Modified immune cells and their use

Abstract

Provided are modified immune cells, including tumor-infiltrating lymphocytes (TILs) or B cells, compositions comprising the immune cells, and methods of treating neoplastic or cancerous conditions comprising administering the immune cells to a subject. [Solution] Provided are modified T cells that specifically bind to neoantigens, wherein the modified T cells comprise a switch molecule, wherein the switch molecule comprises the extracellular domain (ECD) of a protein that induces an immune cell activation signal in an unmodified T cell upon binding to its ligand, wherein the ECD is fused to the intracellular domain (ICD) of a costimulatory molecule that mediates the 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 modified T cell, and the T cells are cells that express endogenous PD-1 obtained from peripheral blood mononuclear cells.

Description

Technical Field

[0001] [Cross - reference to Related Applications] This application claims the benefit and priority of Chinese Patent Application No. 201711101450.X, filed on November 10, 2017, Chinese Patent Application No. 201810017770.5, filed on January 9, 2018, Chinese Patent Application No. 201810037682.1, filed on January 16, 2018, PCT International Application PCT / CN2018 / 090638, filed on June 11, 2018, PCT International Application PCT / CN2018 / 094126, filed on July 2, 2018, and PCT International Application PCT / CN2018 / 114897, filed on November 9, 2018, the entire contents of which are incorporated herein by reference.

Background Art

[0002] Immunotherapy involves modifying a patient's own immune cells to redirect cytotoxicity towards target cells, such as cancer cells. Modified immune cells, such as T cells, expressing a chimeric antigen receptor (CAR) can utilize endogenous immune cell signaling for immune cytotoxicity.

[0003] Conventional immunotherapies suffer from various deficiencies. Such deficiencies include insufficient signaling from co - stimulatory receptors for a sustained and / or appropriate immune response for therapeutic effect, insufficient specificity of modified immune cells for diseased cells such as cancer cells (e.g., on - target off - tumor effects and toxicity, etc.), and activation of immunosuppressive mechanisms, all of which can minimize the effect 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 an immune cell activation signal by signaling through binding to a ligand that would normally induce an immune cell inactivation signal. The compositions and methods can also induce an immune cell activation signal through binding to a B cell surface protein.

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

[0006] In some embodiments, the modified immune cell is a tumor infiltrating lymphocyte (TIL), where optionally the TIL may express at least one of PD-1, CD137, and TIM-3.

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

[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, the neoantigen may comprise a peptide fragment of a protein encoded by a mutated gene, where the gene is selected from 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 selected from 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,Selected from 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, FLNB, PRPF8, RNF43, MSH6, FGFR2, SMAD4, JAK3, USP8, DLC1, ESRP1, LRP1B, MYH11, BRCA1, CARD11, HSP90AB1, MAP3K9, ADAMTSL3, PDGFRB, RPTOR, ROS1, NFKBIE, AMER1, KLF4, RAC1, TERT, MYOD1, ATP1A1, CSF3R, NOTCH2, CCR4, PAX5, SPTAN1, MLH1, CUBN, RNF213, SMO, ABCC4, AXIN2, CSF1R, PER1, PKHD1, IL7R, RB1, ARID1A, ATM, FES, MTHFR, PTCH2, 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, MUC1, NFKBIZ, NRP2, CTCF, HIST1H3B, KEAP1, SLC22A2, ABCC2, EED, GATA1, 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, the neoantigen can be selected based on the gene profile of a tumor sample from an individual. In some embodiments, the neoantigen 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. 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 checkpoint receptor, cytokine receptor, chemokine receptor, growth factor receptor, or hormone receptor. 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 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 co-stimulatory 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 activation signal can 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 here, 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 the clonal expansion of modified TILs or modified T cells, cytokine release by modified TILs or modified T cells, cytotoxicity of modified TILs or modified T cells, proliferation of modified TILs or modified T cells, differentiation, dedifferentiation, transdifferentiation of modified TILs or modified T cells, migration and / or transport of modified TILs or modified T cells, exhaustion and / or reactivation of modified TILs or modified T cells, and release of other intercellular molecules, metabolites, compounds, or combinations thereof by modified TILs or modified T cells.

[0014] In some embodiments, upon binding of the switch molecule to the ligand, the modified TILs or modified T cells can exhibit enhanced neoantigen binding as compared to unmodified TILs or unmodified T cells.

[0015] In some embodiments, the modified TILs or modified T cells can exhibit increased cytotoxicity against target cells as compared to unmodified TILs or unmodified T cells when the switch molecule binds to the ligand and the modified TILs or modified T cells bind to the neoantigens present on the target cells.

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

[0017] In one aspect, the present disclosure provides a modified immune cell comprising a chimeric antigen receptor (CAR) and a T cell receptor (TCR) complex that exhibit specific binding to a neoantigen, wherein the CAR comprises (a) an antigen interaction 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 cell can be a tumor infiltrating lymphocyte (TIL). In some embodiments, the TIL can be a triple positive T cell expressing PD-1, CD137, and TIM-3. In some embodiments, the TCR complex that exhibits specific binding to the neoantigen can be an endogenous TCR complex. In some embodiments, the TCR complex that exhibits specific binding to the neoantigen can be an exogenous TCR complex.

[0019] In some embodiments, the neoantigen comprises a peptide fragment of a protein encoded by a mutated gene, where the gene is selected from 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, the neoantigen can be selected based on the gene profile of a tumor sample from an individual. In some embodiments, the neoantigen 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 include an immunoreceptor activation tyrosine motif (ITAM). In some embodiments, the intracellular signaling domain may include an immunoreceptor inhibitory tyrosine motif. In some embodiments, the intracellular signaling domain may include the intracellular domain of a molecule selected from the group consisting of 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 (also known as CD154), CD45, CD66d, CD79a, CD79b, CD80, CD86, CD278 (also referred to as ICOS), CD247 ζ, CD247 η, 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 include the intracellular domain of CD3 ζ. In some embodiments, the CAR may further include a co-stimulatory domain. In some embodiments, the co-stimulatory domain may include signaling 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 activated NK cell receptor, or a toll ligand receptor.

[0024] In some embodiments, the co-stimulatory domain is 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, NKp80It may contain a signaling domain of a molecule selected from the group consisting of (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 contact of immune cells with a B cell surface protein, the immune cells may exhibit enhanced proliferation compared to unmodified immune cells. 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 cells may exhibit at least a two-fold increase in proliferation at least about 24, 48, or 96 hours after contact compared to unmodified immune cells.

[0026] In one aspect, the present disclosure provides modified tumor-infiltrating lymphocytes (TILs) that specifically bind to neoantigens, where the modified TILs comprise: (a) a switch molecule comprising an extracellular domain (ECD) of a protein that, upon binding to its ligand, induces an immunosuppressive signal in unmodified TIL cells, wherein the ECD is fused to an intracellular domain (ICD) of a costimulatory molecule that mediates an immune cell activation signal, and wherein binding of the switch molecule to the ligand results in an immune cell activation signal in the modified TILs instead of an immunosuppressive signal; and (b) a chimeric antigen receptor comprising: (i) an antigen interaction 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 present disclosure provides modified immune cells that specifically bind to neoantigens, where the modified immune cells comprise: (a) a switch molecule comprising an extracellular domain (ECD) of a protein that, upon binding to its ligand, induces an immunosuppressive signal in unmodified immune cells, wherein the ECD is fused to an intracellular domain (ICD) of a costimulatory molecule that mediates an immune cell activation signal, and wherein binding of the switch molecule to the ligand results in an immune cell activation signal in the modified immune cells instead of an immunosuppressive signal; and (b) a chimeric antigen receptor comprising: (i) an antigen interaction 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 cells can comprise a T cell receptor (TCR) complex that exhibits specific binding to the neoantigen. In some embodiments, the TCR complex can be an endogenous TCR complex. In some embodiments, the TCR complex can be an exogenous TCR complex.

[0029] In some embodiments, the neoantigen comprises a peptide fragment of a protein encoded by a mutant gene, where the gene is selected from 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, the neoantigen can be selected based on the gene profile of a tumor sample from an individual. In some embodiments, the neoantigen 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. 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 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 TILs or unmodified immune cells upon binding to its ligand may 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 co-stimulatory 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] Immune cell activation signals can be mediated by activators. 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 here, 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, the immune cell activation signal can include clonal expansion of modified TILs or modified immune cells, cytokine release by modified TILs or modified immune cells, cytotoxicity of modified TILs or modified immune cells, proliferation of modified TILs or modified immune cells, differentiation, dedifferentiation, transdifferentiation of modified TILs or modified immune cells, migration and / or transport of modified TILs or modified immune cells, exhaustion and / or reactivation of modified TILs or modified immune cells, and release of other intercellular molecules, metabolites, compounds, or combinations thereof by modified TILs or modified immune cells.

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

[0036] In some embodiments, the intracellular signaling domain may include an immunoreceptor activation tyrosine motif (ITAM). In some embodiments, the intracellular signaling domain may include an immunoreceptor inhibitory tyrosine motif (ITIM). In some embodiments, the intracellular signaling domain may include the intracellular domain of a molecule selected from 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 (also known as CD154), CD45, CD66d, CD79a, CD79b, CD80, CD86, CD278 (also referred to as ICOS), CD247 ζ, CD247 η, 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 include the intracellular domain of CD3 ζ. In some embodiments, the intracellular domain of CD3 ζ may include an immunoreceptor activation tyrosine motif (ITAM). The CAR may further include a co-stimulatory domain. In some embodiments, the co-stimulatory domain may include signaling 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 activated NK cell receptor, or a toll ligand receptor.

[0038] In some embodiments, the co-stimulatory domain is 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, NKp80It may include a signaling domain of a molecule selected from the group consisting of (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 the ligand, the modified TIL or modified immune cell may exhibit enhanced neoantigen binding as compared to an unmodified TIL or unmodified immune cell.

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

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

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

[0043] In one aspect, the present 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 according to any one of claims 21 to 40; (b) exposing the T cell population to a B cell surface protein so as to effect an increase in the T cell population. In some embodiments, the T cell population may be exposed to B cells comprising the B cell surface protein.

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

[0045] In one aspect, the present disclosure provides a composition comprising: (a) a switch molecule comprising an extracellular domain (ECD) of a protein that induces an immunosuppressive signal in an unmodified immune cell upon binding to its ligand, wherein the ECD is fused to an intracellular domain (ICD) of a co-stimulatory molecule that mediates an immune cell activation signal; and (b) one or more polynucleotides encoding 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 interaction domain capable of binding to a B cell surface protein; (ii) a transmembrane domain; and (iii) an intracellular signaling domain.

[0047] In some embodiments, the neoantigen may comprise a peptide fragment of a protein encoded by a mutant gene, where the gene is selected from 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 an immune cell inactivation signal in unmodified TILs or unmodified T cells 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, the co-stimulatory 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 can be selected from CD19, CD20, and CD22. In some embodiments, the intracellular signaling domain may include an immunoreceptor inhibitory tyrosine motif. In some embodiments, the intracellular signaling domain is the intracellular domain of a molecule selected from 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 (also known as CD154), CD45, CD66d, CD79a, CD79b, CD80, CD86, CD278 (also referred to as ICOS), CD247 ζ, CD247 η, 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 co-stimulatory domain. In some embodiments, the co-stimulatory domain is 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, Ly9It may contain a signaling domain of a molecule selected from the group consisting of (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 present disclosure provides a composition comprising one or more polynucleotides encoding one or more chimeric antigen receptors comprising: (a) a switch molecule comprising an extracellular domain (ECD) of a protein that induces an immunosuppressive signal in an unmodified immune cell upon binding to its ligand, 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 of its components; and (c) (i) an antigen interaction 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 present disclosure provides a modified tumor infiltrating lymphocyte (TIL) that specifically binds to a neoantigen, the modified TIL comprising a chimeric stimulatory molecule, wherein the chimeric stimulatory molecule comprises a polypeptide extracellular domain (PED) that binds to the neoantigen, wherein the PED is fused to an intracellular domain (ICD) of a costimulatory molecule that mediates an immune cell activation signal, and binding of the chimeric stimulatory molecule to the neoantigen results in an immune cell activation signal in the modified TIL.

[0054] In one aspect, the present disclosure provides a modified immune cell comprising: (a) a switch molecule comprising an extracellular domain (ECD) of a protein that induces an immune inactivation signal in an unmodified immune cell upon binding to its ligand, wherein the ECD is fused to an intracellular domain (ICD) of a costimulatory molecule that mediates an immune cell activation signal, and binding of the switch molecule to its ligand results in an immune cell activation signal in the modified immune cell instead of an immune cell inactivation signal; and (b) a chimeric antigen receptor (CAR) comprising: (i) an antigen interaction 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 can be obtained from a tumor. In some embodiments, the immune cells may be obtained from the peripheral blood of mononuclear cells. In some embodiments, the immune cells may contain an exogenous TCR complex. In some embodiments, the TCR complex can bind to tumor cells. In some embodiments, the TCR complex may bind to neoantigens. In some embodiments, the protein that induces an immune cell inactivation signal in unmodified immune cells upon binding to its ligand may be a signaling receptor. In some embodiments, the protein that induces an immune cell inactivation signal in unmodified immune cells upon binding to its ligand may be a checkpoint receptor, cytokine receptor, chemokine receptor, growth factor receptor, or 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 co-stimulatory 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 activation signal may be mediated by an activator. In some embodiments, the activator may be a soluble cytokine, soluble chemokine, or growth factor. In some embodiments,The activating factor is a soluble cytokine, and here, 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 modified immune cells, cytokine release by modified immune cells, cytotoxicity of modified immune cells, proliferation of modified immune cells, differentiation, dedifferentiation, transdifferentiation of modified immune cells, migration and / or transport of modified immune cells, exhaustion and / or reactivation of modified immune cells, and release of other intercellular molecules, metabolites, compounds, or combinations thereof by modified immune cells. In some embodiments, the B cell surface protein can be selected from CD19, CD20, and CD22. In some embodiments, the intracellular signaling domain may include an immunoreceptor activation tyrosine motif (ITAM). In some embodiments, the intracellular signaling domain may include an immunoreceptor inhibitory tyrosine motif (ITIM). In some embodiments, the intracellular signaling domain may include the intracellular domain of a molecule selected from 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 (also known as CD154), CD45, CD66d, CD79a, CD79b, CD80, CD86, CD278 (also referred to as ICOS), CD247 ζ, CD247 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 include the intracellular domain of CD3 ζ. In some embodiments, the intracellular domain of CD3 ζ may include an immunoreceptor activation tyrosine motif (ITAM). The CAR furtherIt may contain a co-stimulatory domain. In some embodiments, the co-stimulatory domain may include signaling of MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activated NK cell receptors, or Toll-like receptor receptors. In some embodiments, the co-stimulatory domain is 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,It may include the signaling domain of a molecule selected from the group consisting of 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 contact of 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 herein are hereby 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 present invention are specifically set forth 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 which describes exemplary embodiments in which the principles of the invention are utilized, and to the following appended drawings.

Brief Description of the Drawings

[0058]

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Modes for Carrying Out the Invention

[0059] The practice of the methods disclosed herein, unless otherwise indicated, uses techniques within the skill of the art, including immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA. See, for example, Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012), the series Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds.), the series Methods In Enzymology (Academic Press, Inc.), PCR 2, A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. 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 (R.I. Freshney, ed. (2010)), and the like.

