Modified immune cells and their use

Abstract

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

Description

[Technical Field]

[0001] [Cross-reference of related applications] This application claims the benefits 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, all of which are incorporated herein by reference. [Background technology]

[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, that express chimeric antigen receptors (CARs) can utilize endogenous immune cell signaling for immunocytotoxicity.

[0003] Conventional immunotherapy suffers from various shortcomings. These include insufficient signaling from co-stimulatory receptors for sustained and / or appropriate immune responses for therapeutic effect, insufficient specificity of modified immune cells for disease cells such as cancer cells (e.g., on-target off-tumor effects and toxicity), and activation of immunosuppressive mechanisms, all of which can minimize the effectiveness of the immune response. [Overview of the project]

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

[0005] In one aspect, the present disclosure provides modified immune cells that specifically bind to tumor antigens, wherein the modified immune cells comprise 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 unmodified immune cells 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 chimeric stimulatory molecule to its ligand results in an immune cell activation signal instead of an immune cell inactivation signal in the modified immune cells.

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

[0007] In one aspect, the present disclosure provides modified T cells that specifically bind to neoantigens, wherein the modified T cells comprise a switch molecule, wherein the switch molecule comprises an extracellular domain (ECD) of a protein that induces an immune cell activation signal in unmodified T cells 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 instead of an immune cell inactivation signal in the modified immune cells.

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

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

[0011] In some embodiments, the protein that, upon binding to its ligand, induces an immune cell inactivation signal in an unmodified TIL or unmodified T cell may be a signaling receptor. In some embodiments, the protein that, upon binding to its ligand, induces an immune cell inactivation signal in an unmodified TIL or unmodified T cell may be a checkpoint receptor, a cytokine receptor, a chemokine receptor, a growth factor receptor, or a hormone receptor. In some embodiments, proteins that induce immune cell inactivation signals in unmodified TILs or unmodified T cells upon binding to their ligands 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 attenuators (BTLA), killer cell immunoglobulin-like receptor (KIR), indoleamine-2,3-dioxygenase (IDO), lymphocyte activator 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, immune cell activation signals may be mediated by activators. 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, where 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 functional fragments or variants thereof. In some embodiments, immune cell activation signals may include clonal proliferation 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, when a switch molecule is bound to a ligand, modified TILs or modified T cells may exhibit enhanced neoantigen binding compared to unmodified TILs or unmodified T cells.

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

[0016] In some embodiments, modified TILs or modified T cells may exhibit increased cytokine secretion compared to unmodified TILs or unmodified T cells when a switch molecule binds to a ligand and the modified TILs or modified T cells bind to neoantigens present on target cells. In some embodiments, the cytokine may be IFN-γ or IL-2.

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

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

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

[0020] In some embodiments, neoantigens may be selected based on the genetic profile of tumor samples from an individual. In some embodiments, neoantigens may be selected based on the somatic mutation profile of tumor samples 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-activating tyrosine motif (ITAM). In some embodiments, the intracellular signaling domain may include an immunoreceptor-suppressing tyrosine motif. 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, CD3ζ, CD3γ, CD3δ, CD3ε, CD4, CD5, CD8, CD21, CD22, CD28, CD32, CD40L (CD154), CD45, CD66d, CD79a, CD79b, CD80, CD86, CD278 (also known as ICOS), CD247ζ, CD247η, 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 CD3ζ. In some embodiments, the CAR may further include a co-stimulatory domain. In some embodiments, the co-stimulatory domain may include signaling for MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activators (SLAM proteins), activated NK cell receptors, or Toll ligand receptors.

[0024] In some embodiments, the co-stimulatory domains are 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 Ligands: TNFSF7, CD27 / TNFRSF7, CD28, CD29, CD2F-10 / SLAMF9, CD30; TNFSF8, CD30 / TNFRSF8, CD300a / LMIR1, CD4, CD40; TNFSF5, CD40 / TNFRSF5, CD48 / SLAMF2, CD49a, CD49D, CD49f, CD53, CD58 / LFA-3, CD69, CD7, CD8α, CD8β, CD82 / Kai-1, CD84 / SLAMF5, CD90 / Thy1, CD96, CDDS, 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, NKp80The signaling domain may include 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, contact of immune cells with B cell surface proteins may cause the immune cells to exhibit enhanced proliferation compared to unmodified immune cells. In some embodiments, enhanced proliferation may be confirmed in vitro. In some embodiments, enhanced proliferation may be confirmed in vivo. In some embodiments, immune cells may show at least a twofold increase in proliferation at least about 24, 48, or 96 hours after contact compared to unmodified immune cells.

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

[0027] In one embodiment, the 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, upon binding to its ligand, induces an immune cell inactivation signal in an unmodified immune cell, wherein the ECD is fused to an intracellular domain (ICD) of a co-stimulatory molecule that mediates an immune cell activation signal, and the 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 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.

[0028] In some embodiments, immune cells may include T cell receptor (TCR) complexes that exhibit specific binding to neoantigens. In some embodiments, the TCR complex may be an endogenous TCR complex. In some embodiments, the TCR complex may be an exogenous TCR complex.

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

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

[0031] In some embodiments, the protein that, upon binding to its ligand, induces an immune cell inactivation signal in an unmodified TIL or unmodified immune cell may be a signaling receptor. In some embodiments, the protein that, upon binding to its ligand, induces an immune cell inactivation signal in an unmodified TIL or unmodified immune cell may be a checkpoint receptor, cytokine receptor, chemokine receptor, growth factor receptor, or hormone receptor. In some embodiments, the protein that, upon binding to its ligand, induces an immune cell inactivation signal in unmodified TILs or unmodified immune cells 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 attenuators (BTLA), killer cell immunoglobulin-like receptor (KIR), indoleamine-2,3-dioxygenase (IDO), lymphocyte activator 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 activity signals can be mediated by activators. In some embodiments, the activator may be a soluble cytokine, a soluble chemokine, or a growth factor. In some embodiments, the activator is a soluble cytokine, where the soluble cytokine may be IL-1, IL-2, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IL-21, TNF, TGF, IFN, or functional fragments or variants thereof.

[0034] In some embodiments, the immune cell activation signal may include clonal proliferation and 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 may be selected from CD19, CD20, and CD22.

[0036] In some embodiments, the intracellular signaling domain may include an immunoreceptor-activating tyrosine motif (ITAM). In some embodiments, the intracellular signaling domain may include an immunoreceptor-suppressing 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, CD3ζ, CD3γ, CD3δ, CD3ε, CD4, CD5, CD8, CD21, CD22, CD28, CD32, CD40L (CD154), CD45, CD66d, CD79a, CD79b, CD80, CD86, CD278 (also known as ICOS), CD247ζ, CD247η, 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 CD3ζ. In some embodiments, the intracellular domain of CD3ζ may include an immunoreceptor-activated tyrosine motif (ITAM). The CAR may further include a costimulatory domain. In some embodiments, the costimulatory domain may include signaling for MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte-activating molecules (SLAM proteins), activated NK cell receptors, or Toll ligand receptors.

[0038] In some embodiments, the co-stimulatory domains are 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 Ligands: TNFSF7, CD27 / TNFRSF7, CD28, CD29, CD2F-10 / SLAMF9, CD30; TNFSF8, CD30 / TNFRSF8, CD300a / LMIR1, CD4, CD40; TNFSF5, CD40 / TNFRSF5, CD48 / SLAMF2, CD49a, CD49D, CD49f, CD53, CD58 / LFA-3, CD69, CD7, CD8α, CD8β, CD82 / Kai-1, CD84 / SLAMF5, CD90 / Thy1, CD96, CDDS, 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, NKp80The signaling domain may include 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, when a switch molecule is bound to a ligand, modified TILs or modified immune cells may exhibit enhanced neoantigen binding compared to unmodified TILs or unmodified immune cells.

[0040] In some embodiments, modified TILs or modified T cells may exhibit increased cytotoxicity to target cells compared to unmodified TILs or unmodified T cells when a switch molecule binds to a ligand and the modified TILs or modified immune cells bind to neoantigens present on the target cells.

[0041] In some embodiments, modified TILs or modified immune cells may exhibit increased cytokine secretion compared to unmodified TILs or unmodified immune cells when a switch molecule binds to a ligand and the modified TILs or modified immune cells bind to neoantigens present on target cells. In some embodiments, the cytokines may be IFN-γ or IL-2.

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

[0043] In one embodiment, the disclosure provides a method for increasing a T cell population, the method comprising (a) providing a T cell population comprising at least one modified immune cell as described in any one of claims 21 to 40, and (b) exposing the T cell population to B cell surface proteins such that they are effective in increasing the T cell population. In some embodiments, the T cell population may be exposed to B cells comprising B cell surface proteins.

[0044] In one embodiment, the Disclosure provides a method for increasing a T cell population, comprising the steps of (a) introducing a nucleic acid encoding a chimeric antigen receptor (CAR) into a T cell population to produce a primary CAR-expressing cell population, wherein the CAR comprises (i) an antigen-interacting domain capable of binding to a B cell surface protein; (ii) a transmembrane domain; and (iii) an intracellular signaling domain; and (b) contacting the primary CAR-expressing cell population with a B cell surface protein to produce an increased and / or activated immune cell population.

[0045] In one embodiment, the Disclosure provides a composition comprising (a) a switch molecule comprising an extracellular domain (ECD) of a protein that, upon binding to its ligand, induces an immunoinactivation signal in unmodified immune cells, 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 chimeric antigen receptors, which include (a) an antigen-specific T cell receptor complex or one or more components thereof, and (b) (i) an antigen-interacting domain capable of binding to a B cell surface protein; (ii) a transmembrane domain; and (iii) an intracellular signaling domain.

[0047] In some embodiments, the neoantigen may include a peptide fragment of a protein encoded by a mutant gene, where the gene is ABL1, ACOl 1997, ACVR2A, AFP, AKT1, ALK, ALPPL2, ANAPC1, APC, ARID1A, AR, AR-v7, ASCL2, β2M, BRAF, BTK, C15ORF40, CDH1, CLDN6, CNOT1, CT45A5, CTAG1B, DCT, DKK4, EEF1B2, EEF1DP3, EGFR, EIF2B3, env, EPHB2, ERBB3, ESR1, ESRP1, FAM11 IB, FGFR3, FRG1B, GAGE1, GAGE ​​10, GATA3, GBP3, HER2, IDH1, JAK1, KIT, KRAS, LMAN1, MABEB Selected from 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, upon binding to its ligand, induces an immune cell inactivation signal in an unmodified TIL or unmodified T cell may be a signaling receptor. In some embodiments, the protein may be a checkpoint receptor, cytokine receptor, chemokine receptor, growth factor receptor, or 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 mediating the immune cell activation signal may be interleukin-2 receptor (IL-2R), interleukin-12 receptor (IL-12R), CD2, CD3, CD4, CD7, CD8, CD27, CD28, CD30, CD40, 4-1BB / CD137, ICOS, lymphocyte function-associated antigen-1 (LFA-1), LIGHT, NKG2C, or OX40.

[0050] In some embodiments, the B cell surface protein may be selected from CD19, CD20, and CD22. In some embodiments, the intracellular signaling domain may include an immunoreceptor-suppressive tyrosine motif. 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, CD3ζ, CD3γ, CD3δ, CD3ε, CD4, CD5, CD8, CD21, CD22, CD28, CD32, CD40L (CD154), CD45, CD66d, CD79a, CD79b, CD80, CD86, CD278 (also known as ICOS), CD247ζ, CD247η, 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 include a co-stimulatory domain. In some embodiments, the co-stimulatory domain may be 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 Ligands: TNFSF7, CD27 / TNFRSF7, CD28, CD29, CD2F-10 / SLAMF9, CD30; TNFSF8, CD30 / TNFRSF8, CD300a / LMIR1, CD4, CD40; TNFSF5, CD40 / TNFRSF5, CD48 / SLAMF2, CD49a, CD49D, CD49f, CD53, CD58 / LFA-3, CD69, CD7, CD8α, CD8β, CD82 / Kai-1, CD84 / SLAMF5, CD90 / Thy1, CD96, CDDS, 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 The signaling domain may be a molecule selected from the group consisting of (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 embodiment, the Disclosure provides a composition comprising one or more polynucleotides encoding one or more chimeric antigen receptors comprising (a) a switch molecule, the switch molecule comprising an extracellular domain (ECD) of a protein that, upon binding to its ligand, induces an immunoinactivation signal in unmodified immune cells, the ECD being fused to an intracellular domain (ICD) of a costimulatory protein that mediates an immune cell activation signal; (b) an antigen-specific T cell receptor complex, or one or more components thereof; and (c) one or more polynucleotides encoding one or more chimeric antigen receptors comprising (i) an antigen-interacting domain capable of binding to a B cell surface protein; (ii) a transmembrane domain; and (iii) an intracellular signaling domain.

[0053] In one embodiment, the disclosure provides a modified tumor-infiltrating lymphocyte (TIL) that specifically binds to a neoantigen, comprising a chimeric stimulating molecule, wherein the chimeric stimulating molecule comprises 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 the binding of the chimeric stimulating molecule to the neoantigen results in an immune cell activation signal in the modified TIL.

[0054] In one embodiment, the disclosure provides (a) a switch molecule comprising an extracellular domain (ECD) of a protein that, upon binding to its ligand, induces an immunoinactivation signal in unmodified immune cells, wherein the ECD is fused to an intracellular domain (ICD) of a co-stimulatory molecule that mediates an immune cell activation signal, and the binding of the switch molecule to its ligand results in an immune cell activation signal instead of an immune cell inactivation signal in modified immune cells; and (b) a modified immune cell comprising 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, modified immune cells may express at least one of PD1, CD137, and TIM-3. In some embodiments, immune cells may be obtained from a tumor. In some embodiments, immune cells may be obtained from the peripheral blood of mononuclear cells. In some embodiments, immune cells may contain an exogenous TCR complex. In some embodiments, the TCR complex may bind to tumor cells. In some embodiments, the TCR complex may bind to a neoantigen. 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, a cytokine receptor, a chemokine receptor, a growth factor receptor, or a hormone receptor. In some embodiments, the protein that induces an immune cell inactivation signal in unmodified immune cells upon binding to its ligand may be selected from the group consisting of transforming growth factor-β receptor (TGF-β-R), programmed cell death 1 (PD-1), T cell co-stimulatory receptor 4 (CTLA-4), B and T lymphocyte attenuators (BTLA), killer cell immunoglobulin-like receptor (KIR), indoleamine-2,3-dioxygenase (IDO), lymphocyte activator 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, a soluble chemokine, or a growth factor. In some embodiments,The activator is a soluble cytokine, which may be IL-1, IL-2, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IL-21, TNF, TGF, IFN, or functional fragments or variants thereof. In some embodiments, the immune cell activation signal may include clonal proliferation 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 may be selected from CD19, CD20, and CD22. In some embodiments, the intracellular signaling domain may include an immunoreceptor-activated tyrosine motif (ITAM). In some embodiments, the intracellular signaling domain may include an immunosuppressive tyrosine motif (ITIM). In some embodiments, the intracellular signaling domain may include Fcγ receptor (FcγR), Fcε receptor (FcεR), FCα receptor (FcαR), neonatal Fc receptor (FcRn), CD3, CD3ζ, CD3γ, CD3δ, CD3ε, CD4, CD5, CD8, CD21, CD22, CD28, CD32, CD40L (CD154), CD45, CD66d, CD79a, CD79b, CD80, CD86, CD278 (also known as ICOS), CD247ζ, CD247 The CAR may include intracellular domains of molecules selected from η, DAP10, DAP12, FYN, LAT, Lck, MAPK, MHC complex, NFAT, NF-κB, PLC-γ, iC3b, C3dg, C3d, and Zap70. In some embodiments, the intracellular signaling domain may include the intracellular domain of CD3ζ. In some embodiments, the intracellular domain of CD3ζ may include an immunoreceptor-activated tyrosine motif (ITAM). The CAR may further include:The molecule may also contain a co-stimulatory domain. In some embodiments, the co-stimulatory domain may include signaling for MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activators (SLAM proteins), activated NK cell receptors, or Toll ligand receptors. In some embodiments, the co-stimulatory domains are 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 Ligands: TNFSF7, CD27 / TNFRSF7, CD28, CD29, CD2F-10 / SLAMF9, CD30; TNFSF8, CD30 / TNFRSF8, CD300a / LMIR1, CD4, CD40; TNFSF5, CD40 / TNFRSF5, CD48 / SLAMF2, CD49a, CD49D, CD49f, CD53, CD58 / LFA-3, CD69, CD7, CD8α, CD8β, CD82 / Kai-1, CD84 / SLAMF5, CD90 / Thy1, CD96, CDDS, 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 The system may contain signaling domains of molecules selected from the group consisting of (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, contact of the immune cells with the B cell surface protein may cause the immune cells to 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 show at least a twofold increase in proliferation at least about 24, 48, or 96 hours after contact compared to unmodified immune cells.

[0056] [Inclusion by reference] All publications, patents, and patent applications referenced herein are incorporated by reference to the same degree as each individual publication, patent, or patent application is incorporated by reference specifically and individually.

