, CD8 β, CD82 / Kai-1, CD84 / SLAMF5, CD90 / Thy1, CD96, CDS, CEACAM1, CRACC / SLAMF7, CRTAM, CTLA-4, DAP12, Dectin-1 / CLEC7A, DNAM1 (CD226), DPPIV / CD26, DR3 / TNFRSF25, EphB6, GADS, Gi24 / VISTA / B7-H5, GITR ligand / TNFSF18, GITR / TNFRSF18, HLA class I, HLA-DR, HVEM / TNFRSF14, IA4, ICAM-1, ICOS / CD278, Ikaros, IL2R β, IL2R γ, IL7R α, integrin α4 / CD49d, integrin α4β1, integrin α4β7 / LPAM-1, IPO-3, ITGA4, ITGA6, ITGAD, ITGAE,ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIRDS2, LAG-3, LAT, LIGHT / TNFSF14, LTBR, ​​Ly108, Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1), lymphotoxin-α / TNF-β, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), NTB-A / SLAMF6, OX40 ligand / TNFSF4, OX40 / TNFRSF4, PAG / Cbp, PD-1, PDCD6, PD-L2 / B7-DC, PSGL1, RELT / TNFRSF19L, SELPLG (CD162), SLAM (SLAMF1), SLAM / CD150, SLAMF4 (CD244), SLAMF6 (NTB-A), SLAMF7, SLP-76, TACI / TNFRSF13B, TCL1A, TCL1B, TIM-1 / KIM-1 / HAVCR, TIM-4, TL1A / TNFSF15, TNF RII / TNFRSF1B, TNF-α, TRANCE / RANKL, TSLP, TSLP R, VLA1, and VLA-6. In some embodiments, upon contacting the immune cell with the B cell surface protein, the immune cell may exhibit enhanced proliferation compared to an unmodified immune cell. In some embodiments, the enhanced proliferation may be confirmed in vitro. In some embodiments, the enhanced proliferation may be confirmed in vivo. In some embodiments, the immune cell may exhibit at least a two-fold increase in proliferation at least about 24, 48, or 96 hours after contact compared to an unmodified immune cell.

[0056] [Incorporation by Reference] All publications, patents, and patent applications referenced in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0057] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings. [Brief description of the drawings]

[0058] [Figure 1] FIG. 1 shows T cell receptor (TCR) expression in T cells with or without transduction of the NY-ESO-1 TCR gene. [Diagram 2] FIG. 2 is a diagram showing the process of preparing neoantigen-reactive (or recognizable) T cells. [Figure 3A] FIG. 3A shows lentiviral titration of PD1 / CD28 switch molecules and shows flow cytometric detection of PD1 expression. [Figure 3B] FIG. 3B is a diagram showing lentivirus titration of PD1 / CD28 switch molecules, showing titration curves. [Figure 4] FIG. 4 shows the expression of PD1 / CD28 switch molecules in TDR-1, TIL and neoT cells. [Diagram 5] FIG. 5 shows an assay of the J82-NY-ESO-1-PDL1 bladder cancer cell line. [Figure 6A] FIG. 6A shows expression of CD107a in T cells cultured with tumor cells in an in vitro assay of TCR-T cells expressing a PD1 / CD28 switch molecule targeting NY-ESO-1. [Figure 6B] FIG. 6B shows the release of IFN-γ in T cells cultured with tumor cells in an in vitro assay of TCR-T cells expressing a PD1 / CD28 switch molecule targeting NY-ESO-1. [Figure 6C]FIG. 6C shows the release of IL-2 in T cells cultured with tumor cells in an in vitro assay of TCR-T cells expressing PD1 / CD28 switch molecules targeting NY-ESO-1. [Figure 7A] FIG. 7A shows the release of IFN-γ by TIL cells with or without PD1 / CD28 switch molecules when cultured in the presence or absence of tumor cells. [Figure 7B] FIG. 7B shows the release of IL-2 by TIL cells, with or without PD1 / CD28 switch molecules, when cultured in the presence or absence of tumor cells. [Figure 8A] FIG. 8A shows the release of IFN-γ in neoantigen-reactive T cells (neoT) with or without PD1 / CD28 switch molecules when cultured in the presence or absence of tumor cells. [Figure 8B] FIG. 8B shows the release of IL-2 in neoantigen-reactive T cells (neoT) with or without PD1 / CD28 switch molecules when cultured in the presence or absence of tumor cells. [Figure 9] FIG. 9 shows the expression of chimeric antigen receptor targeting B cell surface protein (BCAR) in TCR-T, TIL, and neoT. [Figure 10] FIG. 10 shows the in vitro efficacy and expansion of NY-ESO-1-TCR-T cells expressing BCAR. [Figure 11] FIG. 11 shows the in vitro efficacy and expansion of NY-ESO-1-TCR-T cells expressing BCAR. [Figure 12A] FIG. 12A shows the in vitro efficacy and expansion of TIL cells expressing BCAR. [Figure 12B] FIG. 12B shows the in vitro efficacy and expansion of TIL cells expressing BCAR. [Figure 13A]FIG. 13A shows the in vitro efficacy and expansion of NeoT cells expressing BCAR. [Figure 13B] FIG. 13B shows the in vitro efficacy and expansion of NeoT cells expressing BCAR. [Figure 14] FIG. 14 shows the expression of PD1sw-BCAR, TIM3sw-BCAR, and TGFBR2sw-BCAR in TILs. [Figure 15] FIG. 15 shows the expression of PD1sw-BCAR, TIM3sw-BCAR, and TGFBR2sw-BCAR in pTILs. [Figure 16A] FIG. 16A shows the release of IFN-γ from various TILs in the absence of B cells. [Figure 16B] FIG. 16B shows the release of IL-2 from various TILs in the absence of B cells. [Figure 17A] FIG. 17A shows the tumor-killing effect of various TILs in the absence (group A) or presence (group B) of B cells. [Figure 17B] FIG. 17B shows the in vitro expansion of various TILs in the absence (group A) or presence (group B) of B cells. [Figure 18A] FIG. 18A shows the release of IFN-γ from various TILs in the absence of B cells. [Figure 18B] FIG. 18B shows the release of IL-2 from various TILs in the absence of B cells. [Figure 19A] FIG. 19A shows the tumor-killing effect of various pTILs in the absence (group A) or presence (group B) of B cells. [Figure 19B] FIG. 17B shows the in vitro expansion of various pTILs in the absence (group A) or presence (group B) of B cells. [Figure 20] FIG. 20 shows the killing effect of BCAR-TCRT against J82-NY-ESO1 tumor cells in the absence or presence of B cells. [Figure 21A] FIG. 21A shows tumor image analysis of subjects 1 to 4. [Figure 21B] FIG. 21B shows tumor image analysis of subjects 1 to 4. [Figure 21C] FIG. 21C shows tumor image analysis of subjects 1 to 4. [Figure 21D] FIG. 21D shows tumor image analysis of subjects 1 to 4. [Figure 22A] FIG. 22A shows tumor image analysis of subjects 1 to 4. [Figure 22B] FIG. 22B shows tumor image analysis of subjects 1 to 4. [Figure 22C] FIG. 22C shows tumor image analysis of subjects 1 to 4. [Figure 22D] FIG. 22D shows tumor image analysis of subjects 1 to 4. [Figure 23A] FIG. 23A shows tumor image analysis of subjects 1 to 4. [Figure 23B] FIG. 23B shows tumor image analysis of subjects 1 to 4. [Figure 23C] FIG. 23C shows tumor image analysis of subjects 1 to 4. [Figure 23D] FIG. 23D shows tumor image analysis of subjects 1 to 4. [Figure 24A] FIG. 24A shows tumor image analysis of subjects 1 to 4. [Figure 24B] FIG. 24B shows tumor image analysis of subjects 1 to 4. [Figure 24C] FIG. 24C shows tumor image analysis of subjects 1 to 4. [Diagram 25] FIG. 25 shows changes in the number of circulating tumor cells (CTCs) in peripheral blood after injection of STIL or SpTIL. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0059] The implementation of the methods disclosed herein utilizes immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, recombinant DNA, which are within the skill of the art, unless otherwise indicated.See, for example, Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012), the series Current Protocols in Molecular Biology (FM Ausubel, et al. eds.), the series Methods In Enzymology (Academic Press, Inc.), PCR 2, A Practical Approach (MJ MacPherson, BD Hames and GR Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Culture of Animal Cells, A Manual of Basic Technique and Specialized Applications, 6th Edition (RI Freshney, ed. (2010)), etc.

[0060] As used in the specification and claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a switch molecule" includes a plurality of switch molecules.

[0061] 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 1 or more than 1 standard deviation per run in the art. Alternatively, "about" can mean up to 20%, up to 10%, up to 5%, or up to 1% of a value. Alternatively, especially with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5 times the value, and more preferably within 2 times the value. When specific values ​​are described in the specification and claims, unless otherwise stated, the term "about" should be presumed to mean within an acceptable error range of the particular value.

[0062] As used herein, "cell" generally refers to a biological cell. A cell may be the basic structural, functional and / or biological unit of a living organism. A cell may be from any organism having one or more cells. Some non-limiting examples include prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, cells of unicellular eukaryotes, protozoan cells, cells from plants (e.g., cells from plants such as crops, fruits, vegetables, cereals, soybeans, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin, hay, potatoes, cotton, hemp, tobacco, flowering plants, conifers, gymnosperms, ferns, club mosses, hornworts, liverworts, mosses, etc.), algal cells (e.g., Botryococcus braunii, green algae, Nannochloropsis gaditana, Chlorella vulgaris, etc.), and the like. Examples of cells that may be used include cells from various organisms, such as mammals, mammalian species, mammalian organisms, and mammalian species. Examples of cells that may be used include cells from various organisms, such as mammals, mammalian species, mammalian species, and mammalian species. Examples of cells that may be used include cells from various organisms, such as mammals ...

[0063] The term "antigen" as used herein refers to a molecule or fragment thereof that can be bound by a selective binding agent. For example, an antigen can be a ligand that can be bound by a selective binding agent, such as a receptor. As another example, an antigen can be an antigenic molecule that can be bound by a selective binding agent, such as an immunological protein (such as an antibody). An antigen can also refer to a molecule or fragment thereof that can be used in an article to generate antibodies that can bind to the antigen.

[0064] As used herein, the term "neoantigen" generally refers to tumor-specific antigens that arise from genetic mutations. The resulting mutant proteins, or fragments thereof, are capable of triggering antitumor T cell responses.

[0065] The term "gene" as used herein refers to a nucleic acid (e.g., DNA, such as genomic DNA and cDNA) and its corresponding nucleotide sequence involved in encoding an RNA transcript. The term used herein in reference to genomic DNA includes the control region with intervening untranslated regions and may include the 5' and 3' ends. In some cases, the term includes the transcribed region, including the 5' and 3' untranslated regions (5'-UTR and 3'-UTR), exons and introns. In some genes, the transcribed region will include an "open reading frame" that encodes a polypeptide. In some uses of the term, a "gene" includes only coding sequences (e.g., "open reading frame" or "coding region") necessary to encode a polypeptide. In some cases, a gene does not encode a polypeptide, such as, for example, ribosomal RNA genes (rRNA) and transfer RNA (tRNA) genes. In some cases, the term "gene" includes not only the transcribed sequence, but also additionally includes untranscribed regions, including upstream and downstream regulatory sequences, enhancers and promoters. A gene may refer to an "endogenous gene" or a naturally occurring gene in its natural location in the genome of an organism. A gene may refer to an "exogenous gene" or a non-native gene. A non-native gene may refer to a gene that is not normally found in the host organism, but is introduced into the host organism by gene transfer. A non-native gene may also refer to a gene that is not in its natural location in the genome of an organism. A non-native gene may also refer to a nucleic acid of natural origin or a polypeptide that contains mutations, insertions and / or deletions (e.g., a non-native sequence).

[0066] The term "antibody" as used herein refers to a proteinaceous binding molecule with immunoglobulin-like functions. The term antibody includes antibodies (such as monoclonal and polyclonal antibodies) and derivatives, variants and fragments thereof. Antibodies include, but are not limited to, immunoglobulins (IG's) of various classes (i.e., IgA, IgG, IgM, IgD and IgE) and subclasses (e.g., IgG1, IgG2, etc.). Derivatives, variants and fragments thereof may refer to functional derivatives or fragments that retain the binding specificity (e.g., complete and / or partial) of the corresponding antibody. Antigen-binding fragments include Fab, Fab', F(ab')2, variable fragment (Fv), single-chain variable fragment (scFv), minibodies, bispecific antibodies, and single domain antibodies ("sdAb" or "nanobodies" or "camelids"). The term antibody includes antibodies and antigen-binding fragments of antibodies that are optimized, engineered or chemically bound. Examples of optimized antibodies include affinity matured antibodies. Examples of engineered antibodies include Fc-optimized antibodies (such as antibodies optimized in the crystallizable fragment region) and multispecific antibodies (such as bispecific antibodies).

[0067] The term "nucleotide" as used herein generally refers to a base-sugar-phosphate combination. Nucleotides include synthetic nucleotides, and nucleotides include synthetic nucleotide analogs. Nucleotides can be monomeric units of nucleic acid sequences, such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The term nucleotide can include ribonucleoside triphosphates, adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP), and deoxyribonucleoside triphosphates, such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, and derivatives thereof. Such derivatives include, for example, "αS" dATP, 7-deasa-dGTP, and 7-deaza-dATP, nucleotide derivatives that confer nuclease resistance to nucleic acid molecules that contain them, and the like. The term nucleotide as used herein may refer to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Illustrative examples of dideoxyribonucleoside triphosphates include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. Nucleotides may be unlabeled or detectably labeled by known methods. Labeling may be performed using quantum dots. Detectable labels include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels.

[0068] The terms "polynucleotide", "oligonucleotide" and "nucleic acid" are used interchangeably to mean a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, in either single-stranded, double-stranded, or multistranded form. A polynucleotide can be endogenous or exogenous to a cell. A polynucleotide can be present in a cell-free environment. A polynucleotide can be a gene or a fragment thereof. A polynucleotide can be DNA. A polynucleotide can be RNA. A polynucleotide can have any three-dimensional structure and can perform any function, known or unknown. A polynucleotide can contain one or more analogs, such as altered backbones, sugars, or bases. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. Some non-limiting examples of analogs include 5-bromouracil, peptide nucleic acid, heterologous nucleic acid, morpholino, bridged nucleic acid, glycol nucleic acid, threose nucleic acid, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (such as rhodamine or fluorescein-conjugated sugars), thiol-containing nucleotides, biotin-conjugated nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queosine, and wyosine.Non-limiting examples of polynucleotides include coding or non-coding regions of genes or gene fragments, loci (locus) defined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small interfering RNA (siRNA), small hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotides such as cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes, and primers. The sequence of nucleotides may be interrupted by non-nucleotide compounds.

[0069] The term "expression" refers to one or more processes by which a polynucleotide is transcribed from a DNA template (e.g., into an mRNA or other RNA transcript) and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. The transcript and the encoded polypeptide may collectively be referred to as a "gene product." If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. With respect to expression, "up-regulated" generally refers to an increased expression level of a polynucleotide (e.g., an RNA such as an mRNA) and / or a polypeptide relative to its expression level in the wild-type state, whereas "down-regulated" generally refers to a decreased expression level of a polynucleotide (e.g., an RNA such as an mRNA) and / or a polypeptide relative to its expression level in the wild-type state.

[0070] As used herein, the term "regulating" in reference to expression or activity means altering the level of expression or activity. Regulation can occur at the transcriptional and / or translational level.

[0071] The terms "peptide", "polypeptide", and "protein" are used interchangeably herein to mean a polymer of at least two amino acids linked by peptide bonds (peptide bonds). The term does not imply a specific length of the polymer, nor is it intended to imply or distinguish whether the peptide is produced using recombinant techniques, chemical or enzymatic synthesis, or is of natural origin. The term applies to amino acid polymers of natural origin as well as to amino acid polymers that contain at least one modified amino acid. The term includes amino acid chains of any length, including full-length proteins, or proteins (e.g., domains) with or without secondary and / or tertiary structure. The term also includes amino acid polymers that have been modified, for example, by disulfide bond formation, glycosylation, lipid modification, acetylation, phosphorylation, oxidation, and any other manipulation, such as, for example, association with a labeling moiety. As used herein, the terms "amino acid" and "amino acids" generally refer to natural or unnatural amino acids, including, but not limited to, modified amino acids and amino acid analogs. Modified amino acids can include natural amino acids and unnatural amino acids that have been chemically modified to include groups or chemical moieties not naturally occurring in amino acids. Amino acid analogs can refer to amino acid derivatives. The term "amino acid" includes both D- and L-amino acids.

[0072] The terms "derivative," "variant," and "fragment," as used herein with respect to a polypeptide, refer to a polypeptide that is related to a wild-type polypeptide either by amino acid sequence, structure (e.g., secondary and / or tertiary), activity (e.g., enzymatic activity), and / or function. Polypeptide derivatives, variants, and fragments can contain one or more differences (e.g., mutations, insertions, deletions, and the like), truncations, modifications, or combinations thereof, as compared to the wild-type polypeptide.

[0073] As used herein, "fusion" may refer to a protein and / or nucleic acid that includes one or more non-native sequences (e.g., sites, etc.). A fusion may include one or more identical non-native sequences. A fusion may include one or more different non-native sequences. A fusion may be chimeric. A fusion may include a nucleic acid affinity tag. A fusion may include a barcode. A fusion may include a peptide affinity tag. A fusion may provide site-specific subcellular localization of a polypeptide (e.g., a nuclear localization signal (NLS) for targeting to the nucleus, a mitochondrial localization signal for targeting to the mitochondria, a chloroplast localization signal for targeting to the chloroplast, an endoplasmic reticulum (ER) retention signal, etc.). A fusion may provide a non-native sequence (e.g., an affinity tag, etc.) that can be used for tracking or purification. A fusion may be a small molecule such as biotin or a dye such as an Alexa fluor® dye, a Cyanine 3 dye, a Cyanine 5 dye, etc.

[0074] As used herein, the phrase "exogenous T cell receptor (TCR) complex" or "exogenous TCR complex" refers to a TCR complex in which one or more chains of the TCR have been introduced into the genome of an immune cell that may or may not endogenously express the TCR. In some cases, an exogenous TCR complex may refer to a TCR complex in which one or more chains of the endogenous TCR complex have one or more mutated sequences, e.g., at either the nucleic acid level or the amino acid level. Expression of an exogenous TCR on an immune cell may confer specificity of binding to an epitope or antigen, such as an epitope or antigen that is preferentially present on the surface of a cancer cell or other pathogenic cell or particle. An exogenous TCR complex may include a TCR-α, TCR-β chain, CD3-γ chain, CD3-δ chain, CD3-ζ chain, or any combination thereof, that is introduced into the genome. In some cases, the strands introduced into the genome may replace endogenously occurring strands.

[0075] The terms "subject," "individual," and "patient" are used herein to refer to a vertebrate, preferably a mammal, such as a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Also included are tissues, cells, and progeny of biological entities obtained in vivo or cultured in vitro.

[0076] As used herein, the terms "treatment" and "treating" refer to an approach for obtaining beneficial or desired results, including, but not limited to, therapeutic benefit and / or prophylactic benefit. For example, treatment may include administering a system or cell population disclosed herein. Therapeutic benefit refers to any treatment-related improvement or effect in one or more diseases, conditions, or symptoms under treatment. For prophylactic benefit, the composition may be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject who has not yet developed the disease, condition, or symptom but has reported one or more physiological symptoms of the disease.

[0077] The term "effective amount" or "therapeutically effective amount" refers to an amount of a composition, e.g., a composition comprising immune cells, e.g., lymphocytes (e.g., T lymphocytes and / or NK cells), of the present disclosure, that is sufficient to result in a desired activity upon administration to a subject in need thereof. Within the context of the present disclosure, the term "therapeutically effective" refers to an amount of a composition that is sufficient to delay the onset, halt the progression, relieve or alleviate at least one symptom of the disease being treated by the methods of the present disclosure.

[0078] The term "genetic profile" as used herein refers to information about a particular gene, including its variability and expression in an individual or in a certain tissue. The term "somatic mutation profile" as used herein refers to information about a particular gene associated with a somatic mutation, such as, but not limited to, a particular gene resulting from a somatic mutation. The somatic mutation profile can be used for neoantigen selection.

[0079] In one aspect, the present disclosure provides modified tumor infiltrating lymphocytes (TILs) that specifically bind to tumor-associated antigens, such as, but not limited to, neoantigens. The modified TILs include chimeric stimulating molecules. The chimeric stimulating molecules include a polypeptide extracellular domain (PED) that binds to neoantigens. The PED can be fused to an intracellular domain (ICD) of a costimulatory molecule that mediates immune cell activation signals. Binding of the chimeric stimulating molecule to the neoantigen can result in immune cell activation signals in the modified TILs. In some embodiments, the PED can be an extracellular domain of a surface protein of an unmodified TIL. In some embodiments, examples of PEDs include antibodies, and derivatives, variants, and fragments thereof.

[0080] In one aspect, the present disclosure provides a modified tumor infiltrating lymphocyte (TIL) that specifically binds to a neoantigen, wherein the modified TIL comprises a switch molecule. The switch molecule may comprise an extracellular domain (ECD) of a protein that induces an immune inactivation signal in an unmodified immune cell upon binding to its ligand. The ECD may be fused to an intracellular domain (ICD) of a costimulatory molecule that mediates an immune cell activation signal. Binding of the switch molecule to a ligand may result in an immune cell activation signal instead of an immune cell inactivation signal in the modified immune cell.

[0081] TILs can be any cells obtained from a tumor. For example, TILs can be cells that have migrated to a tumor. TILs can be cells that have infiltrated a tumor. In some embodiments, TILs are white blood cells that have migrated from the bloodstream of a subject to a tumor. TILs can be, for example, T cells, B cells, monocytes, natural killer (NK) cells. In some cases, modified TILs include CD8+ cytotoxic T cells (lymphocytes), Th1 and Th17 CD4+ T cells, natural killer cells, dendritic cells, or M1 macrophages. The immune cell population that includes TILs can be a mixed population of cells. The population of TILs can include cells of various phenotypes, cells with different degrees of differentiation, cells with various cell lineages, or a combination thereof. TILs can generally be defined either biochemically, using cell surface markers, or functionally, depending on their ability to infiltrate tumors and affect treatment. TILs can be classified based on expression of one or more of the following biomarkers: CD4, CD8, TCR αβ, CD25, CD27, CD28, CD56, CD137, CCR7, CD45Ra, CD95, PD-1, and TIM-3. In some embodiments, the modified TILs express at least one of PD-1, CD137, and TIM-3. In some cases, the TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into the patient. In some cases, the modified TILs include "primary TILs," which refer to TILs obtained from a patient tissue sample. In some cases, the modified TILs include "secondary TILs," which refer to TILs that have been expanded or expanded. The TILs can exhibit specific binding to neoantigens. In some cases, the TCR complex of the TILs confers antigen binding specificity (e.g., neoantigen binding).

[0082] In one embodiment, the disclosure provides an engineered T cell that specifically binds to a neoantigen, wherein the engineered T cell comprises a switch molecule. The switch molecule may comprise an extracellular domain (ECD) of a protein that induces an immune cell activation signal in an unengineered T cell upon binding to its ligand. The ECD may be fused to an intracellular domain (ICD) of a costimulatory molecule that mediates the immune cell activation signal. Binding of the switch molecule to a ligand may result in an immune cell activation signal instead of an immune cell inactivation signal in the engineered immune cell.