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

[0061] The terms "about" or "approximately" mean 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., on the limitations of the measuring system. For example, "about" can mean that the standard deviation for one run in the art is 1 or greater. Alternatively, "about" can mean within a range of up to 20%, 10%, 5%, or 1% of a value. Alternatively, especially with respect to biological systems or processes, the term can mean within one order of magnitude, preferably within five times the value, and more preferably within two times the value. When specific values are recited in the specification and claims, the term "about" should be presumed to mean within an acceptable error range of the specific value, unless otherwise stated.

[0062] As used herein, the term "cell" generally refers to a biological cell. A cell can be a basic structural, functional, and / or biological unit of a living organism. A cell can be derived from any organism having one or more cells. Some relatively non-limiting examples include prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, cells of unicellular eukaryotes, protozoan cells, cells derived from plants (e.g., cells from crops, fruits, vegetables, grains, soybeans, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkins, hay, potatoes, cotton, flax, tobacco, flowering plants, conifers, gymnosperms, ferns, clubmosses, horsetails, mosses, liverworts, etc.), algal cells (e.g., Botryococcus braunii, green algae, Nannochloropsis gaditana, Chlorella pyrenoidosa, Ulva lactuca, etc.), seaweeds (e.g., kelp, etc.), fungal cells (e.g., yeast cells, cells from mushrooms, etc.), animal cells, cells derived from invertebrates (e.g., flies, cnidarians, echinoderms, nematodes, etc.), cells derived from vertebrates (e.g., fish, amphibians, reptiles, birds, mammals), cells derived from mammals (e.g., pigs, cows, goats, sheep, rodents, rats, mice, non-human primates, humans, etc.). Often, a cell is not derived from a natural organism (e.g., a cell may be synthetically produced, which is sometimes referred to as an artificial cell).

[0063] As used herein, the term "antigen" means a molecule or a 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 (e.g., an antibody). An antigen can also mean a molecule or a fragment thereof that can be used in a substance to produce an antibody that can bind to that antigen.

[0064] As used herein, the term "neoantigen" generally refers to a tumor-specific antigen resulting from a genetic mutation. The resulting mutant proteins, or fragments thereof, can trigger an anti-tumor T cell response.

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

[0066] As used herein, the term "antibody" means a proteinaceous binding molecule having immunoglobulin-like function. The term antibody includes multiple antibodies (e.g., monoclonal antibodies and polyclonal antibodies, etc.), and their derivatives, variants and fragments. Antibodies are immunoglobulins (IG's) of various classes (i.e., IgA, IgG, IgM, IgD and IgE) and subclasses (e.g., IgG1, IgG2, etc.), although not limited thereto. Their derivatives, variants and fragments may mean 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 fragments (Fv), single-chain variable fragments (scFv), minibodies, bispecific antibodies, and single-domain antibodies ("sdAb" or "nanobody" or "camelids"). The term antibody includes antibodies and antigen-binding fragments of optimized, engineered or chemically conjugated antibodies. Examples of optimized antibodies include affinity matured antibodies. Examples of engineered antibodies include Fc-optimized antibodies (e.g., antibodies optimized in the crystallizable fragment region) and multispecific antibodies (e.g., bispecific antibodies, etc.).

[0067] As used herein, the term "nucleotide" generally refers to a base-sugar-phosphate combination. Nucleotides include synthetic nucleotides and 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), cytidine triphosphate (CTP), guanosine triphosphate (GTP), and deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, and their derivatives. Such derivatives include, for example, "αS" dATP, 7-deaza-dGTP and 7-deaza-dATP, nucleotide derivatives that confer nuclease resistance to nucleic acid molecules containing them. The term nucleotide as used herein can mean dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Exemplary examples of dideoxyribonucleoside triphosphates include, but are not limited to, for example, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. Nucleotides may or may not be labeled, or may be 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 any length of polymeric form of nucleotides, either deoxyribonucleotides or ribonucleotides, or analogs thereof, in either single-stranded, double-stranded, or multi-stranded form. The polynucleotide can be endogenous or exogenous to the cell. The polynucleotide may be present in a cell-free environment. The polynucleotide may be a gene or a fragment thereof. The polynucleotide may be DNA. The polynucleotide may be RNA. The polynucleotide can have any three-dimensional structure and can perform any function, known or unknown. The polynucleotide may contain one or more analogs (e.g., modified backbone, sugar, or base, etc.). When present, the modification to the nucleotide structure can be given before or after polymerization of the polymer. Some non-limiting examples of analogs include 5-bromouracil, peptide nucleic acid, xeno nucleic acid, morpholino, bridged nucleic acid, glycol nucleic acid, threose nucleic acid, dideoxynucleotide, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein-conjugated sugars, etc.), thiol-containing nucleotides, biotin-conjugated nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queuosine, and wyosine, etc.Examples of non-limiting polynucleotides include coding or non-coding regions of genes or gene fragments, loci 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-chain polynucleotides, plasmids, vectors, isolated DNA having any sequence, isolated RNA having any sequence, cell-free polynucleotides such as cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes, and primers. The nucleotide sequence may be interrupted by non-nucleotide compounds.

[0069] The term "expression" means one or more processes by which a polynucleotide is transcribed from a DNA template (e.g., into mRNA or other RNA transcripts), and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. The transcripts and the encoded polypeptides may together be referred to as "gene products". When 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 means an increased expression level of a polynucleotide (e.g., an RNA such as mRNA) and / or a polypeptide relative to its expression level in the wild-type state, while "down-regulated" generally means a reduced expression level of a polynucleotide (e.g., an RNA such as mRNA) and / or a polypeptide relative to its expression level in the wild-type state.

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

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

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

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

[0074] The phrase "exogenous T cell receptor (TCR) complex" or "exogenous TCR complex" as used herein means 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 mean a TCR complex in which one or more chains of the endogenous TCR complex have one or more mutated sequences, either at the nucleic acid level or at the amino acid level. Expression of an exogenous TCR in an immune cell can confer binding specificity for an epitope or antigen (e.g., an epitope or antigen preferentially present on the surface of cancer cells or other pathogenic cells or particles). An exogenous TCR complex can include TCR-α, TCR-β chains, CD3-γ chains, CD3-δ chains, CD3-ζ chains, or any combination thereof that are introduced into the genome. In some cases, the chains introduced into the genome may replace endogenously occurring chains.

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

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

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

[0078] As used herein, the term "genetic profile" means information regarding a particular gene, including gene expression in an individual or in a certain tissue. As used herein, the term "somatic mutation profile" means information regarding a particular gene associated with somatic mutations, including, but not limited to, particular genes caused by somatic mutations. Somatic mutation profiles 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 a chimeric stimulatory molecule. The chimeric stimulatory molecule includes a polypeptide extracellular domain (PED) that binds to a neoantigen. The PED can be fused to an intracellular domain (ICD) of a costimulatory molecule that mediates an immune cell activation signal. Binding of the chimeric stimulatory molecule to the neoantigen can result in an immune cell activation signal in the modified TILs. In some embodiments, the PED can be an extracellular domain of a surface protein of unmodified TILs. In some embodiments, examples of the PED include antibodies, as well as derivatives, variants, and fragments thereof.

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

[0081] TIL can be any cell obtained from a tumor. For example, TIL may be a cell that has migrated into the tumor. TIL may be a cell that has infiltrated the tumor. In some embodiments, TIL is a white blood cell that has migrated from the bloodstream of a subject into the tumor. TIL can be, for example, a T cell, a B cell, a monocyte, or a natural killer (NK) cell. In some cases, modified TIL includes CD8+ cytotoxic T cells (lymphocytes), Th1 and Th17 CD4+ T cells, natural killer cells, dendritic cells, or M1 macrophages. An immune cell population containing TIL can be a mixed population of cells. A population of TIL can include cells of various phenotypes, cells of different degrees of differentiation, cells having various cell lineages, or combinations thereof. TIL can generally be defined either biochemically using cell surface markers or functionally according to their ability to infiltrate tumors and have an effect on treatment. TIL can be classified based on the 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, modified TIL expresses at least one of PD-1, CD137, and TIM-3. In some cases, TIL can be functionally defined by its ability to infiltrate solid tumors upon reintroduction into a patient. In some cases, modified TIL includes "primary TIL," which means TIL obtained from a patient's tissue sample. In some cases, modified TIL includes "secondary TILs," which means TIL that has been expanded or proliferated. TIL can exhibit specific binding to neoantigens. In some cases, the TCR complex of TIL confers antigen-binding specificity (e.g., neoantigen binding).

[0082] In one aspect, the present disclosure provides a modified T cell that specifically binds to a neoantigen, where the modified 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 unmodified T 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 its ligand may result in an immune cell activation signal instead of an immune cell inactivation signal in the modified immune cell.

[0083] The modified T cell 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. The endogenous or exogenous TCR complex of the modified immune cell, for example, may confer the antigen-binding specificity (e.g., neoantigen binding) of the immune cell. In some embodiments, the present disclosure provides a modified T cell comprising an endogenous TCR complex that specifically binds to a neoantigen, the modified T cell comprising a chimeric stimulatory molecule, where the chimeric stimulatory molecule comprises, but is not limited to, a polypeptide extracellular domain (PED) that binds to a membrane protein of a cell, such as a tumor cell, where the PED is fused to an intracellular domain (ICD) of a costimulatory molecule that mediates an immune cell activation signal, and binding of the chimeric stimulatory molecule to the membrane protein results in the immune cell activation signal in the modified T cell.

[0084] In the present specification, the binding of modified immune cells such as, for example, modified T cells or modified tumor-infiltrating lymphocytes (TILs) to neoantigens can activate the immune cells. The switch molecules of the modified cells can be used, for example but not limited to, to provide further control of immune cell activity such as immune cell activation and proliferation. For example, the binding of a switch molecule to a ligand in modified immune cells such as modified T cells or modified TILs can result in an immune cell activation signal instead of an immune cell inactivation signal in the modified immune cells. Inducing an immune cell activation signal in modified immune cells instead of an immune cell inactivation signal can minimize the immunosuppressive effect in the immune cells. Minimizing the immunosuppressive effect in the immune cells can increase the effect of the immune cells in the immune response, for example, by increasing the immunocytotoxicity of the immune cells against target cells such as tumor cells.

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

[0086] In some embodiments, the switch molecule comprises a fragment of an immune checkpoint receptor that can be involved in the regulation of the immune system. Non-limiting examples of such receptors include, but are not limited to, for example, 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 and mucin-3 (TIM-3), and T cell immune receptor 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 of a target cell (e.g., a cancer cell antigen or a tumor antigen). In some examples, the switch molecule can comprise the extracellular variable region of the TCR α and / or β 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 a derivative, variant, or fragment thereof. Non-limiting examples of such ligands include, for example, but are 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. The cytokine receptor can perform various functions, and non-limiting examples include, for example, immune cell regulation and inflammation mediation. In some embodiments, the switch molecule includes 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 includes 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 includes a type II cytokine receptor, or a derivative, variant, or fragment thereof. In some embodiments, the switch molecule includes 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 referred to as platelet tissue factor), or a derivative, variant, or fragment thereof.

[0090] In some embodiments, the switch molecule comprises at least an extracellular region (such as a ligand-binding domain, etc.) of a catalytic receptor such as a receptor tyrosine kinase (RTK), or derivatives, variants, or fragments thereof. In some embodiments, the switch molecule is a class I RTK (e.g., the epidermal growth factor (EGF) receptor family including EGFR;The ErbB family, including ErbB-2, ErbB-3, and ErbB-4; class II RTKs (e.g., the insulin receptor family including INSR, IGF-1R, and IRR); class III RTKs (e.g., the platelet-derived growth 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 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 including PTK7); class XVI RTKs (e.g., the RYK receptor family including Ryk); class XVII RTKs (e.g., the MuSK receptor family such as MuSK), or derivatives, variants, or fragments thereof.;

[0091] A switch molecule comprising an RTK, or a derivative, variant, or fragment thereof, can 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 α or TGF-α, amphiregulin or AR, epiregulin or EPR, epigen, 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., 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 factor, relaxin family peptides including relaxin 1, relaxin 2, relaxin 3, Leydig cell-specific insulin-like peptide (gene INSL3), early placenta 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 (such as a ligand-binding domain, etc.) of a catalytic receptor such as a receptor-type threonine / serine kinase (RTSK), or derivatives, variants, or fragments thereof. The switch molecule can comprise a type I RTSK, a type II RTSK, or derivatives, variants, or fragments 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 derivatives, variants, or fragments thereof. The switch molecule comprises a type II receptor selected from the group consisting of TGFβR2, BMPR2, ACVR2A, ACVR2B, and AMHR2 (AMHR), or derivatives, variants, or fragments thereof.

[0093] The switch molecule of RTSK, or derivatives, variants, or fragments thereof, can bind to an antigen comprising any suitable RTSK ligand, or derivatives, variants, or fragments thereof.

[0094] The switch molecule may comprise 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 engineered to regulate a proliferation signal and / or a survival signal in immune cells. In some embodiments, the ICD is an intracellular domain of a costimulatory molecule selected from MHC class I protein, MHC class II protein, TNF receptor protein, immunoglobulin-like protein, cytokine receptor, integrin, signaling lymphocyte activation molecule (SLAM protein), activated NK cell receptor, BTLA, or toll-like receptor. In some embodiments, the costimulatory domain is 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, DNAM1It comprises the signaling domain of a molecule selected from the group consisting of (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 linked by a transmembrane domain, for example, by a transmembrane segment. In some embodiments, the transmembrane segment comprises a polypeptide. The transmembrane polypeptide can have any suitable polypeptide sequence. In some cases, the transmembrane polypeptide comprises the polypeptide sequence of the 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 site of an endogenous or wild-type transmembrane protein. In some embodiments, the transmembrane polypeptide comprises a non-natural polypeptide sequence, such as the sequence of a polypeptide linker. 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 modified immune cell. In some embodiments, the immune cell activation signal is mediated by an activator. The activator may 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 immune cells. 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 modified immune cells (e.g., modified TILs or modified T cells), cytokine release by modified immune cells (e.g., modified TILs or modified T cells), cytotoxicity of modified immune cells (e.g., modified TILs or modified T cells), proliferation of modified immune cells (e.g., modified TILs or modified T cells), differentiation, dedifferentiation, or transdifferentiation of modified immune cells (e.g., modified TILs or modified T cells), migration and / or transport of modified immune cells (e.g., modified TILs or modified T cells), exhaustion and / or reactivation of modified immune cells (e.g., modified TILs or modified T cells), and release of other intercellular molecules, metabolites, compounds, or combinations thereof by modified immune cells (e.g., modified TILs or modified T cells).

[0098] In some embodiments, immune cell activity comprises or results in clonal expansion of immune cells. Clonal expansion includes the generation of daughter cells arising from immune cells. Daughter cells obtained from clonal expansion may contain switch molecules. Clonal expansion of modified immune cells can be greater than that of equivalent immune cells without switch molecules. Clonal expansion of 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 400-fold, about 400-fold to about 500-fold, about 500-fold to about 600-fold, about 600-fold to about 700-fold that of equivalent immune cells without switch molecules. In some embodiments, measurement of clonal expansion can include, for example, quantifying the number of immune cells, with and without switch molecules, and after ligand binding to the switch molecule. Quantification of the number of immune cells can be performed by a variety of techniques, non-limiting examples of which include flow cytometry, trypan blue dye exclusion assay, and hemocytometry.