[0057] Novel features of the present invention are specifically described in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by referring 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 explanation of the drawing]

[0058] [Figure 1] Figure 1 shows the T cell receptor (TCR) expression in T cells with and without transduction of the NY-ESO-1 TCR gene. [Figure 2] Figure 2 shows the process for preparing neoantigen-reactive (or recognizable) T cells. [Figure 3A] Figure 3A shows the lentivir titration of the PD1 / CD28 switch molecule and demonstrates flow cytometry detection of PD1 expression. [Figure 3B] Figure 3B shows the lentivir titration of the PD1 / CD28 switch molecule, and displays the titration curve. [Figure 4] Figure 4 shows the expression of PD1 / CD28 switch molecules in TDR-1, TIL, and neoT cells. [Figure 5] Figure 5 shows the assay for the J82-NY-ESO-1-PDL1 bladder cancer cell line. [Figure 6A] Figure 6A shows the expression of CD107a in T cells cultured together with tumor cells in an in vitro assay of TCR-T cells expressing a PD1 / CD28 switch molecule that targets NY-ESO-1. [Figure 6B] Figure 6B shows the release of IFN-γ in T cells cultured with tumor cells in an in vitro assay of TCR-T cells expressing a PD1 / CD28 switch molecule targeting NY-ESO-1. [Figure 6C]Figure 6C shows the release of IL-2 in T cells cultured with tumor cells in an in vitro assay of TCR-T cells expressing a PD1 / CD28 switch molecule that targets NY-ESO-1. [Figure 7A] Figure 7A shows the release of IFN-γ by TIL cells with and without the PD1 / CD28 switch molecule, when cultured in the presence or absence of tumor cells. [Figure 7B] Figure 7B shows the release of IL-2 by TIL cells with and without the PD1 / CD28 switch molecule, when cultured in the presence or absence of tumor cells. [Figure 8A] Figure 8A shows the release of IFN-γ in neoantigen-reactive T cells (neoTs) with and without the PD1 / CD28 switch molecule, when cultured in the presence or absence of tumor cells. [Figure 8B] Figure 8B shows the release of IL-2 in neoantigen-reactive T cells (neoTs) with and without the PD1 / CD28 switch molecule, when cultured in or without tumor cells. [Figure 9] Figure 9 shows the expression of chimeric antigen receptors targeting B cell surface proteins (BCARs) in TCR-T, TIL, and neoT cells. [Figure 10] Figure 10 shows the in vitro effect and increase of NY-ESO-1-TCR-T cells expressing BCAR. [Figure 11] Figure 11 shows the in vitro effect and increase of NY-ESO-1-TCR-T cells expressing BCAR. [Figure 12A] Figure 12A shows the in vitro effect and increase of TIL cells expressing BCAR. [Figure 12B] Figure 12B shows the in vitro effect and increase of TIL cells expressing BCAR. [Figure 13A]Figure 13A shows the in vitro effect and increase of NeoT cells expressing BCAR. [Figure 13B] Figure 13B shows the in vitro effect and increase of NeoT cells expressing BCAR. [Figure 14] Figure 14 shows the expression of PD1sw-BCAR, TIM3sw-BCAR, and TGFBR2sw-BCAR in TIL. [Figure 15] Figure 15 shows the expression of PD1sw-BCAR, TIM3sw-BCAR, and TGFBR2sw-BCAR in pTIL. [Figure 16A] Figure 16A shows the release of IFN-γ from various TILs in the absence of B cells. [Figure 16B] Figure 16B shows the release of IL-2 from various TILs in the absence of B cells. [Figure 17A] Figure 17A shows the tumor-killing effects of various TILs in the absence of B cells (Group A) or in the presence of B cells (Group B). [Figure 17B] Figure 17B shows the in vitro increase of various TILs in the absence of B cells (Group A) or in the presence of B cells (Group B). [Figure 18A] Figure 18A shows the release of IFN-γ from various TILs in the absence of B cells. [Figure 18B] Figure 18B shows the release of IL-2 from various TILs in the absence of B cells. [Figure 19A] Figure 19A shows the tumor-killing effects of various pTILs in the absence of B cells (Group A) or in the presence of B cells (Group B). [Figure 19B] Figure 17B shows the in vitro increase of various pTILs in the absence of B cells (Group A) or in the presence of B cells (Group B). [Figure 20] Figure 20 shows the killing effect of BCAR-TCRT on J82-NY-ESO1 tumor cells in the absence or presence of B cells. [Figure 21A] Figure 21A shows the tumor image analysis of subjects 1-4. [Figure 21B] Figure 21B shows the tumor image analysis of subjects 1-4. [Figure 21C] Figure 21C shows the tumor image analysis of subjects 1-4. [Figure 21D] Figure 21D shows the tumor image analysis of subjects 1-4. [Figure 22A] Figure 22A shows the tumor image analysis of subjects 1-4. [Figure 22B] Figure 22B shows the tumor image analysis of subjects 1-4. [Figure 22C] Figure 22C shows the tumor image analysis of subjects 1-4. [Figure 22D] Figure 22D shows the tumor image analysis of subjects 1-4. [Figure 23A] Figure 23A shows the tumor image analysis of subjects 1-4. [Figure 23B] Figure 23B shows the tumor image analysis of subjects 1-4. [Figure 23C] Figure 23C shows the tumor image analysis of subjects 1-4. [Figure 23D] Figure 23D shows the tumor image analysis of subjects 1-4. [Figure 24A] Figure 24A shows the tumor image analysis of subjects 1-4. [Figure 24B] Figure 24B shows the tumor image analysis of subjects 1-4. [Figure 24C] Figure 24C shows the tumor image analysis of subjects 1-4. [Figure 25] Figure 25 shows the changes in the number of circulating tumor cells (CTCs) in peripheral blood after infusion of STIL or SpTIL. [Modes for carrying out the invention]

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

[0060] As used in specifications and claims, the singular forms “a,” “an,” and “the” also include the plural unless the context clearly indicates otherwise. For example, the term “a switch molecule” includes multiple switch molecules.

[0061] The terms “about” or “approximately” mean within an acceptable margin of error for a particular value as determined by those skilled in the art, and this will depend in part on how the value is measured or determined, i.e., the limits of the measuring system. For example, “about” could mean that the standard deviation per run in the art is 1 or greater than 1. Alternatively, “about” could mean a range of up to 20%, 10%, 5%, or 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term could mean within one order of magnitude, preferably within five times the value, and more preferably within twice the value. Where specific values ​​are given in the specification and claims, unless otherwise stated, the term “about” should be presumed to mean within an acceptable margin of error for the particular value.

[0062] As used herein, “cell” generally refers to a biological cell. A cell can be the basic structural, functional, and / or biological unit of a living organism. A cell can originate from any organism that has one or more cells. Some specific examples include prokaryotic cells; eukaryotic cells; bacterial cells; archaeal cells; cells of unicellular eukaryotes; protozoan cells; cells of plant origin (e.g., cells from crops, fruits, vegetables, grains, soybeans, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkins, hay, potatoes, cotton, hemp, tobacco, flowering plants, conifers, gymnosperms, ferns, clubmosses, hornworts, bryophytes, mosses, etc.); algal cells (e.g., Botryococcus brownii, green algae, Nannochloropsis gaditana, Chlorella). Examples include cells derived from pyrenoids (e.g., Ephemerides), seaweed (e.g., kelp), fungal cells (e.g., yeast cells, fungal fossils), animal cells, cells from invertebrates (e.g., fruit flies, cnidarians, echinoderms, nematodes), cells from vertebrates (e.g., fish, amphibians, reptiles, birds, mammals), and cells from mammals (e.g., pigs, cows, goats, sheep, rodents, rats, mice, non-human primates, humans). Often, cells are not derived from natural organisms (for example, cells may be synthetically produced, which are sometimes called artificial cells).

[0063] As used herein, the term "antigen" means a molecule or fragment thereof that can be bound by a selective binder. For example, an antigen may be a ligand that can be bound by a selective binder, such as a receptor. Another example is an antigenic molecule that can be bound by a selective binder, such as an immunological protein (e.g., an antibody). An antigen may also mean a molecule or fragment thereof that can be used in an object to produce an antibody that can bind to that antigen.

[0064] As used herein, the term "neoantigen" generally refers to tumor-specific antigens resulting from gene mutations. The resulting mutant proteins, or fragments thereof, can trigger an antitumor T cell response.

[0065] As used herein, the term “gene” means a nucleic acid (such as DNA and cDNA, e.g., genomic DNA) and its corresponding nucleotide sequence that is involved in encoding an RNA transcript. In relation to genomic DNA, the term as used herein includes a regulatory region along with intervening untranslated regions, and may include the 5' and 3' ends. In some cases, the term includes the transcription region, including the 5' and 3' untranslated regions (5'-UTR and 3'-UTR), exons, and introns. In some genes, the transcription region will include an “open reading frame” that encodes a polypeptide. In some uses of the term, “gene” includes only the coding sequence (e.g., “open reading frame” or “coding region”) necessary to encode a polypeptide. In some cases, a gene does not encode a polypeptide, such as ribosomal RNA genes (rRNA) and transfer RNA (tRNA) genes. In some cases, the term “gene” includes not only the transcription sequence but also, additionally, an untranslated region, including upper and lower regulatory sequences, enhancers, and promoters. The term "gene" can mean an "endogenous gene," or a naturally occurring gene located in its natural position within the genome of an organism. It can also mean an "exogenous gene," or a non-natural gene. A non-natural gene can mean a gene that is not normally found in a host organism but is introduced into the host organism through gene transfer. A non-natural gene can also mean a gene that is not in its natural position within the genome of an organism. A non-natural gene can also mean a naturally occurring nucleic acid, or a polypeptide (e.g., a non-natural sequence) containing mutations, insertions, and / or deletions.

[0066] As used herein, the term “antibody” refers to a protein-binding molecule having immunoglobulin-like function. The term “antibody” includes multiple antibodies (e.g., monoclonal antibodies and polyclonal antibodies), and their derivatives, variants, and fragments. Antibodies are, but are not limited to, immunoglobulins (IGs) of various classes (i.e., IgA, IgG, IgM, IgD, and IgE) and subclasses (e.g., IgG1, IgG2, etc.). Their derivatives, variants, and fragments may refer to functional derivatives or fragments that retain the binding specificity (e.g., complete and / or partial) of the corresponding antibody. Antigen-binding fragments include Fab, Fab', F(ab')2, variable fragments (Fv), single-chain variable post-fragment evant (scFv), minibodies, bispecific antibodies, and single-domain antibodies ("sdAb", "nanobodies", or "camelids"). The term "antibody" includes antibodies and antigen-binding fragments of optimized, manipulated, or chemically conjugated antibodies. Examples of optimized antibodies include affinity-mature antibodies. Examples of manipulated antibodies include Fc-optimized antibodies (e.g., antibodies optimized in the crystalline fragment region) and multispecific antibodies (e.g., bispecific antibodies).

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

[0068] The terms “polynucleotide,” “oligonucleotide,” and “nucleic acid” are used interchangeably to mean any polymerized form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or their analogues, in single-stranded, double-stranded, or multi-stranded forms. Polynucleotides may be endogenous or exogenous to cells. Polynucleotides may exist in a cell-free environment. Polynucleotides may be genes or fragments thereof. Polynucleotides may be DNA. Polynucleotides may be RNA. Polynucleotides may have any three-dimensional structure and may perform any function, whether known or not. Polynucleotides may contain one or more analogues (e.g., altered backbone, sugars, or bases). If present, modifications to the nucleotide structure may be given before or after polymer association. Some non-exclusive examples of analogs include 5-bromouracil, peptide nucleic acids, heteronucleotides, morpholinos, cross-linked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordicepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein-conjugated sugars), thiol-containing nucleotides, biotin-conjugated nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, cuosin, and waiosin.Examples of non-limiting polynucleotides include coding or non-coding regions of genes or gene fragments, loci (locus) defined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small interfering RNA (siRNA), small hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA with arbitrary sequences, isolated RNA with arbitrary sequences, 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" refers to one or more processes by which polynucleotides are 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 peptides, polypeptides, or proteins. Transcripts and encoded polypeptides may collectively be referred to as "gene products." When polynucleotides are derived from genomic DNA, expression may include the splicing of mRNA within eukaryotic cells. With respect to expression, "up-regulated" generally means an increased level of expression of polynucleotides (e.g., RNA such as mRNA) and / or polypeptides relative to their wild-type expression level, while "down-regulated" generally means a reduced level of expression of polynucleotides (e.g., RNA such as mRNA) and / or polypeptides relative to their wild-type expression level.

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

[0071] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein to mean a polymer of at least two amino acids linked by peptide bonds (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 is produced using recombinant techniques, chemical or enzymatic synthesis, or of natural origin. The term applies to naturally occurring amino acid polymers as well as to amino acid polymers containing at least one modified amino acid. The term includes amino acid chains of any length, including full-length proteins, or proteins with or without secondary and / or tertiary structures (e.g., domains). The term also includes amino acid polymers modified by any other operation, such as disulfide bond formation, glycosylation, lipid modification, acetylation, phosphorylation, oxidation, and, for example, agreement with labeling components. As used herein, the terms “amino acid” and “amino acids” generally refer to natural or non-natural amino acids, and include, but are not limited to, modified amino acids and amino acid analogs. Modified amino acids may include natural amino acids and non-natural amino acids that have been chemically modified to include groups or chemical sites that do not exist naturally. Amino acid analogs may refer to amino acid derivatives. The term “amino acid” includes both D-amino acids and L-amino acids.

[0072] Where used herein in relation to polypeptides, the terms “derivative,” “variant,” and “fragment” mean polypeptides that are related to the wild-type polypeptide by any of the following: amino acid sequence, structure (e.g., secondary and / or tertiary), activity (e.g., enzymatic activity), and / or function. Polypeptide derivatives, variants, and fragments may include one or more differences (e.g., mutations, insertions, and deletions), truncations, modifications, or combinations thereof compared to the wild-type polypeptide.

[0073] As used herein, “fusion” may mean a protein and / or nucleic acid containing one or more non-natural sequences (e.g., sites). A fusion may contain one or more identical non-natural sequences. A fusion may contain one or more different non-natural sequences. A fusion may be a chimera. A fusion may contain nucleic acid affinity tags. A fusion may contain barcodes. A fusion may contain peptide affinity tags. A fusion may provide site-specific intracellular localization of polypeptides (e.g., nuclear localization signals (NLS) targeting the nucleus, mitochondrial localization signals targeting mitochondria, chloroplast localization signals targeting chloroplasts, endoplasmic reticulum (ER) retention signals, etc.). A fusion may provide non-natural sequences (e.g., affinity tags) that can be used for tracking or purification. A fusion may be a biotin or a small molecule such as a dye, such as Alexa fluor® dye, cyanine 3 dye, cyanine 5 dye, etc.

[0074] As used herein, the phrases “exogenous T cell receptor (TCR) complex” or “exogenous TCR complex” mean a TCR complex in which one or more chains of the TCR are 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, for example, at either the nucleic acid level or the amino acid level. Expression of exogenous TCRs in immune cells may confer specificity of binding to epitopes or antigens (e.g., epitopes or antigens that preferentially exist on the surface of cancer cells or other pathogenic cells or particles). An exogenous TCR complex may include TCR-α, TCR-β chains, CD3-γ chains, CD3-δ chains, CD3-ζ chains, or any combination thereof, introduced into the genome. In some cases, the strands introduced into the genome may replace endogenously occurring strands.

[0075] The terms “subject,” “individual,” and “patient” are used herein to mean vertebrates, preferably mammals such as humans. Mammals include, but are not limited to, mice, monkeys, humans, livestock, sports animals, and pets. Tissues, cells, and offspring of biological entities obtained in vivo or cultured in vitro are also included.

[0076] As used herein, the terms “treatment” and “treating” mean an approach to obtain beneficial or desired results, including, but not limited to, therapeutic and / or preventive benefits. For example, treatment may include administering any system or cell population disclosed herein. Therapeutic benefit means any improvement or effect, under treatment, relating to any treatment of one or more diseases, health conditions, or symptoms. For preventive benefit, a composition may be administered to a subject at risk of developing a particular disease, health condition, or symptom, or to a subject who has not yet developed a disease, health condition, or symptom but has reported one or more physiological symptoms of a disease.

[0077] The terms “effective amount” or “therapeutic effective amount” mean an amount of a composition, such as a composition containing immune cells (e.g., T lymphocytes and / or NK cells) as described herein, that is sufficient to produce the desired activity when administered to a subject in need. In the context of this disclosure, the term “therapeutic effective” means an amount of a composition that is sufficient to delay the progression of symptoms, halt exacerbation, or alleviate or reduce at least one symptom of the disease treated by the methods described herein.

[0078] As used herein, the term "genetic profile" refers to information about a particular gene, including its diversity and gene expression in an individual or in certain tissues. As used herein, the term "somatic mutation profile" refers to information about a particular gene associated with a somatic mutation, but is not limited to these two. Somatic mutation profiles may be used in neoantigen selection.

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

[0080] In one embodiment, the disclosure provides a modified tumor-infiltrating lymphocyte (TIL) that specifically binds to a neoantigen, wherein the modified TIL comprises a switch molecule. The switch molecule may comprise an extracellular domain (ECD) of a protein that, upon binding to its ligand, induces an immunoinactivation signal in unmodified immune cells. The ECD may fuse to an intracellular domain (ICD) of a co-stimulatory 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.