[0083] The engineered T cells may comprise a T cell receptor (TCR) complex that exhibits specific binding to a neoantigen. In some embodiments, the TCR complex is an endogenous TCR complex. In some embodiments, the TCR complex is an exogenous TCR complex. For example, an endogenous or exogenous TCR complex of an engineered immune cell may confer antigen binding specificity (e.g., neoantigen binding) to the immune cell. In some embodiments, the present disclosure provides engineered T cells comprising an endogenous TCR complex that specifically binds to a neoantigen, the engineered T cells comprising a chimeric stimulating molecule, the chimeric stimulating molecule comprising a polypeptide extracellular domain (PED) that binds to a membrane protein of a cell, including, but not limited to, a tumor cell, the PED being fused to an intracellular domain (ICD) of a costimulatory molecule that mediates an immune cell activation signal, and the binding of the chimeric stimulating molecule to the membrane protein results in the immune cell activation signal in the engineered T cells.

[0084] The binding of modified immune cells, such as modified T cells or modified TILs provided herein, to neoantigens can activate immune cells. The switch molecules of modified cells can be used to provide further control of immune cell activity, such as, but not limited to, immune cell activation and proliferation. The binding of the switch molecules in modified immune cells, such as modified T cells or modified TILs, to ligands can result in immune cell activation signals instead of immune cell inactivation signals in modified immune cells. Inducing immune cell activation signals instead of immune cell inactivation signals in modified immune cells can minimize the immunosuppressive effects in immune cells. Minimizing the immunosuppressive effects in immune cells can increase the effect of immune cells in immune responses, for example, by increasing immune cytotoxicity against target cells, such as tumor cells.

[0085] The switch molecule may comprise an extracellular domain (ECD) of a protein that induces an immune inactivation signal in unmodified immune cells upon binding to its ligand. The protein may be a signaling receptor or any functional fragment, derivative, or variant thereof. In some cases, the signaling receptor may be a membrane-bound receptor. The signaling receptor may induce one or more signaling pathways in a cell in response to binding of a ligand. In some cases, the signaling receptor may be a non-membrane-bound receptor. The switch molecule may comprise a fragment, such as an extracellular domain, of a receptor selected from a G protein-coupled receptor (GPCR); an integrin receptor; a cadherin receptor; a catalytic receptor (such as a kinase); a death receptor; a checkpoint receptor; a cytokine receptor; a chemokine receptor; a growth factor receptor; a hormone receptor; and an immune receptor.

[0086] In some embodiments, the switch molecule comprises a fragment of an immune checkpoint receptor that may be involved in the control of the immune system. Non-limiting examples of such receptors include, but are not limited to, programmed cell death 1 (PD-1), T cell co-stimulatory receptor 4 (CTLA-4), B and T lymphocyte attenuator (BTLA), killer cell immunoglobulin-like receptor (KIR), indoleamine-2,3-dioxygenase (IDO), lymphocyte activation gene-3 (LAG3), T cell immunoglobulin mucin-3 (TIM-3), and T cell immunoreceptor with Ig and ITIM domains (TIGIT).

[0087] In some embodiments, the switch molecule comprises at least an extracellular fragment of a TCR that can be involved in recognizing a neoantigen (e.g., a cancer cell antigen or a tumor antigen) of a target cell. In some examples, the switch molecule can comprise the extracellular variable region of a TCR alpha and / or beta chain.

[0088] A switch molecule comprising an immune checkpoint receptor or any derivative, variant, or fragment thereof can bind to an antigen comprising any suitable immune checkpoint receptor ligand or derivative, variant, or fragment thereof, such as, but not limited to, B7-1, B7-H3, B7-H4, HVEM (Herpesvirus Entry Mediator), AP2M1, CD80, CD86, SHP-2, PPP2R5A, MHC (e.g., class I, class II), PD-L1, and PD-L2.

[0089] In some embodiments, the switch molecule comprises a fragment of a cytokine receptor. Cytokine receptors can perform a variety of functions, including, but not limited to, immune cell regulation and inflammation mediation. In some embodiments, the switch molecule comprises a cytokine receptor, such as a type I cytokine receptor or a type II cytokine receptor, or a derivative, variant, or fragment thereof. In some embodiments, the switch molecule comprises an interleukin receptor (e.g., IL-2R, IL-3R, IL-4R, IL-5R, IL-6R, IL-7R, IL-9R, IL-11R, IL-12R, IL-13R, IL-15R, IL-21R, IL-23R, IL-27R, and IL-31R), a colony stimulating factor receptor (e.g., erythropoietin receptor, CSF-1R, CSF-2R, GM-CSFR, and G-CSFR), a hormone receptor / neuropeptide receptor (e.g., growth hormone receptor, protein receptor, and leptin receptor), or a derivative, variant, or fragment thereof. In some embodiments, the switch molecule comprises a type II cytokine receptor, or a derivative, variant, or fragment thereof. In some embodiments, the switch molecule comprises an interferon receptor (e.g., and IFNGR), an interleukin receptor (e.g., IL-10R, IL-20R, IL-22R, and IL-28R), a tissue factor receptor (also called platelet tissue factor), or a derivative, variant, or fragment thereof.

[0090] In some embodiments, the switch molecule comprises at least an extracellular region (e.g., a ligand binding domain) of a catalytic receptor, such as a receptor tyrosine kinase (RTK), or a derivative, variant, or fragment thereof. In some embodiments, the switch molecule comprises a class I RTK (e.g., the epidermal growth factor (EGF) receptor family, including EGFR;Class II RTKs (e.g., the insulin receptor family including INSR, IGF-1R, and IRR), Class III RTKs (e.g., the platelet-derived factor (PDGF) receptor family including DGFR-α, PDGFR-β, CSF-1R, KIT / SCFR, and FLK2 / FLT3), Class IV RTKs (e.g., the fibroblast growth factor (FGF) receptor family including FGFR-1, FGFR-2, FGFR-3, and FGFR-4), Class V RTKs (e.g., the vascular endothelial growth factor (VEGF) receptor family including VEGFR1, VEGFR2, and VEGFR3), Class VI RTKs (e.g., the hepatocyte growth factor (HGF) receptor family including hepatocyte growth factor receptor (HGFR / MET) and RON), Class VII RTKs (e.g., the hepatocyte growth factor (HGF) receptor family including HGFR / MET and RON), Class VIII ... Class VIII RTKs (e.g., the tropomyosin receptor kinase (Trk) receptor family, including TRKA, TRKB, and TRKC), Class VIII RTKs (e.g., the ephrin (Eph) receptor family, including EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHB1, EPHB2, EPHB3, EPHB4, EPHB5, and EPHB6), Class IX RTKs (e.g., the AXL receptor family, such as AXL, MER, and TRYO3), Class X RTKs (e.g., the LTK receptor family, such as TK and ALK), Class XI RTKs (e.g., the TIE receptor family, such as TIE and TEK), Class XII RTKs (e.g., the ROR receptor family ROR1 and ROR2), Class XIII RTKs (e.g., the discoidin domain receptor (DDR) family, such as DDR1 and DDR2), class XIV RTKs (e.g., the RET receptor family, such as RET), class XV RTKs (e.g., the KLG receptor family, which includes PTK7), class XVI RTKs (e.g., the RYK receptor family, which includes Ryk), class XVII RTKs (e.g., the MuSK receptor family, such as MuSK), or derivatives, variants, or fragments thereof;

[0091] The switch molecule comprising an RTK, or a derivative, variant, or fragment thereof, may bind to any suitable RTK ligand, or an antigen comprising a derivative, variant, or fragment thereof. Non-limiting examples of RTK ligands include growth factors, cytokines, and hormones. Growth factors include, for example, members of the epidermal growth factor family (e.g., epidermal growth factor or EGF, heparin-binding EGF-like growth factor or HB-EGF, transforming growth factor alpha or TGF-alpha, amphiregulin or AR, epiregulin or EPR, epigene, betacellulin or BTC, neuregulin-1 or NRG1, neuregulin-2 or NRG2, neuregulin-3 or NRG3, and neuregulin-4 or NRG4), fibroblast growth factors (e.g., FG, NF-κB ... FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15 / 19, FGF16, FGF17, FGF18, FGF20, FGF21, and FGF23), vascular endothelial growth factor family (e.g., VEGF-A, VEGF-B, VEGF-C, VEGF-D, and PIGF), and platelet-derived growth factor family (e.g., PDGFA, PDGFB, PDGFC, and PDGFD). Hormones include, for example, members of the insulin / IGF / relaxin family (e.g., insulin, insulin-like growth factors, relaxin family peptides including relaxin 1, relaxin 2, relaxin 3, Leydig cell-specific insulin-like peptide (gene INSL3), early placental insulin-like peptide (ELIP) (gene INSL4), insulin-like peptide 5 (gene INSL5), and insulin-like peptide 6).

[0092] In some embodiments, the switch molecule comprises at least an extracellular region (e.g., a ligand-binding domain, etc.) of a catalytic receptor, such as a receptor threonine / serine kinase (RTSK), or a derivative, variant, or fragment thereof. The switch molecule may comprise a type I RTSK, a type II RTSK, or a derivative, variant, or fragment thereof. The switch molecule comprises a type I receptor selected from the group consisting of ALK1 (ACVRL1), ALK2 (ACVR1A), ALK3 (BMPR1A), ALK4 (ACVR1B), ALK5 (TGFβR1), ALK6 (BMPR1B), and ALK7 (ACVR1C), or a derivative, variant, or fragment thereof. The switch molecule comprises a type II receptor selected from the group consisting of TGFβR2, BMPR2, ACVR2A, ACVR2B, and AMHR2 (AMHR), or a derivative, variant, or fragment thereof.

[0093] The RTSK, or derivative, variant or fragment thereof switch molecule may bind to an antigen, including any suitable RTSK ligand, or derivative, variant or fragment thereof.

[0094] The switch molecule may include an intracellular domain (ICD) of a costimulatory molecule that induces an immune cell activation signal. The costimulatory molecule may bind to a ligand. In some cases, the costimulatory molecule may be activated by a ligand-responsive protein. In some embodiments, the costimulatory molecule may be activated to regulate proliferation and / or survival signals in immune cells. In some embodiments, the ICD is an intracellular domain of a costimulatory molecule selected from an MHC class I protein, an MHC class II protein, a TNF receptor protein, an immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocyte activation molecule (SLAM protein), an activating NK cell receptor, BTLA, or a Toll ligand receptor. In some embodiments, the costimulatory domain is selected from the group consisting of 2B4 / CD244 / SLAMF4, 4-1BB / TNFSF9 / CD137, B7-1 / CD80, B7-2 / CD86, B7-H1 / PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BAFF R / TNFRSF13C, BAFF / BLyS / TNFSF13B, BLAME / SLAMF8, BTLA / CD272, CD100 (SEMA4D), CD103, CD11a, CD11b, CD11c, CD11d, CD150, CD160 (BY55), CD18, CD19, CD2, CD200, CD229 / SLAMF3, CD27 Ligand / TNFSF7, CD27 / TNFRSF7, CD28, CD29, CD2F-10 / SLAMF9, CD30, CD30 Ligand / TNFSF8, CD30 / TNFRSF8, CD300a / LMIR1, CD4, CD40 Ligand / TNFSF5, CD40 / TNFRSF5, CD48 / SLAMF2, CD49a, CD49D, CD49f, CD5, CD53, CD58 / LFA-3, CD69, CD7, CD8 α, CD8 β, CD82 / Kai-1, CD84 / SLAMF5, CD90 / Thy1, CD96, CDS, CEACAM1, CRACC / SLAMF7, CRTAM, CTLA-4, DAP12, Dectin-1 / CLEC7A, DNAM1(CD226), DPPIV / CD26, DR3 / TNFRSF25, EphB6, GADS, Gi24 / VISTA / B7-H5, GITR ligand / TNFSF18, GITR / TNFRSF18, HLA class I, HLA-DR, HVEM / TNFRSF14, IA4, ICAM-1, ICOS / CD278, Ikaros, IL2R β, IL2R γ, IL7R α, IR-12R, integrin α4 / CD49d, integrin α4β1, integrin α4β7 / LPAM-1, IPO-3, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIRDS2, LAG-3, LAT, LIGHT / TNFSF14, LTBR, ​​Ly108, Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1), lymphotoxin-α / TNF-β, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), NTB-A / SLAMF6, OX40 ligand / TNFSF4, OX40 / TNFRSF4, PAG / Cbp, PD-1, PDCD6, PD-L2 / B7-DC, PSGL1, RELT / TNFRSF19L, SELPLG (CD162), SLAM (SLAMF1), SLAM / CD150, SLAMF4 (CD244), SLAMF6 (NTB-A), SLAMF7, SLP-76, TACI / TNFRSF13B, TCL1A, TCL1B, TIM-1 / KIM-1 / HAVCR, TIM-4, TL1A / TNFSF15, TNF RII / TNFRSF1B, TNF-α, TRANCE / RANKL, TSLP, TSLP R, VLA1, and VLA-6.

[0095] The ECD and ICD of the costimulatory molecule may be connected by a transmembrane domain, for example, by a transmembrane segment. In some embodiments, the transmembrane segment comprises a polypeptide. The transmembrane polypeptide may have any suitable polypeptide sequence. In some cases, the transmembrane polypeptide comprises a polypeptide sequence of a transmembrane portion of an endogenous or wild-type transmembrane protein. In some embodiments, the transmembrane polypeptide comprises a polypeptide sequence having at least one (e.g., at least two, three, four, five, six, seven, eight, nine, ten, or more) amino acid substitution, deletion, and insertion compared to the transmembrane portion of an endogenous or wild-type transmembrane protein. In some embodiments, the transmembrane polypeptide comprises a non-natural polypeptide sequence, for example, a polypeptide linker sequence. The polypeptide linker may be flexible or rigid. The polypeptide linker may be structured or unstructured. In some embodiments, the transmembrane polypeptide transmits a signal from the ECD to the ICD, such as a signal indicating ligand binding.

[0096] Binding of the ligand to the switch molecule can result in an immune cell activation signal in the engineered immune cell. In some embodiments, the immune cell activation signal is mediated by an activator. The activator can be an immunomodulatory molecule. The activator can bind, activate, or stimulate T cells or other immune cells to regulate their activity. In some embodiments, the activator can be secreted from the immune cell. The activator can be, for example, a soluble cytokine, a soluble chemokine, or a growth factor molecule. Non-limiting examples of activators that can mediate immune cell activation include, for example, soluble cytokines such as IL-1, IL-2, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IL-21, tumor necrosis factor (TNF), transforming growth factor (TGF), interferon (IFN), or derivatives, variants, or fragments thereof.

[0097] Immune cell activation signals can include or result in clonal expansion of the modified immune cell (e.g., modified TIL or modified T cell), cytokine release by the modified immune cell (e.g., modified TIL or modified T cell), cytotoxicity of the modified immune cell (e.g., modified TIL or modified T cell), proliferation of the modified immune cell (e.g., modified TIL or modified T cell), differentiation, dedifferentiation, or transdifferentiation of the modified immune cell (e.g., modified TIL or modified T cell), migration and / or trafficking of the modified immune cell (e.g., modified TIL or modified T cell), exhaustion and / or reactivation of the modified immune cell (e.g., modified TIL or modified T cell), release of other intercellular molecules, metabolites, compounds, or combinations thereof by the modified immune cell (e.g., modified TIL or modified T cell).

[0098] In some embodiments, the immune cell activity includes or results in clonal expansion of the immune cell. Clonal expansion includes the generation of daughter cells arising from the immune cell. The daughter cells resulting from the clonal expansion may include the switch molecule. The clonal expansion of the modified immune cell may be greater than that of a comparable immune cell that does not have the switch molecule. The clonal expansion of the modified immune cells can be about 5-fold to about 10-fold, about 10-fold to about 20-fold, about 20-fold to about 30-fold, about 30-fold to about 40-fold, about 40-fold to about 50-fold, about 50-fold to about 60-fold, about 60-fold to about 70-fold, about 70-fold to about 80-fold, about 80-fold to about 90-fold, about 90-fold to about 100-fold, about 100-fold to about 200-fold, about 200-fold to about 300-fold, about 300-fold to about 400-fold, about 400-fold to about 500-fold, about 500-fold to about 600-fold, about 600-fold to about 700-fold. In some embodiments, measuring the clonal expansion can include, for example, quantifying the number of immune cells with and without the switch molecule and after ligand binding to the switch molecule. Quantification of immune cell numbers can be performed by a variety of techniques, including, but not limited to, flow cytometry, trypan blue exclusion assay, and blood cell counts.

[0099] In some embodiments, the immune cell activity includes or results in cytokine release by the immune cell. In some embodiments, the immune cell activity includes or results in the release of intercellular molecules, metabolites, compounds, or combinations thereof. Cytokine release by the modified immune cell may include release of IL-1, IL-2, IL-4, IL-5, IL-6, IL-13, IL-17, IL-21, IL-22, IFNγ, TNFalpha, CSF, TGFβ, granzymes, and the like. In some embodiments, cytokine release may be quantified using enzyme-linked immunosorbent assay (ELISA), flow cytometry, Western blot, and the like. Cytokine release by the modified immune cell may be greater than that of a comparable immune cell without the switch molecule. The modified immune cells provided herein may produce about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, 200-fold, 250-fold, or 300-fold or more greater cytokine release compared to a comparable immune cell not having the switch molecule. The modified immune cells may exhibit increased cytokine secretion when the switch molecule binds to a ligand and the modified immune cell binds to a neoantigen present on a target cell compared to a comparable immune cell not having the switch molecule (e.g., unmodified). In some embodiments, the secreted cytokine is IFNγ or IL-2. In some embodiments, cytokine release may be quantified in vitro or in vivo.

[0100] In some embodiments, the cytotoxicity of immune cell activity includes or results from. In some cases, the cytotoxicity of the modified immune cells provided herein can be used to kill target cells. The immune cells or immune cell populations expressing the switch molecule can induce death in target cells. Killing of target cells can be used in a variety of applications, including but not limited to treating diseases or disorders in which it is desirable to eliminate a cell population or inhibit cell proliferation. Cytotoxicity can also refer to the release by immune cells of cytotoxic cytokines, such as IFNγ or granzymes. In some cases, the modified immune cells provided herein can have altered (i) release of cytotoxins, such as perforin, granzymes, and granulysin, and / or (ii) induction of apoptosis via Fas-Fas ligand interaction between T cells and target cells. In some embodiments, cytotoxicity can be quantified by cytotoxicity assays, including co-culture assays, ELISPOT, chromium release cell characteristic assays, and the like. The toxicity of the modified immune cells provided herein may be greater than that of a comparable immune cell that does not have the switch molecule. The modified immune cells may exhibit increased cytotoxicity against target cells when the switch molecule binds to a ligand and the modified immune cells bind to a neoantigen present on the target cell, as compared to a comparable immune cell that does not have the switch molecule (e.g., unmodified). The modified immune cells of the present disclosure may be about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, or 200% or more cytotoxic to target cells as compared to a comparable immune cell that does not have the switch molecule.The modified immune cells of the present disclosure may induce death of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, or 200% more target cells than comparable immune cells without the switch molecule. In some embodiments, the immune cells provided herein may induce apoptosis in target cells presenting target epitopes (such as neoantigens) on their surface. In some embodiments, cytotoxicity may be measured in vitro or in vivo. In some embodiments, measuring cytotoxicity may include measuring the level of disease after administration of modified immune cells provided herein compared to the level of disease before administration. In some embodiments, measuring cytotoxicity can include measuring the level of disease following administration of an engineered immune cell provided herein and the level of disease following administration of a comparable immune cell that does not have the switch molecule.

[0101] In some embodiments, the immune cell activity includes or results in the proliferation of immune cells. The proliferation of immune cells may refer to an increase in immune cells. The proliferation of immune cells may refer to a change in the phenotype of immune cells. The proliferation of the modified immune cells of the present disclosure may be increased over that of a comparable immune cell that does not have the switch molecule. The proliferation of the modified immune cells of the present disclosure may be about 5 to about 10 times, about 10 to about 20 times, about 20 to about 30 times, about 30 to about 40 times, about 40 to about 50 times, about 50 to about 60 times, about 60 to about 70 times, about 70 to about 80 times, about 80 to about 90 times, about 90 to about 100 times, about 100 to about 200 times, about 200 to about 300 times, about 300 to about 400 times, about 400 to about 500 times, about 500 to about 600 times, about 600 to about 700 times. In some embodiments, the proliferation may be measured by quantifying the number of immune cells. Quantifying the number of immune cells may include flow cytometry, trypan blue exclusion test, and hemocytometry. Proliferation may also be determined by phenotypic analysis of immune cells.

[0102] In some embodiments, the activity of the immune cells includes or results in differentiation, dedifferentiation, or transdifferentiation of the immune cells. Differentiation, dedifferentiation, or transdifferentiation of the immune cells can be determined by evaluating the phenotypic expression of differentiation, dedifferentiation, or transdifferentiation markers on the cell surface by flow cytometry. In some embodiments, the modified immune cells provided herein have an enhanced differentiation capacity compared to a comparable immune cell that does not have the switch molecule. In some embodiments, the modified immune cells provided herein have an enhanced dedifferentiation capacity compared to a comparable immune cell that does not have the switch molecule. In some embodiments, the modified immune cells provided herein have a higher transdifferentiation capacity compared to a comparable immune cell that does not have the switch molecule.

[0103] In some embodiments, the activity of immune cells includes or results in the migration and / or trafficking of immune cells. In some embodiments, the migration can be determined by quantifying the localization of immune cells to a target site. For example, modified immune cells provided herein can be quantified at a target site, e.g., at a site that is not a target site, after administration. Quantification can be performed by isolating the lesion and quantifying the number of immune cells, e.g., tumor infiltrating lymphocytes, that contain the switch molecule. The migration and / or trafficking of immune cells that contain the switch molecule is greater than that of comparable immune cells that do not have the switch molecule. In some embodiments, the number of immune cells that contain the switch molecule in a target site, e.g., in a lesion area, can be about 5, 10, 15, 20, 25, 30, 35, or 40 times the number of comparable immune cells that do not have the switch molecule. Trafficking can also be determined in vitro using a transwell migration assay. In some embodiments, for example in a transwell migration assay, the number of immune cells containing a switch molecule at a target site can be about 5, 10, 15, 20, 25, 30, 35, or 40 times the number of equivalent immune cells not bearing the switch molecule.

[0104] In some embodiments, immune cell activity includes or results in exhaustion and / or reactivation of immune cells. Exhaustion and / or reactivation of immune cells can be measured by phenotypic analysis by flow cytometry or microscopic analysis. For example, expression levels of markers of exhaustion, such as programmed cell death protein 1 (PD1), lymphocyte activation gene-3 (LAG3), 2B4, CD160, Tim3, and T cell immune receptor with immunoglobulin and ITIM domains (TIGIT), can be measured quantitatively and / or qualitatively. In some cases, immune cells, such as T cells, can lose effector function in a hierarchical manner and become exhausted. Exhaustion can result in loss of function, such as IL-2 production and cytokine expression, and high proliferation capacity. Exhaustion can also involve defects in IFNγ, TNF, and chemokine production and degranulation. Exhaustion or activation of modified immune cells provided herein is greater than comparable immune cells without the switch molecule. In some embodiments, the immune cells provided herein undergo at least about a 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, 200-fold, 250-fold, or 300-fold or more increase in exhaustion or activation compared to a comparable immune cell that does not have the switch molecule. In some embodiments, the immune cells provided herein undergo at least about a 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, 200-fold, 250-fold, or 300-fold or more decrease in exhaustion or activation compared to a comparable immune cell that does not have the switch molecule.