[0099] In some embodiments, immune cell activity comprises or results in cytokine release by immune cells. In some embodiments, immune cell activity comprises or results in the release of intercellular molecules, metabolites, compounds, or combinations thereof. Cytokine release by modified immune cells can include the release of IL-1, IL-2, IL-4, IL-5, IL-6, IL-13, IL-17, IL-21, IL-22, IFNγ, TNF alpha, CSF, TGFβ, granzyme, 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 modified immune cells can be greater than that of equivalent immune cells without a switch molecule. The modified immune cells provided herein can produce cytokine release that is 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 than that of equivalent immune cells without a switch molecule. Modified immune cells can exhibit increased cytokine secretion when the switch molecule binds to a ligand and the modified immune cells bind to neoantigens present on target cells, as compared to equivalent immune cells without a switch molecule (e.g., unmodified). In some embodiments, the secreted cytokine is IFNγ or IL-2. In some embodiments, cytokine release can be quantified in vitro or in vivo.

[0100] In some embodiments, it comprises, or results in, cytotoxicity of immune cell activity. In some cases, the cytotoxicity of the modified immune cells provided herein can be used to kill target cells. Immune cells or immune cell populations expressing a switch molecule can induce death in target cells. The death of target cells can be used in various applications, for example, but not limited to, treating diseases or disorders where it is desirable to remove a cell population or where it is desirable to suppress cell proliferation. Cytotoxicity can also mean the release by immune cells of cytotoxic cytokines such as IFNγ or granzyme. In some cases, the modified immune cells provided herein may have altered (i) release of cytotoxins such as, for example, perforin, granzyme, and granulysin, and / or (ii) induction of apoptosis via Fas-Fas ligand interactions between T cells and target cells. In some embodiments, cytotoxicity can be quantified by a cytotoxicity assay including, but not limited to, co-culture assays, ELISPOT, chromium release cell characterization assays, etc. The toxicity of the modified immune cells provided herein can be higher than that of equivalent immune cells without a switch molecule. Modified immune cells can exhibit increased cytotoxicity against target cells when the switch molecule binds to a ligand and the modified immune cells bind to neoantigens present on the target cells, as compared to immune cells without a switch molecule (e.g., unmodified). The modified immune cells of the present disclosure can 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% more cytotoxic to target cells as compared to equivalent immune cells without a switch molecule.The modified immune cells of the present disclosure can induce death of target cells that is 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 than that of equivalent immune cells without a switch molecule. In some embodiments, the immune cells provided herein can induce apoptosis in target cells presenting a target epitope (such as a neoantigen, etc.) on their surface. In some embodiments, cytotoxicity can be measured in vitro or in vivo. In some embodiments, measurement of cytotoxicity can include comparing the level of disease after administration of the modified immune cells provided herein with the level of disease before administration. In some embodiments, measurement of cytotoxicity can include measuring the level of disease after administration of the modified immune cells provided herein and the level of disease after administration of equivalent immune cells without a switch molecule.

[0101] In some embodiments, immune cell activity includes or results in the proliferation of immune cells. The proliferation of immune cells may mean an increase in immune cells. The proliferation of immune cells may mean a change in the phenotype of immune cells. The proliferation of the modified immune cells of the present disclosure can be increased compared to that of equivalent immune cells without a switch molecule. The proliferation of the modified immune cells of the present disclosure is 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 400-fold, about 400-fold to about 500-fold, about 500-fold to about 600-fold, about 600-fold to about 700-fold greater than the proliferation of equivalent immune cells without a switch molecule. In some embodiments, proliferation can be measured by quantifying the number of immune cells. Quantifying the number of immune cells can include flow cytometry, trypan blue dye exclusion assay, and hemocytometry. Proliferation may also be determined by phenotypic analysis of immune cells.

[0102] In some embodiments, immune cell activity includes or results in the differentiation, dedifferentiation, or transdifferentiation of immune cells. The differentiation, dedifferentiation, or transdifferentiation of immune cells can be determined by flow cytometry by assessing the phenotypic expression of markers of differentiation, dedifferentiation, or transdifferentiation on the cell surface. In some embodiments, the modified immune cells provided herein have an enhanced differentiation ability compared to equivalent immune cells without a switch molecule. In some embodiments, the modified immune cells provided herein have an enhanced dedifferentiation ability compared to equivalent immune cells without a switch molecule. In some embodiments, the modified immune cells provided herein have a higher transdifferentiation ability compared to equivalent immune cells without a switch molecule.

[0103] In some embodiments, the activation of immune cells comprises or results in the movement and / or transport of immune cells. In some embodiments, the movement can be determined by quantifying the localization of immune cells to a target site. For example, the modified immune cells provided herein can be quantified at a target site, e.g., a site that is not the target site, after administration. Quantification can be performed by isolating the lesion and quantifying the number of immune cells containing the switch molecule, such as tumor infiltrating lymphocytes. The movement and / or transport of immune cells containing the switch molecule is more than that of equivalent immune cells without the switch molecule. In some embodiments, the number of immune cells containing the switch molecule at a target site, such as a lesion area, can be about 5, 10, 15, 20, 25, 30, 35, or 40 times the number of equivalent immune cells without the switch molecule. Transport 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 the switch molecule at the target site can be about 5, 10, 15, 20, 25, 30, 35, or 40 times the number of equivalent immune cells without the switch molecule.

[0104] In some embodiments, immune cell activity comprises or results in immune cell exhaustion and / or reactivation. Immune cell exhaustion and / or reactivation can be measured by phenotypic analysis by flow cytometry or microscopic analysis. For example, the 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 immunoreceptor 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. As a result of exhaustion, functions such as, for example, IL-2 production and cytokine expression, and high proliferative capacity can be lost. Exhaustion can also be accompanied by defects in IFNγ, TNF, and chemokine production and degranulation. The exhaustion or activation of the modified immune cells provided herein is greater than that of equivalent immune cells without a switch molecule. In some embodiments, the immune cells provided herein have at least 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 greater increase in exhaustion or activation compared to equivalent immune cells without a switch molecule. In some embodiments, the immune cells provided herein have at least 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 greater decrease in exhaustion or activation compared to equivalent immune cells without a switch molecule.

[0105] In some embodiments, binding of a switch molecule to a ligand results in modified immune cells (such as modified TILs or modified T cells, etc.) exhibiting enhanced neoantigen binding as compared to equivalent immune cells without the switch molecule.

[0106] In one aspect, the present disclosure provides modified immune cells comprising a chimeric antigen receptor (CAR) and a T cell receptor (TCR) complex that exhibit specific binding to a neoantigen. The CAR can include an antigen interaction 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 a neoantigen can be an endogenous TCR complex or an exogenous TCR complex. The TCR complex, which can be, for example, endogenous or exogenous in the modified immune cells, confers antigen binding specificity (such as neoantigen binding) to the immune cells.

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

[0109] The CAR comprises an antigen interaction domain capable of binding to a B cell surface protein. The B cell surface protein can be any protein that can be found on the surface of a 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 to a surface protein on a non-B cell, provided that binding to the surface protein does not significantly endanger the overall health of the host or the immune system. In some embodiments, the surface protein is a surface protein on an immune cell. In some embodiments, it is a surface protein on a cell other than an immune cell. In some embodiments, the surface protein is 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 (a, b, c, d, e, f), CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD78, CD79, provided that binding to the surface protein does not significantly endanger the overall health of the host or the immune system.(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, CD156It may be selected from (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 can bind to a B cell surface protein or a fragment thereof on dead B cells. B cell apoptosis can occur before or after the initiation of an immune response (e.g., an immune response against tumor cells, etc.). Thus, dead B cells or their debris can still have a B cell surface protein or a fragment thereof presented on their surface. The ability of the CAR to target both live and dead B cells can increase the opportunity for (i) binding of immune cells containing the CAR to the B cell surface protein and (i) initiation of signal transduction of the intracellular signal domain. In some cases, signal transduction of the intracellular signal domain can promote the expansion (proliferation) of immune cells containing the CAR.

[0111] In some embodiments, the antigen - interacting domain of the CAR can bind to a B - cell surface protein or a fragment thereof that is bound (e.g., via covalent and / or non - covalent bonds) to the surface of a particle (e.g., a nanoparticle). The particle can be any particulate material comprising organic and / or inorganic materials. The particle can be, in at least one dimension, from about 1 nanometer (nm) to about 50 micrometers (μm). The particle can be, in at least one dimension, 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. The particle can be, in at least one dimension, at most 50 μm, 10 μm, 5 μm, 1 μm, 500 nm, 100 nm, 50 nm, 10 nm, 5 nm, 1 nm, or less. The particle can be, for example, a nanoparticle, microparticle, nanosphere, microsphere, nanorod, microrod, nanofiber, nanoribbon, etc. Examples of particles include, for example, metal nanoparticles (e.g., gold nanoparticles, silver nanoparticles, and iron nanoparticles, etc.), intermetallic compound nanoparticles, semiconductor nanoparticles, core - shell nanoparticles, particles having an inorganic core and a polymer shell, particles having an organic core and a polymer shell, or mixtures thereof. Alternatively, the particle can be, for example, a cross - linked polymer, a hydrogel polymer, a biodegradable polymer, polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), a copolymer, a polysaccharide, starch, cellulose, chitosan, polyhydroxyalkanoic acid (PHA), PHB, PHV, a lipid, a peptide, a peptide amphiphile, a polypeptide (e.g., a protein, etc.), or combinations thereof. Particles presenting a B - cell surface protein on their surface can be introduced in vitro into immune cells comprising a CAR that binds to the B - cell surface protein. Alternatively, or in addition, particles presenting a B - cell surface protein can be introduced in vivo (e.g., by local or systemic injection) with immune cells comprising a CAR. Such particles can be used in vitro or in vivo to expand a population of immune cells comprising a CAR.

[0112] The antigen-binding domain can include 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, mouse 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 domain (VHH) of camelid-derived nanobodies. In some embodiments, the first antigen-binding domain includes at least one of Fab, Fab’, F(ab’)2, Fv, scFv. In some embodiments, the antigen-binding domain includes an antibody mimetic. An antibody mimetic means a molecule capable of binding to a target molecule with an affinity equivalent to that of an antibody, such as a single-chain binding molecule, a cytochrome b562-based binding molecule, fibronectin or a fibronectin-like protein backbone (such as adnectin), a lipocalin backbone, a calixarene backbone, an A-domain, and other backbones. In some embodiments, the antigen-binding domain includes a transmembrane receptor, or any derivative, variant, or fragment thereof. For example, the antigen-binding domain can include at least the ligand-binding domain of a transmembrane receptor.

[0113] In some embodiments, the antigen-binding domain may comprise a 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 also be obtained from the whole exome sequence of tumor cells or primary cells. In some embodiments, the scFV may be mutated, whereby the scFV may have a higher affinity for its target. In some cases, the affinity of the scFV for its target may be optimized for targets expressed at low levels in normal tissue. This optimization may be performed, for example, to minimize potential toxicities such as hypercytokinemia. In other cases, cloning of an scFV with a higher affinity for a cell membrane-bound form of the target may be preferred over its soluble counterpart. This modification may be performed when some targets can also be detected in soluble form at various levels and their targeting can cause unintended toxicities such as hypercytokinemia, for example.

[0114] The antigen-binding domain of the CAR of the subject system can be bound to the intracellular signaling domain via a transmembrane domain. The transmembrane domain may be a transmembrane segment. The transmembrane domain of the subject CAR can anchor the CAR to the cell membrane of a cell, such as an immune cell. In some embodiments, the transmembrane segment comprises a polypeptide. The transmembrane polypeptide that binds 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 the polypeptide sequence of the 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 substitutions, deletions, and insertions compared to the transmembrane protein of the endogenous or wild-type transmembrane protein. In some embodiments, the transmembrane polypeptide comprises a non-natural polypeptide sequence, such as the sequence of a polypeptide linker. 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 extracellular region to the intracellular region of the cell, for example, via the antigen-binding domain. The native transmembrane site of CD28 can be used in the CAR. In other cases, the native transmembrane site of CD8 alpha can be used in the CAR.

[0115] The CARs of the present disclosure can include signaling domains involved in immune cell signaling, or derivatives, variants, or fragments thereof. The intracellular signaling domain of the CAR can induce the activity of immune cells comprising the CAR. The intracellular signaling domain can transmit effector function signals and direct the cell to perform specific functions. The signaling domain may include the signaling domains of other molecules. In some cases, truncated portions of the signaling domain are used in the CAR.

[0116] In some embodiments, the intracellular signaling domain comprises a plurality of signaling domains involved in immune cell signaling, or derivatives, variants, or fragments thereof. For example, the intracellular signaling domain can comprise at least two immune cell signaling domains, such as at least two, three, four, five, six, seven, eight, nine, ten immune cell signaling domains, etc. The immune cell signaling domain can be involved in the initial activation of the TCR complex in either a stimulating 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 the CAR of interest can comprise the signaling domains of 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 (also known as CD154), CD45, CD66d, CD79a, CD79b, CD80, CD86, CD278 (also referred to as ICOS), CD247 ζ, CD247 η, DAP10, DAP12, FYN, LAT, Lck, MAPK, MHC complex, NFAT, NF-κB, PLC-γ, iC3b, C3dg, C3d, and Zap70. In some embodiments, the signaling domain can comprise an immunoreceptor activation tyrosine motif or ITAM. The signaling domain containing ITAM can 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. The signaling domain containing ITAM can be modified, for example, by phosphorylation when the antigen-binding domain binds to an epitope. Phosphorylated ITAM can function as a docking site for other proteins, such as proteins involved in various signaling pathways.In some embodiments, the primary signaling domain is a modified ITAM domain, such as a mutated, truncated, and / or optimized ITAM domain, including a modified ITAM domain having altered (e.g., increased or decreased) activity compared to the native ITAM domain.

[0117] In some embodiments, the intracellular signaling domain of the CAR of interest includes an FcγR signaling domain (e.g., 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 includes an FcεR signaling domain (e.g., ITAM). The FcεR signaling domain may be selected from FcεRI and FcεRII (CD23). In some embodiments, the intracellular signaling domain includes an FcαR signaling domain (e.g., ITAM). The FcαR signaling domain may be selected from FcεRI (CD89) and Fcα / μR. In some embodiments, the intracellular signaling domain includes a CD3 ζ signaling domain. In some embodiments, the primary signaling domain includes the ITAM of CD3 ζ.