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

[0082] In one embodiment, the disclosure provides a modified T cell that specifically binds to a neoantigen, wherein the modified T cell comprises a switch molecule. The switch molecule may comprise an extracellular domain (ECD) of a protein that, upon binding to its ligand, induces an immune cell activation signal in an unmodified T cell. The ECD may be fused to an intracellular domain (ICD) of a co-stimulatory molecule that mediates the 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] Modified T cells may contain a T cell receptor (TCR) complex that exhibits specific binding to neoantigens. 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 may confer antigen-binding specificity (e.g., neoantigen binding) to the immune cell. In some embodiments, the disclosure provides modified T cells containing an endogenous TCR complex that specifically binds to neoantigens, wherein the modified T cell contains a chimeric stimulating molecule, the chimeric stimulating molecule comprising, but not limited to, an extracellular polypeptide domain (PED) that binds to a membrane protein of a cell, including tumor cells, wherein the PED is fused to an intracellular domain (ICD) of a co-stimulatory molecule that mediates an immune cell activation signal, and the binding of the chimeric stimulating molecule to the membrane protein results in the immune cell activation signal in the modified T cell.

[0084] The binding of modified immune cells, such as modified T cells or modified TILs, to neoantigens, as provided herein, can activate immune cells. Switch molecules for modified cells can be used to provide further control of immune cell activity, such as immune cell activation and proliferation, but are not limited to these. 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 instead of an immune cell inactivation signal in modified immune cells can minimize the immunosuppressive effect in immune cells. Minimizing the immunosuppressive effect in immune cells can increase the effect of immune cells in the immune response, for example, by increasing immunocytotoxicity against target cells, such as tumor cells.

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

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

[0087] In some embodiments, the switch molecule includes at least an extracellular fragment of the TCR that can be involved in recognizing a target cell's neoantigen (e.g., cancer cell antigen or tumor antigen). In some examples, the switch molecule may include extracellular variable regions of the TCR α and / or β chains.

[0088] A switch molecule containing an immune checkpoint receptor, or any derivative, variant, or fragment thereof, can bind to any suitable immune checkpoint receptor ligand or antigen containing its derivative, variant, or fragment. Non-exclusive examples of such ligands include, 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 includes a cytokine receptor fragment. Cytokine receptors can perform a variety of functions, but non-limiting examples include immune cell regulation and inflammation mediation. In some embodiments, the switch molecule may be 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 interleukin receptors (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), colony-stimulating factor receptors (e.g., erythropoietin receptor, CSF-1R, CSF-2R, GM-CSFR, and G-CSFR), hormone receptors / neuropeptide receptors (e.g., growth hormone receptor, protein receptor, and leptin receptor), or derivatives, variants, or fragments thereof. In some embodiments, the switch molecule includes type II cytokine receptors, or derivatives, variants, or fragments thereof. In some embodiments, the switch molecule includes an interferon receptor (e.g., IL-10R, IL-20R, IL-22R, and IL-28R), 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 derivatives, variants, or fragments thereof.

[0090] In some embodiments, the switch molecule comprises at least the extracellular domain (e.g., ligand-binding domain) of a catalytic receptor such as a receptor tyrosine kinase (RTK), or a derivative, variant, or fragment thereof. In some embodiments, the switch molecule comprises a class I RTK (e.g., the epidermal growth factor (EGF) receptor family, including EGFR;Class II RTKs (e.g., the ErbB family including ErbB-2, ErbB-3, and ErbB-4), Class III RTKs (e.g., the insulin receptor family including INSR, IGF-1R, and IRR), Class IV RTKs (e.g., the platelet-derived factor (PDGF) receptor family including DGFR-α, PDGFR-β, CSF-1R, KIT / SCFR, and FLK2 / FLT3), Class IV RTKs (e.g., the fibroblast growth factor (FGF) receptor family including FGFR-1, FGFR-2, FGFR-3, and FGFR-4), Class V RTKs (e.g., the vascular endothelial growth factor (VEGF) receptor family including VEGFR1, VEGFR2, and VEGFR3), Class VI RTKs (e.g., the hepatocyte growth factor (HGF) receptor family including hepatocyte growth factor receptor (HGFR / MET) and RON), Class VII RTKs (e.g., the 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, including AXL, MER, and TRYO3), Class X RTKs (e.g., the LTK receptor family, including TK and ALK), Class XI RTKs (e.g., the TIE receptor family, including TIE and TEK), Class XII RTKs (e.g., the ROR receptor family, including ROR1 and ROR2), Class XIII This includes RTKs (e.g., the discoidine 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] Switch molecules containing RTKs, or their derivatives, variants, or fragments, can bind to any suitable RTK ligand, or antigen containing its derivatives, variants, or fragments. 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, beta-cellulin or BTC, neuregulin-1 or NRG1, neuregulin-2 or NRG2, neuregulin-3 or NRG3, and neuregulin-4 or NRG4), fibroblast growth factor (e.g., FG This includes F1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15 / 19, FGF16, FGF17, FGF18, FGF20, FGF21, and FGF23), the vascular endothelial growth factor family (e.g., VEGF-A, VEGF-B, VEGF-C, VEGF-D, and PIGF), and the platelet-derived growth factor family (e.g., PDDFA, PDDFB, PDGFC, and PDDFD). 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 placental insulin-like peptide (ELIP) (gene INSL4), insulin-like peptide 5 (gene INSL5), and insulin-like peptide 6).

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

[0093] RTSKs, or their derivatives, variants, or fragment switch molecules, can bind to any suitable RTSK ligand, or antigen containing its derivatives, variants, or fragments.

[0094] The switch molecule may include an intracellular domain (ICD) of a costimulatory molecule that triggers an immune cell activation signal. The costimulatory molecule may be bound 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 acted upon to modulate proliferation and / or survival signals in immune cells. In some embodiments, the ICD is an intracellular domain of a costimulatory molecule selected from MHC class I proteins, MHC class II proteins, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activators (SLAM proteins), activated NK cell receptors, BTLA, or Toll ligand receptors. In some embodiments, the co-stimulatory domains are 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 Ligands: TNFSF7, CD27 / TNFRSF7, CD28, CD29, CD2F-10 / SLAMF9, CD30, CD30; Ligands: TNFSF8, CD30 / TNFRSF8, CD300a / LMIR1, CD4, CD40; Ligands: TNFSF5, CD40 / TNFRSF5, CD48 / SLAMF2, CD49a, CD49D, CD49f, CD5, CD53, CD58 / LFA-3, CD69, CD7, CD8α, CD8β, CD82 / Kai-1, CD84 / SLAMF5, CD90 / Thy1, CD96, CDDS, CEACAM1, CRACC / SLAMF7, CRTAM, CTLA-4, DAP12, Dectin-1 / CLEC7A, DNAM1(CD226), DPPIV / CD26, DR3 / TNFRSF25, EphB6, GADS, Gi24 / VISTA / B7-H5, GITR ligand / TNFSF18, GITR / TNFRSF18, HLA class I, HLA-DR, HVEM / TNFRSF14, IA4, ICAM-1, ICOS / CD278, Ikaros, IL2R β, IL2R γ, IL7R α, IR-12R, integrin α4 / CD49d, integrin α4β1, integrin α4β7 / LPAM-1, IPO-3, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIRDS2, LAG-3, LAT, LIGHT / TNFSF14, LTBR, ​​Ly108, Ly9 (CD229), Lymphocyte function-associated antigen-1 (LFA-1), Lymphotoxin-α / TNF-β, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), NTB-A / SLAMF6, OX40 ligand / TNFSF4, OX40 / TNFRSF4, PAG / Cbp, PD-1, PDCD6, PD-L2 / B7-DC, PSGL1, RELT / TNFRSF19L, SELPLG (CD162), SLAM (SLAMF1), SLAM / CD150, SLAMF4 (CD244), SLAMF6 It contains the signaling domain of a molecule selected from the group consisting of (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 co-stimulatory molecules ECD and ICD 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 may have any suitable polypeptide sequence. In some cases, the transmembrane polypeptide comprises the polypeptide sequence of the transmembrane region 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 region of an endogenous or wild-type transmembrane protein. In some embodiments, the transmembrane polypeptide comprises a non-native polypeptide sequence, such as the sequence of a polypeptide linker. The polypeptide linker may be mobile 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 ligand-binding signal.

[0096] The binding of a ligand to a switch molecule can result in an immune cell activation signal in modified immune cells. In some embodiments, the immune cell activation signal is mediated by an activator. The activator may be an immunomodulatory molecule. The activator may bind to, activate, or stimulate T cells or other immune cells to modulate their activity. In some embodiments, the activator may be secreted from immune cells. The activator may 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 their derivatives, variants, or fragments.

[0097] Immune cell activation signals may include or result from the clonal proliferation 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 the 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 includes, or results from, the clonal proliferation of immune cells. Clonal proliferation includes the generation of daughter cells derived from immune cells. The daughter cells obtained from clonal proliferation may contain switch molecules. Clonal proliferation of modified immune cells may be greater than that of equivalent immune cells that do not possess switch molecules. Clonal proliferation of modified immune cells may be approximately 5 to 10 times, 10 to 20 times, 20 to 30 times, 30 to 40 times, 40 to 50 times, 50 to 60 times, 60 to 70 times, 70 to 80 times, 80 to 90 times, 90 to 100 times, 100 to 200 times, 200 to 300 times, 300 to 400 times, 400 to 500 times, 500 to 600 times, and 600 to 700 times compared to equivalent immune cells without the switch molecule. In some embodiments, the measurement of clonal proliferation may include, for example, quantifying the number of immune cells with and without the switch molecule, and after ligand binding to the switch molecule. The number of immune cells can be quantified using various methods, including, but not limited to, flow cytometry, trypan blue exclusion tests, and blood cell counts.

[0099] In some embodiments, immune cell activity includes, or results from, cytokine release by immune cells. In some embodiments, immune cell activity includes, or results from, the release of intercellular molecules, metabolites, compounds, or combinations thereof. Cytokine release by modified immune cells may 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, granzymes, etc. In some embodiments, cytokine release may be quantified using enzyme-linked immunosorbent assay (ELISA), flow cytometry, Western blotting, etc. Cytokine release by modified immune cells may be greater than that of equivalent immune cells without switch molecules. Modified immune cells provided herein may exhibit cytokine release approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, or 300 times greater than equivalent immune cells without the switch molecule. Modified immune cells may show increased cytokine secretion when the switch molecule binds to a ligand and the modified immune cells bind to neoantigens present on target cells, compared to equivalent immune cells without the 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, the cytotoxicity of immune cell activity is included or results from it. 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 switch molecules can induce death in target cells. The death of target cells can be used in a variety of applications, for example, to treat diseases or disorders in which it is desirable to eliminate a cell population or to suppress cell proliferation, although these are not limited to these. Cytotoxicity can also mean the release of cytotoxic cytokines by immune cells, such as IFNγ or granzymes. In some cases, the modified immune cells provided herein may have altered (i) release of cytotoxic substances such as perforin, granzymes, and granulysin, and / or (ii) induction of apoptosis via Fas-Fas ligand interaction between T cells and target cells. In some embodiments, cytotoxicity can be quantified by cytotoxicity assays, including co-culture assays, ELISPOT, and chromium-releasing cell characterization assays. The toxicity of the modified immune cells provided herein may be higher than that of equivalent immune cells without the switch molecule. Modified immune cells may exhibit increased cytotoxicity to target cells compared to immune cells without the switch molecule (e.g., unmodified immune cells) when the switch molecule binds to the ligand and when the modified immune cells bind to neoantigens present on the target cells. The modified immune cells of this disclosure may be approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, or 200% or more cytotoxic to target cells compared to equivalent immune cells without the switch molecule.The modified immune cells of this disclosure may induce at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, or 200% more target cell death than equivalent immune cells without the switch molecule. In some embodiments, the immune cells provided herein may induce apoptosis in target cells that present a target epitope (e.g., neoantigen) on their surface. In some embodiments, cytotoxicity may be measured in vitro or in vivo. In some embodiments, the measurement of cytotoxicity may include measuring the level of disease after administration of the modified immune cells provided herein compared to the level of disease before administration. In some embodiments, the measurement of cytotoxicity may 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 the switch molecule.

[0101] In some embodiments, immune cell activity includes, or results from, the proliferation of immune cells. Immune cell proliferation may mean an increase in the size of the immune cells. Immune cell proliferation may also mean a change in the phenotype of the immune cells. The proliferation of the modified immune cells of this disclosure may be increased compared to that of equivalent immune cells without the switch molecule. The proliferation of the modified immune cells of this disclosure may be approximately 5 to 10 times, 10 to 20 times, 20 to 30 times, 30 to 40 times, 40 to 50 times, 50 to 60 times, 60 to 70 times, 70 to 80 times, 80 to 90 times, 90 to 100 times, 100 to 200 times, 200 to 300 times, 300 to 400 times, 400 to 500 times, 500 to 600 times, and 600 to 700 times greater than the proliferation of equivalent immune cells without the switch molecule. Proliferation may also be determined by phenotypic analysis of immune cells.

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

[0103] In some embodiments, immune cell activity includes, or results from, the migration and / or transport of immune cells. In some embodiments, migration can be determined by quantifying the localization of immune cells to target sites. For example, modified immune cells provided herein can be quantified after administration at target sites, and, for example, at non-target sites. Quantification can be performed by isolating lesions and by quantifying the number of immune cells containing switch molecules, such as tumor-infiltrating lymphocytes. The migration and / or transport of immune cells containing switch molecules is greater than that of equivalent immune cells without switch molecules. In some embodiments, the number of immune cells containing switch molecules at a target site, such as a lesion area, may be about 5, 10, 15, 20, 25, 30, 35, or 40 times the number of equivalent immune cells without switch molecules. 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 may be approximately 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 includes, or results from, 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 immune receptor (TIGIT) having immunoglobulin and ITIM domains, 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 IL-2 production and cytokine expression, and high proliferative capacity may be lost. Exhaustion may also be accompanied by deficiencies in IFNγ, TNF, and chemokine production, as well as degranulation. The exhaustion or activation of the modified immune cells provided herein is greater than that of equivalent immune cells without switch molecules. In some embodiments, the immune cells provided herein undergo at least a 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 11x, 12x, 13x, 14x, 15x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 150x, 200x, 250x, or 300x or more increase in exhaustion or activation compared to equivalent immune cells without the switch molecule. In some embodiments, the immune cells provided herein undergo at least a 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 11x, 12x, 13x, 14x, 15x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 150x, 200x, 250x, or 300x or greater reduction in exhaustion or activation compared to equivalent immune cells lacking the switch molecule.

[0105] In some embodiments, binding of a switch molecule to a ligand causes modified immune cells (e.g., modified TILs or modified T cells) to exhibit enhanced neoantigen binding compared to equivalent immune cells without the switch molecule.

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

[0107] T cell receptor (TCR) complexes that exhibit specific binding to neoantigens can be endogenous or exogenous TCR complexes. Modified immune cells, whether endogenous or exogenous, confer antigen-binding specificity (e.g., neoantigen binding) to the immune cell.

[0108] In some embodiments, the immune cells are tumor-infiltrating lymphocytes (TILs). TILs may be, for example, T cells, B cells, monocytes, or 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 may express at least one of PD-1, CD137, and TIM-3. In some cases, modified TILs include “secondary TILs,” meaning amplified or proliferated TILs.

[0109] CAR contains an antigen interaction domain capable of binding to a B cell surface protein. The B cell surface protein may 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-interacting domain of the CAR can bind to surface proteins on non-B cells, provided that binding to the surface protein does not significantly jeopardize the host's overall health or immune system. In some embodiments, the surface protein is a surface protein on an immune cell. In some embodiments, the surface protein is a surface protein on a cell other than an immune cell. In some embodiments, the surface proteins are 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(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、CD156You may also choose 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-interacting domain of a CAR may be able to bind B cell surface proteins or fragments thereof on dead B cells. B cell apoptosis can occur before or after the onset of an immune response (e.g., an immune response against tumor cells). Therefore, dead B cells or their debris may still have B cell surface proteins or fragments thereof presented on their surface. The ability of CARs to target both living and dead B cells may increase the chances for immune cells containing the CAR to (i) bind to B cell surface proteins and (i) initiate signal transduction of the intracellular signaling domain. In some cases, signal transduction of the intracellular signaling domain may promote the growth (proliferation) of immune cells containing the CAR.

[0111] In some embodiments, the antigen interaction domain of the CAR can bind a B cell surface protein or a fragment thereof to the surface of a particle (e.g., nanoparticles) (e.g., via conjugated and / or non-conjugated bonds). The particles can be any particulate material, including organic and / or inorganic materials. The particles can be about 1 nanometer (nm) to about 50 micrometers (μm) in at least one dimension. The particles can be at least about 1 nm, 5 nm, 10 nm, 50 nm, 100 nm, 500 nm, 1 μm, 5 μm, 10 μm, 50 μm, or more in at least one dimension. The particles can be at most 50 μm, 10 μm, 5 μm, 1 μm, 500 nm, 100 nm, 50 nm, 10 nm, 5 nm, 1 nm, or less in at least one dimension. The particles may be, for example, nanoparticles, microparticles, nanospheres, microspheres, nanorods, microrods, nanofibers, nanoribbons, etc. Examples of particles include, for example, metal nanoparticles (e.g., gold nanoparticles, silver nanoparticles, and iron nanoparticles), intermetallic compound nanosemiconductor 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, particles may be, for example, crosslinked polymers, hydrogel polymers, biodegradable polymers, polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), copolymers, polysaccharides, starch, cellulose, chitosan, polyhydroxyalkanoic acid (PHA), PHB, PHV, lipids, peptides, peptide amphiphiles, polypeptides (e.g., proteins), or combinations thereof. Particles displaying B cell surface proteins on their surface may be introduced in vitro into immune cells containing CARs that bind to B cell surface proteins. Alternatively, or additionally, particles displaying B cell surface proteins may be introduced in vivo (e.g., by localized or systemic injection) together with immune cells containing CARs. Such particles can be used in vitro or in vivo to increase populations of immune cells containing CARs.