[0105] In some embodiments, upon binding of the switch molecule to a ligand, the engineered immune cell (such as an engineered TIL or engineered T cell) exhibits enhanced neoantigen binding compared to a comparable immune cell that does not possess the switch molecule.

[0106] In one embodiment, the present disclosure provides modified immune cells comprising a chimeric antigen receptor (CAR) and a T cell receptor (TCR) complex that exhibits specific binding to a neoantigen. The CAR can include an antigen-interacting domain capable of binding a B cell surface protein, a transmembrane domain, and an intracellular signaling domain.

[0107] The T cell receptor (TCR) complex that exhibits specific binding to the neoantigen can be an endogenous TCR complex or an exogenous TCR complex. The TCR complex, e.g., endogenous or exogenous, of the engineered immune cell confers antigen-binding specificity (e.g., neoantigen binding) to the immune cell.

[0108] In some embodiments, the immune cells are tumor infiltrating lymphocytes (TILs). The TILs can be, for example, T cells, B cells, monocytes, natural killer (NK) cells. In some cases, the TILs include CD8+ cytotoxic T cells (lymphocytes), Th1 and Th17 CD4+ T cells, natural killer cells, dendritic cells, or M1 macrophages. In some embodiments, the TILs can express at least one of PD-1, CD137, and TIM-3. In some cases, the modified TILs include "secondary TILs," which refers to expanded or expanded TILs.

[0109] CAR comprises an antigen interaction domain that can bind B cell surface protein. B cell surface protein can be any protein that can be found on the surface of B cell. Non-limiting examples include CD1d, CD5, CD10, CD11a, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD28, CD29, CD34, CD37, CD38, CD40, CD44, CD45, CD49b, CD69, CD72, CD74, CD80, CD83, CD84, CD86, CD93, CD95, CD117, CD127, CD138, CD147, CD148, CD185, CD270, CD284 and CD360. In some embodiments, the antigen interaction domain of the CAR can bind a surface protein on a non-B cell, so long as binding to the surface protein does not significantly compromise the overall health or immune system of the host. In some embodiments, the surface protein is a surface protein on an immune cell. In some embodiments, the surface protein is a surface protein on a cell other than an immune cell. In some embodiments, the surface protein is selected from the group consisting of CD31, CD32, A, B, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42, a, b, c, d, CD43, CD44, CD45, CD46, CD47, CD48, CD49 (a, b, c, d, e, f), CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD61, CD62 (E, L, P), CD63, CD64 (A, B, C), CD66, provided that binding to the surface protein does not significantly compromise the overall health or immune system of the host. (a, b, c, d, e, f), CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD78, CD79(a、b)、CD80、CD81、CD82、CD83、CD84、CD85(a、d、e、h、j、k)、CD86、CD87、CD88、CD89、CD90、CD91、CD92、CD93、CD94、CD95、CD96、CD97、CD98、CD99、CD100、CD1(a-c)、1A、1D、1E、CD2、CD3(γ、δ、ε)、CD4、CD5、CD6、CD7、CD8、a、CD9、CD10、CD11 (a、b、c、d)、CD13、CD14、CD15、CD16、A、B、CD18、CD19、CD20、CD21、CD22、CD23、CD24、CD25、CD26、CD27、CD28、CD29、CD30、CD101、CD102、CD103、CD104、CD105、CD106、CD107 (a、b)、CD108、CD109、CD110、CD111、CD112、CD113、CD114、CD115、CD116、CD117、CD118、CD119、CD120 (a、b)、CD121(a、b)、CD122、CD123、CD124、CD125、CD126、CD127、CD129、CD130、CD131、CD132、CD133、CD134、CD135、CD136、CD137、CD138、CD140b、CD141、CD142、CD143、CD144、CD146、CD147、CD148、CD150、CD191、CD192、CD193、CD194、CD195、CD196、CD197、CDw198、CDw199、CD200、CD201、CD202b、CD204、CD205、CD206、CD207、CD208、CD209、CDw210(a、b)、CD212、CD213a(1、2)、CD217、CD218、(a、b)、CD220、CD221、CD222、CD223、CD224、CD225、CD226、CD227、CD228、CD229、CD230、CD233、CD234、CD235(a、b)、CD236、CD238、CD239、CD240CE、CD240D、CD241、CD243、CD244、CD246、CD247、CD248、CD249、CD252、CD253、CD254、CD256、CD257、CD258、CD261、CD262、CD263、CD264、CD265、CD266、CD267、CD268、CD269、CD271、CD272、CD273、CD274、CD275、CD276、CD278、CD279、CD280、CD281、CD282、CD283、CD284、CD286、CD288、CD289、CD290、CD292、CDw293、CD294、CD295、CD297、CD298、CD299、CD300A、CD301、CD302、CD303、CD304、CD305、CD306、CD307、CD309、CD312、CD314、CD315、CD316、CD317、CD318、CD320、CD321、CD322、CD324、CD325、CD326、CD328、CD329、CD331、CD332、CD333、CD334、CD335、CD336、CD337、CD338、CD339、CD340、CD344、CD349、CD350、CD151、CD152、CD153、CD154、CD155、CD156(a,b,c), CD157, CD158, (a,d,e,i,k), CD159(a,c), CD160, CD161, CD162, CD163, CD164, CD166, CD167 (a,b), CD168, CD169, CD170, CD171, CD172 (a,b,g), CD174, CD177, CD178, CD179 (a,b), CD180, CD181, CD182, CD183, CD184, CD185, and CD186.

[0110] In some embodiments, the antigen interaction domain of the CAR may be able to bind a B cell surface protein or a fragment thereof on a dead B cell. B cell apoptosis may occur before or after the occurrence of an immune response (such as an immune response against a tumor cell). Thus, a dead B cell or its debris may still have a B cell surface protein or a fragment thereof displayed on its surface. The ability of the CAR to target both live and dead B cells may increase the chances of the immune cell comprising the CAR to (i) bind to the B cell surface protein and (i) initiate signal transduction of the intracellular signal domain. In some cases, the signal transduction of the intracellular signal domain may promote the expansion (proliferation) of the immune cell comprising the CAR.

[0111] In some embodiments, the antigen-interacting domain of the CAR may be capable of binding a B cell surface protein or fragment thereof that is bound (e.g., via covalent bonds and / or via non-covalent bonds) to the surface of a particle (e.g., nanoparticle). The particle may be any particulate material, including organic and / or inorganic materials. The particle may be from about 1 nanometer (nm) to about 50 micrometers (μm) in at least one dimension. The particle may be at least about 1 nm, 5 nm, 10 nm, 50 nm, 100 nm, 500 nm, 1 μm, 5 μm, 10 μm, 50 μm, or more in at least one dimension. The particle may be at most 50 μm, 10 μm, 5 μm, 1 μm, 500 nm, 100 nm, 50 nm, 10 nm, 5 nm, 1 nm, or less in at least one dimension. The particle may be, for example, a nanoparticle, a microparticle, a nanosphere, a microsphere, a nanorod, a microrod, a nanofiber, a nanoribbon, etc. Examples of particles include, for example, metal nanoparticles (e.g., gold nanoparticles, silver nanoparticles, and iron nanoparticles), intermetallic nanosemiconductor nanoparticles, core-shell nanoparticles, particles with inorganic cores and polymer shells, particles with organic cores and polymer shells, or mixtures thereof. Alternatively, the particles can be, for example, crosslinked polymers, hydrogel polymers, biodegradable polymers, polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), copolymers, polysaccharides, starches, cellulose, chitosan, polyhydroxyalkanoic acid (PHA), PHB, PHV, lipids, peptides, peptide amphiphiles, polypeptides (e.g., proteins, etc.), or combinations thereof. Particles presenting B cell surface proteins on their surface can be introduced in vitro into immune cells that contain a CAR that binds to the B cell surface protein. Alternatively, or in addition, particles presenting B cell surface proteins can be introduced in vivo (e.g., by localized or systemic injection) with immune cells that contain a CAR. Such particles can be used to expand populations of immune cells containing a CAR in vitro or in vivo.

[0112] The antigen-binding domain may comprise any protein or molecule capable of binding to an antigen, such as a B-cell surface protein. Non-limiting examples of antigen-binding domains include, but are not limited to, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, murine antibodies, or functional derivatives, variants, or fragments thereof, including, but not limited to, Fab, Fab', F(ab')2, Fv, single chain Fv (scFv), minibodies, bispecific antibodies, and single domain antibodies, such as the heavy chain variable region (VH), light chain variable region (VL), and variable region (VHH) of camelid nanobodies. In some embodiments, the first antigen-binding domain comprises at least one of Fab, Fab', F(ab')2, Fv, and scFv. In some embodiments, the antigen-binding domain comprises an antibody mimetic. Antibody mimetics refer to molecules that can bind to target molecules with affinity comparable to antibodies, including, for example, single chain binding molecules, cytochrome b562-based binding molecules, fibronectin or fibronectin-like protein scaffolds (such as adnectins), lipokine scaffolds, calixarene scaffolds, A-domains and other scaffolds. In some embodiments, the antigen binding domain comprises a transmembrane receptor, or any derivative, variant, or fragment thereof. For example, the antigen binding domain may comprise at least the ligand binding domain of a transmembrane receptor.

[0113] In some embodiments, the antigen-binding domain may comprise an scFV. The scFV may be derived from an antibody whose variable region sequence is known. In some embodiments, the scFV may be derived from an antibody sequence obtained from an available mouse hybridoma. The scFV may be obtained from whole exome sequencing of tumor cells or primary cells. In some embodiments, the scFV may be mutated, so that the scFV has a higher affinity for its target. In some cases, the affinity of the scFV for its target may be optimized for targets that are expressed at low levels in normal tissues. This optimization may be performed to minimize potential toxicity, such as hypercytokinemia. In other cases, cloning of an scFV with a higher affinity for a target in a cell membrane-bound form may be preferred over its counterpart in a soluble form. This modification may be performed when some targets may also be detected in a soluble form at various levels, and their targeting may cause unintended toxicity, such as hypercytokinemia.

[0114] The antigen-binding domain of the CAR of the subject system can be linked to the intracellular signaling domain via a transmembrane domain. The transmembrane domain can be a transmembrane segment. The transmembrane domain of the subject CAR can anchor the CAR to the cell membrane of a cell, e.g., an immune cell. In some embodiments, the transmembrane segment comprises a polypeptide. The transmembrane polypeptide linking the antigen-binding domain and the intracellular signaling domain of the CAR can have any suitable polypeptide sequence. In some cases, the transmembrane polypeptide comprises a polypeptide sequence of a transmembrane site of an endogenous or wild-type transmembrane protein. In some embodiments, the transmembrane polypeptide comprises a polypeptide sequence having at least one (e.g., at least two, three, four, five, six, seven, eight, nine, ten, or more) amino acid substitutions, deletions, and insertions compared to the transmembrane protein of an endogenous or wild-type transmembrane protein. In some embodiments, the transmembrane polypeptide comprises a non-natural polypeptide sequence, such as a polypeptide linker sequence. The polypeptide linker can be flexible or rigid. The polypeptide linker may be structured or unstructured. In some embodiments, the transmembrane polypeptide transmits a signal from the extracellular region of the cell to the intracellular region, for example, via the antigen binding domain. The natural transmembrane site of CD28 may be used in the CAR. In other cases, the natural transmembrane site of CD8 alpha may be used in the CAR.

[0115] The CAR of the present disclosure may comprise a signaling domain involved in immune cell signaling, or a derivative, variant, or fragment thereof. The intracellular signaling domain of the CAR may induce the activity of the immune cell comprising the CAR. The intracellular signaling domain may transmit effector function signals and instruct the cell to perform a specific function. The signaling domain may comprise the signaling domain of another molecule. In some cases, the cleaved portion of the signaling domain is used in the CAR.

[0116] In some embodiments, the intracellular signaling domain comprises multiple signaling domains involved in immune cell signaling, or derivatives, variants, or fragments thereof. For example, the intracellular signaling domain comprises at least two immune cell signaling domains, such as at least 2, 3, 4, 5, 6, 7, 8, 9, 10 immune cell signaling domains, etc. The immune cell signaling domain can be involved in the initial activation of the TCR complex, either in a stimulatory or inhibitory direction. The intracellular signaling domain can be the intracellular signaling domain of the T cell receptor (TCR) complex. The intracellular signaling domain of a subject CAR can include the signaling domains of Fc gamma receptor (FcγR), Fc epsilon receptor (FcεR), Fca receptor (FcαR), neonatal Fc receptor (FcRn), CD3, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD4, CD5, CD8, CD21, CD22, CD28, CD32, CD40L (CD154), CD45, CD66d, CD79a, CD79b, CD80, CD86, CD278 (also referred to as ICOS), CD247 zeta, CD247 eta, DAP10, DAP12, FYN, LAT, Lck, MAPK, MHC complex, NFAT, NF-κB, PLC-γ, iC3b, C3dg, C3d, and Zap70. In some embodiments, the signaling domain may comprise an immunoreceptor tyrosine-based activation motif or ITAM. An ITAM-containing signaling domain may comprise two repeats of the amino acid sequence YxxL / I, separated by 6-8 amino acids, where x is independently any amino acid, forming the conserved motif YxxL / Ix(6-8)YxxL / I. An ITAM-containing signaling domain may be modified, for example, by phosphorylation upon binding of the antigen-binding domain to an epitope. The phosphorylated ITAM may serve as a docking site for other proteins, such as proteins involved in various signaling pathways.In some embodiments, the primary signaling domain comprises a modified ITAM domain, e.g., a mutated, truncated, and / or optimized ITAM domain, that has altered (e.g., increased or decreased) activity compared to the native ITAM domain.

[0117] In some embodiments, the intracellular signaling domain of the subject CAR comprises an FcγR signaling domain (e.g., an ITAM). The FcγR signaling domain may be selected from FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16a), and FcγRIIIB (CD16b). In some embodiments, the intracellular signaling domain comprises an FcεR signaling domain (e.g., an ITAM). The FcεR signaling domain may be selected from FcεRI and FcεRII (CD23). In some embodiments, the intracellular signaling domain comprises an FcαR signaling domain (e.g., an ITAM). The FcαR signaling domain may be selected from FcεRI (CD89) and Fcα / μR. In some embodiments, the intracellular signaling domain comprises a CD3 ζ signaling domain. In some embodiments, the primary signaling domain comprises an ITAM of CD3 zeta.

[0118] In some embodiments, the intracellular signaling domain of the subject CAR comprises an immunoreceptor tyrosine-based activation motif or ITAM. Signaling domains that contain ITIMs comprise a conserved amino acid sequence (S / I / V / LxYxxI / V / L) found in the cytoplasmic tail of several inhibitory receptors of the immune system. Primary signaling domains that contain ITIMs can be modified, e.g., phosphorylated, by enzymes such as, for example, Src kinase family members (e.g., Lck, etc.). Following phosphorylation, other proteins, including enzymes, can be recruited to the ITIMs. These other proteins include, but are not limited to, enzymes such as the phosphotyrosine phosphatases SHP-1 and SHP-2, the inositol monophosphate degrading enzymes referred to as SHIPs, and proteins with one or more SH2 domains (e.g., ZAP70, etc.). The intracellular signaling domains are BTLA, CD5, CD31, CD66a, CD72, CMRF35H, DCIR, EPO-R, FcγRIIB (CD32), Fc receptor-like protein 2 (FCRL2), Fc receptor-like protein 3 (FCRL3), Fc receptor-like protein 4 (FCRL4), Fc receptor-like protein 5 (FCRL5), Fc receptor-like protein 6 (FCRL6), protein G6b (G6B), interleukin 4 receptor (IL4R), immunoglobulin superfamily receptor translocation associated 1 (IRTA1), immunoglobulin superfamily receptor translocation associated 2 (IRTA2), killer cell immunoglobulin-like receptor 2DL1 (KIR2DL1), and killer cell immunoglobulin-like receptor 2DL2 (KIR2DL2), killer cell immunoglobulin-like receptor 2DL3 (KIR2DL3), killer cell immunoglobulin-like receptor 2DL4 (KIR2DL4), killer cell immunoglobulin-like receptor 2DL5 (KIR2DL5), killer cell immunoglobulin-like receptor 3DL1 (KIR3DL1), killer cell immunoglobulin-like receptor 3DL2 (KIR3DL2), leukocyte immunoglobulin-like receptor superfamily B member1 (LIR1), leukocyte immunoglobulin-like receptor superfamily B member 2 (LIR2), leukocyte immunoglobulin-like receptor superfamily B member 3 (LIR3), leukocyte immunoglobulin-like receptor superfamily B member 5 (LIR5), leukocyte immunoglobulin-like receptor superfamily B member 8 (LIR8), leukocyte-associated immunoglobulin-like receptor 1 (LAIR-1), mast cell function-associated antigen (MAFA), NKG2A, natural cytotoxicity triggering receptor 2 (NKp44), NTB-A, programmed cell death protein 1 (PD-1), PILR, SIGLECL1, sialic acid-binding immunoglobulin-like lectin 2 (SIGLEC2 or CD22), sialic acid-binding immunoglobulin-like lectin n and signaling domains (e.g., ITIMs) of signal transduction pathway-regulating protein 1 (SIGLEC1), signal transduction pathway-regulating protein 1 (SIGLEC2), signal transduction pathway-regulating protein 2 (SIGLEC3), signal transduction pathway-regulating protein 3 (SIGLEC3 or CD33), sialic acid-binding immunoglobulin-like lectin 5 (SIGLEC5 or CD170), sialic acid-binding immunoglobulin-like lectin 6 (SIGLEC6), sialic acid-binding immunoglobulin-like lectin 7 (SIGLEC7), sialic acid-binding immunoglobulin-like lectin 10 (SIGLEC10), sialic acid-binding Ig-like lectin 11 (SIGLEC11), sialic acid-binding Ig-like lectin 4 (SIGLEC4), sialic acid-binding Ig-like lectin 8 (SIGLEC8), sialic acid-binding Ig-like lectin 9 (SIGLEC9), platelet and endothelial cell adhesion molecule 1 (PECAM-1), signal regulatory protein (SIRP 2), and signal threshold-regulated transmembrane adaptor 1 (SIT). In some embodiments, the intracellular signaling domain comprises a modified ITAM domain, e.g., a mutated, truncated, and / or optimized ITAM domain, that has altered (e.g., increased or decreased) activity compared to the native ITAM domain.

[0119] In some embodiments, the intracellular signaling domain comprises at least two ITAM domains (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 ITAM domains). In some embodiments, the intracellular signaling domain comprises at least two ITIM domains (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 ITIM domains) (e.g., at least two primary signaling domains). In some embodiments, the intracellular signaling domain comprises both an ITAM domain and an ITIM domain.

[0120] In some cases, the intracellular signaling domain of the subject CAR may comprise a costimulatory domain. In some embodiments, the costimulatory domain, e.g., from a costimulatory molecule, may provide a costimulatory signal for immune cell signaling, e.g., for activation and / or inactivation of immune cell activity, e.g., signaling from an ITAM and / or ITIM domain. In some embodiments, the costimulatory domain is operable to regulate proliferation and / or survival signals in immune cells. In some embodiments, the costimulatory signaling domain comprises a signaling domain of an MHC class I protein, an MHC class II protein, a TNF receptor protein, an immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocyte activation molecule (SLAM protein), an activating NK cell receptor, BTLA, or a Toll ligand receptor. In some embodiments, the costimulatory domain is selected from the group consisting of 2B4 / CD244 / SLAMF4, 4-1BB / TNFSF9 / CD137, B7-1 / CD80, B7-2 / CD86, B7-H1 / PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BAFF R / TNFRSF13C, BAFF / BLyS / TNFSF13B, BLAME / SLAMF8, BTLA / CD272, CD100 (SEMA4D), CD103, CD11a, CD11b, CD11c, CD11d, CD150, CD160 (BY55), CD18, CD19, CD2, CD200, CD229 / SLAMF3, CD27 Ligand / TNFSF7, CD27 / TNFRSF7, CD28, CD29, CD2F-10 / SLAMF9, CD30 Ligand / TNFSF8, CD30 / TNFRSF8, CD300a / LMIR1, CD4, CD40 Ligand / TNFSF5, CD40 / TNFRSF5, CD48 / SLAMF2, CD49a, CD49D, CD49f, CD53, CD58 / LFA-3, CD69, CD7, CD8 α, CD8β, CD82 / Kai-1, CD84 / SLAMF5, CD90 / Thy1, CD96, CDS, CEACAM1, CRACC / SLAMF7, CRTAM, CTLA-4, DAP12; Dectin-1 / CLEC7A, DNAM1(CD226), DPPIV / CD26, DR3 / TNFRSF25, EphB6, GADS, Gi24 / VISTA / B7-H5, and GITR LINK / TNFSF18, GITR / TNFRSF18, HLA, I, HLA-DR, HVEM / TNFRSF14, IA4, ICAM-1, ICOS / CD278, Ikaros, IL2R β, IL2R γ, IL7R α-mediated α4 / CD49d and α4β1 α4β7 / LPAM-1, IPO-3, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, I TGB1, ITGB2, ITGB7, KIRDS2, LAG-3, LAT, LIGHT / TNFSF14, LTBR, ​​Ly108, Ly9 (CD229) Lipoprotein Factor Activator-1(LFA-1) Fan Factor-α / TNF-β NKG2C NK G2D, NKp30, NKp44, NKp46, NKp80(KLRF1), NTB-A / SLAMF6, OX40 LINK / TNFSF4, OX40 / TNFRSF4, PAG / Cbp, PD-1, PDCD6, PD-L2 / B7-DC, PSGL1, RELT / TNFRSF19L, SELPLG (CD162), SLAM (SLAMF1) SLAM / CD150 SLAMF4 (CD244) SLAMF6 (NTB-A), SLAMF7, SLP-76, TACI / TNFRSF13B, TCL1A, TCL1B, TIM-1 / KIM-1 / HAVCR, TIM-4, TL1A / TNFSF15, TNF RII / TNFRSF1B, TNF-α, TRANCE / RANKL, TSLP, and TSLPThe intracellular signaling domain comprises a signaling domain of a molecule selected from the group consisting of VLA-R, VLA1, and VLA-6. In some embodiments, the intracellular signaling domain comprises multiple costimulatory domains, for example at least two, for example at least three, four, or five costimulatory domains. The costimulatory signaling region can provide a signal synergistic with the primary effector activation signal and can meet the requirements for T cell activation. In some embodiments, the addition of a costimulatory domain to the CAR can enhance the effect and persistence of the immune cells provided herein.