[0118] In some embodiments, the intracellular signaling domain of the CAR of interest comprises an immunoreceptor activation tyrosine motif or ITAM. The signaling domain comprising an ITIM contains a conserved amino acid sequence (S / I / V / LxYxxI / V / L) found at the cytoplasmic terminus of some inhibitory receptors of the immune system. The primary signaling domain comprising an ITIM can be modified, for example, by phosphorylation by an enzyme such as a Src kinase family member (such as Lck). Following phosphorylation, other proteins, including enzymes, can be recruited to the ITIM. These other proteins include, but are not limited to, enzymes such as the phosphorylated tyrosine phosphatases SHP-1 and SHP-2, the inositol monophosphate phosphatase called SHIP, and proteins having one or more SH2 domains (such as ZAP70). The intracellular signaling domain is 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), 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 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 a signaling domain (e.g., ITIM) of signal threshold control transmembrane adapter 1 (SIT). In some embodiments, the intracellular signaling domain is a modified ITAM domain, such as a mutated, truncated, and / or optimized ITAM domain, having 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 CAR of interest may include a co-stimulatory domain. In some embodiments, the co-stimulatory domain from a co-stimulatory molecule, for example, may provide a co-stimulatory signal for immune cell signaling such as signaling from ITAM and / or ITIM domains, for example, for activation and / or inactivation of immune cell activity. In some embodiments, the co-stimulatory domain is operable to regulate proliferation and / or survival signals in immune cells. In some embodiments, the co-stimulatory signaling domain includes the 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 activated NK cell receptor, BTLA, or a toll-like receptor. In some embodiments, the co-stimulatory domain is 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, 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, TSLPIt comprises a signaling domain of a molecule selected from the group consisting of R, VLA1, and VLA-6. In some embodiments, the intracellular signaling domain comprises a plurality of co-stimulatory domains, such as at least two, such as at least three, four, or five co-stimulatory domains. The co-stimulatory signaling region may provide a signal synergistic with the primary effector activation signal and may meet the requirements for T cell activation. In some embodiments, the addition of co-stimulatory domains to the CAR may enhance the effect and persistence of the immune cells provided herein.

[0121] Binding of the CAR to the B cell surface protein can enhance the proliferation of immune cells as compared to immune cells without the CAR. Proliferation of immune cells can mean an increase in the number of immune cells. Proliferation of immune cells may also mean a change in the phenotype of the immune cells. The proliferation of immune cells provided herein comprising a CAR can be increased as compared to that of equivalent immune cells without a CAR that exhibits binding to the B cell surface protein. The proliferation of immune cells comprising 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 400-fold, about 400-fold to about 500-fold, about 500-fold to about 600-fold, about 600-fold to about 700-fold as compared to that of equivalent immune cells without a CAR. The proliferation of immune cells comprising 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 400-fold, about 400-fold to about 500-fold, about 500-fold to about 600-fold, about 600-fold to about 700-fold, and the proliferation is confirmed 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, proliferation comprises quantifying the number of immune cells. Quantifying the number of immune cells can include flow cytometry, trypan blue dye exclusion assay, and hemocytometry. Proliferation may also be determined by phenotypic analysis of the immune cells.

[0122] In one aspect, the present disclosure provides a modified immune cell that specifically binds to a neoantigen, wherein the modified immune cell comprises: (a) a chimeric stimulatory molecule comprising a polypeptide extracellular domain (PED) that binds to the neoantigen, wherein the PED is fused to an intracellular domain (ICD) of a co-stimulatory molecule that mediates an immune cell activation signal, and binding of the chimeric stimulatory molecule to the neoantigen results in the immune cell activation signal in the modified immune cell; and (b) a chimeric antigen receptor comprising: (i) an antigen interaction 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 the PED include antibodies, as well as their derivatives, variants, and fragments.

[0123] In one aspect, the present disclosure provides a modified immune cell that specifically binds to a neoantigen, wherein the modified immune cell comprises: (a) a switch molecule comprising an extracellular domain (ECD) of a protein that induces an immune inactivation signal in an unmodified immune cell upon binding to its ligand, wherein the ECD is fused to an intracellular domain (ICD) of a co-stimulatory molecule that mediates an immune cell activation signal, and binding of the switch molecule to its ligand results in an immune cell activation signal in the modified immune cell instead of an immune cell inactivation signal; and (b) a chimeric antigen receptor comprising: (i) an antigen interaction 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 present disclosure provides modified tumor infiltrating lymphocytes (TILs) that specifically bind to neoantigens, where the modified immune cells comprise: (a) a switch molecule comprising an extracellular domain (ECD) of a protein that induces an immune cell inactivation signal in unmodified immune cells upon binding to its ligand, wherein the ECD is fused to an intracellular domain (ICD) of a costimulatory molecule that mediates an immune cell activation signal, and wherein binding of the switch molecule to the ligand results in an immune cell activation signal in the modified TILs instead of an immune cell inactivation signal; and (b) a chimeric antigen receptor comprising: (i) an antigen interaction domain capable of binding to a B cell surface protein; (ii) a transmembrane domain; and (iii) an intracellular signaling domain.

[0125] In one aspect, the present disclosure provides modified immune cells that overexpress a cytokine, such as a chemokine, wherein 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 of the modified immune cells provided herein.

[0126] A cytokine means a protein (e.g., chemokine, interferon, lymphokine, interleukin, and tumor necrosis factor, etc.) that is released by cells and can affect the behavior of cells. 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 local immune regulatory effects.

[0127] Certain cytokines can function as pro-inflammatory cytokines. A pro-inflammatory cytokine means a cytokine that is involved in inducing or amplifying an inflammatory response. Pro-inflammatory cytokines can work together 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. An anti-inflammatory cytokine means a cytokine that is involved in reducing an inflammatory response. 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 having pro-inflammatory and / or chemotactic functions can be upregulated in immune cells. Upregulation of the expression of cytokines having pro-inflammatory and / or chemotactic functions can be beneficial, for example, in immunotherapy to stimulate an immune response against target cells.

[0129] Examples of cytokines that can be overexpressed by the immune cells provided in this specification include, but are not limited to, lymphokines, monokines, and normal polypeptide hormones. The cytokines included are growth hormones such as human growth hormone, N-methionyl human growth hormone, bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), luteinizing hormone (LH); liver growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-α; Müllerian duct inhibitory factor; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-α; platelet growth factor; 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; stem cell factor (SCF); bone inductive factor; interferons (IFNs) such as IFN-α, IFN-β, 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); interleukins (ILs) such as IL-1, IL-1a, IL-1b, IL-1RA, IL-18, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, 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). A cytokine receptor means 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 (such as a ligand), an IL-1 receptor family member, an interleukin-6 (IL-6) family member (such as a ligand), an IL-6 receptor, an interleukin-10 (IL-10) family member (such as a ligand), an IL-10 receptor, an interleukin-12 (IL-12) family member (such as a ligand), an IL-12 receptor, an interleukin-17 (IL-17) family member (such as 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 selected from 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 / TNFSF7, TNFSF10 / TRAIL / APO-2L(CD253), RANKL / OPGL / TNFSF11(CD254), TNFSF12, TNF-α / TNFA,TNFSF13, TL1A / TNFSF15, OX-40L / TNFSF4 / CD252, CD40L / CD154 / TNFSF5, IFNA1, IFNA10, IFNA13, IFNA14, IFNA2, IFNA4, IFNA7, IFNB1, IFNE, 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 / MDC, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL3L3, CCL4, CCL4L1 / LAG-1, CCL5, CCL6, CCL7, CCL8, CCL9, CX3CL1, 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 evaluated using a variety of methods. Cytokine expression can be evaluated by assaying a cell culture supernatant in which modified immune cells are grown (e.g., in vitro production) for the presence of one or more cytokines, or by assaying serum obtained from a subject having the modified immune cells (e.g., in vivo production). Cytokine levels can be quantified using any suitable assay in any suitable units such as concentration. In some embodiments, cytokine protein is detected. In some embodiments, cytokine mRNA transcripts are detected. Examples of cytokine assays include, for example, enzyme-linked immunosorbent assay (ELISA), immunoblotting, immunofluorescence, radioimmunoassay, antibody arrays that allow simultaneous detection of various cytokines in a sample, bead-based arrays, quantitative PCR, microarrays, and the like. Other suitable methods include proteomics approaches (2-D gels, MS analysis, etc.).

[0133] In some embodiments, the cytokine overexpressed by the modified immune cells provided herein is a chemokine. The chemokine can be, for example, a CC chemokine, a CXC chemokine, a C chemokine, and a 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 modified 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 present disclosure provides a method for treating cancer in a subject, comprising: (a) administering to the subject any one of the modified TILs, modified T cells, or modified immune cells of various embodiments of the aspects herein; (b) contacting the modified TILs, modified T cells, or modified immune cells with target cancer cells expressing a neoantigen under conditions that induce cytotoxicity of the modified TILs, modified T cells, or modified immune cells against the target cancer cells, thereby inducing death of the target cancer cells.

[0135] In one aspect, the present 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 of the various embodiments of the aspects herein; (b) exposing the T cell population to a B cell surface protein so as to effect an increase in the T cell population. In some embodiments, in (b), the T cell population is exposed to B cells comprising the B cell surface protein.

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

[0137] In one aspect, the present disclosure provides a composition comprising: (a) a switch molecule comprising an extracellular domain (ECD) of a protein that induces an immunosuppressive signal in an unmodified immune cell upon binding to its ligand, wherein 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 of its components.

[0138] In one aspect, 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 of its components; and (b) a chimeric antigen receptor comprising: (i) an antigen interaction 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 present 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 induces an immunosuppressive signal in an unmodified immune cell upon binding to its ligand, wherein the ECD is fused to an 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 interaction 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, a promoter 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 cytomegalovirus (CMV) immediate early, herpes simplex virus thymidine kinase, early and late SV40, retrovirus-derived long terminal repeats (LTRs), human elongation factor 1 promoter (EF1), hybrid constructs containing the cytomegalovirus (CMV) enhancer fused to the chicken β-actin promoter (CAG), murine stem cell virus promoter (MSCV), phosphoglycerate kinase-1 locus promoter (PGK), and those derived from murine metallothionein-I. The promoter may be a fungal promoter. The promoter may be a plant promoter. Databases of plant promoters can be found, for example, at PllantProm. The expression vector may also contain a ribosome binding site for translation initiation and a transcription termination codon. The expression vector may also contain appropriate sequences for amplifying expression.

[0142] In various embodiments of the aspects herein, the modified immune cells can specifically bind to neoantigens and / or neoepitopes. Neoantigens and neoepitopes generally refer to tumor-specific mutations that trigger an antitumor T cell response in some cases. For example, these endogenous mutations can 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). Modified immune cells (e.g., modified TILs or modified T cells) containing a switch molecule exhibit specific binding to tumor-specific neoantigens. The neoantigens bound by the immune cells can be expressed on the target cells and are encoded, for example, as mutations in endogenous genes. In some cases, the neoantigens or neoepitopes specifically bound by the immune cells can be encoded by mutant genes.The gene can be selected from the group consisting of ABL1, ACOl1997, 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, FAM11IB, FGFR3, FRG1B, GAGE1, GAGE10, GATA3, GBP3, HER2, IDH1, JAK1, KIT, KRAS, LMAN1, MABEB16, MAGEA1, MAGEA10, MAGEA4, MAGEA8, MAGEB17, 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, SF3B1, SLC35F5, SLC45A2, SMAP1, SMAP1, SPOP, TFAM, TGFBR2, THAP5, TP53, TTK, TYR, UBR5, VHL, and XPOT. In some embodiments, the neoantigen can be selected based on the gene profile of a tumor sample from an individual. In some embodiments, the neoantigen 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 include a kill switch. The kill switch can be activated to eliminate the immune cells, for example, in cases of severe toxicity such as hypercytokinemia. This can occur when the immune system has a strong response with the release of many inflammatory cytokines, causing mild to severe symptoms such as fever, headache, rash, rapid heartbeat, low blood pressure, and respiratory disorders. The kill switch can be a drug-inducible kill switch. The kill switch may include inducible caspase 9.

[0144] Various embodiments of the aspects herein include cells, such as modified immune cells. Cells, such as immune cells (such as 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 their transgenic species. Examples of samples from the subject from which the cells are derived include, but are not limited to, for example, skin, heart, lung, kidney, bone marrow, breast, pancreas, liver, muscle, smooth muscle, bladder, gallbladder, colon, intestine, brain, prostate, esophagus, thyroid, serum, saliva, urine, gastric juice and digestive juice, tears, feces, semen, vaginal fluid, interstitial fluid from tumor tissue, intraocular fluid, sweat, mucus, earwax, oil, glandular secretions, cerebrospinal fluid, hair, nails, plasma, nasal swab or nasopharyngeal wash, cerebrospinal fluid, cerebrospinal fluid, tissue, throat swab, biopsy, placental fluid, amniotic fluid, umbilical cord blood, emphatic fluids, gingival crevicular fluid, sputum, pus, bacterial flora, meconium, breast milk, and / or other excretions or body tissues, etc. can be mentioned.

[0145] In some cases, the cells can be a population such as T cells, NK cells, B cells, etc. obtained from a subject. T cells can be obtained from a number of sources including PBMCs, bone marrow, lymph node tissue, cord blood, thymus tissue, and tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, the 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. In one embodiment, the cells from an individual's circulating blood are obtained by apheresis. The product of apheresis typically contains lymphocytes such as T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. The cells collected by apheresis may be washed to remove the plasma fraction and to place the appropriate cells in an appropriate buffer or solvent for subsequent processing steps.

[0146] Any of a variety of immune cells can be utilized in the aspects herein. In some embodiments, the 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. An immune effector cell means an immune cell that can perform specific functions in response to stimulation. In some embodiments, the immune cells are immune effector cells capable of inducing 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 cells 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 T cells (NKT cells), tumor-infiltrating lymphocytes (TILs), lymphokine-activated killer cells (LAKs), αβ T cells, γδ T cells, and similar specific classes of T cell lineages. T cells can be classified into two major categories, CD8+ T cells and CD4+ T cells, based on what proteins are present on the cell surface. T cells expressing the subject system can perform multiple functions including killing infected cells and activating or replenishing other immune cells. CD8+ T cells are also referred to as cytotoxic T cells or cytotoxic T lymphocytes (CTLs). CTLs expressing the subject system can be involved in recognizing and eliminating virus-infected cells and cancer cells. CTLs have specific compartments or granules containing cytotoxins that cause apoptosis, e.g., programmed cell death.CD4+ T cells can be subdivided into four subsets, Th1, Th2, Th17, and Treg, although there may be additional subsets, where Th means T helper cell. Th1 cells can coordinate the immune response against intracellular microorganisms, particularly bacteria. They produce and secrete molecules that send warnings to and activate other immune cells such as macrophages that phagocytose bacteria. Th2 cells are involved in coordinating the immune response against extracellular pathogens such as helminths (parasites) by warning 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 replenishing neutrophils.