[0112] The antigen-binding domain may 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, and these include, but are not limited to, Fab, Fab', F(ab')2, Fv, single-chain Fv(scFv), minibodies, bispecific antibodies, and single-domain antibodies, such as the heavy chain variable region (VH), light chain variable region (VL), and variable region (VHH) of camel-derived nanobodies. In some embodiments, the first antigen-binding domain includes at least one of Fab, Fab', F(ab')2, Fv, or scFv. In some embodiments, the antigen-binding domain includes an antibody mimetic. Antibody imitators are molecules capable of binding to target molecules with affinity equivalent to that of antibodies, and include, for example, single-chain binding molecules, cytochrome b562-based binding molecules, fibronectin or fibronectin-like protein backbones (e.g., adnectin), lipokine backbones, calixarene backbones, A-domains, 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 may include at least the ligand-binding domain of a transmembrane receptor.

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

[0114] The antigen-binding domain of the CAR of the system of interest may be bound to the intracellular signaling domain via a transmembrane domain. The transmembrane domain may also be a transmembrane segment. The transmembrane domain of the CAR of interest may immobilize 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 binding the antigen-binding domain and the intracellular signaling domain of the CAR may have any suitable polypeptide sequence. In some cases, the transmembrane polypeptide comprises the polypeptide sequence of the transmembrane region of an endogenous or wild-type transmembrane protein. In some embodiments, the transmembrane polypeptide comprises a polypeptide sequence having at least one (e.g., at least two, three, four, five, six, seven, eight, nine, ten, or more) amino acid substitutions, deletions, and insertions compared to the transmembrane protein of an endogenous or wild-type transmembrane protein. In some embodiments, the transmembrane polypeptide comprises a non-native polypeptide sequence, such as the sequence of a polypeptide linker. The polypeptide linker may be mobile or rigid. Polypeptide linkers may or may not be structured. In some embodiments, transmembrane polypeptides transmit signals from the extracellular to the intracellular region of a cell, for example, via an antigen-binding domain. The native transmembrane domain of CD28 may be used in CARs. In other cases, the native transmembrane domain of CD8 alpha may be used in CARs.

[0115] The CARs of this disclosure may include signaling domains involved in immune cell signaling, or derivatives, variants, or fragments thereof. The intracellular signaling domains of the CARs may induce the activity of immune cells containing the CARs. The intracellular signaling domains may transmit effector function signals and instruct cells to perform specific functions. The signaling domains may include signaling domains of other molecules. In some cases, cleaved sites of signaling domains are used in the CARs.

[0116] In some embodiments, the intracellular signaling domain comprises multiple signaling domains involved in immune cell signaling, or derivatives, variants, or fragments thereof. For example, the intracellular signaling domain comprises at least two immune cell signaling domains, e.g., at least two, three, four, five, six, seven, eight, nine, or ten immune cell signaling domains. The immune cell signaling domain may be involved in the initial activation of the TCR complex, either in a stimulating or repressive manner. The intracellular signaling domain may also be the intracellular signaling domain of a T cell receptor (TCR) complex. The intracellular signaling domains of the target CAR may include the signaling domains of Fcγ receptor (FcγR), Fcε receptor (FcεR), FCα receptor (FcαR), neonatal Fc receptor (FcRn), CD3, CD3ζ, CD3γ, CD3δ, CD3ε, CD4, CD5, CD8, CD21, CD22, CD28, CD32, CD40L (CD154), CD45, CD66d, CD79a, CD79b, CD80, CD86, CD278 (also known as ICOS), CD247ζ, CD247η, DAP10, DAP12, FYN, LAT, Lck, MAPK, MHC complex, NFAT, NF-κB, PLC-γ, iC3b, C3dg, C3d, and Zap70. In some embodiments, the signaling domain may contain an immunoreceptor-activating tyrosine motif or ITAM. The ITAM-containing signaling domain may contain two repeats of the amino acid sequence YxxL / I, separated by 6-8 amino acids, where x is any amino acid independently, forming the conserved motif YxxL / Ix(6-8)YxxL / I. The ITAM-containing signaling domain may be modified by phosphorylation, for example, 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, cleaved, and / or optimized ITAM domain, which has altered (e.g., increased or decreased) activity compared to the native ITAM domain.

[0117] In some embodiments, the intracellular signaling domain of the target CAR 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 CD3ζ.

[0118] In some embodiments, the intracellular signaling domain of the target CAR contains an immunoreceptor-activating tyrosine motif or ITAM. The signaling domain containing ITIM contains a conserved amino acid sequence (S / I / V / LxYxxI / V / L) found at the cytoplasmic end of several repressive receptors in the immune system. The primary signaling domain containing ITIM can be modified, for example, by an enzyme such as a member of the Src kinase family (e.g., Lck), such as by phosphorylation. Following phosphorylation, other proteins, including enzymes, may be added to ITIM. These other proteins include, but are not limited to, the phosphorylated tyrosine phosphatases SHP-1 and SHP-2, an inositol monophosphate-degrading enzyme called SHIP, and proteins having one or more SH2 domains (e.g., ZAP70). The intracellular signaling domains include 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-related 1 (IRTA1), immunoglobulin superfamily receptor translocation-related 2 (IRTA2), killer cell immunoglobulin-like receptor 2DL1 (KIR2DL1), and killer cell immunoglobulin-like receptor 2DL2 (KIR2DL2), killer cell immunoglobulin-like receptor; 2DL3 (KIR2DL3), killer cell immunoglobulin-like receptor; 2DL4 (KIR2DL4), killer cell immunoglobulin-like receptor; 2DL5 (KIR2DL5), killer cell immunoglobulin-like receptor; 3DL1 (KIR3DL1), killer cell immunoglobulin-like receptor; 3DL2 (KIR3DL2), leukocyte immunoglobulin-like receptor; Superfamily B member1 (LIR1), Leukocyte Immunoglobulin-like Receptor Superfamily B Member 2 (LIR2), Leukocyte Immunoglobulin-like Receptor Superfamily B Member 3 (LIR3), Leukocyte Immunoglobulin-like Receptor Superfamily B Member 5 (LIR5), Leukocyte Immunoglobulin-like Receptor Superfamily B Member 8 (LIR8), Leukocyte-associated Immunoglobulin-like Receptor 1 (LAIR-1), Mast Cell Function-Associated Antigen (MAFA), NKG2A, Natural Cytotoxic Trigger Receptor 2 (NKp44), NTB-A, Programmed Cell Death Protein 1 (PD-1), PILR, SIGLECL1, Sialic Acid-Conjugated Immunoglobulin-like Lectin 2 (SIGLEC2 or CD22), Sialic Acid-Conjugated Immunoglobulin-like Lectin It may include 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 the signaling domain (e.g., ITIM) of signal threshold-regulating transmembrane adapter 1 (SIT). In some embodiments, the intracellular signaling domain includes a modified ITAM domain, such as a mutated, cleaved, and / or optimized ITAM domain, which has altered (e.g., increased or decreased) activity compared to the native ITAM domain.

[0119] In some embodiments, the intracellular signaling domain includes at least two ITAM domains (e.g., at least three, four, five, six, seven, eight, nine, or ten ITAM domains). In some embodiments, the intracellular signaling domain includes at least two ITIM domains (e.g., at least three, four, five, six, seven, eight, nine, or ten ITIM domains) (e.g., at least two primary signaling domains). In some embodiments, the intracellular signaling domain includes both ITAM and ITIM domains.

[0120] In some cases, the intracellular signaling domain of the target CAR may include a co-stimulatory domain. In some embodiments, a co-stimulatory domain from, for example, a co-stimulatory molecule may provide a co-stimulatory signal for immune cell signaling, such as signaling from the 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 a signaling domain of an MHC class I protein, an MHC class II protein, a TNF receptor protein, an immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocyte activator molecule (SLAM protein), an activated NK cell receptor, BTLA, or a Toll ligand receptor. In some embodiments, the co-stimulatory domains are 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 Ligands: TNFSF7, CD27 / TNFRSF7, CD28, CD29, CD2F-10 / SLAMF9, CD30; Ligands: TNFSF8, CD30 / TNFRSF8, CD300a / LMIR1, CD4, CD40; Ligands: TNFSF5, CD40 / TNFRSF5, CD48 / SLAMF2, CD49a, CD49D, CD49f, CD53, CD58 / LFA-3, CD69, CD7, CD8α, CD8β, CD82 / Kai-1, CD84 / SLAMF5, CD90 / Thy1, CD96, CDS, CEACAM1, CRACC / SLAMF7, CRTAM, CTLA-4, DAP12; Dectin-1 / CLEC7A, DNAM1(CD226), DPPIV / CD26, DR3 / TNFRSF25, EphB6, GADS, Gi24 / VISTA / B7-H5, and GITR LINK / TNFSF18, GITR / TNFRSF18, HLA, I, HLA-DR, HVEM / TNFRSF14, IA4, ICAM-1, ICOS / CD278, Ikaros, IL2R β, IL2R γ, IL7R α-mediated α4 / CD49d and α4β1 α4β7 / LPAM-1, IPO-3, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, I TGB1, ITGB2, ITGB7, KIRDS2, LAG-3, LAT, LIGHT / TNFSF14, LTBR, ​​Ly108, Ly9 (CD229) Lipoprotein Factor Activator-1(LFA-1) Fan Factor-α / TNF-β NKG2C NK G2D, NKp30, NKp44, NKp46, NKp80(KLRF1), NTB-A / SLAMF6, OX40 LINK / TNFSF4, OX40 / TNFRSF4, PAG / Cbp, PD-1, PDCD6, PD-L2 / B7-DC, PSGL1, RELT / TNFRSF19L, SELPLG (CD162), SLAM (SLAMF1) SLAM / CD150 SLAMF4 (CD244) SLAMF6 (NTB-A), SLAMF7, SLP-76, TACI / TNFRSF13B, TCL1A, TCL1B, TIM-1 / KIM-1 / HAVCR, TIM-4, TL1A / TNFSF15, TNF RII / TNFRSF1B, TNF-α, TRANCE / RANKL, TSLP, and TSLPThe intracellular signaling domain comprises a signaling domain of a molecule selected from the group consisting of R, VLA1, and VLA-6. In some embodiments, the intracellular signaling domain comprises multiple co-stimulatory domains, e.g., at least two, e.g., at least three, four, or five co-stimulatory domains. The co-stimulatory signaling region may provide a synergistic signal with the primary effector activation signal and may satisfy the requirements for T cell activation. In some embodiments, the addition of a co-stimulatory domain to the CAR may enhance the effects and persistence of the immune cells provided herein.

[0121] The binding of CARs to B cell surface proteins may enhance the proliferation of immune cells compared to immune cells without CARs. Immune cell proliferation may mean an increase in the size of the immune cells. Immune cell proliferation may also mean a change in the phenotype of the immune cells. The proliferation of immune cells containing CARs provided herein may be increased compared to that of equivalent immune cells without CARs that exhibit binding to B cell surface proteins. The proliferation of immune cells containing CARs is increased by approximately 5 to 10 times, 10 to 20 times, 20 to 30 times, 30 to 40 times, 40 to 50 times, 50 to 60 times, 60 to 70 times, 70 to 80 times, 80 to 90 times, 90 to 100 times, 100 to 200 times, 200 to 300 times, 300 to 400 times, 400 to 500 times, 500 to 600 times, and 600 to 700 times compared to the proliferation of equivalent immune cells without CARs. The proliferation of immune cells containing CARs was increased by approximately 5 to 10 times, 10 to 20 times, 20 to 30 times, 30 to 40 times, 40 to 50 times, 50 to 60 times, 60 to 70 times, 70 to 80 times, 80 to 90 times, 90 to 100 times, 100 to 200 times, 200 to 300 times, 300 to 400 times, 400 to 500 times, 500 to 600 times, and 600 to 700 times compared to the proliferation of equivalent immune cells without CARs, and the proliferation was observed at least approximately 12, 24, 36, 48, 60, 72, 84, or 96 hours after contact of B cells with B cell surface proteins. Increased proliferation can be confirmed either in vitro or in vivo. In some embodiments, proliferation involves quantifying the number of immune cells. Quantifying the number of immune cells may include flow cytometry, trypan blue exclusion tests, and hematological counts. Proliferation may also be determined by phenotypic analysis of immune cells.

[0122] In one embodiment, the disclosure provides a modified immune cell that specifically binds to a neoantigen, wherein the modified immune cell comprises (a) a chimeric stimulating 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-stimulating molecule that mediates an immune cell activation signal, and the binding of the chimeric stimulating molecule to the neoantigen results in the chimeric stimulating molecule constituting 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 PEDs include antibodies, as well as their derivatives, variants, and fragments.

[0123] In one embodiment, the 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, upon binding to its ligand, induces an immunoinactivation signal in an unmodified immune cell, wherein the ECD is fused to an intracellular domain (ICD) of a co-stimulatory molecule that mediates an immune cell activation signal, and the binding of the switch molecule to its ligand results in an immune cell activation signal instead of an immune cell inactivation 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.

[0124] In one embodiment, the disclosure provides a modified tumor-infiltrating lymphocyte (TIL) 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, upon binding to its ligand, induces an immune cell inactivation signal in an unmodified immune cell, wherein the ECD is fused to an intracellular domain (ICD) of a co-stimulatory molecule that mediates an immune cell activation signal, and the 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 TIL; and (b) a chimeric antigen receptor comprising (i) an antigen-interacting domain capable of binding to a B cell surface protein; (ii) a transmembrane domain; and (iii) an intracellular signaling domain.

[0125] In one embodiment, the disclosure provides modified immune cells that overexpress cytokines, such as chemokines, wherein the immune cells are (i) tumor-infiltrating lymphocytes (TILs); (ii) stromal tumor-infiltrating lymphocytes (sTILs); or (iii) T cells exhibiting specific binding to antigens. Modified immune cells that overexpress chemokines may be any modified immune cells provided herein.

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

[0127] Certain cytokines can function as pro-inflammatory cytokines. Pro-inflammatory cytokines are cytokines that are involved in inducing or amplifying inflammatory responses. 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. Anti-inflammatory cytokines are cytokines that are involved in reducing inflammatory responses. In some cases, anti-inflammatory cytokines can modulate pro-inflammatory cytokine responses. Some cytokines can function as both pro-inflammatory and anti-inflammatory cytokines. Certain cytokines, such as chemokines, can function in chemomigration. Chemokines can induce directional chemomigration in nearby responsive cells.

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

[0129] Examples of cytokines that can be overexpressed by immune cells provided herein include, but are not limited to, lymphokines, monokines, and conventional polypeptide hormones. The cytokines included are growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH); liver growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-α; Müllerian 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 factor-I and -II; erythropoietin (EPO); Flt-3L; and stem cell factor (SCF). ; Bone-inducing factors; 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); IL-1, IL-1a, IL-1b, IL-1RA, IL-18, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, These include interleukins (ILs) such as IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, and 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, and TRANCE; and other polypeptide factors including LIF, oncostatin M (OSM), and Kit ligand (KL). A cytokine receptor refers to a receptor protein that binds to cytokines. Cytokine receptors can be both membrane-bound and soluble.

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

[0131] In some embodiments, the overexpressed cytokines are interleukin (IL-1) family members or related proteins; tumor necrosis factor (TNF) family members or related proteins; interferon (IFN) family members or related proteins; interleukin-6 (IL-6) family members or related proteins; or chemokines or related proteins.In some embodiments, the cytokines are IL18, IL18BP, IL1A, IL1B, IL1F10, IL1F3 / IL1RA, IL1F5, IL1F6, IL1F7, IL1F8, IL1RL2, IL1F9, IL33, BAFF / BLyS / TNFSF138, 4-1BBL, CD153 / CD30L / TNFSF8, CD40LG, CD70, Fas Ligand / FASLG / CD95L / CD178, EDA-A1, TNFSF14 / LIGHT / CD258, TNFA, LTA / TNFB / TNFSF1, LTB / TNFC, CD70 / CD27L / TN FSF7, TNFSF10 / TRAIL / APO-2L(CD253), RANKL / OPGL / TNFSF11(CD254), TNFSF12, TNF-α / TNFA, TNFSF13, TL1A / TNFSF 15, OX-40L / TNFSF4 / CD252, CD40L / CD154 / TNFSF5, IFNA1, IFNA10, IFNA13, IFNA14, IFNA2, IFNA4, IFNA7, IFNB1, IF NE, IFNG, IFNZ, IFNA8, IFNA5 / IFNaG, IFNω / IFNW1, CLCF1, CNTF, IL11, IL31, IL6, leptin, LIF, OSM, CCL1 / TCA3, CCL11, CCL12 / MCP-5, CCL13 / MCP-4, CCL14, CCL15, CCL16, CCL17 / TARC, CCL18, CCL19, CCL2 / MCP-1, CCL20, CCL21, CCL22 / M DC, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL3L3, CCL4, CCL4L1 / LAG-1, CCL5, CCL6, CCL7, CCL8, CCL9, CX Selected from 3CL1, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, CXCL2 / MIP-2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7 / Ppbp, CXCL9, IL8 / CXCL8, XCL1, XCL2, FAM19A1, FAM19A2, FAM19A3, FAM19A4, and FAM19A5.