[0121] The binding of CAR to B cell surface protein can enhance the proliferation of immune cells compared to immune cells that do not have CAR. The proliferation of immune cells can refer to the increase of immune cells. The proliferation of immune cells can refer to the change in phenotype of immune cells. The proliferation of immune cells comprising CAR provided herein can be increased compared to that of comparable immune cells that do not have CAR that exhibits binding to B cell surface protein. The proliferation of immune cells containing a CAR can be increased by about 5 to about 10 times, about 10 to about 20 times, about 20 to about 30 times, about 30 to about 40 times, about 40 to about 50 times, about 50 to about 60 times, about 60 to about 70 times, about 70 to about 80 times, about 80 to about 90 times, about 90 to about 100 times, about 100 to about 200 times, about 200 to about 300 times, about 300 to about 400 times, about 400 to about 500 times, about 500 to about 600 times, or about 600 to about 700 times compared to the proliferation of equivalent immune cells not having a CAR. The proliferation of immune cells containing a CAR can be increased by about 5-fold to about 10-fold, about 10-fold to about 20-fold, about 20-fold to about 30-fold, about 30-fold to about 40-fold, about 40-fold to about 50-fold, about 50-fold to about 60-fold, about 60-fold to about 70-fold, about 70-fold to about 80-fold, about 80-fold to about 90-fold, about 90-fold to about 100-fold, about 100-fold to about 200-fold, about 200-fold to about 300-fold, about 300-fold to about 400-fold, about 400-fold to about 500-fold, about 500-fold to about 600-fold, or about 600-fold to about 700-fold over the proliferation of comparable immune cells without a CAR, and the proliferation is observed for at least about 12, 24, 36, 48, 60, 72, 84, or 96 hours after contact of the B cells with the B cell surface protein. The increased proliferation can be confirmed either in vitro or in vivo. In some embodiments, the proliferation includes quantifying the number of immune cells. Quantifying the number of immune cells can include flow cytometry, trypan blue exclusion, and hemocytometry. Proliferation can also be determined by phenotypic analysis of immune cells.

[0122] In one aspect, the disclosure provides an engineered immune cell that specifically binds to a neoantigen, wherein the engineered immune cell comprises (a) a chimeric stimulating molecule comprising a polypeptide extracellular domain (PED) that binds to a neoantigen, wherein the PED is fused to an intracellular domain (ICD) of a costimulatory molecule that mediates an immune cell activation signal, and wherein binding of the chimeric stimulating molecule to the neoantigen results in the immune cell activation signal in the engineered immune cell, and (b) a chimeric antigen receptor comprising (i) an antigen interacting domain capable of binding to a B cell surface protein; (ii) a transmembrane domain; and (iii) an intracellular signaling domain. In some embodiments, examples of PEDs include antibodies, as well as derivatives, variants, and fragments thereof.

[0123] In one aspect, the disclosure provides an engineered immune cell that specifically binds a neoantigen, wherein the engineered immune cell comprises: (a) a switch molecule comprising an extracellular domain (ECD) of a protein that upon binding to its ligand induces an immune inactivation signal in a non-engineered immune cell, wherein the ECD is fused to an intracellular domain (ICD) of a costimulatory molecule that mediates an immune cell activation signal, wherein binding of the switch molecule to the ligand results in an immune cell activation signal instead of an immune cell inactivation signal in the engineered immune cell; and (b) a chimeric antigen receptor comprising: (i) an antigen interacting domain capable of binding to a B cell surface protein; (ii) a transmembrane domain; and (iii) an intracellular signaling domain.

[0124] In one aspect, the disclosure provides an engineered tumor infiltrating lymphocyte (TIL) that specifically binds a neoantigen, wherein the engineered immune cell comprises (a) a switch molecule comprising an extracellular domain (ECD) of a protein that upon binding to its ligand induces an immune cell inactivation signal in a non-engineered immune cell, where the ECD is fused to an intracellular domain (ICD) of a costimulatory molecule that mediates an immune cell activation signal, and where binding of the switch molecule to the ligand results in an immune cell activation signal instead of an immune cell inactivation signal in the engineered TIL, and (b) a chimeric antigen receptor comprising: (i) an antigen-interacting domain capable of binding to a B cell surface protein; (ii) a transmembrane domain; and (iii) an intracellular signaling domain.

[0125] In one embodiment, the present disclosure provides modified immune cells that overexpress a cytokine, such as a chemokine, where the immune cells are (i) tumor infiltrating lymphocytes (TILs); (ii) stromal tumor infiltrating lymphocytes (sTILs); or (iii) T cells that exhibit specific binding to an antigen. The modified immune cells that overexpress a chemokine can be any modified immune cell provided herein.

[0126] Cytokines refer to proteins (e.g., chemokines, interferons, lymphokines, interleukins, and tumor necrosis factors) that are released by cells and can affect cell behavior. Cytokines are produced by a wide range of cells, including immune cells such as macrophages, B lymphocytes, T lymphocytes, and mast cells, as well as endothelial cells, fibroblasts, and various stromal cells. Cytokines can be involved in systemic or localized immunomodulatory effects.

[0127] Certain cytokines can function as pro-inflammatory cytokines. Pro-inflammatory cytokines refer to cytokines that are involved in inducing or amplifying inflammatory responses. Pro-inflammatory cytokines can work with various cells of the immune system, such as neutrophils and leukocytes, to generate an immune response. Certain cytokines can function as anti-inflammatory cytokines. Anti-inflammatory cytokines refer to cytokines that are involved in reducing inflammatory responses. In some cases, anti-inflammatory cytokines can regulate pro-inflammatory cytokine responses. Some cytokines can function as both pro-inflammatory and anti-inflammatory cytokines. Certain cytokines, such as chemokines, can function in chemotaxis. Chemokines can induce directed chemotaxis in nearby responsive cells.

[0128] In some embodiments, the expression of cytokines with pro-inflammatory and / or chemotaxis functions can be upregulated in immune cells.Upregulation of the expression of cytokines with pro-inflammatory and / or chemotaxis functions can be beneficial, for example, in immunotherapy, to stimulate immune responses against target cells.

[0129] Examples of cytokines that can be overexpressed by immune cells provided herein include, but are not limited to, lymphokines, monokines, and general polypeptide hormones. The cytokines included are growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factors; fibroblast growth factors; prolactin; placental lactogen; tumor necrosis factor-α; Müllerian inhibitory factor; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrins; thrombopoietin (TPO); nerve growth factors such as NGF-α; platelet growth factors; transforming growth factors (TGFs) such as TGF-α, TGF-β, TGF-β1, TGF-β2, and TGF-β3; insulin-like growth factors-I and -II; erythropoietin (EPO); Flt-3L; and stem cell factor (SCF). ;bone morphogenetic factors;interferons (IFNs) such as IFN-α, IFN-β, and IFN-γ;colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF);granulocyte-CSF (G-CSF);macrophage stimulating factor (MSP);IL-1, IL-1a, IL-1b, IL-1RA, IL-18, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, Interleukins (ILs), such as IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-20; tumor necrosis factors, such as CD154, LT-β, ​​TNF-α, TNF-β, 4-1BBL, APRIL, CD70, CD153, CD178, GITRL, LIGHT, OX40L, TALL-1, TRAIL, TWEAK, TRANCE; and other polypeptide factors, including LIF, oncostatin M (OSM) and Kit ligand (KL). Cytokine receptor refers to a receptor protein that binds to a cytokine. Cytokine receptors can be both membrane-bound and soluble.

[0130] In some embodiments, the overexpressed cytokine is an interleukin (IL-1) family member (e.g., a ligand), an IL-1 receptor family member, an interleukin-6 (IL-6) family member (e.g., a ligand), an IL-6 receptor, an interleukin-10 (IL-10) family member (e.g., a ligand), an IL-10 receptor, an interleukin-12 (IL-12) family member (e.g., a ligand), an IL-12 receptor, an interleukin-17 (IL-17) family member (e.g., a ligand), or an IL-17 receptor.

[0131] In some embodiments, the overexpressed cytokine is an interleukin (IL-1) family member or related protein; a tumor necrosis factor (TNF) family member or related protein; an interferon (IFN) family member or related protein; an interleukin-6 (IL-6) family member or related protein; or a chemokine or related protein.In some embodiments, the cytokine is IL18, IL18BP, IL1A, IL1B, IL1F10, IL1F3 / IL1RA, IL1F5, IL1F6, IL1F7, IL1F8, IL1RL2, IL1F9, IL33, BAFF / BLyS / TNFSF138, 4-1BBL, CD153 / CD30L / TNFSF8, CD40LG, CD70, Fas Ligand / FASLG / CD95L / CD178, EDA-A1, TNFSF14 / LIGHT / CD258, TNFA, LTA / TNFB / TNFSF1, LTB / TNFC, CD70 / CD27L / TN FSF7, TNFSF10 / TRAIL / APO-2L(CD253), RANKL / OPGL / TNFSF11(CD254), TNFSF12, TNF-α / TNFA, TNFSF13, TL1A / TNFSF 16 NE, IFNG, IFNZ, IFNA8, IFNA5 / IFNaG, IFNω / IFNW1, CLCF1, CNTF, IL11, IL31, IL6, leptin, LIF, OSM, CCL1 / TCA3, CCL11, CCL12 / MCP-5, CCL13 / MCP-4, CCL14, CCL15, CCL16, CCL17 / TARC, CCL18, CCL19, CCL2 / MCP-1, CCL20, CCL21, CCL22 / M DC, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL3L3, CCL4, CCL4L1 / LAG-1, CCL5, CCL6, CCL7, CCL8, CCL9, CX 3CL1, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, CXCL2 / MIP-2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7 / Ppbp, CXCL9, IL8 / CXCL8, XCL1, XCL2, FAM19A1, FAM19A2, FAM19A3, FAM19A4, and FAM19A5.

[0132] Cytokine expression can be assessed using a variety of methods. Cytokine expression can be assessed by assaying cell culture medium in which the modified immune cells are grown (e.g., in vitro production) or by assaying serum obtained from a subject with the modified immune cells (e.g., in vivo production) for the presence of one or more cytokines. Cytokine levels can be quantified using any suitable assay, in a variety of suitable units, such as concentration. In some embodiments, cytokine protein is detected. In some embodiments, mRNA transcripts of the cytokine are detected. Examples of cytokine assays include, for example, enzyme-linked immunosorbent assays (ELISAs), immunoblots, immunofluorescence, radioimmunoassays, antibody arrays that allow for simultaneous detection of various cytokines in a sample, bead-based arrays, quantitative PCR, microarrays, and the like. Other suitable methods include proteomic approaches (2-D gels, MS analysis, and the like).

[0133] In some embodiments, the cytokine overexpressed by the modified immune cells provided herein is a chemokine.The chemokine can be, for example, CC chemokine, CXC chemokine, C chemokine, and CX3C chemokine.In some embodiments, the chemokine overexpressed by the modified immune cells is a CC chemokine selected from CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, and CCL28. The chemokine is a CXC chemokine selected from CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, and CXCL17. In some embodiments, the chemokine overexpressed by the engineered immune cells is a C chemokine selected from XCL1 and XCL2. In some embodiments, the chemokine overexpressed by the immune cells is a CX3C chemokine, and the CX3C chemokine is CX3CL1.

[0134] In one aspect, the disclosure provides a method of treating cancer in a subject, comprising: (a) administering to the subject an engineered TIL, engineered T cell, or engineered immune cell of any one of the various embodiments of the aspects herein; and (b) contacting a target cell that is a cancer expressing a neoantigen with the engineered TIL, engineered T cell, or engineered immune cell under conditions that induce cytotoxicity of the engineered TIL, engineered T cell, or engineered immune cell against the target cell that is a cancer, thereby inducing death of the target cell that is a cancer.

[0135] In one aspect, the disclosure provides a method for expanding a T cell population, the method comprising: (a) providing a T cell population comprising at least one modified immune cell of any one of the various embodiments of the aspects herein; and (b) exposing the population of T cells to a B cell surface protein to effect expansion of the population of T cells. In some embodiments, in (b), the population of T cells is exposed to B cells comprising the B cell surface protein.

[0136] In one aspect, the disclosure provides a method for expanding a T cell population comprising: (a) introducing into a T cell population a nucleic acid encoding a chimeric antigen receptor (CAR), thereby creating a first CAR-expressing cell population, wherein the CAR comprises (i) an antigen interacting domain capable of binding to a B cell surface protein; (ii) a transmembrane domain; and (iii) an intracellular signaling domain; and (b) contacting the first CAR-expressing cell population with the B cell surface protein, thereby creating an expanded and / or activated immune cell population.

[0137] In one aspect, the disclosure provides a composition comprising one or more polynucleotides encoding one or more of: (a) a switch molecule comprising an extracellular domain (ECD) of a protein that, upon binding to its ligand, induces an immune inactivation signal in an unmodified immune cell, where the ECD is fused to an intracellular domain (ICD) of a costimulatory molecule that mediates an immune cell activation signal; and (b) an antigen-specific T cell receptor complex, or one or more components thereof.

[0138] In one aspect, the disclosure provides a composition comprising one or more polynucleotides encoding one or more of: (a) an antigen-specific T cell receptor complex, or one or more components thereof; and (b) a chimeric antigen receptor comprising: (i) an antigen interacting domain capable of binding to a B cell surface protein; (ii) a transmembrane domain; and (iii) an intracellular signaling domain.

[0139] In one aspect, the disclosure provides a composition comprising one or more polynucleotides encoding one or more of: (a) a switch molecule comprising the extracellular domain (ECD) of a protein that, upon binding to its ligand, induces an immune inactivation signal in an unmodified immune cell, where the ECD is fused to the intracellular domain (ICD) of a costimulatory molecule that mediates an immune cell activation signal; (b) an antigen-specific T cell receptor complex, or one or more components thereof; and (c) a chimeric antigen receptor comprising: (i) an antigen interacting domain capable of binding to a B cell surface protein; (ii) a transmembrane domain; and (iii) an intracellular signaling domain.

[0140] In various embodiments of the aspects herein, promoters can be used with the compositions of the present disclosure. Examples of promoters include those that are active in eukaryotic cells, mammalian cells, non-human mammalian cells, or human cells. The promoter can be an inducible or constitutively active promoter. Alternatively, or in addition, the promoter can be tissue or cell specific.

[0141] Non-limiting examples of suitable eukaryotic promoters (i.e., promoters that are functional in eukaryotic cells) include those derived from cytomegalovirus (CMV) immediate early, herpes simplex virus thymidine kinase, early and late SV40, long terminal repeats (LTRs) from retroviruses, human elongation factor 1 promoter (EF1), a hybrid construct containing the cytomegalovirus (CMV) enhancer fused to the avian β-actin promoter (CAG), mouse stem cell virus promoter (MSCV), phosphoglycerate kinase-1 locus promoter (PGK), and mouse metallothionein-I. The promoter may be a fungal promoter. The promoter may be a plant promoter. A database of plant promoters can be found, for example, at PlantProm. The expression vector may also include a ribosome binding site for translation initiation and a transcription termination codon. The expression vector may also include appropriate sequences for amplifying expression.

[0142] In various embodiments of the aspects herein, the engineered immune cells may specifically bind neoantigens and / or neoepitopes. Neoantigens and neoepitopes generally refer to tumor-specific mutations that in some cases trigger anti-tumor T cell responses. For example, these endogenous mutations may be identified using a whole exome sequencing approach. See Tran E et al., "Cancer immunotherapy based on mutation-specific CD4+ T cells in a patient with epithelial cancer", Science 344: 641-644 (2014). Engineered immune cells (e.g., engineered TILs or engineered T cells) containing switch molecules exhibit specific binding to tumor-specific neoantigens. The neoantigens bound by the immune cells may be expressed on the target cell and encoded, for example, as mutations in an endogenous gene. In some cases, the neoantigens or neoepitopes that are specifically bound by the immune cells may be encoded by a mutant gene.The genes are ABL1, ACOL 1997, ACVR2A, AFP, AKT1, ALK, ALPPL2, ANAPC1, APC, ARID1A, AR, AR-v7, ASCL2, β2M, BRAF, BTK, C15ORF40, CDH1, CLDN6, CNOT1, CT45A5, CTAG1B (encoding NY-ESO-1), DCT, DKK4, EEF1B2, EEF1DP3, EGFR, EIF2B3, env, EPHB2, ERBB3, ESR1, ESRP1, FAM11 IB, FGFR3, FRG1B, GAGE1, GAGE ​​10, GATA3, GBP3, HER2, IDH1, JAK1, KIT, KRAS, LMAN1, MABEB 16, MAGEA1, MAGEA10, MAGEA4, MAGEA8, MAGEB 17, MAGEB4, MAGEC1, MEK, MLANA, MLL2, MMP13, MSH3, MSH6, MYC, NDUFC2, NRAS, NY-ESO, PAGE2, PAGE5, PDGFRa, PIK3CA, PMEL, pol protein, POLE, PTEN, RAC1, RBM27, RNF43, RPL22, RUNX1, SEC31A, SEC63, SF3B 1, SLC35F5, SLC45A2, SMAP1, SMAP1, SPOP, TFAM, TGFBR2, THAP5, TP53, TTK, TYR, UBR5, VHL, and XPOT. In some embodiments, neoantigens may be selected based on a genetic profile of a tumor sample from an individual. In some embodiments, neoantigens can be selected based on the somatic mutation profile of a tumor sample from an individual.

[0143] In various embodiments of the aspects herein, the modified immune cells may further comprise a kill switch. The kill switch may be activated to eliminate immune cells in the case of severe toxicity, such as hypercytokinemia. This may occur when the immune system has a strong response in which many inflammatory cytokines are released, causing mild to severe symptoms such as fever, headache, rash, fast heartbeat, low blood pressure and respiratory problems. The kill switch may be a drug-inducible kill switch. The kill switch may comprise an inducible caspase 9.

[0144] Various embodiments of the aspects herein include cells, such as modified immune cells. The cells, such as immune cells (e.g., lymphocytes, such as T cells and NK cells), can be obtained from a subject. Non-limiting examples of subjects include, for example, humans, dogs, cats, mice, rats, and transgenic species thereof. Examples of samples from subjects from which cells can be derived include, but are not limited to, for example, These may include skin, heart, lungs, kidneys, bone marrow, breast, pancreas, liver, muscle, smooth muscle, bladder, gallbladder, colon, intestine, brain, prostate, esophagus, thyroid, serum, saliva, urine, gastric and digestive fluids, tears, stool, semen, vaginal fluid, interstitial fluid from neoplastic tissue, ocular fluid, sweat, mucus, earwax, oil, glandular secretions, spinal fluid, hair, nails, plasma, nasal swabs or nasopharyngeal washings, spinal fluid, cerebrospinal fluid, tissue, throat swabs, biopsies, placental fluid, amniotic fluid, umbilical cord blood, emphatic fluids, cavity fluid, sputum, pus, bacterial flora, meconium, breast milk, and / or other excretions or body tissues.

[0145] In some cases, the cells may be a population of T cells, NK cells, B cells, etc. obtained from a subject. T cells may be obtained from a number of sources, including PBMCs, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, and tissue from a site of infection, retina, pleural effusion, splenic tissue, and tumors. In some embodiments, T cells may be obtained from a unit of blood drawn from a subject using any of a number of techniques, such as, for example, Ficoll® separation. In some embodiments, cells from an individual's circulating blood are obtained by apheresis. The product of apheresis typically includes lymphocytes, including T cells, monocytes, granulocytes, B cells, etc., other nucleated white blood cells, red blood cells, and platelets. Cells collected by apheresis may be washed to remove plasma fractions and to place the cells in an appropriate buffer or solvent suitable for subsequent processing steps.

[0146] Any of a variety of immune cells may be utilized in the embodiments herein. In some embodiments, immune cells include granulocytes, such as basophils, eosinophils, and neutrophils; mast cells; monocytes that can develop into macrophages; antigen-presenting cells, such as dendritic cells; and lymphocytes, such as natural killer cells (NK cells), B cells, and T cells. In some embodiments, the immune cells are immune effector cells. By immune effector cells is meant immune cells that can perform a specific function in response to a stimulus. In some embodiments, the immune cells are immune effector cells that can induce cell death. In some embodiments, the immune cells are lymphocytes. In some embodiments, the lymphocytes are NK cells. In some embodiments, the lymphocytes are T cells. In some embodiments, the T cells are activated T cells. T cells include both naive and immune memory (e.g., central memory or TCM, effector memory or TEM and effector memory RA or TEMRA) cells, effector cells (e.g., cytotoxic T cells or CTLs or Tc cells), helper cells (e.g., Th1, Th2, Th3, Th9, Th7, TFH), regulatory cells (e.g., Treg and Trl cells), natural killer cells (NKT cells), tumor infiltrating lymphocytes (TILs), lymphocyte-activated killer cells (LAKs), αβ T cells, γδ T cells, and similar unique classes of T cell lineages. T cells can be classified into two broad categories, CD8+ T cells and CD4+ T cells, based on what proteins are present on the cell surface. T cells expressing the system of interest can perform multiple functions, including killing infected cells and activating or recruiting other immune cells. CD8+ T cells are also referred to as cytotoxic T cells or cytotoxic T lymphocytes (CTLs). CTLs expressing the target system can be involved in recognizing and eliminating virus-infected and cancer cells. CTLs are equipped with specific compartments or granules that contain cytotoxins that induce apoptosis, e.g., programmed cell death.CD4+ T cells are subdivided into four subsets, Th1, Th2, Th17, and Treg, although there may be additional subsets as well, where Th stands for T helper cell. Th1 cells can coordinate immune responses against intracellular microorganisms, especially bacteria. They produce and secrete molecules that alert and activate other immune cells, such as macrophages, which phagocytose bacteria. Th2 cells are involved in coordinating immune responses against extracellular pathogens, such as helminths (parasites), by alerting B cells, granulocytes, and mast cells. Th17 cells can produce interleukin 17 (IL-17), a signaling molecule that activates immune and non-immune cells. Th17 cells are important for recruiting neutrophils.

[0147] In some embodiments, the immune cell population provided herein may be heterogeneous. In some embodiments, the cells used may be composed of a heterogeneous mixture of CD4 and CD8 T cells. The CD4 and CD8 cells may have phenotypic characteristics of circulating effector T cells. In some embodiments, the cells may be central memory cells.

[0148] In some embodiments, the cells are peripheral blood mononuclear cells (PBMCs), peripheral blood lymphocytes (PBLs), and other blood cell subsets, including but not limited to T cells, natural killer cells, monocytes, natural killer T cells, monocyte precursors, hematopoietic stem cells, or non-pluripotent stem cells. In some cases, the cells can be any immune cell, such as T cells, such as CD3+ T cells, CD4+ T cells, CD8+ T cells, or any type of T cell, such as tumor infiltrating lymphocytes (TILs). T cells also include memory T cells, memory stem T cells, or other effector T cells. T cells can also be selected from a mixed population, such as, for example, selection of T cells from whole blood. T cells can also be expanded from a mixed population. T cells can also be skewed to a specific population and phenotype. For example, T cells can be phenotypically skewed to include CD45RO(-), CCR7(+), CD45RA(+), CD62L(+), CD27(+), CD28(+) and / or IL-7Rα(+). Appropriate cells can be selected that include one or more markers selected from the list including CD45RO(-), CCR7(+), CD45RA(+), CD62L(+), CD27(+), CD28(+) and / or IL-7Rα(+). Cells also include stem cells, such as, for example, embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, neuronal stem cells, and mesenchymal stem cells. Cells can include any number of primary cells, such as, for example, human cells, non-human cells, and / or mouse cells. Cells can be progenitor cells. Cells can be derived from the subject (e.g., patient) to be treated. Cells can be derived from a human donor. The host cells may be TSCM memory stem cells, comprised of CD45RO(-), CCR7(+), CD45RA(+), CD62L(+), CD27(+), CD28(+) and / or IL-7Rα(+), which may also express CD95, IL-2Rβ, CXCR3, and LFA-1, and may exhibit a number of functional characteristics characteristic of such memory stem cells.The host cell may be a central memory TCM cell that contains L-selectin and CCR7, and the central memory cell may secrete, for example, IL-2, but not IFNα or IL-4. The cell may also be an effector memory TEM cell that contains L-selectin and CCR7, and may produce effector cytokines such as, for example, IFNγ and IL-4.