[0147] In some embodiments, the immune cell populations provided herein may be heterogeneous. In some embodiments, the cells used may be composed of a heterogeneous mixture of CD4 and CD8 T cells. CD4 and CD8 cells may have the 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 progenitor cells, hematopoietic stem cells, or non-pluripotent stem cells. In some cases, the cells can be any immune cells, such as T cells including CD3+ T cells, CD4+ T cells, CD8+ T cells, such as tumor-infiltrating lymphocytes (TILs), or any type of T cells. 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 by selection of T cells from whole blood. T cells can also be expanded from a mixed population. T cells can also be skewed towards a particular population and phenotype. For example, T cells can be phenotypically skewed to include CD45RO(-), CCR7(+), CD45RA(+), CD62L(+), CD27(+), CD28(+) and / or IL-7Rα(+). Suitable cells can be selected that contain one or more markers selected from the list including CD45RO(-), CCR7(+), CD45RA(+), CD62L(+), CD27(+), CD28(+) and / or IL-7Rα(+). The cells also include stem cells such as, by way of example, embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, neural stem cells, and mesenchymal stem cells. The cells can include any number of primary cells, such as, for example, human cells, non-human cells, and / or mouse cells. The cells can be progenitor cells. The cells can be from the subject (e.g., patient) being treated. The cells can be from a human donor. The host cell can be a TSCM memory stem cell composed of CD45RO(-), CCR7(+), CD45RA(+), CD62L(+), CD27(+), CD28(+) and / or IL-7Rα(+), and the memory stem cell can also express CD95, IL-2Rβ, CXCR3, and LFA-1, and can exhibit a number of functional characteristics characteristic of the memory stem cell.The host cell may be a central memory TCM cell containing 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 containing L-selectin and CCR7 and may produce effector cytokines such as 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, 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, heterologous cells can also be used. T cells can be obtained from a unit of blood collected from a subject using any of a number of techniques such as Ficoll® separation. Cells from an individual's circulating blood can be obtained by apheresis or leukapheresis. The product by apheresis typically includes lymphocytes such as T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. The cells collected by apheresis may be washed to remove the plasma fraction and to place the appropriate cells in a suitable buffer or solvent such as phosphate buffered saline (PBS) for subsequent processing steps. After washing, the cells can be resuspended in various biocompatible buffers such as Ca-free, Mg-free PBS. Alternatively, unwanted components of the apheresis sample can be removed and the cells can be resuspended directly in the culture solution. The sample can be provided directly by the subject or indirectly through one or more intermediaries such as a sample collection service provider or a healthcare provider (e.g., a physician or a nurse). In some embodiments, separating T cells from peripheral blood leukocytes includes lysing red blood cells and separating the peripheral blood leukocytes from monocytes by centrifugation, for example, via a PERCOL gradient.

[0150] Subpopulations of specific T cells, such as CD4 or CD8, can further be separated by positive or negative selection techniques. Negative selection of a T cell population can be achieved, for example, by a combination of antibodies oriented to surface markers specific to the cells being negatively selected. One suitable technique is cell sorting via negative magnetic immunoadsorption, which utilizes a cocktail of monoclonal antibodies oriented to cell surface markers on the cells being 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 primarily to create a homogeneous desired T cell population. In some embodiments, the composition includes a mixture of two or more (e.g., two, three, four, five, 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 can be enriched by any suitable method, non-limiting examples of such methods including treating the cell population to trigger growth and / or differentiation into the desired cell type, treating to arrest the growth of undesired cell types, treating to kill or lyse undesired cell types, and purification of the desired cell type (e.g., purification on an affinity column that retains desired or undesired cell types based on one or more cell surface markers). In some embodiments, the enriched population of cells is an enriched population of cytotoxic lymphocytes selected from cytotoxic T cells (also 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 the separation of a desired cell population by positive or negative selection, the concentration of cells and surfaces (such as particles like beads) is varied. In certain 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 beads and the cells. 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. Concentrations of 10 million, 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 45 million, or 50 million cells / mL may also be used. In yet another embodiment, concentrations of 75 million, 80 million, 85 million, 90 million, 95 million, or 100 million cells / mL may be used. In further embodiments, concentrations of 125 million, or 150 million cells / mL may be used. Using high concentrations can result in increased cell yields, cell activation, and cell proliferation.

[0153] Various target cells can be killed using the systems and methods of the present disclosure. The target cells to which this method can be applied can include a variety of broad 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 can be isolated cells. The target cells may be cells inside 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 archaebacterial cells or derived from archaebacterial 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 cells may be vertebrate cells or derived from vertebrate cells. The target cells may be microbial cells or derived from microbial cells. The target cells may be fungal cells or derived from fungal cells. The target cells may be derived from a specific organ or tissue.

[0154] The target cells may be stem cells or derived from progenitor cells. The target cells can include stem cells (e.g., adult stem cells, embryonic stem cells, induced pluripotent stem (iPS) cells, etc.) and progenitor cells (e.g., cardiac progenitor cells, neural progenitor cells, etc.). The target cells can include mammalian stem cells and progenitor cells such as, for example, rodent stem cells, rodent progenitor cells, human stem cells, human progenitor cells, etc. Clonal cells can include descendant cells. The target cells can contain a target nucleic acid. The target cells may be within an organism. The target cells can be genetically modified cells. The target cells may be host cells.

[0155] The target cells may be primary cells. For example, the primary cells can be passaged 0 times, 1 time, 2 times, 4 times, 5 times, 10 times, 15 times or more. The cells may be single-celled organisms. The cells can be grown in a culture medium.

[0156] The target cells may be diseased cells. Diseased cells can change metabolism, gene expression, and / or morphological characteristics. Diseased cells can be cancer cells, diabetic cells, and apoptotic cells. Diseased cells can be cells derived from a subject suffering from a disease. Exemplary diseases can 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. For example, 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 solutions can generally be balanced salt solutions (e.g., normal saline, phosphate buffered saline (PBS), Hank's balanced salt solution, etc.) supplemented with fetal bovine serum or other natural factors in combination with a low-concentration acceptable buffer. Buffers can include HEPES, phosphate buffer, lactate buffer, etc. The cells may be used immediately or stored (e.g., by freezing). The frozen cells can be thawed and may be reusable. The cells can be frozen in DMSO, serum, solvent buffer (e.g., 10% DMSO, 50% serum, 40% buffer solvent, etc.) and / or other known solutions used to store cells at the freezing temperature.

[0158] Non-limiting examples of cells that can be target cells include, but are not limited to, for example, B cells, T cells (cytotoxic T cells, natural killer T cells, regulatory T cells, helper T cells), natural killer cells, cytokine-induced killer (CIK) cells (see, for example, U.S. Patent No. 20080241194), and other lymphocyte cells; for example, granulocytes (basophilic granulocytes, eosinophilic granulocytes, neutrophilic granulocytes / hyper-segmented neutrophils), monocytes / macrophages, erythrocytes (reticulocytes), mast cells, platelets / megakaryocytes, dendritic cells, and other myeloid cells; cells derived from the endocrine system including cells of the thyroid (thyroid epithelial cells, parafollicular cells), parathyroid (chief cells of the parathyroid gland, oxyphil cells), adrenal gland (chromaffin cells), pineal gland (pinealocytes); cells of the nervous system including glial cells (astrocytes, microglia), magnocellular neurosecretory cells, stellate cells, Betz cells, and cells of the pituitary gland (gonadotropin-secreting cells, adrenocorticotropic hormone-secreting cells, thyroid-stimulating hormone-producing cells, growth hormone-producing cells, mammotropic hormone-secreting cells); cells of the respiratory system including lung cells (type I lung cells, type II lung cells), Clara cells, goblet cells, dust cells; cells of the respiratory system including cardiomyocytes, pericytes; cells of the digestive system including cells of the stomach (chief cells of the stomach, parietal cells), goblet cells, Paneth cells, G cells, D cells, ECL cells, I cells, K cells, S cells; enterochromaffin cells, APUD cells, cells of the liver (hepatocytes, Kupffer cells), and enteric endocrine cells such as those of cartilage / bone / muscle; bone cells including osteoblasts, osteocytes, osteoclasts, cells of the teeth (cementoblasts, ameloblasts); chondrocytes including chondroblasts, chondrocytes; cells of the skin including spicules, keratinocytes, melanocytes (nevus cells); muscle cells such as myocytes; cells of the urinary system including podocytes, juxtaglomerular cells, mesangial cells within the glomerulus / mesangial cells outside the glomerulus, brush border cells of the proximal renal tubule, macula densa cells; cells of the reproductive system including sperm, Sertoli cells, Leydig cells, oocytes; 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 shaft cells, cortical hair shaft cells, keratinized hair shaft cells, keratinized hair root sheath cells, hair root sheath cells of the Huxley layer, hair root sheath cells of the Henle layer, outer hair root sheath cells, hair matrix cells (stem cells),Epidermal cells of wet stratified barrier epithelium, cornea, tongue, oral cavity, esophagus, anal canal, urinary tract, and vagina terminal; basal cells (stem cells) of epithelium of cornea, tongue, oral cavity, esophagus, anal canal, urinary tract, and vagina terminal; urothelial cells (inner side of bladder and ureter); exocrine epithelial cells; salivary gland mucosal cells (secretion rich in polysaccharides); salivary gland serous cells (secretion rich in glycoprotein enzymes); von Ebner gland cells of tongue (rinsing taste buds); mammary gland cells (milk secretion); lacrimal gland cells (tear secretion); ceruminous glands in ear canal (wax secretion); dark cells of eccrine sweat gland (glycoprotein secretion); clear cells of eccrine sweat gland (low-molecular secretion); apocrine sweat gland cells (aromatic secretion, sex hormone-sensitive); cells of glands in eyelid (special sweat glands); cells of sebaceous glands (secretion of lipid-rich sebum); Bowman's gland cells of nose (rinsing olfactory epithelium); Brunner's gland cells of duodenum (enzymes and alkaline mucus); seminal vesicle cells (secreting semen components containing fructose for swimming sperm); prostate gland cells (secreting semen components); urethral gland cells (mucus secretion); Bartholin gland cells (secreting vaginal lubricant); Littre's gland cells (mucus secretion); endometrial cells (carbohydrate secretion); goblet cells separated from respiratory and digestive tracts (mucus secretion); mucosal cells inside stomach (mucus secretion); gastric gland zymogenic cells (secreting pepsinogen); gastric gland acid-secreting cells (secreting hydrochloric acid); pancreatic acinar cells (secreting bicarbonate and digestive enzymes); Paneth cells of small intestine (secreting lysozyme); type II pneumocytes of lung (secreting surfactant); Clara cells of lung; hormone-secreting cells; anterior pituitary cells; growth hormone-producing cells; prolactin-secreting cells; thyroid-stimulating hormone-producing cells; gonadotropin-producing cells; adrenocorticotropic hormone-producing cells; intermediate pituitary cells; magnocellular neurosecretory cells; intestinal and airway cells; thyroid cells, thyroid epithelial cells, parafollicular cells, parathyroid cells, chief cells of parathyroid, eosinophilic cells, adrenal cells, chromaffin cells, Leydig cells of testis, theca interna cells of follicle, luteal cells of ruptured follicle, granulosa lutein cells, theca lutein cells, juxtaglomerular cells (secreting renin); macular cells of kidney; metabolic and storage cells; barrier function cells (lung, intestine, exocrine glands, and urogenital organs); kidney, type I pneumocytes (inner side of air cavity of lung); pancreatic duct cells (centroacinar cells); nonstriated duct cells (sweat glands, salivary glands, mammary glands, etc.); duct cells (seminal vesicles, prostate glands, etc.); epithelial cells covering closed internal body cavities; ciliated cells with propulsion function; extracellular matrix-secreting cells; contractile cells; skeletal muscle cells, stem cells, cardiomyocytes,Blood and immune system cells, red blood cells (erythrocytes), 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 precursor cells of the blood and immune system (various types), pluripotent stem cells, totipotent stem cells, induced pluripotent stem cells, adult stem cells, sensory transducer cells, autonomic nerve cells, sensory organ and peripheral nerve support cells, central nervous system neurons and glial cells, lens cells, pigment cells, melanocytes, retinal pigment epithelial cells, germ cells, oogonia / oocytes, spermatids, spermatocytes, spermatogonia (stem cells of spermatocytes), sperm, nurse cells, ovarian follicle cells, Sertoli cells (in the testis), thymic epithelial cells, interstitial cells, interstitial renal cells, and other cells including, etc.; are mentioned.

[0159] It should be noted that there are some issues with the original text's grammar and punctuation which have been carried over as accurately as possible in the translation.Cancer cells are a specific object of interest. In some embodiments, the target cells are cancer cells. Non-limiting examples of cancer cells include, for example, acanthoma, acinar cell carcinoma, acoustic neuroma, acral lentiginous melanoma, congenital pulmonary cyst, 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, acute promyelocytic leukemia, adamantinoma, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenomatoid odontogenic tumor, adrenocortical carcinoma, adult T-cell leukemia, aggressive NK-cell leukemia, AIDS-related cancer, AIDS-related lymphoma, alveolar soft part sarcoma, anal cancer, undifferentiated large cell lymphoma, undifferentiated thyroid cancer, angioimmunoblastic T-cell lymphoma, angioleiomyolipoma, angiosarcoma, appendiceal cancer, astrocytoma, atypical rhabdomyosarcoma, basal cell carcinoma, basaloid carcinoma, B-cell leukemia, B-cell lymphoma, Bellini duct carcinoma, biliary tract cancer, bladder cancer, blastoma, bone cancer, bone tumor, brainstem glioma, brain tumor, breast cancer, Brenner tumor, bronchial tumor, bronchioloalveolar carcinoma, brown tumor, Burkitt lymphoma, carcinoma of unknown primary, carcinoid tumor, carcinoma, carcinoma in situ, penile cancer, carcinoma of unknown origin, 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, dermoid cyst, fibroblastic small cell B-cell large cell tumor lymphoma, erythroblastic neuroepithelial tumor, embryonal carcinoma, endodermal sinus tumor, endometrial cancer, endometrial uterine cancer, endometroid tumor, enteropathy-associated T-cell lymphoma, epithelioblastoma, epithelioma, epithelioid sarcoma, erythroleukemia, esophageal tumor, esophageal tumor, esophageal tumor, Ewing family sarcoma, Ewing sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, extramammary Paget disease, fallopian tube cancer, fetal fetus, 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, glucagonoma, gonadoblastoma, granulosa cell tumor, hairy cell leukemia,Hairy cell leukemia, head and neck cancer, head and neck cancer, cardiac cancer, hemangioblastoma, pericytic tumor, angiosarcoma, hematological tumor, hepatocellular carcinoma, hepatosplenic T-cell lymphoma, hereditary breast and ovarian cancer syndrome, Hodgkin lymphoma, Hodgkin lymphoma, hypopharyngeal cancer, hypothalamic glioma, inflammatory breast cancer, intraocular melanoma, islet cell carcinoma, islet cell tumor, juvenile myelomonocytic leukemia, Kaposi Sarcoma, Kaposi's sarcoma, kidney tumor, Krukenberg tumor, Kruckenberg tumor, laryngeal cancer, laryngeal cancer, melanoma derived from malignant nevus, leukemia, leukemia, lip cancer and oral cancer, liposarcoma, lung cancer, corpus luteum cyst, lymphangioma, lymphangiosarcoma, lymphoepithelioma, lymphocytic leukemia, lymphoma, macroglobulinemia, malignant fibrous histiocytoma, 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, medulloblastoma, medulloepithelioma, melanoma, melanoma, meningioma, Merkel cell carcinoma, mesothelioma, mesothelioma, potential metastatic cervical squamous cancer, metastatic urothelial cancer, Müllerian duct mixed tumor, monocytic leukemia, intraoral cancer, mucinous tumor, multiple endocrine neoplasia, multiple myeloma, multiple myeloma, fungating polyp, fungating polyps, myelodysplastic disorder, myelodysplastic syndrome, myeloid leukemia medical disorder, myxoma, nasal cancer, nasopharyngeal cancer, nasopharyngeal cancer, neoplasm, schwannoma, neuroblastoma, neuroblastoma, neurofibroma, nerve tumor, nodular melanoma, non-Hodgkin lymphoma, non-Hodgkin lymphoma, non-melanoma skin cancer, non-small cell lung, eye tumor, oligodendroglioma, anaplastic oligodendroglioma, tumor cell tumor, 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 epitheloid cell tumor, pharyngeal cancer, pheochromocytoma, moderately differentiated pineal parenchymal tumor, pineoblastoma, pituitary cell tumor, pituitary adenoma, pituitary tumor, plasmacytoma, pleuropulmonary blastoma, polyembryoma, precursor T-lymphoblastic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, primary hepatocellular carcinoma, primary liver cancer, primary peritoneal cancerPrimary neuroectodermal tumors, prostate cancer, pseudomyxoma peritonei, rectal cancer, renal cell carcinoma, respiratory cancers involving the NUT gene on chromosome 15, retinoblastoma, rhabdomyoma, rhabdomyosarcoma, Richter transformation, sacrococcygeal teratoma, salivary gland cancer, sarcoma, schwannoma, sebaceous gland cancer, secondary neoplasms, seminoma, serous tumors, sertoli-leydig cell tumors, sex cord-stromal cell tumors, sezary syndrome cancer, skin cancer, blue round cell tumors, small cell cancer, small cell lung cancer, small cell lymphoma, small intestine cancer, soft tissue sarcoma, somatostatin-producing tumors, so-called, spinal cord tumors, spinal cord tumors, splenic marginal zone lymphoma, squamous cell carcinoma, stomach cancer, superficial spreading melanoma, primitive neuroectodermal tumors on the tentorium, surface epithelial stromal tumors, synovial sarcoma, T cell acute lymphoblastic leukemia, T cell large granular lymphocyte leukemia, T cell leukemia, T cell lymphoma, T cell prelymphocytic leukemia, teratoma, terminal lymphoma, testicular cancer, lethargy, pharyngeal cancer, thymic cancer, thymoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, transitional cell carcinoma, urachal carcinoma, urethral cancer, genitourinary tumors, uterine sarcoma, choroidal melanoma, vaginal cancer, verner-morrison syndrome, verrucous carcinoma, visual pathway glioma, vulvar cancer, waldenstrom's hypergammaglobulinemia, warthin tumor, wilms tumor, and combinations thereof, etc. can be mentioned. 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 hematopoietic origin such as lymphoma. The antigen may be a tumor-associated antigen.,