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

[0133] In some embodiments, the cytokines overexpressed by the modified immune cells provided herein are chemokines. The chemokines may be, for example, CC chemokines, CXC chemokines, C chemokines, and CX3C chemokines. In some embodiments, the chemokines overexpressed by the modified immune cells are CC chemokines 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 embodiment, the Disclosure provides a method for treating cancer in a subject, comprising (a) administering to a subject any one of the various embodiments of the embodiments herein of a modified TIL, modified T cell, or modified immune cell; and (b) contacting the modified TIL, modified T cell, or modified immune cell with cancerous target cells expressing a neoantigen under conditions that induce cytotoxicity of the modified TIL, modified T cell, or modified immune cell against cancerous target cells, thereby inducing the death of the cancerous target cells.

[0135] In one embodiment, the Disclosure provides a method for increasing a T cell population, the method comprising (a) providing a T cell population comprising at least one modified immune cell of any one of the various embodiments of the embodiments herein, and (b) exposing the T cell population to B cell surface proteins to an effect on increasing the T cell population. In some embodiments, in (b), the T cell population is exposed to B cells comprising B cell surface proteins.

[0136] In one embodiment, the Disclosure provides a method for increasing a T cell population, comprising the steps of (a) introducing a nucleic acid encoding a chimeric antigen receptor (CAR) into a T cell population to produce a primary CAR-expressing cell population, wherein the CAR comprises (i) an antigen-interacting domain capable of binding to a B cell surface protein; (ii) a transmembrane domain; and (iii) an intracellular signaling domain; and (b) contacting the primary CAR-expressing cell population with a B cell surface protein to produce an increased and / or activated immune cell population.

[0137] In one embodiment, the Disclosure provides a composition comprising (a) a switch molecule comprising an extracellular domain (ECD) of a protein that, upon binding to its ligand, induces an immunoinactivation signal in unmodified immune cells, 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.

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

[0139] In one embodiment, the Disclosure provides a composition comprising one or more polynucleotides encoding one or more chimeric antigen receptors, which include (a) a switch molecule comprising an extracellular domain (ECD) of a protein that, upon binding to its ligand, induces an immunoinactivation 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; (b) an antigen-specific T cell receptor complex, or one or more components thereof; and (c) an antigen-interacting domain capable of binding to a B cell surface protein; (ii) a transmembrane domain; and (iii) an intracellular signaling domain.

[0140] In various embodiments of the embodiments described herein, promoters may be used in conjunction with the compositions of this disclosure. Examples of promoters include those active in eukaryotic cells, mammalian cells, non-human mammalian cells, or human cells. Promoters may be inductive or constitutively active promoters. Alternatively or additionally, promoters may be tissue or cell-specific.

[0141] Non-limiting examples of suitable eukaryotic cell promoters (i.e., promoters that are functional in eukaryotic cells) include early cytomegalovirus (CMV), herpes simplex virus thymidine kinase, early and late SV40, retroviral terminal repeat sequences (LTRs), human elongation factor 1 promoter (EF1), hybrid constructs including a cytomegalovirus (CMV) enhancer fused to a tri-β-actin promoter (CAG), mouse stem cell virus promoter (MSCV), phosphoglycerate kinase-1 locus promoter (PGK), and those derived from mouse metallothionein-I. Promoter may also be a fungal promoter. Promoter may also be a plant promoter. Databases of plant promoters can be found, for example, in PlantProm. Expression vectors may also contain ribosome-binding sites and transcription termination codons for translation initiation. Expression vectors may also contain appropriate sequences for amplification of expression.

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

[0143] In various embodiments of the embodiments described herein, the modified immune cells may further include a kill switch. The kill switch may be activated to eliminate the immune cells in cases of severe toxicity, such as hypercytokinemia. This can occur when the immune system has a strong response in which many inflammatory cytokines are released, causing mild to severe symptoms such as fever, headache, rash, rapid heartbeat, hypotension, and respiratory distress. The kill switch may be a drug-induced kill switch. The kill switch may include inducible caspase 9.

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

[0145] In some cases, the cells may be a population of T cells, NK cells, B cells, etc., obtained from a subject. T cells can be obtained from numerous sources, including PBMCs, bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, and tissue from infection sites, ascites, pleural fluid, splenic tissue, and tumors. In some embodiments, T cells may be obtained from a unit of blood collected from a subject using any many techniques, such as Ficoll® isolation. In some embodiments, cells from an individual's circulating blood are obtained by apheresis. Products from apheresis typically include lymphocytes, including T cells, monocytes, granulocytes, and B cells, other nucleated leukocytes, erythrocytes, and platelets. Cells collected by apheresis may be washed to remove plasma fractions and to place cells suitable for subsequent processing steps into appropriate buffers or solvents.

[0146] Any variety of immune cells may be used in the embodiments herein. In some embodiments, immune cells include granulocytes such as basophils, eosinophils and neutrophils; mast cells; monocytes that can develop into macrophages; antigen-presenting cells such as dendritic cells; and lymphocytes such as natural killer cells (NK cells), B cells and T cells. In some embodiments, immune cells are immune effector cells. Immune effector cells mean immune cells that can perform specific functions in response to stimuli. In some embodiments, immune cells are immune effector cells that can induce cell death. In some embodiments, immune cells are lymphocytes. In some embodiments, lymphocytes are NK cells. In some embodiments, lymphocytes are T cells. In some embodiments, T cells are activated T cells. T cells include both unsensitized cells and immunological memory cells (e.g., central memory or TCM, effector memory or TEM, and effector memory RA or TEMRA), effector cells (e.g., cytotoxic T cells or CTLs or Tc cells), helper cells (e.g., Th1, Th2, Th3, Th9, Th7, TFH), regulatory cells (e.g., Treg and Trl cells), natural killer cells (NKT cells), tumor-infiltrating lymphocytes (TILs), lymphocyte-activated killer cells (LAKs), αβT cells, γδT 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 the proteins present on their cell surface. T cells expressing the systems in question can perform multiple functions, including killing infected cells and activating or supplementing other immune cells. CD8+ T cells are also called cytotoxic T cells or cytotoxic T lymphocytes (CTLs). CTLs expressing the target system may be involved in recognizing and eliminating virus-infected and cancer cells. CTLs possess specific compartments or granules containing cytotoxins that induce apoptosis, such as programmed cell death.CD4+ T cells are subdivided into four subsets, Th1, Th2, Th17, and Treg, although there may be additional subsets, where Th stands for helper T cell. Th1 cells can coordinate the immune response against intracellular microorganisms, particularly bacteria. They produce and secrete molecules that alert and activate other immune cells, such as macrophages, which phagocytose bacteria. Th2 cells are involved in coordinating the immune response against extracellular pathogens such as helminths (parasites) by alerting B cells, granules, 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 neutrophil replenishment.

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

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

[0149] In various embodiments of the embodiments described herein, immune cells include lymphocytes. In some embodiments, lymphocytes are natural killer cells. In some embodiments, lymphocytes are T cells. T cells can be obtained from numerous sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, umbilical cord blood, and tumors. In some embodiments, any number of available T cell lines can be used. Immune cells, such as lymphocytes (e.g., cytotoxic lymphocytes), are preferably autologous cells, however, heterologous cells can also be used. T cells can be obtained from a unit of blood taken from a subject using any many techniques, such as Ficoll® isolation. Cells from the circulating blood of an individual can be obtained by apheresis or leukocyte removal. Products by apheresis typically include lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. Cells collected by apheresis may be washed to remove plasma fractions and to position cells suitable for subsequent processing steps in a suitable buffer or solvent, such as phosphate-buffered saline (PBS). After washing, cells may be resuspended in various biocompatible buffers, such as Ca-free and Mg-free PBS. Alternatively, undesirable components of the apheresis sample may be removed, and the cells may be resuspended directly in the culture solution. Samples may be provided directly by the subject or indirectly through one or more intermediaries, such as a sample collection service provider or healthcare provider (e.g., a physician or nurse). In some embodiments, the separation of T cells from peripheral blood leukocytes includes lysing the erythrocytes and separating the peripheral blood leukocytes from monocytes by centrifugation, for example, via a PERCOL gradient.

[0150] Specific T cell subpopulations, such as CD4 or CD8, can be further separated by positive or negative selection techniques. Negative selection of a T cell population can be achieved, for example, by a combination of antibodies oriented to surface markers specific to the negatively selected cells. One suitable technique is cell sorting via a negative magnetic immunoadhesion reaction, utilizing a cocktail of monoclonal antibodies oriented to cell surface markers on the negatively selected cells. For example, to separate CD4+ cells, the monoclonal antibody cocktail may include antibodies against CD14, CD20, CD1 lb, CD16, HLA-DR, and CD8. The negative selection process can be used primarily to produce a homogeneous desired T cell population. In some embodiments, the composition comprises a mixture of two or more (e.g., two, three, four, five or more) different types of T cells.

[0151] In some embodiments, immune cells are members of an enriched cell population. One or more desired cell types may be enriched by any suitable method, non-limiting examples of such methods include treating a cell population to trigger proliferation and / or differentiation into a desired cell type, treating to halt the growth of an undesired cell type, treating to kill or lyse an undesired cell type, and purifying a desired cell type (e.g., purification on an affinity column that retains a desired or undesired cell type based on one or more cell surface markers). In some embodiments, the enriched cell population is a cell population enriched in cytotoxic lymphocytes selected from cytotoxic T cells (variously also 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] To separate a desired cell population by positive or negative selection, the concentrations of cells and surfaces (e.g., particles such as beads) are varied. In some embodiments, it may be preferable to significantly reduce the volume in which the beads and cells are mixed together (i.e., increase the cell concentration) to ensure maximum contact between cells and beads. For example, a concentration of 2 billion cells / mL may be used. In some embodiments, a concentration of 1 billion cells / mL may be used. In some embodiments, a concentration of 100 million cells / mL may be used. Concentrations of 10 million, 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 45 million, or 50 million cells / mL may 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 higher concentrations may result in increased cell yield, cell activation, and cell proliferation.

[0153] Various target cells can be killed using the systems and methods covered by this disclosure. Target cells to which this method can be applied may include a wide range of cell types. Target cells may be in vitro. Target cells may be in vivo. Target cells may be ex vivo. Target cells may be isolated cells. Target cells may be cells within organs. Target cells may be organs. Target cells may be mammalian cells or derived from mammalian cells. Target cells may be human cells or derived from human cells. Target cells may be prokaryotic cells or derived from prokaryotic cells. Target cells may be bacterial cells or derived from bacterial cells. Target cells may be archaeal cells or derived from archaeal cells. Target cells may be eukaryotic cells or derived from eukaryotic cells. Target cells may be pluripotent stem cells. Target cells may be plant cells or derived from plant cells. Target cells may be animal cells or derived from animal cells. 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 originate from a specific organ or tissue.

[0154] Target cells may be stem cells or derived from progenitor cells. Target cells may include stem cells (e.g., adult stem cells, embryonic stem cells, induced pluripotent stem (iPS) cells, etc.) and progenitor cells (e.g., cardiomyocyte progenitor cells, neural progenitor cells, etc.). Target cells may include mammalian stem cells and progenitor cells such as rodent stem cells, rodent progenitor cells, human stem cells, human progenitor cells, etc. Cloned cells may include progenitor cells. Target cells may contain target nucleic acids. Target cells may be within a living organism. Target cells may be genetically modified cells. Target cells may be host cells.

[0155] The target cells may be primary cells. For example, primary cells may be cultured 0, 1, 2, 4, 5, 10, 15 times or more. The cells may be unicellular organisms. The cells may grow in a culture medium.

[0156] Target cells may be disease cells. Disease cells may have altered metabolism, gene expression, and / or morphological characteristics. Disease cells may be cancer cells, diabetic cells, and apoptotic cells. Disease cells may be cells derived from a subject suffering from a disease. Exemplary diseases may include vascular disorders, cancer, metabolic disorders, eye diseases, organ disorders, musculoskeletal disorders, and heart disease.

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

[0158] Non-limiting examples of cells that can be target cells include, but are not limited to, lymphoid cells such as B cells, T cells (cytotoxic T cells, natural killer T cells, regulatory T cells, helper T cells), natural killer cells, cytokine-induced killer (CIK) cells (see, for example, U.S. Patent No. 20080241194); myeloid cells such as granulocytes (basophilic granulocytes, eosinophilic granulocytes, neutrophil granulocytes / hypersegmented neutrophils), monocytes / macrophages, erythrocytes (reticulocytes), mast cells, platelets / megakaryocytes, dendritic cells; A Cells derived from the endocrine system, including cells of the thyroid gland (thyroid epithelial cells, parafollicular cells), parathyroid gland (parathyroid chief cells, eosinophilic cells), adrenal gland (chromaffin cells), and pineal gland (pineal cells); cells of the nervous system, such as glial cells (astrocytes, microglia), giant neurosecretory cells, astrocytes, Betschel cells, and pituitary gland (gonadotropin-secreting cells, adrenocorticotropin-secreting cells, thyroid-stimulating hormone-producing cells, growth hormone-producing cells, mammary gland-stimulating hormone-secreting cells); lung cells (type I lung cells, type II lung cells), Clara cells, goblet cells, and dust cells. Cells of the respiratory system such as vesicles; cells of the respiratory system such as cardiomyocytes and pericytes; cells of the digestive system such as stomach (gastrocnemiocytes, parietal cells), goblet cells, Paneth cells, G cells, D cells, ECL cells, I cells, K cells, and S cells; enteroendocrine cells such as enterochromaffin cells, APUD cells, liver (hepatocytes, Kupffer cells), cartilage / bone / muscle; osteocytes such as osteoblasts, osteocytes, osteoclasts, and teeth (cementoblasts, ameloblasts); chondrocytes such as chondrocytes and chondrocytes; skin cells such as filaments, keratinocytes, and melanocytes (nevus cells); muscle cells such as myocytes. Cells; cells of the urinary system such as podocytes, juxtaglomerular cells, intraglomerular mesangial cells / extraglomerular mesangial cells, renal proximal tubular brush border cells, macula compacta cells; cells of the reproductive system such as sperm, Sertoli cells, Leydig cells, oocytes; and adipocytes, fibroblasts, tendinocytes, epidermal keratinocytes (differentiated epithelial cells), epithelial basal cells (stem cells), nail and toenail keratinocytes, nail bed basal cells (stem cells), medullary hair stem cells, cortical hair stem cells, keratinized hair stem cells, keratinized root sheath cells, root sheath cells of the layer of Hakusuri, root sheath cells of the layer of Henle, outer root sheath cells, hair matrix cells (stem cells),Moisture-laden barrier epithelial cells, epidermal cells of stratified squamous epithelium of the cornea, tongue, oral cavity, esophagus, anal canal, urinary tract and vaginal terminal, basal cells (stem cells) of epithelium of the cornea, tongue, oral cavity, esophagus, anal canal, urinary tract and vaginal terminal, urothelial cells (inside of the bladder and ureters), exocrine epithelial cells, salivary gland mucosal cells (secreting polysaccharides), salivary gland serous cells (secreting glycoprotein enzymes), von Ebner gland cells of the tongue (washing away taste buds) mammary gland cells (milk secretion), lacrimal gland cells (tear secretion), ear canal glands (wax secretion), eccrine sweat gland dark cells (glycoprotein secretion), eccrine sweat gland clear cells (low molecular weight secretion), apocrine sweat gland cells (aromatic secretion, sex hormone sensitive), eyelid gland cells (special sweat glands), sebaceous gland cells (lipid-rich sebum secretion), Bowman's gland cells in the nose (washes the olfactory epithelium), Brunner's gland cells in the duodenum (enzymes and alkaline mucus), seminal vesicles Cells (secreting seminal fluid components including fructose from swimming sperm), prostate cells (secreting seminal fluid components), urethral gland cells (mucus secretion), Bartholin's gland cells (vaginal lubrication secretion), Little cell gland (mucus secretion), endometrial cells (carbohydrate secretion), isolated goblet cells of the respiratory and digestive tract (mucus secretion), mucosal cells of the stomach lining (mucus secretion), gastric gland enzyme progenitor cells (pepsinogen secretion), gastric gland acid secreting cells (hydrochloric acid secretion), pancreatic acinar cells (bicarbonate and digestive enzyme secretion), Paneth cells of the small intestine (lysozyme secretion), lung cells of type II lung (surfactant secretion), Clara cells of the lung, hormone-secreting cells, anterior pituitary cells, growth hormone-producing cells, mammogenic hormone-secreting cells, thyroid-stimulating hormone-producing cells, gonadotropin-producing cells, adrenocorticotropin-producing cells, pituitary middle lobe cells, giant cell neurosecretory cells, intestinal and airway cells, thyroid cells Thyroid epithelial cells, parafollicular cells, parathyroid cells, parathyroid chief cells, eosinophils, adrenal cells, chromaffin cells, Reidig cells of the testis, tetracellular cells of follicular cells, luteal cells of ruptured follicular cells, granulosa lutein cells, tetracellular cells, juxtaglomerular cells (renin secretion), macular cells of the kidney, metabolic and storage cells, barrier function cells (lungs, intestines, exocrine glands and urogenital tract), kidney, type I lung cells (inside the air space of the lung), pancreatic duct cells (central acinar cells), non-striatal duct cells (sweat glands, salivary glands, mammary glands, etc.), ductal cells (seminal vesicles, prostate, etc.), epithelial cells lining closed internal cavities, ciliated cells with propulsive function, extracellular matrix secretory cells, contractile cells; skeletal muscle cells, stem cells, cardiomyocytes,Blood and immune system cells, red blood cells, megakaryocytes (platelet progenitor cells), monocytes, connective tissue macrophages (various types), epidermal Langerhans cells, osteoclasts (in bone), dendritic cells (in lymphoid tissue), microglia (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 blood and immune system (various types) Other cells include progenitor cells, pluripotent stem cells, totipotent stem cells, induced pluripotent stem cells, adult stem cells, sensory transducer cells, autonomic nerve cells, sensory organ and peripheral nerve supporting cells, central nervous system neurons and glial cells, lens cells, pigment cells, melanocytes, retinal pigment epithelial cells, germ cells, oogonia / oocytes, spermatids, spermatocytes, spermatogonia (spermatocyte stem cells), sperm, nurse cells, ovarian follicular cells, Sertoli cells (in the testes), thymic epithelial cells, stromal cells, interstitial kidney cells, etc.