[0149] In various embodiments of the aspects herein, the immune cells include lymphocytes. In some embodiments, the lymphocytes are natural killer cells. In some embodiments, the lymphocytes are T cells. T cells can be obtained from a number of sources, including peripheral blood, mononuclear cells, bone marrow, lymph node tissue, spleen tissue, umbilical cord blood, and tumors. In some embodiments, any number of available T cell lines can be used. Immune cells, such as lymphocytes (e.g., cytotoxic lymphocytes, etc.), can preferably be autologous cells, however, xenogeneic cells can also be used. T cells can be obtained from a unit of blood drawn from a subject using any of a number of techniques, such as, for example, Ficoll® separation. Cells from an individual's circulating blood can be obtained by apheresis or leukapheresis. Apheresis products typically include lymphocytes, including T cells, monocytes, granulocytes, B cells, etc., other nucleated white blood cells, red blood cells, and platelets. Cells collected by apheresis may be washed to remove plasma fractions and to place the cells in a suitable buffer or solvent, such as, for example, phosphate-buffered saline (PBS), suitable for subsequent processing steps. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg-free PBS. Alternatively, undesirable components of the apheresis sample may be removed and the cells may be resuspended directly in culture solution. The sample may be provided directly by the subject or indirectly through one or more intermediaries, such as a sample collection service provider or a health care provider (e.g., a doctor or nurse). In some embodiments, separating T cells from peripheral blood leukocytes includes lysing red blood cells and separating peripheral blood leukocytes from monocytes, such as by centrifugation through a PERCOL gradient.

[0150] Specific T cell subpopulations, such as CD4 or CD8, can be further separated by positive or negative selection techniques. Negative selection of T cell populations can be achieved, for example, by a combination of antibodies directed to surface markers specific to the cells to be negatively selected. One suitable technique includes cell sorting via negative magnetic immunoadhesion, utilizing a cocktail of monoclonal antibodies directed to cell surface markers on the cells to be negatively selected. For example, to separate CD4+ cells, the monoclonal antibody cocktail can include antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. The process of negative selection can be used to create a desired T cell population that is largely homogenous. In some embodiments, the composition comprises a mixture of two or more (e.g., 2, 3, 4, 5 or more) different types of T cells.

[0151] In some embodiments, the immune cells are members of an enriched cell population. One or more desired cell types may be enriched by any suitable method, including, but not limited to, treating the cell population to trigger proliferation and / or differentiation into the desired cell type, treating to stop the growth of undesired cell types, treating to kill or lyse undesired cell types, purifying the desired cell type (e.g., purifying on an affinity column to retain desired or undesired cell types based on one or more cell surface markers, etc.). In some embodiments, the enriched population of cells is a cell population enriched in cytotoxic lymphocytes selected from cytotoxic T cells (variously known as cytotoxic lymphocytes, CTLs, T killer cells, cytotoxic T cells, CD8+ T cells, and killer T cells), natural killer (NK) cells, and lymphokine-activated killer (LAK) cells.

[0152] For separation of the desired cell population by positive or negative selection, the concentration of cells and surfaces (e.g., particles such as beads) is varied. In some embodiments, it may be preferable to significantly reduce the volume in which the beads and cells are mixed together (i.e., increase the concentration of cells) to ensure maximum contact between the cells and beads. For example, a concentration of 2 billion cells / mL may be used. In some embodiments, a concentration of 1 billion cells / mL may be used. In some embodiments, a concentration of 100 million cells / mL may be used. A concentration of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / mL may be used. In yet another embodiment, a concentration of 75, 80, 85, 90, 95, or 100 million cells / mL may be used. In further embodiments, concentrations of 125 million or 150 million cells / mL may be used. Using higher concentrations may result in increased cell yield, cell activation, and cell proliferation.

[0153] A variety of target cells may be killed using the systems and methods of the presently disclosed subject matter. Target cells to which the methods may be applied may include a wide variety of cell types. The target cells may be in vitro. The target cells may be in vivo. The target cells may be ex vivo. The target cells may be isolated cells. The target cells may be cells within an organ. The target cells may be an organ. The target cells may be mammalian cells or derived from mammalian cells. The target cells may be human cells or derived from human cells. The target cells may be prokaryotic cells or derived from prokaryotic cells. The target cells may be bacterial cells or derived from bacterial cells. The target cells may be archaeal cells or derived from archaeal cells. The target cells may be eukaryotic cells or derived from eukaryotic cells. The target cells may be pluripotent stem cells. The target cells may be plant cells or derived from plant cells. The target cells may be animal cells or derived from animal cells. The target cells may be invertebrate cells or derived from invertebrate cells. The target cell may be or be derived from a vertebrate cell. The target cell may be or be derived from a microbial cell. The target cell may be or be derived from a fungal cell. The target cell may be or be derived from a specific organ or tissue.

[0154] The target cell may be a stem cell or derived from a progenitor cell. The target cell may include stem cells (e.g., adult stem cells, embryonic stem cells, induced pluripotent stem (iPS) cells, etc.) and progenitor cells (e.g., cardiomyocyte progenitor cells, neural progenitor cells, etc.). The target cell may include mammalian stem cells and progenitor cells, such as rodent stem cells, rodent progenitor cells, human stem cells, human progenitor cells, etc. The cloned cell may include a progeny cell. The target cell may include a target nucleic acid. The target cell may be in an organism. The target cell may be a genetically modified cell. The target cell may be a host cell.

[0155] The target cell may be a primary cell. For example, a culture of primary cells may be passaged 0, 1, 2, 4, 5, 10, 15 or more times. The cell may be a single-cell organism. The cell may be grown in culture.

[0156] The target cell may be a diseased cell. The diseased cell may have altered metabolism, gene expression, and / or morphological characteristics. The diseased cell may be a cancer cell, a diabetic cell, and an apoptotic cell. The diseased cell may be a cell from a subject suffering from a disease. Exemplary diseases may include vascular disorders, cancer, metabolic disorders, eye diseases, organ disorders, musculoskeletal disorders, heart diseases, etc.

[0157] When the target cells are primary cells, they can be obtained from an individual by any method. For example, white blood cells can be obtained by apheresis, leukapheresis, density gradient separation, etc. Cells from tissues such as skin, muscle, bone marrow, spleen, liver, kidney, lung, intestine, stomach, etc. can be obtained by biopsy. An appropriate solution can be used for dispersion or suspension of the obtained cells. Such a solution can generally be a balanced salt solution (e.g., normal saline, phosphate buffered saline (PBS), Hank's balanced salt solution, etc.), appropriately supplemented with fetal bovine serum or other natural factors, together with a low concentration of an acceptable buffer. Buffers can include HEPES, phosphate buffer, lactate buffer, etc. The cells can be used immediately or stored (e.g., by freezing). Frozen cells can be thawed and can be reused. Cells may be frozen in DMSO, serum, solvent buffer (e.g., 10% DMSO, 50% serum, 40% buffered solvent, etc.), and / or other known solutions used to preserve cells at freezing temperatures.

[0158] Non-limiting examples of cells that can be target cells include, but are not limited to, lymphoid cells, such as B cells, T cells (cytotoxic T cells, natural killer T cells, regulatory T cells, helper T cells), natural killer cells, and cytokine-induced killer (CIK) cells (see, e.g., U.S. Patent No. 20080241194); myeloid cells, such as granulocytes (basophilic granulocytes, eosinophilic granulocytes, neutrophilic granulocytes / hypersegmented neutrophils), monocytes / macrophages, red blood cells (reticulocytes), mast cells, platelets / megakaryocytes, and dendritic cells; Cells from the endocrine system, including cells of the thyroid gland (thyroid epithelial cells, parafollicular cells), parathyroid gland (chief parathyroid cells, eosinophilic cells), adrenal gland (chromaffin cells), and pineal gland (pineal cells); cells of the nervous system, such as glial cells (astrocytes, microglia), magnocellular neurosecretory cells, astrocytes, Bechtel cells, and pituitary gland (gonadotropes, corticotropes, thyrotropes, somatotropes, lactotropes); lung cells (type I lung cells, type II lung cells), Clara cells, goblet cells, dust cells, and thyroid gland (thyrotropes). Cells of the respiratory system such as cysts; cells of the respiratory system such as cardiomyocytes, pericytes; cells of the digestive system such as stomach (chief cells, parietal cells), goblet cells, Paneth cells, G cells, D cells, ECL cells, I cells, K cells, S cells; enteroendocrine cells such as enterochromaffin cells, APUD cells, liver (hepatocytes, Kupffer cells), cartilage / bone / muscle; bone cells such as osteoblasts, osteocytes, osteoclasts, teeth (cementoblasts, ameloblasts); cartilage cells such as chondroblasts, chondrocytes; skin cells such as cysts, keratinocytes, melanocytes (nevus cells); muscle cells such as myocytes Cells of the urinary system such as podocytes, juxtaglomerular cells, intraglomerular / extraglomerular mesangial cells, renal proximal tubule brush border cells, macula densa cells; cells of the reproductive system such as sperm, Sertoli cells, Leydig cells, and eggs; and adipocytes, fibroblasts, tendon cells, epidermal keratinocytes (differentiated epithelial cells), epithelial basal cells (stem cells), keratinocytes of the nails and toenails, nail bed basal cells (stem cells), medullary hair stem cells, cortical hair stem cells, keratinocyte hair stem cells, keratinocyte root sheath cells, root sheath cells of the layer of Huxley, root sheath cells of the layer of Henle, outer root sheath cells, hair matrix cells (stem cells),Wetting stratified barrier epithelial cells, epithelial cells of the stratified squamous epithelium of the cornea, tongue, oral cavity, esophagus, anal canal, urinary tract and distal vagina, basal cells (stem cells) of the epithelium of the cornea, tongue, oral cavity, esophagus, anal canal, urinary tract and distal vagina, urothelial cells (lining the bladder and ureters), exocrine epithelial cells, salivary gland mucosa cells (secreting polysaccharides rich), salivary gland serous cells (secreting glycoprotein enzymes rich), von Ebner's gland cells of the tongue (washing the taste buds) , mammary gland cells (secrete milk), lacrimal gland cells (secrete tears), ear canal glands in the ear (secrete wax), eccrine sweat gland dark cells (secrete glycoproteins), eccrine sweat gland clear cells (secrete small molecules), apocrine sweat gland cells (secrete aroma, sensitive to sex hormones), cells of the glands in the eyelids (specialized sweat glands), cells of the sebaceous glands (secrete lipid-rich sebum), Bowman's gland cells in the nose (cleans the olfactory epithelium), Brunner's gland cells in the duodenum (enzymes and alkaline mucus), and seminal vesicles cells (secrete seminal fluid components including fructose from swimming sperm), prostate cells (secrete seminal fluid components), urethral gland cells (secrete mucus), Bartholin's gland cells (secrete vaginal lubrication), Little cell gland (secrete mucus), endometrial cells (secrete carbohydrates), isolated goblet cells of the respiratory and digestive tract (secrete mucus), mucosal cells lining the stomach (secrete mucus), gastric gland zymogen cells (secrete pepsinogen), gastric gland acid secreting cells (secrete hydrochloric acid), pancreatic acinar cells (secrete bicarbonate and digestive enzymes), Paneth cells of the small intestine (secrete lysozyme), type II pneumocytes of the lung (secrete surfactant), Clara cells of the lung, hormone secreting cells, anterior pituitary cells, growth hormone producing cells, lactotrophic cells, thyrotrophic cells, gonadotrophic cells, adrenocorticotrophic cells, intermediate pituitary cells, giant cell neurosecretory cells, intestinal and airway cells, thyroid cells , thyroid epithelial cells, parafollicular cells, parathyroid cells, parathyroid chief cells, eosinophil cells, adrenal cells, chromaffin cells, Leydig cells of the testis, theca cells of the ovarian follicle, luteal cells of ruptured follicles, granulosa lutein cells, theca lutein cells, juxtaglomerular cells (secreting renin), macular cells of the kidney, metabolic and storage cells, barrier function cells (lung, intestine, exocrine glands and urogenital tract), kidney, type I pneumocytes (inner side of air spaces of the lung), pancreatic ductal cells (central acinar cells), non-striated ductal cells (sweat glands, salivary glands, mammary glands, etc.), ductal cells (seminal vesicles, prostate, etc.), epithelial cells lining closed internal body cavities, ciliated cells with propulsive functions, extracellular matrix secreting cells, contractile cells; skeletal muscle cells, stem cells, cardiac myocytes,Blood and immune system cells, erythrocytes (red blood cells), megakaryocytes (platelet precursor cells), monocytes, connective tissue macrophages (various types), epidermal Langerhans cells, osteoclasts (in bone), dendritic cells (in lymphoid tissue), microglial cells (in the central nervous system), neutrophil granulocytes, eosinophil granulocytes, basophil granulocytes, mast cells, helper T cells, suppressor T cells, cytotoxic T cells, natural killer T cells, B cells, natural killer cells, reticulocytes, stem cells and of the blood and immune system (various types) Other cells including progenitor cells, pluripotent stem cells, totipotent stem cells, induced pluripotent stem cells, adult stem cells, sensory transducer cells, autonomic nerve cells, sensory and peripheral nerve supporting cells, central nervous system neurons and glial cells, lens cells, pigment cells, melanocytes, retinal pigment epithelial cells, germ cells, oogonia / oocytes, sperm cells, spermatocytes, spermatogonia (stem cells of spermatocytes), spermatozoa, nurse cells, ovarian follicular cells, Sertoli cells (in the testis), thymic epithelial cells, interstitial cells, interstitial kidney cells, etc.;

[0159] Cancer cells are of particular interest. In some embodiments, the target cells are cancer cells. Non-limiting examples of cancer cells include, for example, acanthoma, acinic cell carcinoma, acoustic neuroma, acral lentigo melanoma, congenital pneumonia, acute eosinophilic leukemia, acute lymphoblastic leukemia, acute megakaryoblastic leukemia, mature monocytic leukemia, acute myeloid leukemia with maturation, acute myeloid leukemia, acute myeloid leukemia, acute myeloid leukemia promyelocytic leukemia, adamantinoma, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenoid odontogenic tumor, adrenocortical carcinoma, adult T-cell leukemia, progressive NK cell leukemia, AIDS-related cancer, AIDS-related lymphoma, alveolar soft part sarcoma, ameloblastoma anal carcinoma, anaplastic leukemia, pulmonary ... large cell lymphoma, anaplastic thyroid carcinoma, angioimmunoblastic T-cell lymphoma, angiomyolipoma, angiosarcoma, appendix cancer, astrocytoma, atypical teratoid rhabdomyoma, basal cell carcinoma, basal-like carcinoma, B-cell leukemia, B-cell lymphoma, Bellini duct carcinoma, biliary tract cancer, bladder cancer, blastoma, bone cancer, bone tumor, brain stem glioma, brain tumor, breast cancer, Brenner tumor, bronchial tumor, bronchoalveolar carcinoma, brown tumor, Burkitt lymphoma, cancer of unknown primary, carcinoid tumor, cancer, carcinoma in situ, penile cancer, cancer of unknown primary, carcinosarcoma, Castleman disease, central nervous system embryonal tumor, cerebellar astrocytoma, cerebral astrocytoma, cervical Cancer, cholangiocarcinoma, chondroma, chondrosarcoma, chordoma, choriocarcinoma, choroid plexus papilloma, chronic lymphocytic leukemia, leukemic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorder, chronic neutrophilic leukemia, clear cell tumor, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, Degos disease, dermatofibrosarcoma, nodular cyst, desmoplastic small cell B-cell large cell neoplasm lymphoma, erythroblastic neuroepithelial tumor, embryonal carcinoma, endodermal sinus tumor, endometrial carcinoma, endometrioid uterine carcinoma, endometrioid tumor, enteropathy-associated T-cell lymphoma, ependymoblastoma, ependymoma, epithelioid sarcoma, erythroleukemia, esophageal tumor, esophageal tumor Tumors Ewing family sarcoma, Ewing sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, extramammary Paget's disease, fallopian tube cancer, fetal, fibroma, fibrosarcoma, follicular lymphoma, follicular thyroid cancer, gallbladder cancer, gallbladder cancer, ganglioglioma, ganglioneuroma, gastric cancer, gastric lymphoma, gastrointestinal cancer, gastrointestinal carcinoid, gastrointestinal stromal tumor, gastrointestinal stromal tumor, germ cell tumor, germinoma, gestational choriocarcinoma, gestational trophoblastic tumor, giant cell tumor of bone, glioblastoma, glioma, glioma, glioma, glioma glucagonoma, gonadoblastoma, granulosa cell tumor, hairy cell leukemia,Hairy cell leukemia, Head and neck cancer, Head and neck cancer, Cardiac cancer, Hemangioblastoma, Hemangiopericytoma, Hemangiosarcoma, Hematologic tumors, Hepatocellular carcinoma, Hepatosplenic T-cell lymphoma, Hereditary breast and ovarian cancer syndrome, Hodgkin's lymphoma, Hodgkin's lymphoma, Hypopharyngeal cancer, Hypothalamic glioma, Inflammatory breast cancer, Intraocular melanoma, Pancreatic islet cell carcinoma, Pancreatic islet cell tumor, Juvenile myelomonocytic leukemia, Kaposi's sarcoma, Kidney tumor, Klatzkin tumor, Krukenberg tumor, Laryngeal cancer, Laryngeal cancer cancer), lentigo maligna melanoma, leukemia, leukemia, lip and oral cancer, liposarcoma, lung cancer, luteoma, lymphangioma, lymphangiosarcoma, lymphoepithelioma, lymphocytic leukemia, lymphoma, macroglobulinemia, malignant fibrous histiocytoma, malignant fibrous histiocytoma of bone, malignant glioma, malignant mesothelioma, malignant peripheral nerve sheath tumor, malignant rhabdoid tumor, malignant triton tumor, MALT lymphoma, mantle cell lymphoma, Mast cell leukemia, mediastinal germ cell tumor, mediastinal tumor, medullary thyroid carcinoma, medulloblastoma, medulloepithelioma, melanoma, melanoma, meningioma, Merkel cell carcinoma, mesothelioma, mesothelioma, occult metastatic squamous cervical carcinoma, metastatic urothelial carcinoma, mixed mullerian tumor, monocytic leukemia, oral cancer, mucinous tumor, multiple endocrine neoplasia, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic syndrome, myelogenous leukemia, myxoma, myel ... , nasal cavity cancer, nasopharyngeal cancer, nasopharyngeal cancer, neoplasm, schwannoma, neuroblastoma, neuroblastoma, neurofibroma, neuroma, nodular melanoma, non-Hodgkin's lymphoma, non-Hodgkin's lymphoma, non-melanoma skin cancer, non-small cell lung, eye tumor, oligoastrocytoma, oligodendroglioma, oncocytoma, optic nerve sheath meningioma, oral cancer, oral cancer, oropharyngeal cancer, osteosarcoma, osteosarcoma, ovarian cancer, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian tumor, breast disease, Pancoast tumor , pancreatic cancer, pancreatic cancer, papillary thyroid cancer, papillomatosis, paraganglioma, paranasal sinus cancer, parathyroid cancer, penile cancer, perivascular epithelioid cell tumor, pharyngeal cancer, pheochromocytoma, moderately differentiated pineal parenchymal tumor, pineoblastoma, pituitary cell tumor, pituitary adenoma, pituitary tumor, plasma cell neoplasm, pleuropulmonary blastoma, polygerminoma, precursor T-lymphoblastic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, primary hepatocellular carcinoma, primary liver cancer, primary peritoneal cancer,Primary neuroectodermal tumors, prostate cancer, pseudomyxoma peritonei, rectal cancer, renal cell carcinoma, respiratory cancer involving the NUT gene on chromosome 15, retinoblastoma, rhabdomyoma, rhabdomyosarcoma, Richter transformation, sacrococcygeal teratoma, salivary gland cancer, sarcoma, neurinoma, sebaceous gland cancer, secondary neoplasms, seminoma, serous tumor, Sertoli-Leydig cell tumor, sex cord stromal cell tumor, Sezary syndrome cancer, skin cancer, small blue round cell tumor, small cell carcinoma, small cell lung cancer, small cell lymphoma, small intestine cancer, soft tissue sarcoma, somatostatin-producing tumor, sooty wart, spinal cord tumor, spinal cord tumor, splenic marginal zone lymphoma, squamous cell carcinoma, gastric cancer, superficial spreading melanoma, The targeted cancer cells include cells of cancers such as supratentorial primitive neuroectodermal tumor, surface epithelial stromal tumor, synovial sarcoma, T-cell acute lymphoblastic leukemia, T-cell large granular lymphocytic leukemia, T-cell leukemia, T-cell lymphoma, T-cell prolymphocytic leukemia, teratoma, end-stage lymphoid cancer, testicular cancer, coma, pharyngeal cancer, thymic cancer, thymoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, transitional cell carcinoma, urachal cancer, urethral cancer, genitourinary tumors, uterine sarcoma, uveal melanoma, vaginal cancer, Berner-Morrison syndrome, verrucous carcinoma, visual pathway glioma, vulvar cancer, Waldenstrom's hypergammaglobulinemia, Warthin's tumor, Wilms' tumor, and combinations thereof. In some embodiments, the targeted cancer cells represent a subpopulation within a cancer cell population, such as cancer stem cells. In some embodiments, the cancer is of the hematopoietic lineage, such as lymphoma. The antigen may be a tumor-associated antigen. ,

[0160] In some embodiments, the target cells form tumors. Tumors treated using the methods herein may result in stabilized tumor growth (e.g., one or more tumors do not increase in size by more than 1%, 5%, 10%, 15%, or 20% and / or do not metastasize). In some embodiments, the tumor is stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more weeks. In some embodiments, the tumor is stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more months. In some embodiments, the tumor is stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more years. In some embodiments, the size of the tumor or the number of tumor cells is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more. In some embodiments, the tumor is completely eliminated or reduced to below detection levels. In some embodiments, the subject remains tumor-free (e.g., in remission) for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more weeks after treatment. In some embodiments, the subject remains tumor-free for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more months after treatment. In some embodiments, the subject remains tumor-free for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more years following treatment.

[0161] The death of target cells can be detected by any suitable method, including but not limited to, counting cells before and after treatment, or measuring the level of a marker associated with live or dead cells (e.g., live or dead target cells). The extent of cell death can be measured by any suitable method. In some embodiments, the extent of cell death is measured relative to an initial condition. For example, an individual has a known initial amount of target cells, such as an initial cell mass of known size or a known concentration of circulating target cells. In such cases, the extent of cell death can be expressed as a ratio of surviving cells after treatment to the initial cell population. In some embodiments, the extent of cell death can be measured by a suitable cell death assay. A variety of cell death assays are available, and a variety of detection methodologies can be utilized. Examples of detection methodologies include, but are not limited to, the use of cell staining, microscopy, flow cytometry, cell sorting, and combinations thereof.

[0162] When the tumor is subjected to surgical resection following completion of the treatment period, the effectiveness of the treatment in reducing tumor size can be measured by measuring the percentage of resected tissue that is necrotic (i.e., dead). In some embodiments, the treatment is therapeutically effective when the necrotic percentage of the resected tissue is greater than about 20% (e.g., at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). In some embodiments, the necrotic percentage of the resected tissue is 100%, i.e., no viable tumor tissue is present or detectable.