[0160] In some embodiments, the target cells form a tumor. The tumors treated using the methods herein can 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 below the level of detection. In some embodiments, the subject maintains a tumor-free state (e.g., is 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 maintains a tumor-free state 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 maintains a tumor-free state for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more years after treatment.

[0161] Death of the target cells can be detected by any suitable method, for example, but not limited to, measuring the cells before and after treatment, or measuring the level of markers associated with live or dead cells (e.g., live or dead target cells). The degree of cell death can be measured by any suitable method. In some embodiments, the degree of cell death is measured in relation to an initial condition. For example, an individual has a known initial amount of target cells, such as an initial cell mass of a known size or a known concentration of circulating target cells. In such cases, the degree of cell death can be expressed as the ratio of the surviving cells after treatment to the initial cell population. In some embodiments, the degree 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, for example, but not limited to, the use of cell staining, microscopy, flow cytometry, cell sorting, and combinations thereof.

[0162] If the tumor is subjected to surgical resection following completion of the treatment period, the effect of the treatment that causes the tumor size to regress can be measured by measuring the proportion of necrotic (i.e., dead) excised tissue. In some embodiments, the treatment is therapeutically effective if the necrotic proportion of the excised 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 proportion of the excised tissue is 100%, which means that there is no or undetectable viable tumor tissue.

[0163] Exposing the target cells to the immune cells or immune cell populations disclosed herein can be done either in vitro or in vivo. Exposing the target cells to the immune cells or immune cell populations means bringing the target cells into contact with the immune cells and / or in their sufficient vicinity such that an antigen of the target cells (e.g., cell membrane-bound or unbound) can bind to a switch molecule expressed on the immune cells. Exposing the target cells to the immune cells or immune cell populations also generally means bringing the target cells into contact with the immune cells and / or in their sufficient vicinity such that an antigen of the target cells (e.g., cell membrane-bound or unbound) can bind to a CAR expressed on the immune cells. Exposing the target cells to the immune cells or immune cell populations in vitro can be achieved by co-culturing the target cells and the immune cells. The target cells and the immune cells can be co-cultured, for example, as adherent cells or alternatively in suspension. The target cells and the immune cells can be co-cultured in various suitable types of cell culture media, for example, together with adjuvants, growth factors, ions, etc. Exposing the target cells to the immune cells or immune cell populations in vivo can in some cases be achieved by administering the immune cells to a subject, e.g., a human subject, such that the immune cells can localize to the target cells via the circulatory system. In some cases, the immune cells can be delivered immediately to the region where the target cells are localized, e.g., by direct injection, etc.

[0164] The exposure can be carried out for any suitable period of time, e.g., 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 longer.

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

[0166] Any suitable delivery method can be used to introduce the compositions and molecules of the present disclosure (such as polypeptides and / or nucleic acids encoding polypeptides, etc.) into host cells such as immune cells. Various components can be delivered simultaneously or separately at different times. The choice of method may 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] The delivery method includes contacting one or more nucleic acids containing the nucleotide sequence encoding the composition of the present disclosure with the target polynucleotide or introducing it into a cell (or a cell population such as an immune cell). A suitable nucleic acid containing the nucleotide sequence encoding the composition of the present disclosure may include an expression vector, where the expression vector containing the 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, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI) transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun method, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, and the like.

[0169] In some embodiments, the present disclosure provides a method comprising 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 aspects, the present disclosure further provides cells produced by such methods, organisms (such as animals, plants, fungi, etc.) containing or produced from such cells.

[0170] Conventional viral and non-viral based gene delivery methods can be used to introduce nucleic acids into mammalian cells or target tissues. Such methods can be used to administer nucleic acids encoding the compositions of the present disclosure to cells or host organisms in culture. Non-viral vector delivery systems can include DNA plasmids, RNA (e.g., transcripts of vectors described herein), naked nucleic acids, and nucleic acids complexed with delivery vehicles 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 can include lipofection, nucleofection, microinjection, particle bombardment, virosomes, liposomes, immunoliposomes, polycations, or lipid:nucleic acid complexes, naked DNA, artificial virus particles, and drug-enhanced DNA uptake. Cationic and neutral lipids appropriate for receptor recognition lipofection of effective polynucleotides can be used. Delivery can be to cells (e.g., in vitro or ex vivo administration) or to target tissues (e.g., in vivo administration). Preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipids, can be used.

[0172] RNA or DNA virus-based systems can be used to target specific cells of the body and to transport the viral payload to the cell nucleus. Viral vectors may be administered directly (in vivo) or they may be used to treat cells in vitro, and the modified cells may optionally be 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 retroviral, lentiviral, and adeno-associated virus gene transfer methods, which can result in long-term expression of the inserted transgene. High transformation efficiencies can be observed in many different cell types and target tissues.

[0173] The affinity of retroviruses is 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 can produce high viral titers. The choice of retroviral gene transfer can depend on the target tissue. Retroviral vectors can contain cis-acting terminal repeat sequences with the ability to package exogenous sequences up to 6 - 10 kb. The minimal cis-acting LTR is sufficient for vector replication and packaging and can be used for the incorporation of 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] An adenovirus-based system can be used. An adenovirus-based system can lead to transient expression of a transgene. An adenovirus-based vector can have high transduction efficiency in cells and may not require cell division. High titers and levels of expression can be obtained in adenovirus-based vectors. Adeno-associated virus (「AAV」) vectors can be used to transduce cells having a target nucleic acid, for example, in the 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 virus particles capable of infecting host cells. Such cells include 293 cells (e.g., for adenovirus packaging), and Psi2 cells or PA317 cells (e.g., for retrovirus packaging). Virus vectors can be created by producing cell lines that package nucleic acids into virus particles. The vector can contain the minimal viral sequences necessary for packaging and subsequent integration into the host. The vector may also contain other viral sequences that can be replaced by an expression cassette for the polynucleotide(s) to be expressed. The missing viral functions can be supplied in trans by the packaging cell line. For example, an AAV vector can contain the ITR sequences from the AAV genome that are necessary for packaging and integration into the host genome. The viral DNA can be packaged in the cell line, which can contain a helper plasmid that encodes other AAV genes, namely rep and cap, but does not have the ITR sequences. The cell line can also be infected with adenovirus as a helper. The helper virus can promote the replication of the AAV vector and the expression of the AAV genes from the helper plasmid. Contamination by adenovirus can be reduced, for example, by heat treatment, where adenovirus is more sensitive than AAV. Additional methods for delivery of nucleic acids to cells, such as those described in U.S. Patent Application 20030087817, may be used, and these are incorporated herein by reference.

[0176] The host cell is transiently or non-transiently transfected with one or more of the vectors described herein. The cell can be transfected in a subject as it occurs naturally. The cell can be harvested from or be derived from a subject and then transfected. The cell can be derived from a cell harvested from a subject, i.e., a cell line. In some embodiments, a cell transfected with one or more of the vectors described herein is used to establish a new cell line containing sequences from the one or more vectors. In some embodiments, a cell transiently transfected (e.g., transient transfection with one or more vectors or transfection with RNA) with the compositions of the present disclosure is used to establish a new cell line containing cells that contain the modification but no other exogenous sequences.

[0177] Any suitable vector compatible with the host cell can be used in the methods 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] Contact of the cells with the compositions of the present disclosure can occur in any culture medium and under any culture conditions that promote the survival of the cells. For example, the cells can be suspended in a convenient, suitable nutritive solvent such as Iscove’s modified DMEM or RPMI 1640, supplemented with fetal bovine serum or heat-inactivated goat serum (about 5-19%), L-glutamine, thiol, particularly 2-mercaptoethanol, and antibiotics such as, for example, penicillin and streptomycin. The culture medium can contain growth factors to which the cells are responsive. Growth factors as defined herein are molecules that can promote the survival, growth, and / or differentiation of cells, either in the culture medium or in untreated tissue, through specific effects on transmembrane receptors. Growth factors can include polynucleotide factors and non-polynucleotide factors.

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

[0180] Pharmaceutical compositions containing the molecules (e.g., polypeptides and / or nucleic acids encoding polypeptides) or immune cells described herein can be administered for prophylactic and / or therapeutic treatment. In therapeutic applications, the composition can be administered to a subject already suffering from a disease or medical condition in an amount sufficient to cure or at least partially arrest the symptoms of the disease or medical condition, or to cure, heal, ameliorate, or remit the medical condition. The effective amount for this use can vary based on the severity and course of the disease or medical condition, previous treatments, the health, weight, and responsiveness of the subject to the agent, as well as the judgment of the person administering the treatment.

[0181] Multiple therapeutic agents can be administered in any order or simultaneously. When administered simultaneously, the multiple therapeutic agents can be provided in a single, integrated form, or in multiple forms, such as multiple separate pills, for example. The molecules can be filled together or separately, in a single package or in multiple packages. When not simultaneous, the timing of the multiple doses can vary over periods as long as about a month.

[0182] The molecules described herein can be administered before, during, or after the onset of a disease or medical condition, and the timing of administration of the composition containing the compound can vary. For example, the pharmaceutical composition can be used as a prophylactic agent and can be continuously administered to a subject having a predisposition to a medical condition or disease to prevent the onset of the disease or medical condition. The molecules and pharmaceutical compositions can be administered to the subject as soon as practicable during or after the onset of symptoms. Administration of the molecule can be initiated within the first 48 hours, within the first 24 hours, within the first 6 hours, or within the first 3 hours of the onset of symptoms. The first administration can be via any practical route, using any of the formulations described herein, such as by any of the routes described herein. The molecule can be administered as soon as practicable after the onset of the disease or medical condition has been detected or suspected, and for the length of time necessary for the treatment of the disease (e.g., 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 the subject. Examples of biological compartments include, but are not limited to, viruses (retroviruses, adenoviruses), nanospheres, liposomes, quantum dots, nanoparticles, microparticles, nanocapsules, vehicles, polyethylene glycol particles, hydrogels, and micelles.

[0184] For example, a biological compartment may include liposomes. A liposome can be a self-assembling structure that includes one or more lipid bilayers, and each layer can include two monolayers that contain amphiphilic lipid molecules oriented opposite to each other. The amphiphilic lipid can include a polar (hydrophilic) head conjugated to two or more non-polar (hydrophobic) acyl or alkyl chains. The energetically unfavorable contact between the hydrophobic acyl chains and the surrounding aqueous solvent causes the amphiphilic lipid molecules to arrange themselves such that the polar heads can be oriented towards the surface of the bilayer and the acyl chains can be oriented towards the interior of the bilayer, effectively shielding the acyl chains from contact with the aqueous environment.

[0185] Examples of preferred amphiphilic compounds used within liposomes include phospholipids and sphingolipids, and representative examples include, for example, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, palmitoyl oleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylcholine, distearoyl phosphatidylcholine (DSPC), dilinoleoyl phosphatidylcholine, and egg yolk sphingomyelin, or any combination thereof.

[0186] The biochemical compartment may contain nanoparticles. The nanoparticles can have diameters ranging from about 40 nm to about 1.5 μm, from about 50 nm to about 1.2 μm, from about 60 nm to about 1 μm, from about 70 nm to about 800 nm, from about 80 nm to about 600 nm, from about 90 nm to about 400 nm, and from about 100 nm to about 200 nm.

[0187] In some cases, as the size of the nanoparticles increases, the release rate can decrease or become longer, and as the size of the nanoparticles decreases, the release rate can increase.

[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 contain up to 30, 40, 50, 60, 70, 80% or more nanoparticles. In some cases, the nucleic acid molecules of the present disclosure can be bound to the surface of the nanoparticles.

[0189] The biochemical compartment may contain a virus. The virus can 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). The pharmaceutical composition of the present disclosure can be delivered to cells using a virus. The virus can be infected and transduced in vivo, ex vivo, or in vitro. In ex vivo and in vitro delivery, the transduced cells can be administered to a subject in need of treatment.

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

[0191] Viral delivery systems (such as viruses containing the pharmaceutical compositions of the present disclosure) can be administered to the cells, tissues, and organs of a subject in need thereof by direct injection, stereotactic injection, by an intracerebroventricular, mini-pump injection system, by convection, by catheter, intravenously, parenterally, intraperitoneally, and / or subcutaneously. In some cases, cells can be transduced in vitro or ex vivo using a 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 a pharmaceutical composition, and the stem cells can be transplanted into a patient to treat a disease. In some examples, the dose of transduced cells administered to a subject can be, per single dose, about 1×10 5 cells / kg, about 5×10 5 cells / kg, about 1×10 6 cells / kg, about 2×10 6 cells / kg, about 3×10 6 cells / kg, about 5×10 6 cells / kg, about 5×10 6 cells / kg, about 6×10 6 cells / kg, about 7×10 6 cells / kg, about 8×10 6 cells / kg, about 9×10 6 cells / kg, about 1×10 7 cells / kg, about 5×10 6 cells / kg, and about 1×10 8 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, polyethyleneimine (PEI) transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun method, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, and the like.