[0159] Cancer cells are of particular interest. In some embodiments, the target cells are cancer cells. Non-limiting examples of cancer cells include, for example, acanthoma, acinar cell carcinoma, acoustic neuroma, lentigo melanoma, congenital pulmonary malformation, acute eosinophilic leukemia, acute lymphoblastic leukemia, acute megakaryoblastic leukemia, mature monocytic leukemia, mature acute myeloid leukemia, acute myeloid leukemia, acute myeloid leukemia, acute myeloid leukemia, promyelocytic leukemia, adamantinoma, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenoid odontogenic tumor, adrenocortical carcinoma, adult T-cell leukemia, progressive NK-cell leukemia, AIDS-related cancer, AIDS-related lymphoma, alveolar soft tissue sarcoma, ameloblastoma, anal carcinoma, and undifferentiated cancer. Large cell lymphoma, undifferentiated thyroid cancer, angioimmunoblastic T-cell lymphoma, angiomyolipoma, angiosarcoma, appendiceal cancer, astrocytoma, malformed rhabdomyomas, 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, bronchoalveolar carcinoma, pheochromocytoma, Burkitt lymphoma, cancer of unknown primary origin, carcinoid tumor, cancer, carcinoma in situ, penile cancer, cancer of unknown primary origin, carcinosarcoma, Castleman disease, central nervous system embryonic tumor, cerebellar astrocytoma, cerebral astrocytoma, cervical Cancer, cholangiocarcinoma, chondroma, chondrosarcoma, chordoma, choriocarcinoma, choroid plexus papilloma, chronic lymphocytic leukemia, leukocyte leukemia, chronic myeloid leukemia, chronic myeloproliferative disorder, chronic neutrophilic leukemia, clear cell tumor, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, Degos disease, dermatofibrosarcoma, nodular cyst, fibroplastic small cell B-cell large cell tumor lymphoma, erythroblastic neuroepithelial tumor, embryonic carcinoma, endodermal sinus tumor, endometrial cancer, endometrial uterine cancer, endometrioid tumor, intestinal disease-associated T-cell lymphoma, ependymoblastoma, ependymoma, epithelioid sarcoma, erythroleukemia, esophageal tumor, esophageal tumor, esophagus Tumors: Ewing family sarcoma, Ewing sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic cholangiocarcinoma, extramammary Paget's disease, fallopian tube cancer, fetal fibroma, fibrosarcoma, follicular lymphoma, follicular thyroid cancer, gallbladder cancer, ganglion glioma, ganglion neuroma, gastric cancer, gastric lymphoma, gastrointestinal cancer, gastrointestinal carcinoid, gastrointestinal stromal tumor, germ cell tumor, germ blastoma, gestational trophoblastic carcinoma, giant cell tumor of bone, glioblastoma, glioma, glioma, glioma glucagonoma, gonadoblastoma, granulosa cell tumor, hairy cell leukemia,Hair cell leukemia, head and neck cancer, heart cancer, hemangioblastoma, hemangiopericytoma, angiosarcoma, hematological malignancies, hepatocellular carcinoma, hepatosplenic T-cell lymphoma, hereditary breast and ovarian cancer syndrome, Hodgkin lymphoma, hypopharyngeal cancer, hypothalamic glioma, inflammatory breast cancer, intraocular melanoma, islet cell carcinoma, islet cell tumor, juvenile myelomonocytic leukemia, Kaposi's sarcoma, kidney tumor, Kratzkin's tumor, Krukenberg's tumor, laryngeal cancer cancer), melanoma originating from malignant lentigo, leukemia, leukemia, lip cancer and oral cancer, liposarcoma, lung cancer, luteal malformation, lymphangioma, lymphangiosarcoma, lymphoepithelioma, lymphocytic leukemia, lymphoma, macroglobulinemia, malignant fibrous histiocytoma, malignant fibrous histiocytoma of bone, malignant glioma, malignant mesothelioma, malignant peripheral nerve schwannoma, malignant rhabdoid tumor, malignant triton tumor, MALT lymphoma, mantle cell lymphoma, Mast cell leukemia, mediastinal germ cell tumor, mediastinal tumor, medullary thyroid carcinoma, medulloblastoma, medullary cystoma, melanoma, meningioma, Merkel cell carcinoma, mesothelioma, occult metastatic cervical squamous carcinoma, metastatic urothelial carcinoma, Müllerian mixed tumor, monocytic leukemia, oral cancer, myxoid neoplasm, multiple endocrine neoplasia, multiple myeloma, multiple myeloma, mycosis fungoides, myelodysplastic disease, myelodysplastic syndrome, myeloid leukemia, myxoma Nasal cavity cancer, nasopharyngeal cancer, neoplasms, schwannoma, neuroblastoma, neurofibroma, neuroma, nodular melanoma, non-Hodgkin lymphoma, non-melanoma skin cancer, non-small cell lung cancer, eye tumors, oligostellate tumor, oligodendroglioma, tumor cell tumor, optic nerve sheath meningioma, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, ovarian epithelial carcinoma, ovarian germ cell tumor, breast diseases, Pancoast tumor , pancreatic cancer, pancreatic cancer, papillary thyroid carcinoma, papillomatosis, paraganglioma, sinus cancer, parathyroid cancer, penile cancer, perivascular epithelioid cell tumor, pharyngeal cancer, pheochromocytoma, moderately differentiated pineal parenchymal tumor, pineoblastoma, pituitary cell tumor, pituitary adenoma, pituitary tumor, plasma cell tumor, pleublastoma, polygermoma, progenitor T lymphoblastic lymphoma, primary central nervous system lymphoma, primary exudative lymphoma, primary hepatocellular carcinoma, primary liver cancer, primary peritoneal cancer,Primary 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, Sertli-Leydig cell tumors, sex cord-stromal cell tumors, Sézary syndrome cancer, skin cancer, small blue round cell tumors, small cell carcinoma, small cell lung cancer, small cell lymphoma, small intestine cancer, soft tissue sarcoma, somatostatin-producing tumors, rhizomatous warts, spinal cord tumors, spinal cord tumors, splenic marginal zone lymphoma, squamous cell carcinoma, gastric cancer, superficial spreading melanoma, Examples of cancer cells include supratentorial primitive neuroectodermal tumors, surface epithelial stromal tumors, synovial sarcomas, T-cell acute lymphoblastic leukemia, T-cell macrogranular lymphocytic leukemia, T-cell leukemia, T-cell lymphoma, T-cell prelymphocytic leukemia, teratomas, end-stage lymphoma, testicular cancer, coma, pharyngeal cancer, thymic cancer, thymoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, transitional cell carcinoma, urachal cancer, urethral cancer, urogenital tumors, uterine sarcomas, uveal melanoma, vaginal cancer, Berner-Morrison syndrome, verrucous cancer, visual pathway glioma, vulvar cancer, Waldenström hypergammaglobulinemia, Warthin's tumor, Wilms' tumor, and combinations thereof. In some embodiments, targeted cancer cells represent a subpopulation within a cancer cell population, such as cancer stem cells. In some embodiments, the cancer is of hematopoietic lineage, such as lymphoma. Antigens may be tumor-associated antigens. ,

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

[0161] Target cell death can be detected by any suitable method, for example, by measuring cells before and after treatment, or by measuring the levels of markers associated with live or dead cells (e.g., live or dead target cells), although these methods are not limited to those mentioned above. 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 initial conditions. 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 viable 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 are available. Examples of detection methodologies include, but are not limited to, cell staining, microscopy, flow cytometry, cell sorting, and combinations thereof.

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

[0163] Exposure of target cells to immune cells or immune cell populations disclosed herein may be carried out either in vitro or in vivo. Exposure of target cells to immune cells or immune cell populations means contacting the target cells with and / or in close proximity to them so that the antigens of the target cells (e.g., cell membrane-bound or non-bound) can bind to switch molecules expressed on the immune cells. Exposure of target cells to immune cells or immune cell populations also generally means contacting the target cells with and / or in close proximity to them so that the antigens of the target cells (e.g., cell membrane-bound or non-bound) can bind to CARs expressed on the immune cells. Exposure of target cells with immune cells or immune cell populations in vitro can be achieved by co-culturing the target cells with the immune cells. Target cells and immune cells can be co-culturned, for example, as adherent cells, or alternatively, in suspension. Target cells and immune cells can be co-culturned in various suitable types of cell culture solvents, for example, with adjuvants, growth factors, ions, etc. Exposing target cells to immune cells or immune cell populations in vivo can, in some cases, be achieved by administering immune cells to a subject, such as a human subject, and allowing the immune cells to localize to 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, for example, by direct injection.

[0164] Exposure may occur for any appropriate period of time, for example, 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 may be linked by chemical bonds such as amide or disulfide bonds; low molecular weight organic molecules (e.g., hydrocarbon chains); amino acid sequences such as peptide linkers (e.g., amino acid sequences of about 3 to 200 amino acids in length); or by combinations of low molecular weight organic molecules and peptide linkers. The peptide linker may provide the desired flexibility to enable the desired expression, activity, and / or steric position of the chimeric polypeptide. The peptide linker may be of any length suitable for linking at least two target domains and is preferably sufficiently flexible and designed to enable proper folding and / or function and / or activity of one or both of the linked domains. The peptide linker may have a length of at least 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids. In some embodiments, the peptide linker has an amino acid length of about 0–200, about 10–190, about 20–180, about 30–170, about 40–160, about 50–150, about 60–140, about 70–130, about 80–120, and about 90–110. In some embodiments, the linker sequence may include an endogenous protein sequence. In some embodiments, the linker sequence includes glycine, alanine, and / or serine amino acid residues. In some embodiments, the linker may include motifs such as multiple or repeating motifs, for example, GS, GGS, GGGGS, GGSG, or SGGG. The linker sequence may include any native amino acids, non-native amino acids, or combinations thereof.

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

[0167] A delivery method includes contacting a target polynucleotide with one or more nucleic acids containing nucleotide sequences encoding the compositions of the Disclosure, or introducing them into a cell (or a population of cells such as immune cells). A suitable nucleic acid containing nucleotide sequences encoding the compositions of the Disclosure may include an expression vector, wherein the expression vector containing nucleotide sequences encoding one or more compositions of the Disclosure is a recombinant expression vector.

[0168] Non-limiting examples of delivery methods or transformations include, for example, viral or bacteriophage infection, gene transfer, binding, plasmofusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI) transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun method, calcium phosphate precipitation, direct microinjection, and nanoparticle-mediated nucleic acid delivery.

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

[0170] Conventional viral and nonviral-based gene transfer methods may be used to introduce nucleic acids into mammalian cells or target tissues. Such methods may be used to administer nucleic acids encoding the compositions of this disclosure to cells or host organisms in culture. Nonviral vector delivery systems may include nucleic acids in complex with a DNA plasmid, RNA (e.g., transcripts of vectors described herein), naked nucleic acids, and a delivery vehicle such as a liposome. Viral vector delivery systems include DNA and RNA viruses, which may have genomes that are either episomal or integrated after delivery to cells.

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

[0172] RNA or DNA virus-based systems can be used to target specific cells in the body and to deliver 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 modified cells may optionally be administered (ex vivo). Virus-based systems may include retroviruses, lentiviruses, adenoviruses, and adeno-associated and herpes simplex virus vectors for gene transfer. Integration into the host genome can occur with retrovirus, lentivirus, 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 can be modified by incorporating exogenous envelope proteins, expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors capable of transforming or infecting non-dividing cells and producing high viral titers. The choice of retroviral gene delivery may depend on the target tissue. Retroviral vectors may contain cis-acting terminal repeat sequences with the ability to package exogenous sequences up to 6–10 kb. Minimal cis-acting LTRs are sufficient for vector replication and packaging, and can be used to incorporate therapeutic genes into target cells to provide persistent transgene expression. Retroviral vectors may include those based on mouse leukemia virus (MuLV), gibbon leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof.

[0174] Adenovirus-based systems may be used. Adenovirus-based systems can induce transient expression of transgenes. Adenovirus-based vectors may have high transduction efficiency in cells and may not require cell division. High titers and levels of expression can be obtained with adenovirus-based vectors. Adeno-associated virus ("AAV") vectors may be used to transduce cells containing target nucleic acids, 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 viral particles capable of infecting host cells. Examples of such cells include 293 cells (e.g., for adenovirus packaging) and Psi2 or PA317 cells (e.g., for retrovirus packaging). Viral vectors can be created by producing cell lines that package nucleic acids into viral particles. The vector may contain the minimum viral sequence necessary for packaging and subsequent integration into a host. The vector may also contain other viral sequences that can be replaced by expression or sets for the polynucleotides (multiple polynucleotides) to be expressed. Missing viral functions can be supplied trans by the packaging cell line. For example, an AAV vector may contain ITR sequences from the AAV genome required for packaging and integration into the host genome. Viral DNA can be packaged within a cell line, which may contain helper plasmids encoding other AAV genes, namely rep and cap, but without ITR sequences. Cell lines can also be infected with adenovirus as helpers. Helper viruses can facilitate the replication of AAV vectors and the expression of AAV genes from helper plasmids. Adenovirus contamination can be reduced, for example, by heat treatment, in which adenoviruses are more susceptible than AAV. Additional methods for delivering nucleic acids to cells, such as those described in U.S. Patent Application No. 20030087817, are also used and are incorporated herein by reference.

[0176] Host cells are transiently or nontransiently transfected with one or more vectors described herein. Cells may be transfected in a manner that occurs naturally in a subject. Cells may be taken from or derived from a subject and then transfected. Cells may be derived from cells taken from a subject, i.e., a cell line. In some embodiments, cells transfected with one or more vectors described herein are used to establish a new cell line containing sequences derived from one or more vectors. In some embodiments, cells transiently transfected with the compositions of this disclosure (e.g., transient transfection with one or more vectors or transfection with RNA) are used to establish a new cell line containing cells that include modifications but do not contain other exogenous sequences.

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

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

[0180] Pharmaceutical compositions comprising molecules (e.g., polypeptides and / or nucleic acids encoding polypeptides) or immune cells as described herein may be administered for prophylactic and / or therapeutic purposes. In therapeutic applications, a composition may be administered to a subject already suffering from a disease or condition in an amount sufficient to cure or at least partially cessate the symptoms of the disease or condition, or to cure, heal, improve, or induce remission of the condition. The effective amount for this use may vary based on the severity and course of the disease or condition, previous treatments, the subject's health status, weight, and responsiveness to the drug, as well as the subject's decision to administer the treatment.

[0181] Multiple therapeutic agents may be administered in any order or simultaneously. When administered simultaneously, multiple therapeutic agents may be provided in a single, integrated form or in multiple forms, such as multiple separate pills. Molecules may be filled together or separately in a single package or in multiple packages. When not administered simultaneously, the timing of multiple doses may vary by up to approximately months.

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

[0183] Molecules can be packaged within biological compartments. Biological compartments containing molecules can be administered to subjects. Examples of biological compartments, but not limited to, include viruses (lentiviruses, adenoviruses), nanospheres, liposomes, quantum dots, nanoparticles, microparticles, nanocapsules, vehicles, polyethylene glycol particles, hydrogels, and micelles.

[0184] For example, a biological compartment may include liposomes. Liposomes can be self-assembled structures comprising one or more lipid bilayers, each layer of which may contain two monolayers containing amphiphilic lipid molecules oriented opposite each other. The amphiphilic lipids may contain polar (hydrophilic) heads conjugated to two or more nonpolar (hydrophobic) acyl or alkyl chains. Energically unfavorable contact between the hydrophobic acyl chains and the surrounding aqueous solvent causes the amphiphilic lipid molecules to orient themselves such that the polar heads face the surface of the bilayer and the acyl chains face inward, effectively shielding the acyl chains from contact with the aqueous environment.