[0163] Exposing a target cell to an immune cell or immune cell population disclosed herein can be performed either in vitro or in vivo. Exposing a target cell to an immune cell or immune cell population means contacting the target cell with and / or in sufficient proximity to the immune cell so that the antigen of the target cell (e.g., membrane-bound or non-bound) can bind to the switch molecule expressed on the immune cell. Exposing a target cell to an immune cell or immune cell population also generally means contacting the target cell with and / or in sufficient proximity to the immune cell so that the antigen of the target cell (e.g., membrane-bound or non-bound) can bind to the CAR expressed on the immune cell. Exposing a target cell to an immune cell or immune cell population in vitro can be achieved by co-culturing the target cell with the immune cell. The target cell and the immune cell can be co-cultured, for example, as adherent cells or alternatively, in suspension. The target cell and the immune cell can be co-cultured in various suitable types of cell culture media, for example, with supplements, growth factors, ions, etc. Exposing a target cell to an immune cell or population of immune cells in vivo can, in some cases, be accomplished by administering the immune cells to a subject, e.g., a human subject, and allowing the immune cells to localize to the target cell via the circulatory system. In some cases, the immune cells can be delivered immediately, e.g., by direct injection, to the area where the target cell is localized.

[0164] Exposure can be for any suitable period of time, such as at least 1 minute, at least 5 minutes, at least 10 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 12 hours, at least 16 hours, at least 20 hours, at least 24 hours, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, or more.

[0165] The various domains of the switch molecules and CARs provided herein can be linked by chemical bonds, such as amide bonds or disulfide bonds; small organic molecules (e.g., hydrocarbon chains); amino acid sequences, such as peptide linkers (e.g., amino acid sequences of about 3 to 200 amino acids in length), or a combination of small organic molecules and peptide linkers. The peptide linker can provide the desired flexibility to allow for the desired expression, activity, and / or conformation of the chimeric polypeptide. The peptide linker can be of any length suitable for linking at least two domains of interest, and is preferably designed to be sufficiently flexible to allow for proper folding and / or function and / or activity of one or both of the linked domains. The peptide linker can have a length of at least 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids. In some embodiments, the peptide linker has a length of about 0-200 amino acids, about 10-190 amino acids, about 20-180 amino acids, about 30-170 amino acids, about 40-160 amino acids, about 50-150 amino acids, about 60-140 amino acids, about 70-130 amino acids, about 80-120 amino acids, about 90-110 amino acids. In some embodiments, the linker sequence may include an endogenous protein sequence. In some embodiments, the linker sequence includes glycine, alanine, and / or serine amino acid residues. In some embodiments, the linker may include a motif, such as, for example, multiple or repeated motifs such as GS, GGS, GGGGS, GGSG, or SGGG. The linker sequence may include any natural amino acid, unnatural amino acid, or combinations thereof.

[0166] Any suitable delivery method can be used to introduce the compositions and molecules of the present disclosure (e.g., polypeptides and / or nucleic acids encoding polypeptides) into host cells, such as immune cells. The various components can be delivered simultaneously or temporally separately. The choice of method can depend on the type of cell being transformed or the conditions under which transformation occurs (e.g., in vitro, ex vivo, or in vivo).

[0167] Methods of delivery include contacting one or more nucleic acids comprising a nucleotide sequence encoding a composition of the present disclosure with a target polynucleotide or introducing the nucleic acid into a cell (or a population of cells, such as an immune cell). Suitable nucleic acids comprising a nucleotide sequence encoding a composition of the present disclosure may comprise an expression vector, where an expression vector comprising a nucleotide sequence encoding one or more compositions of the present disclosure is a recombinant expression vector.

[0168] Non-limiting examples of delivery methods or transformation include, for example, viral or bacteriophage infection, gene transfer, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethylenimine (PEI) transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, and the like.

[0169] In some embodiments, the disclosure provides methods that include delivering one or more polynucleotides, or one or more vectors described herein, or one or more transcripts thereof, and / or proteins translated therefrom, to a host cell. In some embodiments, the disclosure further provides cells produced by such methods, organisms (e.g., animals, plants, fungi, etc.) comprising or produced from such cells.

[0170] Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acid into mammalian cells or target tissues. Such methods can be used to administer nucleic acid encoding the composition of the present disclosure to cells in culture or host organisms. Non-viral vector delivery systems can include DNA plasmids, RNA (e.g., transcripts of vectors described herein), naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as liposomes. Viral vector delivery systems include DNA and RNA viruses, which can have either episomal or integrated genomes after delivery to cells.

[0171] Non-viral nucleic acid delivery methods may include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycations or lipid:nucleic acid complexes, naked DNA, artificial virus particles, and drug-enhanced DNA uptake. Cationic and neutral lipids suitable for effective receptor-recognition lipofection of polynucleotides may be used. Delivery may be to cells (e.g., in vitro or ex vivo administration) or to target tissues (e.g., in vivo administration). Preparations of lipid:nucleic acid complexes may be used, including targeted liposomes such as immunolipids.

[0172] RNA or DNA virus-based systems can be used to target specific cells in the body and deliver the viral payload to the cell nucleus. Viral vectors can be administered directly (in vivo) or they can be used to treat cells in vitro and the modified cells can be optionally administered (ex vivo). Virus-based systems can include retroviruses, lentiviruses, adenoviruses, adeno-associated and herpes simplex virus vectors for gene transfer, etc. Integration into the host genome can occur with retroviruses, lentiviruses and adeno-associated virus gene transfer methods, which can result in long-term expression of the inserted transgene. High transformation efficiency can be observed in many different cell types and target tissues.

[0173] The tropism of retroviruses can be modified by incorporating foreign envelope proteins, expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors that can transform or infect non-dividing cells and produce high viral titers. The choice of retroviral gene transfer can depend on the target tissue. Retroviral vectors can contain cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. Minimal cis-acting LTRs are sufficient for vector replication and packaging, which can be used to incorporate therapeutic genes into target cells to provide persistent transgene expression. Retroviral vectors can include those based on murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof.

[0174] Adenovirus-based systems may be used. Adenovirus-based systems may lead to transient expression of the transgene. Adenovirus-based vectors may have high transduction efficiency in cells and may not require cell division. High titers and levels of expression may be obtained in adenovirus-based vectors. Adeno-associated virus ("AAV") vectors may be used to transduce cells with target nucleic acids, for example, in in vitro production of nucleic acids and peptides, and for in vivo or ex vivo gene therapy methods.

[0175] Packaging cells can be used to form viral particles capable of infecting host cells. Such cells include 293 cells (e.g., for packaging adenovirus), and Psi2 or PA317 cells (e.g., for packaging retrovirus). Viral vectors can be made by producing cell lines that package nucleic acids into viral particles. The vectors can contain minimal viral sequences necessary for packaging and subsequent integration into the host. The vectors can contain other viral sequences that can be replaced by an expression vector for the polynucleotide(s) to be expressed. Missing viral functions can be supplied in trans by the packaging cell line. For example, AAV vectors can contain ITR sequences from the AAV genome that are necessary for packaging and integration into the host genome. Viral DNA can be packaged into cell lines that can contain helper plasmids encoding other AAV genes, i.e., rep and cap, but without ITR sequences. The cell lines can also be infected with adenovirus as a helper. Helper virus can promote the replication of AAV vector and the expression of AAV gene from helper plasmid.Adenovirus contamination can be reduced, for example, by heat treatment, to which adenovirus is more sensitive than AAV.Additional methods for the delivery of nucleic acid to cells can be used, for example, as described in US Patent Application 20030087817, which are incorporated herein by reference.

[0176] Host cells are transiently or non-transiently transfected with one or more vectors described herein. Cells can be transfected as they naturally occur in a subject. Cells can be taken or derived from a subject and transfected. Cells can be derived from cells taken from a subject, i.e., cell lines. In some embodiments, cells transfected with one or more vectors described herein are used to establish new cell lines that contain sequences from one or more vectors. In some embodiments, cells transiently transfected with a composition of the present disclosure (e.g., transient transfection with one or more vectors or transfection with RNA) are used to establish new cell lines that include cells that contain the modifications but not other exogenous sequences.

[0177] Any suitable vector compatible with the host cell can be used in the method of the present disclosure. Non-limiting examples of vectors for eukaryotic host cells include, for example, pXT1, pSG5 (Stratagene®), pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia®).

[0178] Contacting the cells with the compositions of the present disclosure may be performed in any culture medium and under any culture conditions that promote cell survival. For example, the cells may be suspended in a convenient suitable nutrient medium such as Iscove's modified DMEM or RPMI 1640, supplemented with fetal bovine serum or heat-inactivated goat serum (about 5-19%), L-glutamine, thiols, particularly 2-mercaptoethanol, and antibiotics, such as penicillin and streptomycin. The culture medium may contain growth factors to which the cells are responsive. Growth factors, as defined herein, are molecules that can promote cell survival, growth, and / or differentiation in culture or in intact tissues through specific effects on transmembrane receptors. Growth factors may include polynucleotide and non-polynucleotide factors.

[0179] In many embodiments, the system selected is targeted to a specific tissue or cell type.In some cases, the tissue or cell targeting of the delivery system is achieved by the binding of the delivery system to tissue or cell specific markers, such as cell surface proteins.Viral or non-viral delivery systems can be customized for the target tissue or cell type of interest.

[0180] Pharmaceutical compositions comprising the molecules (e.g., polypeptides and / or nucleic acids encoding the polypeptides) or immune cells described herein can be administered for prophylactic and / or therapeutic treatments. In therapeutic applications, the compositions can be administered to a subject already suffering from a disease or condition in an amount sufficient to cure or at least partially arrest the symptoms of the disease or condition, or to cure, heal, improve, or ameliorate the condition. Amounts effective for this use can vary based on the severity and course of the disease or condition, previous treatments, the subject's health, weight, and responsiveness to drugs, and the determination of willingness to treat.

[0181] The multiple therapeutic agents can be administered in any order or simultaneously. If administered simultaneously, the multiple therapeutic agents can be provided in a single, integrated form, or in multiple forms, such as multiple separate pills. The molecules can be packaged together or separately, in a single package, or in multiple packages. If not administered simultaneously, the timing of the multiple doses can vary by as much as about months.

[0182] The molecules described herein can be administered before, during, or after the onset of a disease or condition, and the timing of administering the composition containing the compound can vary. For example, the pharmaceutical composition can be used as a prophylactic and can be administered continuously to a subject prone to a condition or disease to prevent the onset of the disease or condition. The molecules and pharmaceutical compositions can be administered to the subject during or as soon as possible after the onset of symptoms. Administration of the molecules can begin within the first 48 hours of the onset of symptoms, within the first 24 hours of the onset of symptoms, within the first 6 hours of the onset of symptoms, within the first 3 hours of the onset of symptoms. The initial administration can be via any practical route, such as by any route described herein, using any formulation described herein. The molecules can be administered as soon as practical after the onset of a disease or condition is detected or suspected, and for the length of time required to treat the disease (e.g., from about 1 month to about 3 months). The length of treatment can vary for each subject.

[0183] The molecule can be packaged in a biological compartment. The biological compartment containing the molecule can be administered to a subject. The biological compartment can include, but is not limited to, viruses (lentivirus, adenovirus), nanospheres, liposomes, quantum dots, nanoparticles, microparticles, nanocapsules, vehicles, polyethylene glycol particles, hydrogels, and micelles.

[0184] For example, the biological compartment may include a liposome. A liposome may be a self-assembled structure that includes one or more lipid bilayers, each layer may include two monolayers that include amphiphilic lipid molecules in opposite orientation. The amphiphilic lipid may include a polar (hydrophilic) head that is covalently bonded to two or more non-polar (hydrophobic) acyl or alkyl chains. Energetically unfavorable contact between the hydrophobic acyl chains and the surrounding aqueous solvent causes the amphiphilic lipid molecule to arrange itself such that the polar heads may be oriented toward the surface of the bilayer and the acyl chains are oriented toward the interior of the bilayer, effectively shielding the acyl chains from contact with the aqueous environment.

[0185] Examples of preferred amphipathic compounds for use in liposomes include phosphoglycerides and sphingolipids, representative examples of which include, for example, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine, distearoylphosphatidylcholine (DSPC), dilinoleoylphosphatidylcholine, and egg yolk sphingomyelin, or any combination thereof.

[0186] The biochemical compartment may comprise a nanoparticle having a diameter of about 40 nm to about 1.5 μm, about 50 nm to about 1.2 μm, about 60 nm to about 1 μm, about 70 nm to about 800 nm, about 80 nm to about 600 nm, about 90 nm to about 400 nm, or about 100 nm to about 200 nm.

[0187] In some cases, as the size of the nanoparticles increases, the release rate may be slowed or prolonged, and as the size of the nanoparticles decreases, the release rate may be increased.

[0188] The amount of albumin in the nanoparticles can range from about 5% to about 85% albumin (v / v), about 10% to about 80%, about 15% to about 80%, about 20% to about 70% albumin (v / v), about 25% to about 60%, about 30% to about 50%, or about 35% to about 40%. The pharmaceutical composition can include up to 30, 40, 50, 60, 70, 80% or more nanoparticles. In some cases, the nucleic acid molecules of the present disclosure can be attached to the surface of the nanoparticles.

[0189] The biochemical compartment may include a virus. The virus may be a delivery system for the pharmaceutical composition of the present disclosure. Exemplary viruses include lentivirus, retrovirus, adenovirus, herpes simplex virus I or II, parvovirus, reticuloendotheliosis virus, and adeno-associated virus (AAV), etc. The pharmaceutical composition of the present disclosure may be delivered to cells using a virus. The virus may infect and transduce in vivo, ex vivo, or in vitro. In ex vivo and in vitro delivery, the transduced cells may be administered to a subject in need of treatment.

[0190] The pharmaceutical composition may be packaged in a viral delivery system, for example, the composition may be packaged in an HSV-1 helper virus-free packaging system.

[0191] The viral delivery system (e.g., a virus containing the pharmaceutical composition of the present disclosure) can be administered to cells, tissues, organs of a subject in need thereof by direct injection, stereotactic injection, intracerebroventricularly, by mini-pump infusion system, by convection, by catheter, intravenous, parenteral, intraperitoneal, and / or subcutaneous injection. In some cases, cells can be transduced in vitro or ex vivo using the viral delivery system. The transduced cells can be administered to a subject suffering from a disease. For example, stem cells can be transduced using a viral delivery system containing the pharmaceutical composition, and the stem cells can be transplanted into a patient to treat the disease. In some examples, the dose of transduced cells given to a subject is about 1×10 per dose. 5 cells / kg, approximately 5×10 5 cells / kg, approximately 1×10 6 cells / kg, approximately 2×10 6 cells / kg, approximately 3×10 6 cells / kg, approximately 5×10 6 cells / kg, approximately 5×10 6 cells / kg, approximately 6×10 6 cells / kg, approximately 7×10 6 cells / kg, approximately 8×10 6 cells / kg, approximately 9×10 6 cells / kg, approximately 1×10 7 cells / kg, approximately 5×10 6 cells / kg, and approximately 1 × 10 8 It can be cells / kg or more.

[0192] Introduction of biochemical compartments into cells can occur by viral or bacteriophage infection, gene transfer, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethylenimine (PEI) transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, etc.

[0193] In some embodiments, immune cells expressing the system of interest are administered. The immune cells expressing the system of interest may be administered before, during, or after the onset of a disease or condition, and the timing of administering the composition comprising the compound may vary. For example, the immune cells expressing the system of interest may be used as a prophylactic and may be administered continuously to subjects prone to a condition or disease to prevent the onset of the disease or condition. The immune cells may be administered to the subject during or as soon as possible after the onset of symptoms. Administration may begin within the first 48 hours of the onset of symptoms, within the first 24 hours of the onset of symptoms, within the first 6 hours of the onset of symptoms, within the first 3 hours of the onset of symptoms. The initial administration may be via any practical route, such as by any route described herein, using any formulation described herein. The immune cells may be administered as soon as practical after the onset of a disease or condition is detected or suspected, and for the length of time required to treat the disease (e.g., from about 1 month to about 3 months). The length of treatment may vary for each subject.

[0194] The molecules described herein (e.g., polypeptides and / or nucleic acids, etc.) may be present in an amount of about 1 mg to about 2000 mg, about 5 mg to about 1000 mg, about 10 mg to about 25 mg to 500 mg, about 50 mg to about 250 mg, about 100 mg to about 200 mg, about 1 mg to about 50 mg, about 50 mg to about 100 mg, about 100 mg to about 150 mg, about 150 mg to about 200 mg, about 200 mg to about 250 mg, about 250 mg to about 300 mg, about 300 mg to about 350 mg , about 350 mg to about 400 mg, about 400 mg to about 450 mg, about 450 mg to about 500 mg, about 500 mg to about 550 mg, about 550 mg to about 600 mg, about 600 mg to about 650 mg, about 650 mg to about 700 mg, about 700 mg to about 750 mg, about 750 mg to about 800 mg, about 800 mg to about 850 mg, about 850 mg to about 900 mg, about 900 mg to about 950 mg, or about 950 mg to about 1000 mg of the composition.

[0195] The molecules described herein (e.g., polypeptides and / or nucleic acids) may be administered at a concentration of about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg , about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg, about 900 mg, about 950 mg, about 1000 mg, about 1050 mg, about 1100 mg, about 1150 mg, about 1200 mg, about 1250 mg, about 1300 mg, about 1350 mg, about 1400 mg, about 1450 mg, about 1500 mg, about 1550 mg, about 1600 mg, about 1650 mg, about 1700 mg, about 1750 mg, about 1800 mg, about 1850 mg, about 1900 mg, about 1950 mg, or about 2000 mg.

[0196] The molecules described herein (e.g., polypeptides and / or nucleic acids) can be present in a composition that provides at least 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 5.6, 6, 6.5, 10, or more activity units per mg of molecule. The activity can be modulation of gene expression. In some embodiments, the total amount of active units of the molecules delivered to the subject is at least 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 60,000, 70,000, 80,000, 90,000, 110,000, 120,000, 130,000, 140,000, 150,000, 160,000, 170,000, 180,000, 190,000, 200,000, 210,000, 220,000, 230,000, or 250,000 or more units. In some embodiments, the total amount of active units of molecules delivered to a subject is at most 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 60,000, 70,000, 80,000, 90,000, 110,000, 120,000, 130,000, 140,000, 150,000, 160,000, 170,000, 180,000, 190,000, 200,000, 210,000, 220,000, 230,000, or 250,000 or more units.

[0197] Various aspects of the present disclosure are further illustrated by the following non-limiting examples.

[0198] Example 1: NY-ESO-1 targeting TCR T cells Tumor cells from apheresis were NY-ESO-1 positive and the subject had HLA-A:0201 leukocytes. Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll lymphocyte separation. After 2 hours of adherent culture, T cells were removed. NY-ESO-1 TCR lentivirus was added at a multiplicity of infection (MOI) of 1. T cells were then cultured and expanded. TCR expression was measured by flow cytometry. In Figure 1, the left panel shows TCR expression in T cells and the right panel shows expression in transduced T cells. The histogram plot in Figure 1 shows more TCR expression in T cells transduced with the NY-ESO-1 TCR gene.

[0199] Example 2: Preparation of triple positive T cells Tumor tissue in an amount of 10 g or more was removed from the patient. Cells were isolated by enzymatic digestion. CD3 positive T cells were isolated using CD3 magnetic beads. Other cells were grown in adherent culture to provide tumor cells from the patient. T cells were then isolated using magnetic beads. Triple positive T cells for PD-1, CD137 and TIM-3 were sorted via flow cytometry and further cultured and expanded.

[0200] Example 3: Neoantigen-active T cells The preparation is shown in Figure 2. PBMCs or tumor cells from surgery were subjected to whole exome sequencing or RNA transcriptome sequencing. 20 mutations were selected based on affinity prediction in terms of the patient's HLA typing. Neoantigen-encoding genes were synthesized and transcribed into RNA. PBMCs were isolated and subjected to adherent culture for 2 hours. Adherent monocytes were harvested. Cytokines were added to promote dendritic cell differentiation and maturation. RNA was transfected into dendritic cells by electroporation. Suspension cells were obtained as primarily T cells and cultured with dendritic cells. CD137+ positive cells were then isolated to provide neoantigen-reactive (i.e., recognizing) T cells ("neoT"). The neoT were then expanded.

[0201] Example 4: Preparation of lentivirus for PD1 / CD28 switch molecule (PD1sw, SEQ ID NO:2) A fourth generation lentiviral vector system was used. PD1 / CD28 vector, packaging vector pMDL-gag, Rev, and envelope vector pMD2.G were co-transfected into HEK293T cells using calcium phosphate or liposome-PEI. Supernatants were harvested 48 hours later and centrifuged to concentrate the lentivirus.

[0202] Titration of lentivirus was performed with 3-fold serial dilutions. After 48-72 h transduction with 50 μL of lentivirus, HEK293T cells were harvested and then stained for PD-1. PD-1 positive rate (PD-1+%) was analyzed by flow cytometry, and titers were calculated based on the following: Titer (TU / mL) = 40,000-45,000 (this is the starting number of HEK293T cells) x PD1 + % × dilution factor × 20 (initial PD1 + %<20%

[0203] Figures 3A and 3B show the calculation of PD1 / CD28 lentiviral titers. 3×10 7 Larger titers may be used further.

[0204] Example 5: Transduction of PD1 / CD28 into neoantigen-active T cells Three types of T cells were obtained: Switch-NY-ESO-1-TCR-T, Switch-TIL, and Switch-neoT. Switch-NY-ESO-1-TCR-T cells were obtained by expressing switch molecules in ESO-1-TCR-T cells of Example 1. Switch-TIL cells were obtained by expressing switch molecules in triple positive T cells of Example 2. Switch-neoT cells were obtained by expressing switch molecules in neoT cells of Example 3. Flow cytometry shows that the expression rate of Switch is about 60% in all three types of T cells. See Figure 4.

[0205] Example 6: In vitro assay of NY-ESO-1-targeted TCR-T cells expressing PD1 / CD28 switch molecules J82-NY-ESO-1-PD-L1 tumor cells were constructed, with HLA typing of A:0201. Lentiviral vectors were added to J82 (bladder, transitional cell carcinoma) at an infection efficiency of MOI=5 to transduce PD-L1 and NY-ESO-1 transgenes. G418 and puromycin were added 72 hours later to screen positive cells. Flow cytometry was performed to measure the expression of PD-L1 and NY-ESO-1 about 2 weeks later (Figure 5). As shown in Figure 5, more than 95% of the transduced J82 cells simultaneously expressed PDL1 and NY-ESO-1, confirming the success of cell construction. Figures 6A-6C show data from an in vitro cell killing assay, in which J82 or J82-NY-ESO-1-PD-L1 bladder cancer cells were contacted with T cells, NY-ESO1-TCR T cells, or Switch-NY-ESO1-TCR T cells. T cells, NY-ESO1-TCR T cells, or Switch-NY-ESO1-TCR T cells were gated by expression of CD8 and CD107a. Surface localization of CD107a (also known as LAMP-1), a degranulation marker that is normally found inside the granules of T cells, is a sign of cytotoxic activity as T cells release perforin and granzymes from granules to kill target cells. As shown in Figure 6A, the data show greater cell killing activity of Switch-NY-ESO1-TCR T cells compared to NY-ESO1-TCR T cells. Figures 6B and 6C show that exposure to J82 bladder cancer cells does not induce secretion of IFN-γ and IL-2 in T cells, NY-ESO1-TCR T cells, or Switch-NY-ESO1-TCR T cells, whereas exposure to J82-NY-ESO1-PDL1 bladder cancer cells induces secretion of IFN-γ and IL-2 in both NY-ESO1-TCR T cells and Switch-NY-ESO1-TCR T cells, with the secretion levels of IFN-γ and IL-2 being higher in Switch-NY-ESO1-TCR T cells.