[0193] In some embodiments, immune cells expressing the target system are administered. The immune cells expressing the target system can be administered before, during, or after the occurrence of a disease or medical condition, and the timing of administering the composition containing the compound can vary. For example, the immune cells expressing the target system can be used as a prophylactic agent and can be continuously administered to a subject having a tendency for a medical condition or disease to prevent the occurrence of the disease or medical condition. The immune cells can be administered to the subject as soon as possible during or after the onset of symptoms. Administration can be initiated within the first 48 hours, within the first 24 hours, within the first 6 hours, or within the first 3 hours of the occurrence of symptoms. The first administration can be via any practical route using any of the formulations described herein, such as by any of the routes described herein. The immune cells can be administered as soon as practically possible after the occurrence or suspicion of the occurrence of a disease or medical condition and for the length of time required for the treatment of the disease (e.g., about 1 month to about 3 months). The length of treatment can vary for each subject.

[0194] The molecules described herein (such as polypeptides and / or nucleic acids, etc.) can be present in a composition in the range of about 1 mg to about 2000 mg, about 5 mg to about 1000 mg, about 10 mg to about 25 mg - 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 m 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.

[0195] The molecules described in this specification (such as polypeptides and / or nucleic acids, etc.) can be present in a composition in an amount 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 (such as polypeptides and / or nucleic acids, etc.) 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 active units / mg of the molecule. The activity can be the regulation of gene expression. In some embodiments, the total number of active units of the molecule delivered to a subject is at least 25000, 30000, 35000, 40000, 45000, 50000, 60000, 70000, 80000, 90000, 110000, 120000, 130000, 140000, 150000, 160000, 170000, 180000, 190000, 200000, 210000, 220000, 230000, or 250000 or more units. In some embodiments, the total number of active units of the molecule delivered to a subject is at most 25000, 30000, 35000, 40000, 45000, 50000, 60000, 70000, 80000, 90000, 110000, 120000, 130000, 140000, 150000, 160000, 170000, 180000, 190000, 200000, 210000, 220000, 230000, or 250000 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 Targeted 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 separated using Ficoll lymphocyte separation. After 2 hours of adhesion 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 separated by enzymatic digestion. CD3-positive T cells were separated using CD3 magnetic beads. Other cells were grown in adhesion culture to provide tumor cells from the patient. T cells were then separated 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-activated 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. And 20 mutations were selected based on affinity prediction from the perspective of the patient's HLA typing. The gene encoding the neoantigen was synthesized and then transcribed into RNA. PBMCs were isolated and subjected to 2-hour adherent culture. Adherent monocytes were recovered. Cytokines were added to promote dendritic cell differentiation and maturation. RNA was transfected into dendritic cells by electroporation. Suspension cells were mainly obtained as T cells and cultured together with dendritic cells. CD137+ positive cells were then isolated to provide neoantigen-reactive (e.g., recognition) T cells ("neoT"). NeoT was 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. The PD1 / CD28 vector, packaging vector pMDL-gag, Rev, and envelope vector pMD2.G were co-introduced into HEK293T cells using calcium phosphate or liposome-PEI. The supernatant was collected after 48 hours and centrifuged to concentrate the lentivirus.

[0202] Titration of the lentivirus was performed by 3-fold serial dilution. HEK293T cells transfected with 50 μL of lentivirus after 48 - 72 hours were collected and then stained with PD-1. The PD-1 positive rate (PD-1+%) was analyzed by flow cytometry, and the titer was calculated based on the following. Titer (TU / mL) = 40000 - 45000 (this is the number of HEK293T cells at the start) × PD1 + % × dilution factor × 20 (initial PD1 + % < 20%)

[0203] Figures 3A and 3B show the calculation of the PD1 / CD28 lentivirus titer. Titers greater than 3×10 7 can be used further.

[0204] Example 5: Transduction of neoantigen-activated T cells with PD1 / CD28 Three types of T cells were obtained: Switch-NY-ESO-1-TCR-T, Swich-TIL, and Switch-neoT. The Switch-NY-ESO-1-TCR-T cells were obtained by expressing the switch molecule in the ESO-1-TCR-T cells of Example 1. The Swich-TIL cells were obtained by expressing the switch molecule in the triple-positive T cells of Example 2. The Switch-neoT cells were obtained by expressing the switch molecule in the neoT cells of Example 3. Flow cytometry showed a Switch expression rate of approximately 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 the PD1 / CD28 switch molecule HLA typing was performed, and J82-NY-ESO-1-PD-L1 tumor cells with A:0201 were constructed. A lentiviral vector was added to J82 (bladder, transitional cell carcinoma) at an infection efficiency MOI = 5 to transduce the PD-L1 and NY-ESO-1 transgenes. G418 and puromycin were added 72 hours later to screen for positive cells. Flow cytometry was performed approximately 2 weeks later to measure the expression of PD-L1 and NY-ESO-1 (Figure 5). As shown in Figure 5, more than 95% of the transduced J82 cells expressed PDL1 and NY-ESO-1 simultaneously, which confirmed the success of cell construction. Figures 6A - 6C show data from an in vitro cell killing assay, where 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 the expression of CD8 and CD107a. The surface localization of CD107a (also known as LAMP-1), a degranulation marker normally found inside the granules of T cells, is a sign of cytotoxic activity when T cells release perforin and granzyme from their 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 did not induce the secretion of IFN-γ and IL-2 in T cells, NY-ESO1-TCR T cells, or Switch-NY-ESO1-TCR T cells. On the other hand, exposure to J82-NY-ESO1-PDL1 bladder cancer cells induced the secretion of IFN-γ and IL-2 in both NY-ESO1-TCR T cells and Switch-NY-ESO1-TCR T cells, and the secretion levels of IFN-γ and IL-2 were higher in Switch-NY-ESO1-TCR T cells.

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

[0207] Example 8: In vitro assay of neoT expressing PD1 / CD28 switch molecule 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 indicate 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 that by neoT cells without the PD1 / CD28 switch molecule.

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

[0209] Mice in the control group are treated with PBS by subcutaneous injection (A0). There are 5 treatment groups, which are the untreated group PBS (A1), T cells (A2), switch-T cells (A3), NY-ESO1-TCR-T cell group (A4), and Switch-NY-ESO1-TCR T cell group. The cells are 1×10 into the tail vein 7administered by intravenous injection of individual cells.

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

[0211] The experiment is expected to show that the tumor size of group A5 decreases, or remains approximately constant, or at least the growth rate decreases, 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 treatment groups A3 - A5 on the 10th day after administration, and tumor cells are isolated to obtain TIL. Flow cytometry is performed to measure the total amount of T cells present at the tumor site. Treatment with Switch - NY - ESO1 - TCR T cells (A5) is expected to result in a greater presence of T cells at the tumor site.

[0213] Example 10: Preparation of a Lentiviral CAR Targeting B - Cell Surface Protein (BCAR) CD19 was selected as the B - CAR target, and an anti - CD19 scFv having the sequence shown in SEQ ID NO: 1 was used to construct the B - CAR. A fourth - generation lentiviral vector system was used. The 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 ultra - centrifuged to concentrate the lentivirus.

[0214] Titration of the lentivirus was performed by 3 - fold serial dilution. 293T cells after transduction with 50 μL of lentivirus for 48 - 72 hours were collected and then stained for CAR expression. The CAR positive rate (CAR%) was analyzed by flow cytometry, and the titer was calculated based on the following. Titer (TU / mL) = number of 293T cells at the start × CAR + % × dilution factor × 20 (initial CAR + % < 20%)

[0215] The lentivirus titer was calculated. 3 × 10 7 Titers greater than this were considered 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 showed an expression rate of B - CAR of approximately 60% in all three types of T cells. See Figure 9. The three types of T cells were grown 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 - targeted TCR - T cells, a 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 individual J82 - NY - ESO - 1 - Luc cells were seeded in a 24 - well plate and cultured overnight to adhere. The cells were divided into four groups, A, B, C, and D, and each well also contained 5 × 10 4 individual B cells. Group A was co - cultured with 2 × 10 5 individual T cells. Group B was co - cultured with 2 × 10 5 individual BCAR - T cells (CAR positive rate 60%). Group C was co - cultured with 2 × 10 5 individual NY - ESO1 - TCR - T cells. Group D was co - cultured with 2 × 105 They were co-cultured with individual BCAR-NY-ESO1-TCR-T cells. The following assays were performed.

[0218] Increase in T cells. T cell numbers were measured after 48 and 96 hours of culture. As shown in Figure 10, after 4 days of culture, T cells in Group A increased approximately 3-fold, BCAR-T cells in Group B increased 12-fold, NY-ESO1-TCR-T cells in Group C increased approximately 10-fold, and BCAR-NY-ESO1-TCR-T cells increased approximately 27-fold.

[0219] Proliferation of tumor cells. Supernatants were collected from the 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 the survival rate of tumor cells. See Figure 11. The data show that after 48 hours of culture, the average amount of protein from tumor cells (and thus the number of tumor cells present) was lowest in the treatment with BCAR-NY-ESO1-TCR-T cells compared to other treatments.

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

[0221] Increase in T cells. After 96 hours of co-culture with tumor cells, as shown in Figure 12A, TIL increased approximately 10-fold and BCAR-TIL increased approximately 25-fold. The results indicate that when co-cultured with B cells and tumor cells, the increase in BCAR-TIL was greater compared to TIL without BCAR.

[0222] Proliferation of tumor cells. After 96 hours, the supernatant was recovered 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 the survival rate of tumor cells. The luciferase levels are shown in Figure 12B. The data indicate that after 96 hours of culture, the average amount of protein from tumor cells (and thus the number of tumor cells present) is lower when treated with BCAR-TIL cells compared to treatment with TIL 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 adhere. neoT and BCAR-neoT cells were added to the culture medium. The same amount of B cells was added to the wells, and the following assays were performed.

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

[0225] Proliferation of tumor cells. After 96 hours, the supernatant was recovered 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 the survival rate of tumor cells. The luciferase levels are shown in Figure 13B. The data indicate that after 96 hours of culture, the average amount of protein from tumor cells (and thus the number of tumor cells present) is lower when treated with BCAR-neoT cells compared to treatment with neoT without BCAR.

[0226] Example 15: Animal Experiment of NY-ESO-1 TCR-T Cells Expressing BCAR Animal model: 1×10 6 Individual tumor cells (J82-NY-ESO-1) are inoculated subcutaneously into NSG mice. As a blank control, animals in group A0 are injected subcutaneously with PBS. Tumors are formed in the animals approximately two weeks after the injection of tumor cells. Tumor size is measured 23 days later. 30 mice are selected.

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

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

[0229] Change in tumor volume

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

[0231] Change in the total amount of injected T cells

[0232] Peripheral blood is extracted from the animals in groups A2 - A5 10 days after administration. The total number of CD3+ T cells is measured using flow cytometry, and it is expected that the number of T cells in groups A3 - A5 with BCAR is higher than that in group A4. Both BCAR T cells and dual-target T cells are expected to increase in vivo.

[0233] Example 16: Animal Experiment of TIL Expressing BCAR Animal Model: Tumor cells are isolated from new tumor tissues and injected subcutaneously into NSG mice at a dose of 1×10 6 per animal. As a blank control, the animals in group A0 are injected subcutaneously with PBS. Tumors are formed in the animals approximately 2 weeks after the injection of tumor cells. Tumor size is measured 25 days later. 30 mice are selected.

[0234] Administration: The animals in the blank control (A0) group are injected with PBS via the tail vein. The tumor-forming group is divided into 5 groups, namely, the PBS group (A1), the T cell group (A2), the BCAR-T group (A3), the TIL group (A4), and the BCAR TIL cell group (A5). The animals in groups A1 - A5 are injected with 1×10 4 T cells via the tail vein, and all groups are given 1×10 7 B cells by injection.

[0235] Tumor size and the general 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 × major axis × minor axis × minor axis

[0236] Change in Tumor Volume

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

[0238] Change in the Total Amount of Injected T Cells

[0239] Peripheral blood is extracted from the animals in groups A2 - A5 10 days after administration. The total number of CD3+ T cells is measured using flow cytometry, and it is expected that the number of T cells in groups A3 - A5 with BCAR is higher than that in group A4. It is expected that both BCAR T cells and dual - target T cells increase in vivo.

[0240] Example 17: Animal experiment of neoT expressing BCAR Animal model: Tumor cells are isolated from fresh tumor tissue and injected subcutaneously into NSG mice at a dose of 1×10 6 per animal. As a blank control, the animals in group A0 are injected subcutaneously with PBS. Tumors are formed in the animals approximately 2 weeks after the injection of tumor cells. The tumor size is measured 25 days later. 30 mice are selected.

[0241] Administration: The animals in the blank control (A0) group are injected with PBS via the tail vein. The tumor - forming group is divided into 6 groups, which are the PBS group (A1), the T - cell group (A2), the BCAR - T group (A3), the normal neoT group (A4), the BCAR neoT group (A5), and the high - dose normal neoT cell group (A6). The animals in groups A1 - A5 are injected with 1×10 4 T cells via the tail vein, and group A6 is injected with 1×10 7 T cells. And all groups are given 1×10 7 B cells by injection.

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

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

[0244] Change in tumor volume

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

[0246] Change in the total amount of injected T cells

[0247] Peripheral blood is extracted from the animals of groups A2 - A5 10 days after administration. The total number of CD3+ T cells is measured using flow cytometry, and it is expected to show that the number of T cells in groups A3 - A5 with BCAR is higher than that in group A4. Both BCAR T cells and dual - target T cells have been shown to increase 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 immunosuppressive proteins of their respective switch molecules, and CD28 was used as a co - stimulatory signaling protein.

[0249] Taking PD1sw as an example, a fourth - generation lentiviral vector system was used. The 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. The supernatant was collected after 48 hours and centrifuged to concentrate the lentivirus.

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

[0251] The lentivirus titer was calculated. A titer greater than 3×10 7 was considered 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"), TGFBR2sw-2A-CD19 CAR (hereinafter, TGFBR2sw-BCAR)), lentiviruses were constructed according to the method of Example 18, respectively.

[0254] Example 20: Transduction of TIL and pTIL with Vectors of Switch and BCAR The lentiviruses for the switch and BCAR of Example 19 and their combinations were transduced into TIL and depleted TIL. The following cells were prepared. (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 TIL to provide SuperTIL, hereinafter referred to as "PD1-STILs") 6. TIM3sw-BCAR-TIL (TIM3sw-CD19 CAR lentivirus transduced into TIL to provide SuperTIL, hereinafter referred to as "TIM3-STIL") 7. TGFBR2sw-BCAR-TIL (TGFBR2sw-CD19 CAR lentivirus transduced into TIL to provide SuperTIL, hereinafter referred to as "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 pTIL to provide Super-pTIL, hereinafter referred to as "PD1-SpTILs") 6. TIM3sw-BCAR-pTIL (TIM3sw-CD19 CAR lentivirus transduced into pTIL to provide Super-pTIL, hereinafter referred to as "TIM3-SpTIL") 7. TGFBR2sw-BCAR-pTIL (TGFBR2sw-CD19 CAR lentivirus transduced into pTIL to provide Super-pTIL, hereinafter referred to as "TGFBR2-SpTIL") 8. PD1-SpTIL, TIM3-SpTIL, and TGFBR2-SpTIL were mixed together to provide "XSpTIL".