[0185] Preferred amphiphilic compounds used within liposomes include phosphoglycerides and sphingolipids, with representative examples including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine, distearoylphosphatidylcholine (DSPC), dilinoleoylphosphatidylcholine, and egg yolk sphingomyelin, or any combination thereof.

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

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

[0188] The amount of albumin in the nanoparticles may 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 may contain up to 30, 40, 50, 60, 70, 80% or more of nanoparticles. In some cases, the nucleic acid molecules of this disclosure may be bound to the surface of the nanoparticles.

[0189] The biochemical compartment may contain a virus. The virus may be a delivery system for the pharmaceutical compositions of this disclosure. Exemplary viruses include lentiviruses, retroviruses, adenoviruses, herpes simplex virus I or II, parvoviruses, reticuloendotheliosis virus, and adeno-associated viruses (AAVs). The pharmaceutical compositions of this disclosure may be delivered to cells using a virus. The virus may be infected and transduced in vivo, ex vivo, or in vitro. In ex vivo and in vitro delivery, the transduced cells may be administered to subjects in need of treatment.

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

[0191] A virus delivery system (e.g., a virus including the pharmaceutical composition of the present disclosure) can be administered to the cells, tissues, and organs of a subject as needed by direct injection, stereotactic injection, by an intraventricular, mini-pump injection system, by convection, by catheter, intravenous, parenteral, intraperitoneal, and / or subcutaneous injection. In some cases, cells can be transduced in vitro or ex vivo using a virus delivery system. The transduced cells can be administered to a subject suffering from a disease. For example, stem cells can be transduced using a virus delivery system including 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 is, 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 system of interest are administered. These immune cells may be administered before, during, or after the onset of the disease or condition, and the timing of administration of compositions containing the compounds may vary. For example, immune cells expressing the system of interest may be used as a prophylactic agent and may be continuously administered to subjects prone to a condition or disease to prevent the onset of the disease or condition. The immune cells may be administered to subjects during or as soon as possible after the onset of symptoms. Administration may be initiated within the first 48 hours of symptom onset, within the first 24 hours of symptom onset, within the first 6 hours of symptom onset, or within the first 3 hours of symptom onset. The initial administration may be via any practical route, such as by any route described herein, using any formulation described herein. The immune cells may be administered as soon as practically possible after the onset of the disease or condition is detected or suspected, and for a period of time necessary for the treatment of the disease (e.g., about 1 month to about 3 months). The length of treatment may vary for each subject.

[0194] The molecules described herein (e.g., polypeptides and / or nucleic acids) are available in doses of approximately 1 mg to 2000 mg, 5 mg to 1000 mg, 10 mg to 25 mg to 500 mg, 50 mg to 250 mg, 100 mg to 200 mg, 1 mg to 50 mg, 50 mg to 100 mg, 100 mg to 150 mg, 150 mg to 200 mg, 200 mg to 250 mg, 250 mg to 300 mg, and 300 mg to 350 mg. It may be present in compositions ranging from approximately 350 mg to approximately 400 mg, approximately 400 mg to approximately 450 mg, approximately 450 mg to approximately 500 mg, approximately 500 mg to approximately 550 mg, approximately 550 mg to approximately 600 mg, approximately 600 mg to approximately 650 mg, approximately 650 mg to approximately 700 mg, approximately 700 mg to approximately 750 mg, approximately 750 mg to approximately 800 mg, approximately 800 mg to approximately 850 mg, approximately 850 mg to approximately 900 mg, approximately 900 mg to approximately 950 mg, or approximately 950 mg to approximately 1000 mg.

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

[0196] The molecules described herein (e.g., polypeptides and / or nucleic acids) may be present in compositions providing 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 molecules. The activity may be the regulation of gene expression. In some embodiments, the total number of active units of the molecule delivered to the subject is at least 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 60,000, 70,000, 80,000, 90,000, 110,000, 120,000, 130,000, 140,000, 150,000, 160,000, 170,000, 180,000, 190,000, 200,000, 210,000, 220,000, 230,000, or 250,000 or more units. In some embodiments, the total number of active units of the molecule delivered to the subject is at most 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 60,000, 70,000, 80,000, 90,000, 110,000, 120,000, 130,000, 140,000, 150,000, 160,000, 170,000, 180,000, 190,000, 200,000, 210,000, 220,000, 230,000, or 250,000 or more units.

[0197] Various aspects of this 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 subjects had HLA-A:0201 leukocytes. Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll lymphocyte isolation. After 2 hours of adherent culture, T cells were removed. NY-ESO-1 TCR lentivirus was added at an infection efficiency (MOI) of 1. T cells were then cultured and proliferated. 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 higher TCR expression in T cells transduced with the NY-ESO-1 TCR gene.

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

[0200] Example 3: Neoantigen-activated T cells The preparation is shown in Figure 2. PBMCs or tumor cells from surgical procedures were subjected to whole-exome sequencing or RNA transcriptome sequencing. Twenty mutations were then selected based on affinity predictions in relation to the patient's HLA typing. Genes encoding neoantigens were synthesized and transcribed into RNA. PBMCs were isolated and subjected to 2 hours of adherent culture. Adherent monocytes were harvested. Cytokines were added to promote dendritic cell differentiation and maturation. RNA was introduced into dendritic cells by electroporation. Suspended cells were obtained primarily as T cells and cultured together with dendritic cells. CD137+ positive cells were then isolated to provide neoantigen-reactive (e.g., recognizable) T cells ("neoTs"). The neoTs were then propagated.

[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, the packaging vector pMDL-gag, Rev, and the envelope vector pMD2.G were co-introduced into HEK293T cells using calcium phosphate or liposome-PEI. The supernatant was collected after 48 hours and then centrifuged to concentrate the lentiviruses.

[0202] Lentivirus titration was performed using 3-fold serial dilutions. HEK293T cells were transduced with 50 μL of lentivirus for 48–72 hours, then harvested and subsequently stained with PD-1. The PD-1 positivity rate (PD-1+%) was analyzed by flow cytometry, and titers were calculated based on the following criteria. Titer (TU / mL) = 40000~45000 (This is the initial HEK293T cell count) × PD1 + % × Dilution ratio × 20 (initial PD1 + %<20%

[0203] Figures 3A and 3B show the calculation of PD1 / CD28 lentiviral titers. 3 × 10 7 A higher potency allows for further use.

[0204] Example 5: Transduction of PD1 / CD28 into neoantigen-activated T cells Three types of T cells were obtained: Switch-NY-ESO-1-TCR-T, Switch-TIL, and Switch-neoT. Switch-NY-ESO-1-TCR-T cells were obtained by expressing the switch molecule in ESO-1-TCR-T cells from Example 1. Switch-TIL cells were obtained by expressing the switch molecule in triple-positive T cells from Example 2. Switch-neoT cells were obtained by expressing the switch molecule in neoT cells from Example 3. Flow cytometry showed an expression rate of approximately 60% for Switch in all three types of T cells. See Figure 4.

[0205] Example 6: In vitro assay of NY-ESO-1 targeted TCR-T cells expressing PD1 / CD28 switch molecule J82-NY-ESO-1-PD-L1 tumor cells were constructed using HLA typing A:0201. A lentiviral vector was added to J82 (bladder, transitional cell carcinoma) cells with an infection efficiency MOI of 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 two 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 simultaneously expressed PD-L1 and NY-ESO-1, confirming the success of the cell construction. Figures 6A–6C show data from in vitro cell-killing assays, 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 CD8 and CD107a expression. Surface localization of CD107a (also known as LAMP-1), a degranulation marker normally found inside T cell granules, is a sign of cytotoxic activity as T cells release perforin and granzymes from their granules to kill target cells. As shown in Figure 6A, the data demonstrate greater cell-killing activity of Switch-NY-ESO1-TCR T cells compared to NY-ESO1-TCR T cells. Figures 6B and 6C show that exposure to J82 bladder cancer cells does not induce IFN-γ and IL-2 secretion 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 induces IFN-γ and IL-2 secretion in both NY-ESO1-TCR T cells and Switch-NY-ESO1-TCR T cells, with higher levels of IFN-γ and IL-2 secretion in Switch-NY-ESO1-TCR T cells.

[0206] Example 7: In vitro efficacy assay of TIL expressing PD1 / CD28 molecule Figures 7A and 7B show the release of IFN-γ and IL-2 in TILs (and TILs expressing the PD1 / CD28 switch molecule (Switch-TILs)) 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 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 (neoTs) 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 neoT cells without the PD1 / CD28 switch molecule.

[0208] Example 9: Animal experiments with NY-ESO1-TCR T cells expressing the PD1 / CD28 switch molecule. 1 x 10 6 Individual tumor cells, J82-NY-ESO1-PDL1, are inoculated subcutaneously into NSG mice. Tumor development is expected approximately two weeks later. Tumor size is measured at 23 weeks, and 30 mice are used.

[0209] Control mice are treated with PBS by subcutaneous injection (A0). There are five treatment groups: untreated PBS (A1), T cells (A2), switch-T cells (A3), NY-ESO1-TCR-T cells (A4), and switch-NY-ESO1-TCR-T cells. The cells are injected into the tail vein at a rate of 1 × 10⁶ 7It is administered by intravenous injection of individual cells.

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

[0211] The experiment is expected to show that tumor size in group A5 decreases, remains nearly constant, or at least decreases in rate of increase compared to groups A1-A3.

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

[0213] Example 10: Preparation of lentiviral CARs targeting B cell surface proteins (BCARs) CD19 was selected as the B-CAR target, and 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-introduced into HEK293T cells using calcium phosphate or liposome-PEI. The supernatant was collected after 48 hours and ultracentrifuged to concentrate the lentivirus.

[0214] Lentivirus titration was performed using 3-fold serial dilutions. 293T cells were transduced with 50 μL of lentivirus for 48–72 hours, then harvested and subsequently stained for CAR expression. The CAR positivity rate (CAR%) was analyzed by flow cytometry, and the titer was calculated based on the following: Potency (TU / mL) = Initial number of 293 T cells × CAR+% × Dilution factor × 20 (Initial CAR+% < 20%)

[0215] Lentivirus titers were calculated. 3 × 10 7 A higher potency was considered more 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 a B-CAR expression rate 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 target function of BCAR-NY ESO1-TCR-T cells, 1 × 10⁶ cells were used. 5 Individual J82-NY-ESO-1-Luc cells were seeded in 24-well plates and cultured overnight to allow adhesion. The cells were divided into four groups, A, B, C, and D, and each well was also divided into 5 × 10⁶ cells. 4 It contained 10 B cells. Group A was 2 × 10 5 It was co-cultured with 2 × 10 T cells. Group B was 2 × 10 5 It was co-cultured with 2 × 10 BCAR-T cells (CAR-positive rate 60%). Group C was 2 × 10 5 It was co-cultured with NY-ESO1-TCR-T cells. Group D was 2 × 105 The cells were co-cultured with individual BCAR-NY-ESO1-TCR-T cells. The following assays were performed.

[0218] Increase in T cells. T cell counts 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] Tumor cell proliferation. Supernatants were collected from cultures of groups A-D at 48 and 96 hours. The cultures were then washed three times with PBS. Adherent tumor cells were lysed, and luciferase activity was measured as an indicator of tumor cell viability. See Figure 11. The data show that after 48 hours of culture, the average amount of protein from tumor cells (and therefore the number of tumor cells present) was lowest with treatment using 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 TILs, tumor cells were isolated from new tumor tissue of human subjects and seeded in 24-well plates. The cells were cultured overnight to allow adhesion. TILs and BCAR-TILs were added to the wells. The same amount of B cells were added to the wells, and the following assays were performed.

[0221] Increase in T cells. After 96 hours of co-culture with tumor cells, TILs increased approximately 10-fold, and BCAR-TILs increased approximately 25-fold, as shown in Figure 12A. The results indicate that the increase in BCAR-TILs was greater when co-cultured with B cells and tumor cells compared to BCAR-free TILs.

[0222] Tumor cell proliferation. After 96 hours, the supernatant was collected from the culture mixture. The culture was then washed three times with PBS. Adherent tumor cells were lysed, and luciferase activity was measured as an indicator of tumor cell viability. Luciferase levels are shown in Figure 12B. The data show that after 96 hours of culture, the average amount of protein from tumor cells (and therefore the number of tumor cells present) was lower when treated with BCAR-TIL cells compared to when treated with BCAR-free TILs.

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

[0224] Increase in T cells. After 96 hours of co-culture with tumor cells, neoT cells increased approximately 9-fold, and BCAR-neoT cells increased approximately 23-fold, as shown in Figure 13A. The results indicate that the increase in BCAR-neoT cells was greater than that of BCAR-free neoT cells when co-cultured with B cells and tumor cells.

[0225] Tumor cell proliferation. After 96 hours, the supernatant was collected from the culture mixture. The culture was then washed three times with PBS. Adherent tumor cells were lysed, and luciferase activity was measured as an indicator of tumor cell viability. Luciferase levels are shown in Figure 13B. The data show that after 96 hours of culture, the average amount of protein from tumor cells (and therefore the number of tumor cells present) was lower when treated with BCAR-neoT cells compared to when treated with BCAR-free neoT cells.

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

[0227] Administration: Animals in the blank control (A0) group were infused with PBS via the tail vein. The tumorigenetic group was divided into six groups: the PBS group (A1), the T cell group (A2), the BCAR-T group (A3), the NY-ESO-1 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-A5 were infused via the tail vein at a rate of 1 × 10⁶ 4 Individual cells were injected, and group A6 was 1 × 10 7 Each cell is injected. All groups are 1 × 10⁶ 7 Individual B cells are administered by injection.

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

[0229] Changes in tumor volume

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

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

[0232] Peripheral blood is extracted from animals in groups A2 to 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 to A5 with BCAR will be 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 fresh tumor tissue and injected subcutaneously into NSG mice at a dose of 1×10 6 per animal. 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. 30 mice are selected.

[0234] Administration: Animals in the blank control (A0) group are injected with PBS via the tail vein. The tumor formation 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). Animals in groups A1 to 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 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

[0236] Change in Tumor Volume

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

[0238] [[ID= Peripheral blood will be extracted from animals in groups A2–A5 10 days after administration. The total number of CD3+ T cells will be measured using flow cytometry, and it is expected that the number of T cells in groups A3–A5, which possess BCAR, will be greater than that in group A4. Both BCAR T cells and dual-target T cells are expected to increase in vivo.

[0240] Example 17: Animal experiments with neoT expressing BCAR Animal model: Tumor cells are isolated from new tumor tissue, and 1 × 10⁶ cells are used per animal. 6 The drug is injected subcutaneously into NSG mice in individual doses. As a blank control, animals in group A0 were subcutaneously injected with PBS. Tumors form in the animals approximately two weeks after injection of tumor cells. Tumor size is measured after 25 days. Thirty mice are selected.

[0241] Administration: Animals in the blank control (A0) group were infused with PBS via the tail vein. The tumorigenetic group was divided into six groups: 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). Animals in groups A1-A5 were infused with 1 × 10⁶ via the tail vein. 4 Group A6 was injected with 1 x 10 T cells. 7 Each group is injected with 1 x 10⁶ T cells. 7 Individual B cells are administered by injection.

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

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

[0244] Changes in tumor volume

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

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

[0247] Peripheral blood is extracted from 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, which possess BCAR, will be greater than that in group A4. Both BCAR T cells and dual-target T cells are shown to increase in vivo.

[0248] Example 18: Preparation of lentivirus for three switch molecules Three switch molecules were constructed: 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). The extracellular domains of PD1, TIM3, and TGFBR2 were used as immunoinhibitory proteins for their respective switch molecules, and CD28 was used as a co-stimulatory signaling protein.

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

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

[0251] Lentivirus titers were calculated. 3 × 10 7 A higher potency was considered more suitable for further use.

[0252] TIM3sw and TGFBR2sw were prepared using the same method.

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

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

[0255] Super-pTILs should also be understood as SuperTILs. Super-pTILs are specifically named to identify their different cellular origins.

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

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

[0258] TIM3sw-TIL / pTIL, TGFBR2sw-TIL / pTIL, and BCAR-TIL / pTIL were prepared using the same method.

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

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

[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 proportions so that each was present in amounts between 0% and 100%. In this embodiment, the proportion was 1:1:1.

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

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

[0266] Tumor cells were isolated from the patient's new tumor tissue and seeded in 24-well plates with a luciferase marker. The cells were cultured overnight to allow adhesion. The cells were divided into 10 groups and co-cultured with controls or the following T cells: The following assays were performed: control group (group A1) without T cell-CK supplementation; 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).

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

[0268] 2. Tumor cell proliferation. Supernatant was collected from the culture at 48 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 tumor cell viability. As shown in Figure 17A, the number of tumor cells in each group (groups A2-A10) was reduced compared to the control group (group A1), and the groups with the switch (groups A4-A10) showed an even more significant reduction in the number of tumor cells than the groups without the switch (groups A2 and A3).