[0206] Example 7: In vitro efficacy assay of TIL expressing PD1 / CD28 molecule Figures 7A and 7B show the release of IFN-γ and IL-2 in TILs (and TILs expressing PD1 / CD28 switch molecules (Switch-TILs)) when cultured with tumor cells. The data show that exposure to tumor cells induces higher secretion of IFN-γ and IL-2 by Switch-TIL cells expressing PD1 / CD28 switch molecules compared to TIL cells without PD1 / CD28 switch molecules.

[0207] Example 8: In vitro assay of neoT expressing PD1 / CD28 switch molecules Figures 8A and 8B show the release of IFN-γ and IL-2 in neoantigen-activated T cells (neoT) and neoT cells expressing the PD1 / CD28 switch molecule when cultured with tumor cells. The data show that exposure to tumor cells induces higher secretion of IFN-γ and IL-2 by Switch-neoT cells expressing the PD1 / CD28 switch molecule compared to neoT cells without the PD1 / CD28 switch molecule.

[0208] Example 9: Animal experiments of NY-ESO1-TCR T cells expressing PD1 / CD28 switch molecules 1×10 6 Tumor cells, J82-NY-ESO1-PDL1, are inoculated subcutaneously into NSG mice. Tumors are expected to develop after approximately 2 weeks. Tumor size is measured at 23 weeks, and 30 mice are used.

[0209] The control group of mice is treated with PBS subcutaneously (A0). There are five treatment groups, which are untreated PBS (A1), T cells (A2), switch-T cells (A3), NY-ESO1-TCR-T cells (A4), and Switch-NY-ESO1-TCR T cells. Cells are injected at 1×10 into the tail vein. 7The cells are given by intravenous injection.

[0210] The tumor size is measured every 2-3 days for 30 days, and the general condition of the mice is observed. The tumor size is measured according to the following formula: Tumor size = 1 / 2 x major axis x minor axis x minor axis

[0211] It is expected that the experiment will show that tumor size in group A5 decreases, or remains approximately constant, or at least increases at a reduced rate, compared to groups A1-A3.

[0212] The amount of T cells at the tumor site after treatment is analyzed. Mice are randomly selected from each of the treatment groups A3-A5 on day 10 after dosing, and tumor cells are isolated to obtain TILs. Flow cytometry is performed to measure the amount of total T cells present at the tumor site. It is expected that treatment with Switch-NY-ESO1-TCR T cells (A5) will result in a greater presence of T cells at the tumor site.

[0213] Example 10: Preparation of lentiviral CAR targeting B cell surface protein (BCAR) CD19 was selected as the B-CAR target, and anti-CD19 scFv with the sequence shown in SEQ ID NO:1 was used to construct the B-CAR. A fourth generation lentiviral vector system was used. CA19 CAR vector, packaging vector pMDL-gag, Rev, and envelope vector pMd2.G were co-transfected into HEK293T cells using calcium phosphate or liposome-PEI. The supernatant was collected after 48 hours and ultracentrifuged to concentrate the lentivirus.

[0214] Titration of the lentivirus was performed in 3-fold serial dilutions. 293T cells were harvested after 48-72 h transduction with 50 μL of lentivirus and then stained for CAR expression. The CAR positivity rate (CAR%) was analyzed by flow cytometry and the titer was calculated based on the following: Titer (TU / mL) = initial 293T cell number x CAR+% x dilution factor x 20 (initial CAR+% < 20%)

[0215] The lentivirus titer was calculated to be 3×10 7 The larger titers were deemed suitable for further use.

[0216] Example 11: Transduction of BCAR into tumor-recognizing T cells Three types of T cells, NY-ESO-1-TCR-T, TIL, and neoT, were obtained by transduction with BCAR lentivirus (BCAR-NY-ESO-1-TCR-T, BCAR-TIL, and BCAR-neoT). Flow cytometry shows the expression rate of B-CAR in all three types of T cells to be about 60%. See FIG. 9. The three types of T cells were expanded separately.

[0217] Example 12: In vitro assay of NY-ESO-1 TCR-T cells expressing BCAR To confirm the function of BCAR in NY-ESO-1-targeting TCR-T cells, the J82-NY-ESO-1-Luc tumor cell line with HLA typing A:0201 was constructed. To confirm the dual targeting function of BCAR-NY ESO1-TCR-T cells, 1 × 10 5 J82-NY-ESO-1-Luc cells were seeded in a 24-well plate and cultured overnight to allow attachment. The cells were divided into four groups, A, B, C, and D, and each well also contained 5 × 10 4 Group A contained 2 × 10 B cells. 5 Group B was co-cultured with 2 × 10 T cells. 5 Group C was co-cultured with 2 × 10 BCAR-T cells (CAR positive rate 60%). 5 Group D was co-cultured with 2 × 10 NY-ESO1-TCR-T cells.5 The following assays were performed:

[0218] Expansion of T cells. The number of T cells was counted after 48 and 96 hours of culture. As shown in FIG. 10, after 4 days of culture, T cells in group A were expanded about 3-fold, BCAR-T cells in group B were expanded 12-fold, NY-ESO1-TCR-T cells in group C were expanded about 10-fold, and BCAR-NY-ESO1-TCR-T cells were expanded about 27-fold.

[0219] Tumor cell proliferation. Supernatants were collected from cultures of groups A-D after 48 and 96 hours, and the cultures were washed three times with PBS. Adherent tumor cells were lysed, and luciferase activity was measured as an indicator of tumor cell viability. See FIG. 11. The data show that after 48 hours of culture, the average amount of protein from tumor cells (and therefore the number of tumor cells present) was lowest in treatment with BCAR-NY-ESO1-TCR-T cells compared to the other treatments.

[0220] Example 13: In vitro assay of TILs expressing BCAR To confirm the function of CD19 CAR (BCAR) in TILs, tumor cells were isolated from fresh tumor tissues of human subjects and seeded in 24-well plates. The cells were cultured overnight to allow for attachment. TILs and BCAR-TILs were added into the wells. The same amount of B cells was added to the wells, and the following assays were performed.

[0221] Expansion of T cells. After 96 hours of co-culture with tumor cells, TILs were increased approximately 10-fold and BCAR-TILs were increased approximately 25-fold, as shown in FIG. 12A. The results show that when co-cultured with B cells and tumor cells, there was a greater increase in BCAR-TILs compared to TILs without BCAR.

[0222] Tumor cell proliferation. After 96 hours, the supernatant was collected from the culture mixture, and the cultures were washed three times with PBS. Adherent tumor cells were lysed, and luciferase activity was measured as an indicator of tumor cell viability. Luciferase levels are shown in FIG. 12B. The data show that after 96 hours of culture, the average amount of protein from tumor cells (and therefore the number of tumor cells present) was lower with treatment with BCAR-TIL cells compared to treatment with TILs without BCAR.

[0223] Example 14: In vitro assay of neoantigen-reactive T cells expressing BCAR To confirm the function of CD19 CAR (e.g. BCAR) in neoT cells, tumor cells were isolated from fresh tumor tissue of human subjects and seeded in 24-well plates. The cells were cultured overnight to allow for attachment. In the culture medium, neoT and BCAR-neoT cells were added. The same amount of B cells was added to the wells, and the following assays were performed.

[0224] Expansion of T cells. After 96 hours of co-culture with tumor cells, neoT was increased approximately 9-fold and BCAR-neoT was increased approximately 23-fold, as shown in FIG. 13A. The results show that there was a greater increase in BCAR-neoT compared to neoT without BCAR when co-cultured with B cells and tumor cells.

[0225] Tumor cell proliferation. After 96 hours, the supernatant was collected from the culture mixture, and the cultures were washed three times with PBS. Adherent tumor cells were lysed, and luciferase activity was measured as an indicator of tumor cell viability. Luciferase levels are shown in FIG. 13B. The data show that after 96 hours of culture, the average amount of protein from tumor cells (and therefore the number of tumor cells present) was lower when treated with BCAR-neoT cells compared to when treated with neoT without BCAR.

[0226] Example 15: Animal experiments with NY-ESO-1 TCR-T cells expressing BCAR Animal model: 1×10 6 Tumor cells (J82-NY-ESO-1) are inoculated subcutaneously into NSG mice. As a blank control, animals in group A0 were subcutaneously injected with PBS. Tumors form in the animals about 2 weeks after tumor cell injection. Tumor size is measured after 23 days. 30 mice are selected.

[0227] Administration: Animals in the blank control (A0) group were injected with PBS via the tail vein. The tumorigenic groups were divided into six groups: PBS group (A1), T cell group (A2), BCAR-T group (A3), NY-ESO1 TCR-T group (A4), BCAR&NY-ESO-1 TCR-T dual target T cell group (A5), and high dose NY-ESO-1 TCR-T group (A6). Animals in groups A1-A5 were injected with 1×10 4 Group A6 was injected with 1 x 10 cells; 7 All groups were injected with 1 x 10 cells. 7 The B cells are given by injection.

[0228] Tumor size and the overall condition of the mice are measured every 2-3 days for 28 days after administration. Tumor size is measured according to the following formula: Tumor size = 1 / 2 x major axis x minor axis x minor axis

[0229] Changes in tumor burden

[0230] This experiment is expected to show that mice in group 5 will exhibit the smallest tumor size among all groups.

[0231] Changes in total amount of T cells infused

[0232] Peripheral blood will be drawn from animals in groups A2-A5 10 days after dosing. Total CD3+ T cell counts will be measured using flow cytometry and are expected to show that the number of T cells with BCAR in groups A3-A5 is greater than that in group A4. Both BCAR T cells and dual targeting T cells are expected to expand in vivo.

[0233] Example 16: Animal experiments with BCAR-expressing TILs Animal model: Tumor cells were isolated from fresh tumor tissue and then cultured at 1 × 10 per animal. 6 The tumor cells are injected subcutaneously into NSG mice at a dose of 100 mg / kg / day. As a blank control, animals in group A0 are injected subcutaneously with PBS. Tumors form in the animals about 2 weeks after tumor cell injection. Tumor size is measured after 25 days. 30 mice are selected.

[0234] Administration: Animals in the blank control (A0) group were injected with PBS via the tail vein. The tumorigenic groups were divided into five groups: PBS group (A1), T cell group (A2), BCAR-T group (A3), TIL group (A4), and BCAR TIL cell group (A5). The animals in groups A1 to A5 were injected with 1 × 10 4 T cells were injected at 1 x 10, and all groups were 7 The B cells are given by injection.

[0235] Tumor size and the overall condition of the mice are measured every 2-3 days for 28 days after administration. Tumor size is measured according to the following formula: Tumor size = 1 / 2 x major axis x minor axis x minor axis

[0236] Changes in tumor burden

[0237] This experiment is expected to show that mice in group 5 will exhibit the smallest tumor size among all groups.

[0238] Changes in total amount of T cells infused

[0239] Peripheral blood will be drawn from animals in groups A2-A5 10 days after dosing. Total CD3+ T cell counts will be measured using flow cytometry and are expected to show that the number of T cells with BCAR in groups A3-A5 is greater than that in group A4. Both BCAR T cells and dual targeting T cells are expected to expand in vivo.

[0240] Example 17: Animal experiments with neoT expressing BCAR Animal model: Tumor cells were isolated from fresh tumor tissue and then cultured at 1 × 10 per animal. 6 The tumor cells are injected subcutaneously into NSG mice at a dose of 100 mg / kg / day. As a blank control, animals in group A0 are injected subcutaneously with PBS. Tumors form in the animals about 2 weeks after tumor cell injection. Tumor size is measured after 25 days. 30 mice are selected.

[0241] Administration: Animals in the blank control (A0) group were injected with PBS via the tail vein. The tumorigenic groups were divided into six groups: PBS group (A1), T cell group (A2), BCAR-T group (A3), normal neoT group (A4), BCAR neoT group (A5), and high dose normal neoT cell group (A6). Animals in groups A1-A5 were injected with 1 × 10 4 Group A, 1 x 10 T cells were injected; Group B, 1 x 10 7 All groups were infused with 1 x 10 T cells. 7 The B cells are given by injection.

[0242] Tumor size and the overall condition of the mice are measured every 2-3 days for 28 days after administration. Tumor size is measured according to the following formula:

[0243] Tumor size = 1 / 2 x major axis x minor axis x minor axis

[0244] Changes in tumor burden

[0245] This experiment is expected to show smaller tumor sizes in the A4, A5 and / or A6 groups compared to the tumor sizes in the control A1, A2 and / or A3 groups.

[0246] Changes in total amount of T cells infused

[0247] Peripheral blood will be extracted from animals in groups A2-A5 10 days after dosing. The total number of CD3+ T cells will be measured using flow cytometry, which is expected to show that the number of T cells with BCAR in groups A3-A5 is greater than that in group A4. Both BCAR T cells and dual targeting T cells will be shown to expand in vivo.

[0248] Example 18: Preparation of lentivirus for three switch molecules Three switch molecules, PD1 / CD28 (hereinafter referred to as PD1sw, SEQ ID NO: 2), TIM3 / CD28 (hereinafter referred to as PD1sw, SEQ ID NO: 2), and TGFBR2 / CD28 (hereinafter referred to as TGFBR2sw, SEQ ID NO: 4), were constructed. The extracellular domains of PD1, TIM3, and TGFBR2 were used as immune inhibitory proteins of each switch molecule, and CD28 was used as a costimulatory signaling protein.

[0249] Taking PD1sw as an example, a fourth generation lentiviral vector system was used. PD1 / CD28 vector, packaging vector pMDL-gag, Rev, and envelope vector pMD2.G were co-transfected into HEK293T cells using calcium phosphate or liposome-PEI. Supernatants were collected after 48 hours and centrifuged to concentrate the lentivirus.

[0250] The titer of PD1sw lentivirus was measured in 3-fold serial dilutions. HEK293T cells were harvested after transduction with 50 μL of lentivirus for 48-72 hours and then stained for PD-1. PD-1+ (CAR+%) cells were analyzed by flow cytometry and titers were calculated based on the following: Titer (TU / mL) = initial 293T cell number × PD1+% × dilution factor × 20 (initial PD1+% < 20%).

[0251] The lentivirus titer was calculated to be 3×10 7 The larger titers were deemed suitable for further use.

[0252] TIM3sw and TGFBR2sw were prepared in a similar manner.

[0253] Example 19: Preparation of lentivirus loaded with Switch+BCAR For PD1sw-2A-CD19 CAR (hereinafter, "PD1sw-BCAR"), TIM3sw-2A-CD19 CAR (hereinafter, "TIM3sw-BCAR"), and TGFBR2sw-2A-CD19 CAR (hereinafter, "TGFBR2sw-BCAR"), lentiviruses were constructed according to the method of Example 18, respectively.

[0254] Example 20: Transduction of Switch and BCAR vectors into TILs and pTILs The lentiviruses for switch and BCAR in Example 19 and their combinations were transduced into TILs and peripheral TILs. The following cells were generated: (1) TILs 1. PD1sw-TIL (PD1sw lentivirus transduced into TIL) 2. TIM3sw-TIL (TIM3sw lentivirus transduced into TIL) 3. TGFBR2sw-TIL (TGFBR2sw lentivirus transduced into TIL) 4. BCAR-TIL (CD19 CAR lentivirus transduced into TIL) 5. PD1sw-BCAR-TIL (PD1sw-CD19 CAR lentivirus transduced into TILs to provide SuperTILs, hereafter "PD1-STILs") 6. TIM3sw-BCAR-TIL (TIM3sw-CD19 CAR lentivirus transduced into TILs to provide SuperTILs, hereafter "TIM3-STIL") 7. TGFBR2sw-BCAR-TIL (TGFBR2sw-CD19 CAR lentivirus transduced into TILs to provide SuperTILs, hereafter "TGFBR2-STIL") 8. PD1-STIL, TIM3-STIL and TGFBR2-STIL were mixed together to provide "XSTIL". (2) pTILs 1. PD1sw-pTIL (PD1sw lentivirus transduced into pTIL) 2. TIM3sw-pTIL (TIM3sw lentivirus transduced into pTIL) 3. TGFBR2sw-pTIL (TGFBR2sw lentivirus transduced into pTIL) 4. BCAR-pTIL (CD19 CAR lentivirus transduced into pTIL) 5. PD1sw-BCAR-pTIL (PD1sw-CD19 CAR lentivirus transduced into pTILs to provide Super-pTILs, hereafter "PD1-SpTILs") 6. TIM3sw-BCAR-pTIL (TIM3sw-CD19 CAR lentivirus transduced into pTIL to provide Super-pTIL, hereafter "TIM3-SpTIL") 7. TGFBR2sw-BCAR-pTIL (TGFBR2sw-CD19 CAR lentivirus transduced into pTIL to provide Super-pTIL, hereafter "TGFBR2-SpTIL") 8. PD1-SpTIL, TIM3-SpTIL and TGFBR2-SpTIL were mixed together to provide "XSpTIL".

[0255] It should be understood that Super-pTILs are also SuperTILs, specifically named to identify their distinct cellular origin.

[0256] (1) Preparation of PD1sw-TILs / pTILs

[0257] Based on the titer of PD1sw lentivirus, lentivirus was added to TILs / pTILs at MOI = 5. Flow cytometry assay was performed to sort PD1sw-TILs / pTILs with a PD1 expression rate of approximately 60%.

[0258] TIM3sw-TIL / pTIL, TGFBR2sw-TIL / pTIL, and BCAR-TIL / pTIL were prepared in a similar manner.

[0259] (2) Preparation of PD1-STILs / SpTILs

[0260] Based on the titer of PD1sw-CD19 CAR lentivirus, the lentivirus was added to TILs / pTILs at an MOI of 5. Flow cytometry assay was performed to sort PD1+ cells with an expression rate of PD1 and CD19 CAR of approximately 30%.

[0261] TIM3-STIL / SpTIL and TGFBR2-STIL / SpTIL were prepared in a similar manner to PD1-STIL / SpTIL.

[0262] (3) Preparation of XSTIL / XsTIL

[0263] PD1-STIL / SpTIL, TIM3-STIL / SpTIL, and TGFBR2-STIL / SpTIL were mixed according to a specific ratio so that each was contained at 0 to 100%. In this example, the ratio was 1:1:1.

[0264] Example 21: In vitro assay of SuperTIL effect To confirm the effect of SuperTILs, the effect is observed separately when B cells are not added and when B cells are added.

[0265] (1) Comparison of cell killing effects when B cells are not added

[0266] Tumor cells are isolated from fresh tumor tissue from patients and seeded in 24-well plates with a luciferase marker. Cells are cultured overnight to allow for attachment. Cells are split into groups of 10 and co-cultured with control or with the following T cells: Control group (group A1), normal TIL (group A2), BCAR-TIL (group A3), PD1sw-TIL (group A4), TIM3sw-TIL (group A5), TGFBR2sw-TIL (group A6), PD1-STIL (group A7), TIM3-STIL (group A8), TGFBR2-STIL (group A9), and XSTIL (group A10) without the addition of T cell-CK. The following assays are performed:

[0267] 1. Cytokine secretion by T cells. Secretion of IFN-γ and IL-2 is measured for each group using ELISA after co-culture with tumor cells for 24 hours. As shown in Figures 16A and 16B, no secretion of IFN-γ and IL-2 is observed in the control A1 group, while secretion of IFN-γ and IL-2 is observed in all other groups (groups A2-A10). Among all groups, those with Switch (groups A4-A10) show higher secretion of IFN-γ and IL-2 than those without Switch (groups A2 and A3).

[0268] 2. Proliferation of tumor cells. After 48 hours and 96 hours, the supernatant was collected from the cultures. The cultures were then washed three times with PBS. The adherent tumor cells were lysed, and luciferase activity was measured to measure the amount of protein as an index of the survival rate of tumor cells. As shown in FIG. 17A, the number of tumor cells in each group (groups A2 to A10) was reduced compared to the control group (group A1), and the groups with Switch (groups A4 to A10) showed a more significant reduction in the number of tumor cells than the groups without Switch (groups A2 and A3).

[0269] 3. Increase in T cells. After 48 hours of culture, the number of T cells is counted. As shown in FIG. 17B, the T cells in the groups without Switch (groups A2 and A3) show a slight increase, while the groups with Switch (groups A4 to A10) show a significant increase.

[0270] (2) Comparison of cell killing in the presence of B cells

[0271] Tumor cells and TILs are isolated from fresh tumor tissue from patients and seeded in 24-well plates with a luciferase marker before being cultured overnight to allow for attachment. To each well, an equal amount of B cells is added. The cultured cells are split into groups of 10 and co-cultured with control or with the following T cells: Control group (group B1), normal TIL (group B2), BCAR-TIL (group B3), PD1sw-TIL (group B4), TIM3sw-TIL (group B5), TGFBR2sw-TIL (group B6), PD1-STIL (group B7), TIM3-STIL (group B8), TGFBR2-STIL (group B9), and XSTIL (group B10) without the addition of T cell-CK. The following assays are performed:

[0272] 1. Tumor cell proliferation. After 48 and 96 hours, the supernatants were collected from the cultures, and the cultures were washed three times with PBS. Adherent tumor cells were lysed, and luciferase activity was measured as an indicator of the survival rate of tumor cells. As shown in Figure 17A, the number of tumor cells in each group (groups B2-B10) was reduced compared to the control group (group B1), and the normal TIL group (group B2) showed the least tumor cell reduction compared to the significant reduction achieved by all other groups (groups B3-B10).

[0273] 2. Increase in T cells. After 48 hours of culture, the number of T cells is counted. As shown in FIG. 17B, the normal TIL group (group B2) shows a small increase, while the groups with Switch (groups B4-B10) show a significant increase, and the groups with BCAR (groups B3 and B7-B10) show even greater increase.

[0274] As shown by in vitro assays, the tumor-killing effect of TILs is enhanced after co-transduction with the Switch molecule. When transduced with both the Switch and BCAR molecules to provide SuperTILs, the SuperTILs further exhibit enhanced proliferation and tumor cell-killing effect in the presence of B cells.

[0275] Example 22: In vitro assay of Super-pTIL effect To confirm the effect of Super-pTIL, an experiment similar to that of Super-TIL will be performed (see Example 21).

[0276] (1) Comparison of cell killing effects when B cells are not added

[0277] The groups are: control group without the addition of T cell-CK (group A1); normal pTIL (group A2); BCAR-pTIL (group A3); PD1sw-pTIL (group A4); TIM3sw-pTIL (group A5); TGFBR2sw-pTIL (group A6); PD1-SpTIL (group A7); TIM3-SpTIL (group A8); TGFBR2-SpTIL (group A9); and XSpTIL (group A10). The following assays are performed:

[0278] 1. Cytokine secretion by T cells. As shown in Figures 18A and 18B, no secretion of IFN-γ and IL-2 was observed in the control A1 group, while secretion of IFN-γ and IL-2 was observed in all other groups (groups A2 to A10). Among all groups, those with the Switch (groups A4 to A10) showed higher secretion of IFN-γ and IL-2 than those without the Switch (groups A2 and A3).