[0255] Super-pTIL should also be understood as SuperTIL. Super-pTIL was specifically named to identify different cell origins.

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

[0257] Based on the titer of the PD1sw lentivirus, the lentivirus was added to TILs / pTILs at MOI = 5. A flow cytometry assay was performed to sort PD1sw-TILs / pTILs to have an expression ratio of approximately 60% of PD1.

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

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

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

[0261] TIM3-STIL / SpTIL and TGFBR2-STIL / SpTIL were prepared in the same manner as PD1-STIL / SpTIL.

[0262] (3) Preparation of XSTIL / XsTIL

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

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

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

[0266] Tumor cells were isolated from a patient's fresh tumor tissue and seeded in a 24-well plate together with a luciferase marker. The cells were cultured overnight to adhere. The cells were divided into 10 groups and co-cultured with a control or the following T cells: Control group without addition of T cell-CK (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). The following assays were performed.

[0267] 1. Cytokine secretion by T cells. The secretion of IFN-γ and IL-2 was measured for each group by 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 was observed in the control A1 group, while secretion of IFN-γ and IL-2 was observed in all other groups (groups A2 - A10). Among all groups, those with Switch (groups A4 - A10) showed higher secretion of IFN-γ and IL-2 than those without Switch (groups A2 and A3).

[0268] 2. Proliferation of tumor cells. The supernatant was collected from the culture after 48 hours and 96 hours. The culture was then washed three times with PBS. Adherent tumor cells were lysed, and luciferase activity was measured to determine the amount of protein as an indicator of the survival rate of tumor cells. As shown in Figure 17A, the number of tumor cells in each group (groups A2 - A10) was decreased compared to the control group (group A1), and the groups with Switch (groups A4 - A10) showed a more significant decrease in the number of tumor cells than those without Switch (groups A2 and A3).

[0269] 3. Increase in T cells. The number of T cells was measured after 48 hours of culture. As shown in Figure 17B, the T cells in the groups without Switch (groups A2 and A3) showed a slight increase, while the groups with Switch (groups A4 - A10) showed a significant increase.

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

[0271] Tumor cells and TILs are isolated from the patient's fresh tumor tissue and seeded into 24-well plates together with the luciferase marker before being cultured overnight for adhesion. The same amount of B cells is added to each well. The cultured cells are divided into 10 groups and co-cultured with the control or the following T cells: Control group without addition of T cells-CK (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). The following assays are performed.

[0272] 1. Proliferation of tumor cells. Supernatants are recovered from the cultures after 48 hours and 96 hours. The cultures are then washed three times with PBS. The adherent tumor cells are lysed and luciferase activity is measured as an indicator of the survival rate of the tumor cells. As shown in Figure 17A, the number of tumor cells in each group (Groups B2 - B10) is decreased compared to the control group (Group B1), and the normal TIL group (Group B2) shows the least decrease in tumor cells compared to the significant decrease 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 measured. As shown in Figure 17B, the normal TIL group (Group B2) shows a slight 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.

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

[0275] Example 22: In Vitro Assay of the Super-pTIL Effect To confirm the effect of Super-pTIL, an experiment similar to that for Super-TIL is conducted (see Example 21).

[0276] (1) Comparison of Cell Killing Effect in the Absence of Added B Cells

[0277] The groups are: control group without 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 conducted.

[0278] 1. Cytokine secretion by T cells. As shown in FIGS. 18A and 18B, in the control Group A1, secretion of IFN-γ and IL-2 is not observed, while in all other groups (Groups A2 - A10), secretion of IFN-γ and IL-2 is observed. Among all the groups, those with Switch (Groups A4 - A10) show higher secretion of IFN-γ and IL-2 than the groups without Switch (Groups A2 and A3).

[0279] 2. Proliferation of tumor cells. As shown in Figure 19A, the number of tumor cells in each group (groups A2 - A10) is decreased compared to the control group (group A1), and the groups with Switch (groups A4 - A10) show a more significant decrease in the number of tumor cells.

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

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

[0282] The groups are: control group without addition of T - cell - 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 assays are performed.

[0283] 1. Proliferation of tumor cells. As shown in Figure 19A, the number of tumor cells in each group (groups B2 - B10) is decreased compared to the control group (group B1), and the normal pTIL group (group B2) shows the least decrease in tumor cells compared to the significant decrease 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) showed a slight increase, while the group with Switch (Groups B4 - B10) showed a significant increase, and furthermore, the group with BCAR (Groups B3 and B7 - B10) showed an even greater increase.

[0285] As shown by in vitro assays, the tumor killing effect of pTILs is improved after transduction with the switch molecule. When transduced with both the Switch and BCAR molecules to provide SuperTIL, the SuperTIL shows an enhanced increase and tumor cell killing effect in the presence of B cells.

[0286] Example 23: Animal Experiment of Super - TIL Animal model: 1 × 10 6 individual tumor cells from a new tumor cell line are inoculated subcutaneously into NSG mice. As a blank control, animals in Group A0 are injected subcutaneously with PBS. Tumors are formed in the animals approximately 2 weeks after injection of the tumor cells. Tumor size is measured 25 days later. 132 mice are selected and divided into 22 groups: Group A (11 subgroups) without B cells and Group B (11 subgroups) with B cells as follows.

[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 are given 1 × 104 T cells via the tail vein.

[0288] Group B consists 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 given 1×10 4 T cells via the tail vein, and all groups were given 1×10 7 B cells by injection.

[0289] Tumor size and the general condition of the mice were measured every 2 - 3 days for 28 days after administration. Tumor size was measured according to the following formula. Tumor size = 1 / 2 × major axis × minor axis × minor axis

[0290] (1) Changes in tumor volume

[0291] This experiment is expected to show that mice injected with TIL having a Switch (Groups A4 - A10 and B4 - B6) exhibit a delay in 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 tumor begins to grow again. The results are expected to show that (i) group B7 (PD1-STIL) tumors do not express PD1 but express TIM3 and TGFBR2; (ii) group B8 (TIM3-STIL) tumors do not express TIM3 but express PD1 and TGFBR2; and (iii) group B9 (TGFBR2-STIL) tumors do not express TGFBR2 but express PD1 and TIM3. In some cases, tumor microenvironment markers can escape, which results in the ineffectiveness of the corresponding Switch.

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

[0295] 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 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 are given 1×10 4 T cells via the tail vein.

[0296] Group B consists 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 given 1×10 4 T cells via the tail vein, and all groups were given 1×10 7 B cells by injection.

[0297] Tumor size and the general condition of the mice were measured every 2 - 3 days for 28 days after administration. Tumor size was measured according to the following formula. Tumor size = 1 / 2×major axis×minor axis×minor axis

[0298] The experiment is expected to show that the SuperTIL / pTIL of the present disclosure has specific tumor recognition and killing effects (from TIL or pTIL) via multiple targets. SuperTIL / pTIL may also show the ability to overcome the tumor environment (from one or more Switches) to improve killing and the self - augmenting ability of BCAR. These modified immune cells provide an effective tumor treatment tool for addressing various problems involved in tumor immunocyte 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 - mediated NY - ESO - 1, the J82 - NY - ESO1 tumor cell line with HLA genotyping of A:0201 was used as a target to measure the killing effect of BCAR - TCR T cells.

[0300] 1×10 5Individual J82-NY-ESO1 tumor cells were seeded onto an RTCA (Real Time Cell Analysis) electrode plate and cultured overnight to allow them to adhere. The cells were divided into three groups, A, B, and C. In group A, 1×10 5 cells, 1×10 4 cells, 1×10 3 cells, 1×10 2 cells of BCAR-TCR T were co-cultured with J82-NY-ESO1 cells, respectively. In group B, 1×10 5 cells, 1×10 4 cells, 1×10 3 cells, 1×10 2 cells of BCAR-TCR T and 1×10 5 cells of B cells were co-cultured with J82-NY-ESO1 cells. Group C was a 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 without B cells, only the highest dose of 1×10 5 cells of BCAR-TCR T showed a significant killing effect on J82-NY-ESO1 tumor cells. On the other hand, in group B, even the lowest dose of 1×10 2 cells of BCAR-TCR T in the presence of B cells showed a significant killing effect on J82-NY-ESO1 tumor cells comparable to that of the 1×10 5 cells of BCAR-TCR T dose in group A, indicating an approximately 1000-fold increase in effect.

[0302] Example 26: Clinical anti-tumor effects of STIL and SpTIL (As shown in Table 1) Five subjects were enrolled in the clinical trial and injected with STIL or SpTIL.

[0303]

Table 1

[0304] As shown in Table 1, the registered subjects have various solid tumors, but all are in the advanced stage, highly progressive with resistance and 3 or more distant metastatic lesions. 3 out of 5 subjects (60%) are resistant to targeted therapy and have developed a poor prognosis TP53 mutation. TMB evaluation 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 before treatment (1) Isolation of TIL / pTIL For Subjects 3, 4, and 5, CD3-positive TILs were isolated from freshly excised tumor tissues after enzymatic digestion using CD3 magnetic beads. For Subjects 1 and 2, PBMCs were separated from the patients, where the amount of PD1 + T cells was 19% and 5% of the total T cells, respectively. Such a high proportion of PD1 + T cells is considered to be tumor tissue-derived TILs (pTILs) in peripheral blood, and PD1 + T cells, i.e., pTILs, were further enriched using PR1 beads to provide them.

[0306] Preparation of STIL / SpTIL A lentiviral vector loaded with PD1sw-CD19 CAR was introduced into TIL / pTIL with an introduction efficiency of 2 - 15%. The cells were encapsulated in an infusion bag without expansion. All processes took 3 - 10 days (except for the process of identifying T cell-recognized neoantigens).

[0307] Treatment with the obtained cells at a dose much lower than the reported dose of 10 8 ~10 9 cells / kg, i.e., at a dose of 10 5 ~10 6 cells / kg, is shown in Table 2.

[0308]

Table 2

[0309] Safety evaluation Among the 5 subjects, grade 1 cytokine release syndrome (CRS) demonstrated by high fever was observed in 3 subjects (incidence 60%, 3 / 5). All 3 subjects recovered, 1 without intervention and the other 2 with treatment by tocilizumab. Both the incidence and grade of CRS were much lower than those of CD19 CAR-T treatment. No autoimmune diseases were observed after treatment.

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

[0311]

Table 3

[0312] Effect of CAR on the in vivo increase of STIL / SpTIL The proportion of CAR+ T cells in the peripheral blood of the subjects was monitored. The magnification of STIL (or SpTIL) increased in the peripheral blood was calculated according to the following formula. Increase rate = number of lymphocytes / L × volume of circulating peripheral blood × proportion of T cells in lymphocytes × proportion of STIL in T cells Here, the number of lymphocytes / L was obtained from a predefined blood test, the proportion of T cells in lymphocytes was determined by flow cytometry as the proportion of CD3+ cells, and the proportion of STIL in T cells was determined by flow cytometry as the proportion of CAR+ cells in CD3+ cells.

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

[0314]

Table 4

[0315] Exogenous immunoglobulins were not administered during the observation period, and no immunodeficiency was observed in any of the subjects.

[0316] Dual-specific recognition of STIL and SpTIL Subjects 1, 2, and 4 were observed for circulating tumor cells (CTCs) in peripheral blood. The number of CTCs 2 months after cell injection was compared with the baseline on the injection day. The results are shown in Table 5 and Figure 25 below, indicating a significant decrease in the number of CTCs.

[0317] [Table 5]

[0318] Improvement of the killing effect by the switch molecule Three subjects (1, 2, and 5) were observed to have decreased peripheral blood T cells and persistent pleural effusion or ascites between days 14 and 28. Subject 4 with pleural metastasis developed pleural effusion, and subjects 1 and 2 with peritoneal metastasis developed ascites. T cells were found in both pleural effusion and ascites at higher concentrations than in peripheral blood, along with IL6. This observation is summarized in Table 6, indicating that the killing effect of STIL / SpTIL was enhanced by the switch molecule.

[0319] [Table 6]

[0320] [Table 7]

[0321] Preferred embodiments of the present invention are shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided for illustrative purposes only. Many variations, modifications, and substitutions can be contemplated by those skilled in the art without departing from the present invention. It should be understood that various alternatives of the embodiments of the present invention described herein can be applied when implementing the present invention. The following claims are intended to define the scope of the present invention, and methods and structures within the scope of these claims and their equivalents are intended to be protected thereby.

Claims

1. A modified T cell that specifically binds to a neoantigen, wherein the modified T cell comprises a switch molecule, wherein the switch molecule comprises an extracellular domain (ECD) of a protein that induces an immune cell inactivation signal in unmodified T cells when bound to its ligand, wherein the ECD is fused to an intracellular domain (ICD) of a costimulatory molecule that mediates an immune cell activation signal, binding of the switch molecule to its ligand results in an immune cell activation signal in the modified T cell instead of the immune cell inactivation signal, the T cell is a cell that expresses endogenous PD-1 obtained from peripheral blood mononuclear cells. Modified T cell.

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

3. The modified T cell according to claim 2, wherein the TCR complex is an endogenous TCR complex.

4. The modified T cell according to claim 2, wherein the TCR complex is an exogenous TCR complex.

5. The modified T cell according to any one of claims 1 to 4, wherein the protein that induces an immune cell inactivation signal in unmodified T cells upon binding to its ligand is a signaling receptor.

6. The modified T cell according to any one of claims 1 to 5, wherein the protein that induces an immune cell inactivation signal in unmodified T cells upon binding to its ligand is a checkpoint receptor, cytokine receptor, chemokine receptor, growth factor receptor, or hormone receptor.

7. The modified T cell according to any one of claims 1 to 6, wherein the protein that induces an immune cell inactivation signal in unmodified T cells upon binding to its ligand is 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.

8. The modified T cell according to any one of claims 1 to 7, 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. (a) An antigen interaction domain capable of binding to a B cell surface protein, (b) A transmembrane domain, and (c) An intracellular signaling domain The modified T cell according to any one of claims 1 to 8, further comprising a chimeric antigen receptor (CAR) containing

10. The modified T cell according to claim 9, wherein the B cell surface protein is selected from CD19, CD20, and CD22.

11. The modified T cell according to claim 9 or 10, wherein upon contact of the T cell with the B cell surface protein, the T cell exhibits enhanced proliferation compared to an unmodified T cell.

12. The modified T cell according to claim 11, wherein the enhanced proliferation is confirmed in vitro or in vivo.

13. (a) Administering the modified T cell to a subject, (b) Contacting the modified T cell-expressing neoantigen with the target cell that is cancer under conditions that induce cytotoxicity of the modified T cell against the target cell that is cancer, thereby inducing death of the target cell that is cancer. The modified T cell according to any one of claims 1 to 12, for use in the treatment of cancer in a subject, comprising

14. (a) Providing a T cell population comprising at least one modified T cell according to any one of claims 9 to 12, (b) Exposing the T cell population to the B cell surface protein so as to effect an increase in the T cell population. A method for expanding a T cell population in vitro, comprising

15. The method according to claim 14, wherein in (b), the T cell population is exposed to B cells containing the B cell surface protein.

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