[0269] 3. Increase in T cells. After 48 hours of culture, the number of T cells was measured. As shown in Figure 17B, the T cells in the groups without switches (groups A2 and A3) showed a slight increase, while the groups with switches (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 new tumor tissue and seeded in 24-well plates with a luciferase marker before being cultured overnight for adhesion. An equal amount of B cells are added to each well. The cultured cells are divided into 10 groups and co-cultured with controls or the following T cells: The following assays were performed: control group (group B1) without T cell-CK supplementation; 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).

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

[0273] 2. Increase in T cells. After 48 hours of culture, the number of T cells was measured. As shown in Figure 17B, the normal TIL group (group B2) showed a slight increase, while the group with Switch (groups B4-B10) showed a significant increase, and the group with BCAR (groups B3 and B7-B10) showed an even greater increase.

[0274] As demonstrated by in vitro assays, the tumor-killing effect of TILs is enhanced after transduction with switch molecules. When transductioned with both switch and BCAR molecules to provide SuperTILs, SuperTILs exhibit further enhanced increases and tumor cell-killing effects in the presence of B cells.

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

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

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

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

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

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

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

[0282] The groups were: a control group (Group B1) with no T cell-CK supplementation; 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 were added to each well. The following assay was performed.

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

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

[0285] As demonstrated by in vitro assays, the tumor-killing effect of pTILs is enhanced after transduction with switch molecules. When transductioned with both switch and BCAR molecules to provide SuperTILs, SuperTILs exhibit further enhanced increases and tumor cell-killing effects in the presence of B cells.

[0286] Example 23: Animal experiments with Super-TIL Animal model: 1 × 10⁶ cells from new tumor cells 6 Nucleic tumor cells were inoculated subcutaneously into NSG mice. As a blank control, animals in group A0 were subcutaneously injected with PBS. Tumors formed in the animals approximately two weeks after tumor cell injection. Tumor size was measured at 25 days. 132 mice were selected and divided into group A (11 subgroups) without B cells and group B (11 subgroups) with B cells (22 groups in total) according to the following:

[0287] Group A consists of a blank control group (A0) injected with PBS via the tail vein, and 10 groups of mice into which tumor cells are seeded: 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 into which tumor cells are seeded: untreated-PBS group (Group B1), normal TIL (Group B2), BCAR-TIL group (Group B3), PD1sw-TIL group (Group B4), TIM3sw-TIL (Group B5), TGFBR2sw-TIL (Group B6), PD1-STIL group (Group B7), TIM3-STIL (Group B8), TGFBR2-STIL (Group B9), and XSTIL (Group B10). Groups B2-B10 were injected via the tail vein at a rate of 1 × 10⁶ 4 Given a number of T cells, all groups were 1 × 10 7 Individual B cells are administered by injection.

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

[0290] (1) Changes in tumor volume

[0291] This experiment is expected to demonstrate that mice injected with TILs containing a switch (groups A4-A10 and B4-B6) exhibit delayed tumor growth.

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

[0293] Tumor tissue from groups B7–B9 is 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 may escape, which results in the ineffectiveness of the corresponding switch.

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

[0295] Group A consists of a blank control group (A0) injected with PBS via the tail vein, and 10 groups of mice into which tumor cells are seeded: untreated-PBS group (Group A1), normal pTIL (Group A2), BCAR-pTIL group (Group A3), PD1sw-pTIL group (Group A4), TIM3sw-pTIL (Group A5), TGFBR2sw-pTIL (Group A6), PD1-pSTIL group (Group A7), TIM3-pSTIL (Group A8), TGFBR2-SpTIL (Group A9), and XSpTIL (Group A10). Groups A2-A10 were injected via the tail vein at a rate of 1 × 10⁶ 4 You are given a number of T cells.

[0296] Group B consists of a blank control group (B0) injected with PBS via the tail vein, and 10 groups of mice into which tumor cells are seeded: untreated-PBS group (Group B1), normal TIL (Group B2), BCAR-pTIL group (Group B3), PD1sw-pTIL group (Group B4), TIM3sw-pTIL (Group B5), TGFBR2sw-pTIL (Group B6), PD1-pSTIL group (Group B7), TIM3-SpTIL (Group B8), TGFBR2-SpTIL (Group B9), and XSpTIL (Group B10). Groups B2-B10 were injected via the tail vein at a rate of 1 × 10⁶ 4 Given a number of T cells, all groups were 1 × 10 7 Individual B cells are administered by injection.

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

[0298] Experiments are expected to demonstrate that the SuperTIL / pTILs of this disclosure possess specific tumor recognition and killing effects via multiple targets (from TILs or pTILs). SuperTIL / pTILs may also exhibit the ability to overcome the tumor environment (from one or more switches) to enhance killing and the self-renewal ability of BCARs. These modified immune cells provide an effective oncological therapeutic tool to address various problems associated with tumor immunotherapy.

[0299] Example 25: In vitro killing effect of BCAR-TCR T on NY ESO1 tumor cells To confirm the function of BCAR in TCR-mediated NY-ESO-1, the J82-NY-ESO1 tumor cell line, HLA genotyping of A:0201, was used as a target to measure the killing effect of BCAR-TCR T cells.

[0300] 1 x 10 5Individual J82-NY-ESO1 tumor cells were seeded onto RTCA (Real-Time Cell Analysis) electrode plates and cultured overnight to allow adhesion. The cells were divided into three groups, A, B, and C. In group A, 1 × 10⁶ cells were present. 5 pieces, 1×10 4 pieces, 1×10 3 pieces, 1×10 2 Each BCAR-TCR T cell was co-cultured with J82-NY-ESO1 cells. In Group B, 1 × 10 5 pieces, 1×10 4 pieces, 1×10 3 pieces, 1×10 2 1 BCAR-TCR T and 1 × 10 5 Individual 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, where B cells are absent, the highest dose of 1 × 10⁶ was used. 5 Only BCAR-TCR T cells showed a significant killing effect against J82-NY-ESO1 tumor cells, whereas in group B, 1 × 10⁶ cells were killed in the presence of B cells. 2 Even the lowest dose of individual BCAR-TCR T was 1 × 10⁶ in Group A. 5 It showed a remarkable killing effect against J82-NY-ESO1 tumor cells comparable to individual BCAR-TCR T doses, representing an approximately 1000-fold increase in efficacy.

[0302] Example 26: Clinical antitumor effects of STILs and SpTILs (As shown in Table 1) Five subjects were enrolled in the clinical trial and received either STILs or SpTILs.

[0303] [Table 1]

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

[0305] Cell preparation before treatment (1) Separation of TIL / pTIL For subjects 3, 4, and 5, CD3-positive TILs were isolated from newly excised tumor tissue using CD3 magnetic beads after enzymatic digestion. For subjects 1 and 2, PBMCs were isolated from the patients, where PD1 + The T cell counts were 19% and 5% of the total T cell count, respectively. + The proportion of T cells is thought to be TILs (pTILs) in peripheral blood derived from tumor tissue, and PD1 + The cells were further enriched using PR1 beads to provide T cells, i.e., pTILs.

[0306] Preparation of STIL / SpTIL Lentiviral vectors loaded with PD1sw-CD19 CAR were transfected into TILs / pTILs with a transduction efficiency of 2–15%. Cells were encapsulated in infusion bags without amplification. The entire process (excluding the T cell-recognized neoantigen identification process) took 3–10 days.

[0307] Using the cells obtained from all 5 subjects, 10 8 ~10 9 This is a much lower dose than the reported dose of individual cells / kg. 5 ~10 6 Treatment doses in terms of individual cells / kg are shown in Table 2.

[0308] [Table 2]

[0309] Safety evaluation Of the five subjects, three experienced grade 1 cytokine release syndrome (CRS) as evidenced by high fever (incidence rate 60%, 3 / 5). All three recovered; one received no intervention, and the other two received treatment with tocilizumab. Both the incidence and grade of CRS were significantly lower than in those treated with CD19 CAR-T. No post-treatment autoimmune diseases were observed.

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

[0311] [Table 3]

[0312] Effect of CAR on the increase of STIL / SpTIL in vivo The percentage of CAR+ T cells in the peripheral blood of the subjects was monitored. The ratio of increased STILs (or SpTILs) in the peripheral blood was calculated according to the following formula. Rate of increase = Lymphocyte count / L × Volume of circulating peripheral blood × Percentage of T cells in lymphocytes × Percentage of STILs in T cells Here, the lymphocyte count / L was obtained from a predefined blood test, the percentage of T cells in the lymphocytes was determined by flow cytometry as the percentage of CD3+ cells, and the percentage of STILs in the T cells was determined by flow cytometry as the percentage of CAR+ cells in the 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] No exogenous immunoglobulins were administered during observation, 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 their peripheral blood. The number of CTCs two months after cell injection was compared to baseline on the day of injection. The results are shown in Table 5 and Figure 25 below, showing a significant decrease in the number of CTCs.

[0317] [Table 5]

[0318] Enhanced killing effect by switch molecules Three subjects (1, 2, and 5) were observed to have reduced peripheral blood T cells and persistent pleural or ascites between days 14 and 28. Subject 4, who had pleural metastases, developed pleural effusion, while subjects 1 and 2, who had peritoneal metastases, developed ascites. T cells were found in both pleural and ascites fluids, along with IL-6 at higher concentrations than in peripheral blood. This observation is summarized in Table 6 and indicates 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 conceived by those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be applicable when carrying out the present invention. The following claims define the scope of the present invention, and methods and structures that fall within the scope of these claims and their contents are intended to be protected thereby.

Claims

1. Modified immune cells that specifically bind to neoantigens, wherein the modified immune cells (a) A switch molecule comprising an extracellular domain (ECD) of a protein that, upon binding to its ligand, induces an immune cell inactivation signal in unmodified immune cells, wherein the ECD is fused to an intracellular domain (ICD) of a co-stimulatory molecule that mediates an immune cell activation signal, and the 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 immune cells. (b) A chimeric antigen receptor (CAR) comprising (i) an antigen interaction domain capable of binding to B cell surface proteins; (ii) a transmembrane domain; and (iii) an intracellular signaling domain, and (c) T cell receptor (TCR) complex that shows specific binding to neoantigens Modified immune cells containing [specific components / synthetic elements].

2. The modified immune cell according to claim 1, wherein the TCR complex is an endogenous TCR complex or an exogenous TCR complex.

3. The neoantigen comprises a peptide fragment of a protein encoded by a mutant gene, wherein the gene is ABL1, 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, FAM11IB, 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, PDG FRa, PIK3CA, PMEL, pol gene, POLE, PTEN, RAC1, RBM27, RNF43, RPL22, RUNX1, SEC31A, SEC63, SF3B 1. Modified immune cells according to claim 1, selected from SLC35F5, SLC45A2, SMAP1, SMAP1, SPOP, TFAM, TGFBR2, THAP5, TP53, TTK, TYR, UBR5, VHL, and XPOT.

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

5. The modified immune cell according to claim 1, wherein the protein that induces an immune cell inactivation signal in unmodified immune cells upon binding to the ligand is a signaling receptor, a checkpoint receptor, a cytokine receptor, a chemokine receptor, a growth factor receptor, or a hormone receptor.

6. The modified immune cell according to claim 1, wherein the protein that induces an immune cell inactivation signal in unmodified immune cells upon binding to the ligand is 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 attenuators (BTLA), killer cell immunoglobulin-like receptor (KIR), indoleamine-2,3-dioxygenase (IDO), lymphocyte activator gene-3 (LAG3), T cell immunoglobulin mucin-3 (TIM-3), and TIGIT.

7. The modified immune cell according to claim 1, wherein the co-stimulatory 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.

8. The modified immune cell according to claim 1, wherein the immune cell activation signal is mediated by an activating factor.

9. The modified immune cell according to claim 8, wherein the activating factor is a soluble cytokine, a soluble chemokine, or a growth factor.

10. The modified immune cell according to claim 9, wherein the activating factor is a soluble cytokine, and the soluble cytokine is IL-1, IL-2, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IL-21, TNF, TGF, IFN, or a functional fragment or variant thereof.

11. The modified immune cell according to claim 1, wherein the immune cell activation signal comprises: clonal proliferation and expansion of the modified immune cell; cytokine release by the modified immune cell; cytotoxicity of the modified immune cell; proliferation of the modified immune cell; differentiation, dedifferentiation, and redifferentiation of the modified immune cell; migration and / or transport of the modified immune cell; exhaustion and / or reactivation of the modified immune cell; and release of other intercellular molecules, metabolites, compounds, or combinations thereof by the modified immune cell.

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

13. The modified immune cell according to claim 1, wherein the intracellular signaling domain comprises an immunoreceptor-activating tyrosine motif (ITAM) or an immunoreceptor-suppressing tyrosine motif (ITIM).

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

15. The modified immune cell according to claim 1, wherein the intracellular signaling domain includes the intracellular domain of CD3ζ.

16. The modified immune cell according to claim 15, wherein the intracellular domain of CD3ζ contains an immune receptor activating tyrosine motif (ITAM).

17. The modified immune cell according to claim 1, wherein the CAR further comprises a co-stimulatory domain.

18. The modified immune cell according to claim 17, wherein the co-stimulatory domain comprises a signaling domain of an MHC class I molecule, a TNF receptor protein, an immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocyte activating molecule (SLAM protein), an activated NK cell receptor, or a Toll ligand receptor.

19. The co-stimulatory domain is 2B4 / CD244 / SLAMF4, 4-1BB / TNF-SF9 / CD137, B7-1 / CD80, B7-2 / CD86, B7-H1 / PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BAFF R / TNFRSF13C, BAFF / BlyS / TNF-SF13B, BLAME / SLAMF8, BTLA / CD272, CD100 (SEMA4D), CD103, CD11a, CD11b, CD11c, CD11d, CD150, CD160 (BY55), CD18, CD19, CD2, CD200, CD229 / SLAMF3, CD27 ligand / TNF-SF7, CD27 / TNFRSF7, CD28, CD29, CD2F-10 / SLAMF9, CD30 ligand / TNF-SF8, CD30 / TNFRSF8, CD300a / LMIR1, CD4, CD40 ligand / TNF-SF5, CD40 / TNFRSF5, CD48 / SLAMF2, CD49a, CD49D, CD49f, CD53, CD58 / LFA-3, CD69, CD7, CD8 α, CD8 β, CD82 / Kai-1, CD84 / SLAMF5, CD90 / Thy1, CD96, CDS, CEACAM1, CRACC / SLAMF7, CRTAM, CTLA-4, DAP12, Dectin-1 / CLEC7A, DNAM1 (CD226), DPPIV / CD26, DR3 / TNFRSF25, EphB6, GADS, Gi24 / VISTA / B7-H5, GITR ligand / TNF-SF18, GITR / TNFRSF18, HLA class I, HLA-DR, HVEM / TNFRSF14, IA4, ICAM-1, ICOS / CD278, Ikaros, IL2R β, IL2R γ, IL7R α, integrin α4 / CD49d, integrin α4β1, integrin α4β7 / LPAM-1, IPO-3, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIRDS2, LAG-3, LAT, LIGHT / TNF-SF14, LTBR, Ly108, Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1), lymphotoxin-α / TNF-β, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80(KLRF1), NTB-A / SLAMF6, OX40 ligand / TNFSF4, OX40 / TNFRSF4, PAG / Cbp, PD-1, PDCD6, PD-L2 / B7-DC, PSGL1, RELT / TNFRSF19L, SELPLG (CD162), SLAM (SLAMF1), SLAM / CD150, SLAMF4 (CD244), SLAMF6 (NTB-A), SLAMF7, SLP-76, TACI / TNFRSF13B, TCL1A, TCL1B, TIM-1 / KIM-1 / HAVCR, TIM-4, TL1A / TNFSF15, TNF A modified immune cell according to claim 17, comprising a signaling domain of a molecule selected from the group consisting of RII / TNFRSF1B, TNF-α, TRANCE / RANKL, TSLP, TSLP R, VLA1, and VLA-6.

20. The modified immune cells according to claim 1, wherein, upon binding of the switch molecule to the ligand, the modified immune cells exhibit enhanced neoantigen binding compared to unmodified immune cells.

21. The modified immune cells according to claim 1, wherein the modified immune cells exhibit increased cytotoxicity to the target cells compared to unmodified immune cells when the switch molecule binds to the ligand and the modified immune cells bind to the neoantigen present on the target cells.

22. The modified immune cells according to claim 1, wherein the modified immune cells exhibit increased cytokine secretion compared to unmodified immune cells when the switch molecule binds to the ligand and the modified immune cells bind to the neoantigen present on the target cell.

23. The modified immune cell according to claim 22, wherein the cytokine is IFN-γ or IL-2.

24. The modified immune cell according to claim 1, wherein the modified immune cell is a T cell or a tumor-infiltrating lymphocyte (TIL).

25. (a) Administering the modified immune cells described in claim 1, (b) In a condition in which the modified immune cells induce cytotoxicity against cancerous target cells, thereby inducing the death of the cancerous target cells, the modified immune cells are brought into contact with cancerous target cells that express neoantigens. Modified immune cells according to claim 1 for treating cancer in a subject, including

26. The use of modified immune cells according to Claim 1 in the manufacture of a pharmaceutical composition for increasing a T cell population, wherein increasing the T cell population is (a) To provide a T cell population comprising at least the modified immune cells, (b) Exposing the T cell population to the B cell surface protein in such a manner that it is effective in increasing the T cell population. Use including.

27. The use according to claim 26, wherein, in (b) above, the T cell population is exposed to B cells containing the B cell surface protein.

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