[0279] 2. Tumor cell proliferation. As shown in Figure 19A, the number of tumor cells in each group (groups A2-A10) was reduced compared to the control group (group A1), and the groups with Switch (groups A4-A10) showed a more significant reduction in the number of tumor cells.

[0280] 3. Increase in T cells. After 48 hours of culture, the number of T cells is counted. As shown in FIG. 19B, the T cells in the groups without Switch (groups A2 and A3) show a slight increase, while the groups with Switch (groups A4 to A10) show a significant increase.

[0281] (2) Comparison of cell killing in the presence of B cells

[0282] The groups are: control group without the addition of T cells-CK (group B1); normal pTIL (group B2); BCAR-pTIL (group B3); PD1sw-pTIL (group B4); TIM3sw-pTIL (group B5); TGFBR2sw-pTIL (group B6); PD1-SpTIL (group B7); TIM3-SpTIL (group B8); TGFBR2-SpTIL (group B9); and XpSTIL (group B10). The same amount of B cells is added to each well. The following assay is performed:

[0283] 1. Tumor cell proliferation. As shown in Figure 19A, the number of tumor cells in each group (Groups B2-B10) was reduced compared to the control group (Group B1), and the normal pTIL group (Group B2) showed the least tumor cell reduction compared to the significant reduction achieved by all other groups (Groups B3-B10).

[0284] 2. Increase in T cells. As shown in FIG. 19B, the normal pTIL group (group B2) shows a small increase, while the groups with Switch (groups B4-B10) show a significant increase, and the groups with BCAR (groups B3 and B7-B10) show an even greater increase.

[0285] As shown by in vitro assays, the tumor-killing effect of pTILs is enhanced after co-transduction with Switch molecules. When transduced with both Switch and BCAR molecules to provide SuperTILs, the SuperTILs further exhibit enhanced proliferation and tumor cell-killing effect in the presence of B cells.

[0286] Example 23: Animal experiments with Super-TIL Animal model: 1×10 from fresh tumor cells 6 Tumor cells are inoculated subcutaneously into NSG mice. As a blank control, animals in group A0 were injected subcutaneously with PBS. Tumors form in the animals about 2 weeks after tumor cell injection. Tumor size is measured after 25 days. 132 mice were selected and divided into group A without B cells (11 subgroups) and group B with B cells (11 subgroups) (total 22 groups) according to the following:

[0287] Group A consists of a blank control group (A0) injected with PBS via the tail vein, and 10 groups of mice seeded with tumor cells: Untreated-PBS group (group A1), normal TIL (group A2), BCAR-TIL group (group A3), PD1sw-TIL group (group A4), TIM3sw-TIL (group A5), TGFBR2sw-TIL (group A6), PD1-STIL group (group A7), TIM3-STIL (group A8), TGFBR2-STIL (group A9), and XSTIL (group A10). Groups A2-A10 receive 1 x 104 T cells via the tail vein.

[0288] Group B consisted of a blank control group (B0) injected with PBS via the tail vein, and 10 groups of mice seeded with tumor cells: Untreated-PBS group (group B1), normal TIL (group B2), BCAR-TIL group (group B3), PD1sw-TIL group (group B4), TIM3sw-TIL (group B5), TGFBR2sw-TIL (group B6), PD1-STIL group (group B7), TIM3-STIL (group B8), TGFBR2-STIL (group B9), and XSTIL (group B10). Groups B2-B10 were injected with 1 × 10 4 All groups received 1 x 10 T cells. 7 The B cells are given by injection.

[0289] Tumor size and the overall condition of the mice are measured every 2-3 days for 28 days after administration. Tumor size is measured according to the following formula: Tumor size = 1 / 2 x major axis x minor axis x minor axis

[0290] (1) Changes in tumor burden

[0291] This experiment is expected to show that mice injected with TILs carrying the Switch (groups A4-A10 and B4-B6) show delayed tumor growth.

[0292] (3) Expression of PD1, TIM3, and TFGBR2 in tumor cells

[0293] Tumor tissues from groups B7-B9 are assayed before and after the tumors begin to grow again. The results are expected to show that (i) group B7 (PD1-STIL) tumors do not express PD1, but do express TIM3 and TGFBR2; (ii) group B8 (TIM3-STIL) tumors do not express TIM3, but do express PD1 and TGFBR2; and (iii) group B9 (TGFBR2-STIL) tumors do not express TGFBR2, but do express PD1 and TIM3. In some cases, tumor microenvironment markers can escape, resulting in the ineffectiveness of the corresponding Switch.

[0294] Example 24: Animal experiments with Super-pTIL To confirm the in vivo effect of Super-pTIL, a similar animal experiment as SuperTIL was designed. The animals were also divided into groups: group A (in the absence of B cells) and group B (in the presence of B cells), including 22 groups in total.

[0295] Group A consisted of a blank control group (A0) injected with PBS via the tail vein, and 10 groups of mice seeded with tumor cells: Untreated-PBS group (group A1), normal-pTIL (group A2), BCAR-pTIL group (group A3), PD1sw-pTIL group (group A4), TIM3sw-pTIL (group A5), TGFBR2sw-pTIL (group A6), PD1-pSTIL group (group A7), TIM3-pSTIL (group A8), TGFBR2-SpTIL (group A9), and XSpTIL (group A10). Groups A2-A10 were injected with 1 × 10 4 The recipient is given 10 T cells.

[0296] Group B consisted of a blank control group (B0) injected with PBS via the tail vein, and 10 groups of mice seeded with tumor cells: Untreated-PBS group (group B1), normal TIL (group B2), BCAR-pTIL group (group B3), PD1sw-pTIL group (group B4), TIM3sw-pTIL (group B5), TGFBR2sw-pTIL (group B6), PD1-pSTIL group (group B7), TIM3-SpTIL (group B8), TGFBR2-SpTIL (group B9), and XSpTIL (group B10). Groups B2-B10 were injected with 1 × 10 PBS via the tail vein. 4 All groups received 1 x 10 T cells. 7 The B cells are given by injection.

[0297] Tumor size and the overall condition of the mice are measured every 2-3 days for 28 days after administration. Tumor size is measured according to the following formula: Tumor size = 1 / 2 x major axis x minor axis x minor axis

[0298] The experiments are expected to show that the SuperTIL / pTIL of the present disclosure has specific tumor recognition and killing effect through multiple targets (from TIL or pTIL). SuperTIL / pTIL may also show the ability to overcome the tumor environment (from one or more switches) to improve killing and the self-expansion ability of BCAR. These engineered immune cells provide an effective tumor treatment tool to address various problems involved in tumor immune cell therapy.

[0299] Example 25: Killing effect of BCAR-TCR T on NY ESO1 tumor cells in vitro To confirm the function of BCAR in TCR T cells, the J82-NY-ESO1 tumor cell line, HLA genotyped as A:0201, was used as a target to measure the killing effect of BCAR-TCR T cells.

[0300] 1×10 5J82-NY-ESO1 tumor cells were seeded on RTCA (Real Time Cell Analysis) electrode plates and cultured overnight to allow attachment. The cells were divided into three groups, A, B and C. In group A, 1 × 10 5 pieces, 1×10 4 pieces, 1×10 3 pieces, 1×10 2 BCAR-TCR T cells were co-cultured with J82-NY-ESO1 cells, respectively. In group B, 1 × 10 5 pieces, 1×10 4 pieces, 1×10 3 pieces, 1×10 2 1 × 10 BCAR-TCR T 5 Group B cells were co-cultured with J82-NY-ESO1 cells. Group C was the blank control. The RTCA system was used to record the "Cell Index" every 10 minutes for 24 hours.

[0301] As shown in Figure 20, in group A, where B cells were not present, the highest dose of 1 × 10 5 Only 1 × 10 BCAR-TCR T cells showed a significant killing effect against J82-NY-ESO1 tumor cells, whereas in group B, 1 × 10 2 Even at the lowest dose of BCAR-TCR T cells, 1 × 10 5 The results show a remarkable killing effect against J82-NY-ESO1 tumor cells comparable to a single BCAR-TCR T dose, representing an approximately 1000-fold increase in efficacy.

[0302] Example 26: Clinical antitumor effects of STIL and SpTIL Five subjects (as shown in Table 1) enrolled in the clinical trial and were infused with STILs or SpTILs.

[0303] [Table 1]

[0304] As shown in Table 1, the enrolled subjects suffered from various solid tumors, all of which were late-stage, resistant, and highly progressive with 3 or more distant metastatic lesions. Three of the five subjects (60%) developed TP53 mutations that were resistant to targeted therapy and had poor prognosis. TMB assessment and HLA polymorphism indicated that all of these subjects were unlikely to benefit from PD1 / PDL1 monoclonal antibody therapy or conventional neoantigen therapy.

[0305] Cell preparation prior to treatment (1) Isolation of TILs / pTILs For subjects 3, 4, and 5, CD3-positive TILs were isolated from freshly excised tumor tissues using CD3 magnetic beads after enzymatic digestion. For subjects 1 and 2, PBMCs were isolated from the patients, in which PD1 + The amount of T cells was 19% and 5% of the total number of T cells, respectively. + The proportion of T cells was considered to be TILs (pTILs) in peripheral blood derived from tumor tissue, and PD1 + T cells, i.e., pTILs, were further enriched using PR1 beads.

[0306] Preparation of STILs / SpTILs The lentiviral vector loaded with PD1sw-CD19 CAR was transduced into TIL / pTIL with a transduction efficiency of 2-15%. The cells were sealed in an infusion bag without expansion. The whole process took 3-10 days (except for the identification process of T cell-recognized neoantigens).

[0307] 10 8 ~10 9 This is a much lower dose than the reported dose of 10 cells / kg. 5 ~10 6 Treatment doses in units of cells / kg are shown in Table 2.

[0308] [Table 2]

[0309] Safety assessment Of the five subjects, three had grade 1 cytokine release syndrome (CRS) evidenced by high fever (incidence 60%, 3 / 5). All three recovered, one without intervention, and the other two with tocilizumab treatment. Both the incidence and grade of CRS were much lower than those with CD19 CAR-T treatment. No autoimmune disease was observed after treatment.

[0310] Evaluation of effectiveness The efficacy of the cell injection treatment was confirmed by tumor imaging (FIGS. 21-24 for subjects 1-4, respectively), and the results are shown in Table 3 below.

[0311] [Table 3]

[0312] Effect of CAR on in vivo expansion of STIL / SpTIL The percentage of CAR+ T cells in the peripheral blood of the subjects was monitored. The fold increase in STIL (or SpTIL) in the peripheral blood was calculated according to the following formula: Increase rate = lymphocyte count / L × volume of circulating peripheral blood × percentage of T cells among lymphocytes × percentage of STIL among T cells Here, lymphocyte counts / L were obtained from routine blood tests, T cell percentage among lymphocytes was determined by flow cytometry as percentage of CD3+ cells, and STIL percentage among T cells was determined by flow cytometry as percentage of CAR+ cells among CD3+ cells.

[0313] On day 14, the increase in STIL / SpTIL was calculated and the B cell depletion was measured, and the results are shown in Table 4.

[0314] [Table 4]

[0315] No exogenous immunoglobulin was administered during the observation period, and no immunodeficiency was noted in any of the subjects.

[0316] Dual specific recognition of STILs and SpTILs Subjects 1, 2, and 4 were monitored for circulating tumor cells (CTCs) in the peripheral blood. The number of CTCs two months after cell infusion was compared to the baseline on the day of infusion. The results are shown in Table 5 below and in Figure 25, and show a significant decrease in the number of CTCs.

[0317] [Table 5]

[0318] Improving killing effect by using switch molecules It was observed that three subjects (numbers 1, 2, and 5) showed decreased peripheral blood T cells and persistent pleural or ascites effusion between days 14 and 28. Subject number 4 with pleural metastasis developed pleural effusion, and subjects numbers 1 and 2 with peritoneal metastasis developed ascites. In both pleural and ascites fluids, T cells were found with IL6 at higher concentrations than in peripheral blood. This observation, summarized in Table 6, indicates that the killing effect of STIL / SpTIL was enhanced by the switch molecule.

[0319] [Table 6]

[0320] [Table 7]

[0321] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be applied in practicing the invention. It is intended that the following claims define the scope of the invention, and that methods and structures within the scope of these claims and their subdivisions are thereby protected.

Claims

1. 1. A modified immune cell that specifically binds to a neoantigen, the modified immune cell comprising: (a) a switch molecule comprising an extracellular domain (ECD) of a protein that upon binding to its ligand induces an immune cell inactivation signal in an unmodified immune cell, the ECD being fused to an intracellular domain (ICD) of a costimulatory molecule that mediates an immune cell activation signal, and binding of the switch molecule to a ligand results in an immune cell activation signal instead of an immune cell inactivation signal in said modified immune cell; and (b) a chimeric antigen receptor comprising: (i) an antigen interacting domain capable of binding to a B cell surface protein; (ii) a transmembrane domain; and (iii) an intracellular signaling domain. A modified immune cell comprising:

2. The modified immune cell of claim 1 , wherein the immune cell comprises a T cell receptor (TCR) complex that exhibits specific binding to a neoantigen.

3. The modified immune cell of claim 2 , wherein the TCR complex is an endogenous TCR complex or an exogenous TCR complex.

4. The neoantigens comprise peptide fragments of proteins encoded by mutated genes, the genes being ABL1, ACO1 1997, ACVR2A, AFP, AKT1, ALK, ALPPL2, ANAPC1, APC, ARID1A, AR, AR-v7, ASCL2, β2M, BRAF, BTK, C15ORF40, CDH1, CLDN6, CNOT1, CT45A5, CTAG1B, DCT, DKK4, EEF1B2, EEF1DP3, EGFR, EIF2B3, env, EPHB2, ERBB3, ESR1, ESRP1, FAM11 IB, FGFR3, FRG1B, GAGE1, GAGE2, GAGE3, GAGE4, ​​GAGE5, GAGE6, GAGE7, GAGE8, GAGE9, GAGE10, GAGE111, GAGE12, GAGE13, GAGE14, GAGE15, GAGE16, GAGE17, GAGE18, GAGE19, GAGE111, GAGE112, GAGE16, GAGE19, GAGE18, GAGE19, GAGE111, GAGE112, GAGE15, GAGE16, GAGE17, GAGE18, GAGE19, GAGE19, GAGE20, GAGE19, GAGE111, GAGE112, GAGE113, GAGE14, GAGE15, GAGE15, GAGE16, GAGE17, GAGE18, GAGE19, GAGE19, GAGE21, GAGE19, GAGE111, GAGE112, GAGE113, GAGE15, GAGE16, GAGE17, GAGE18, GAGE19, GAGE21, GAGE19, GAGE19, GAGE111, GAGE111, GAGE1 10, GATA3, GBP3, HER2, IDH1, JAK1, KIT, KRAS, LMAN1, MABEB 16, MAGEA1, MAGEA10, MAGEA4, MAGEA8, MAGEB 17, MAGEB4, MAGEC1, MEK, MLANA, MLL2, MMP13, MSH3, MSH6, MYC, NDUFC2, NRAS, PAGE2, PAGE5, PDGF Ra, PIK3CA, PMEL, pol protein, POLE, PTEN, RAC1, RBM27, RNF43, RPL22, RUNX1, SEC31A, SEC63, SF3B 1. The modified immune cell of claim 1, wherein the target polypeptide is selected from the group consisting of SLC35F5, SLC45A2, SMAP1, SMAP1, SPOP, TFAM, TGFBR2, THAP5, TP53, TTK, TYR, UBR5, VHL, and XPOT.

5. The modified immune cell of claim 1, wherein the neoantigen is selected based on a somatic mutation profile of a tumor sample from an individual.

6. The modified immune cell of claim 1, wherein the protein that induces an immune cell inactivation signal in a non-modified immune cell upon binding to its ligand is a signaling receptor, a checkpoint receptor, a cytokine receptor, a chemokine receptor, a growth factor receptor, or a hormone receptor.

7. 2. The modified immune cell of claim 1, wherein the protein that upon binding to its ligand induces an immune cell inactivation signal in a non-modified immune cell is selected from transforming growth factor-β receptor (TGF-β-R), programmed cell death 1 (PD-1), co-stimulatory receptor 4 of T cells (CTLA-4), B- and T-lymphocyte attenuator (BTLA), killer cell immunoglobulin-like receptor (KIR), indoleamine-2,3-dioxygenase (IDO), lymphocyte activation gene-3 (LAG3), T cell immunoglobulin mucin-3 (TIM-3), and TIGIT.

8. 2. The modified immune cell of claim 1, wherein the costimulatory molecule is interleukin-2 receptor (IL-2R), interleukin-12 receptor (IL-12R), CD2, CD3, CD4, CD7, CD8, CD27, CD28, CD30, CD40, 4-1BB / CD137, ICOS, lymphocyte function associated antigen-1 (LFA-1), LIGHT, NKG2C, or OX40.

9. The modified immune cell of claim 1 , wherein the immune cell activation signal is mediated by an activator.

10. The modified immune cell of claim 9 , wherein the activator is a soluble cytokine, a soluble chemokine, or a growth factor.

11. 11. The modified immune cell of claim 10, wherein the activating factor is a soluble cytokine, and the soluble cytokine is IL-1, IL-2, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IL-21, TNF, TGF, IFN, or a functional fragment or variant thereof.

12. 2. The modified immune cell of claim 1, wherein the immune cell activation signal comprises: clonal expansion of the modified immune cell; cytokine release by the modified immune cell; cytotoxicity of the modified immune cell; proliferation of the modified immune cell; differentiation, de-differentiation, transdifferentiation of the modified immune cell; migration and / or trafficking of the modified immune cell; exhaustion and / or reactivation of the modified immune cell; and release of other intercellular molecules, metabolites, compounds, or combinations thereof by the modified immune cell.

13. The modified immune cell of claim 1 , wherein the B cell surface protein is selected from CD19, CD20 and CD22.

14. The modified immune cell of claim 1 , wherein the intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM) or an immunoreceptor tyrosine-based inhibitory motif (ITIM).

15. The intracellular signaling domain is selected from the group consisting of Fcγ receptor (FcγR), Fcε receptor (FcεR), FCa receptor (FcαR), neonatal Fc receptor (FcRn), CD3, CD3ζ, CD3γ, CD3δ, CD3ε, CD4, CD5, CD8, CD21, CD22, CD28, CD32, CD40L (CD154), CD45, CD66d, CD79a, CD79b, CD80, CD86, CD278 (also called ICOS), CD247ζ, CD247 2. The modified immune cell of claim 1, comprising an intracellular domain of a molecule selected from η, DAP10, DAP12, FYN, LAT, Lck, MAPK, MHC complex, NFAT, NF-κB, PLC-γ, iC3b, C3dg, C3d, and Zap70.

16. The modified immune cell of claim 1 , wherein the intracellular signaling domain comprises the intracellular domain of CD3 ζ.

17. The modified immune cell of claim 16 , wherein the intracellular domain of CD3 ζ comprises an immunoreceptor tyrosine-based activation motif (ITAM).

18. The modified immune cell of claim 1 , wherein the CAR further comprises a costimulatory domain.

19. The modified immune cell of claim 18, wherein the costimulatory domain comprises a signaling domain of an MHC class I molecule, a TNF receptor protein, an immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocyte activation molecule (SLAM protein), an activating NK cell receptor, or a Toll ligand receptor.

20. The co-stimulatory domain is 2B4 / CD244 / SLAMF4, 4-1BB / TNF-SF9 / CD137, B7-1 / CD80, B7-2 / CD86, B7-H1 / PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BAFF R / TNFRSF13C, BAFF / BlyS / TNF-SF13B, BLAME / SLAMF8, BTLA / CD272, CD100 (SEMA4D), CD103, CD11a, CD11b, CD11c, CD11d, CD150, CD160 (BY55), CD18, CD19, CD2, CD200, CD229 / SLAMF3, CD27 ligand / TNF-SF7, CD27 / TNFRSF7, CD28, CD29, CD2F-10 / SLAMF9, CD30 ligand / TNF-SF8, CD30 / TNFRSF8, CD300a / LMIR1, CD4, CD40 ligand / TNF-SF5, CD40 / TNFRSF5, CD48 / SLAMF2, CD49a, CD49D, CD49f, CD53, CD58 / LFA-3, CD69, CD7, CD8 α, CD8 β, CD82 / Kai-1, CD84 / SLAMF5, CD90 / Thy1, CD96, CDS, CEACAM1, CRACC / SLAMF7, CRTAM, CTLA-4, DAP12, Dectin-1 / CLEC7A, DNAM1 (CD226), DPPIV / CD26, DR3 / TNFRSF25, EphB6, GADS, Gi24 / VISTA / B7-H5, GITR ligand / TNF-SF18, GITR / TNFRSF18, HLA class I, HLA-DR, HVEM / TNFRSF14, IA4, ICAM-1, ICOS / CD278, Ikaros, IL2R β, IL2R γ, IL7R α, integrin α4 / CD49d, integrin α4β1, integrin α4β7 / LPAM-1, IPO-3, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIRDS2, LAG-3, LAT, LIGHT / TNF-SF14, LTBR, Ly108, Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1), lymphotoxin-α / TNF-β, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80(KLRF1), NTB-A / SLAMF6, OX40 ligand / TNFSF4, OX40 / TNFRSF4, PAG / Cbp, PD-1, PDCD6, PD-L2 / B7-DC, PSGL1, RELT / TNFRSF19L, SELPLG (CD162), SLAM (SLAMF1), SLAM / CD150, SLAMF4 (CD244), SLAMF6 (NTB-A), SLAMF7, SLP-76, TACI / TNFRSF13B, TCL1A, TCL1B, TIM-1 / KIM-1 / HAVCR, TIM-4, TL1A / TNFSF15, TNF 20. The modified immune cell of claim 18, comprising a signaling domain of a molecule selected from the group consisting of RII / TNFRSF1B, TNF-α, TRANCE / RANKL, TSLP, TSLP R, VLA1, and VLA-6.

21. 2. The modified immune cell of claim 1, wherein upon binding of the switch molecule to the ligand, the modified immune cell exhibits enhanced neoantigen binding compared to a non-modified immune cell.

22. The modified immune cell of claim 1, wherein the modified T cell exhibits increased cytotoxicity against a target cell compared to a non-modified T cell when the switch molecule binds to the ligand and the modified immune cell binds to the neoantigen present on the target cell.

23. The modified immune cell of claim 1, wherein the modified immune cell exhibits increased cytokine secretion compared to a non-modified immune cell when the switch molecule binds to the ligand and the modified immune cell binds to the neoantigen present on the target cell.

24. The modified immune cell of claim 23, wherein the cytokine is IFN-γ or IL-2.

25. The modified immune cell of claim 1 , wherein the modified immune cell is a T cell or a tumor infiltrating lymphocyte (TIL).

26. (a) administering the modified immune cells of claim 1; (b) contacting the modified immune cell with a neoantigen-expressing cancer target cell under conditions that induce cytotoxicity of the modified immune cell against the cancer target cell, thereby inducing death of the cancer target cell. The modified immune cell of claim 1 for treating cancer in a subject.

27. (a) providing a T cell population comprising at least the modified immune cell of claim 1; (b) exposing said population of T cells to said B cell surface protein in a manner that effects an expansion of said population of T cells; A method for expanding a T cell population comprising:

28. The method of claim 27, wherein in (b), the population of T cells is exposed to B cells that contain the B cell surface protein.

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