CN117769593A - Immune cells engineered to promote saenox delivery and uses thereof - Google Patents

Abstract

In certain aspects, the disclosure relates to an immune cell that has been engineered to include one or more heterologous polynucleotides that promote sanoχ transfer of the immune cell. The immune cell may also comprise one or more nucleic acid sequences encoding a Chimeric Antigen Receptor (CAR). Methods of using these engineered immune cells to promote saenox transfer, promote immune responses, and treat cancer are also disclosed.

Description

Immune cells engineered to promote saenox delivery and uses thereof

RELATED APPLICATIONS

The present application claims priority from U.S. provisional application Ser. No. 63/308,195 filed on day 2, month 9 of 2022 and U.S. provisional application Ser. No. 63/216,505 filed on day 6, month 29 of 2021, each of which is expressly incorporated herein by reference in its entirety.

Submission of sequence Listing

The sequence listing associated with this application is submitted in electronic format via the EFS-Web and is incorporated herein by reference in its entirety. The name of the text file containing the sequence list is 129983_00820_sequence_listing. The size of the text file is 72,183 bytes and the text file was created at 2022, 6, 28.

Background

In metazoans, programmed cell death is an important genetic programming process that maintains tissue homeostasis and eliminates potentially harmful cells.

Drawings

Fig. 1A shows an exemplary Chimeric Antigen Receptor (CAR) construct for expression in immune cells. Fig. 1B shows an exemplary construct for expressing a protein that promotes saenox transfer (thanototransmision) in immune cells.

FIGS. 2A and 2B show the relative viability of CT-26 mouse colon cancer cells following induction of Sanot delivery.

FIGS. 3A and 3B show the effect of Cell Turnover Factor (CTF) produced by CT-26 mouse colon cancer cells on stimulation of IFN-related gene activation in macrophages after induction of saenopassing polypeptide expression (e.g., TRIF expression alone or in combination with RIPK3 (cR 3) and/or mesothelin E (cGE)). In fig. 3A, tet-inducible RIPK3 is designated "RIPK3", and the RIPK3 construct containing the constitutive PGK promoter is designated "pgk_ripk3". In fig. 3B, for each sanoχ delivery module, the treatment groups were Control (CTL), doxycycline (Dox), and doxycycline+b/B homodimer (dox+dimer), respectively, from left to right.

FIG. 4 shows the effect of Cell Turnover Factor (CTF) generated by CT-26 mouse colon cancer cells upon induction of TRIF, RIPK3 or TRIF and RIPK3 expression on the stimulation of expression of activation markers in bone marrow-derived dendritic cells (BMDC). MFI is the average fluorescence intensity.

FIGS. 5A, 5B and 5C show the effect of saenopassing polypeptide expression on survival of mice implanted with CT-26 mouse colon cancer cells. "CT26-TF" means CT26 cells expressing TRIF alone, and "CT26-P_R3" means cells expressing RIPK3 alone. In fig. 4B, all mice were treated with anti-PD 1 antibodies.

FIG. 6A shows relative NF-kB activity in THP-1Dual cells treated with caspase inhibitors (Q-VD-Oph) alone or in combination with RIPK3 inhibitors (GSK 872) treated with cell cultures from U937 leukemia cells expressing various Sano transfer payloads. FIGS. 6B and 6C show relative IRF activity in THP-1Dual cells treated with caspase inhibitors (Q-VD-Oph) alone or in combination with RIPK3 inhibitors (GSK 872) treated with cell cultures from U937 leukemia cells expressing various Sano transfer payloads. U937 cells were also treated with doxycycline (to induce expression of the sanoxacin) alone or in combination with B/B homodimers (to induce dimerization). In FIGS. 6A-6C, + + represents U937 cells treated with doxycycline, and++represents U937 cells treated with doxycycline and B/B homodimer.

FIG. 7A shows the relative viability of CT-26 mouse colon cancer cells expressing a Sanot transfer polypeptide alone or in combination with a caspase inhibitor. FIG. 7B shows the effect of Cell Turnover Factor (CTF) produced by CT-26 mouse colon cancer cells on stimulation of IFN-related gene activation in macrophages after induction of expression of a Sanot transfer polypeptide alone or in combination with a caspase inhibitor. FIG. 7C shows the effect of TRIF+RIPK3 expression on survival of mice implanted with CT-26 mouse colon cancer cells, alone or in combination with a caspase inhibitor.

Figure 8 shows a schematic representation of the expression of an anti-mesothelin CAR drive-inducible minitri f construct.

Figures 9A to 9C show the percentage of total cell death of Jurkat T cell lines containing anti-mesothelin CAR and/or inducible miniTRIF constructs. Cells were treated with various concentrations of mesothelin or CD3/CD28 activator and incubated for 24, 48 or 72 hours. The numbers on the X-axis represent mesothelin concentration.

Fig. 10A-10C show the ratio of necrotic to apoptotic cell death in Jurkat T cell lines containing anti-mesothelin CAR and/or inducible miniTRIF constructs. Cells were treated with various concentrations of mesothelin or CD3/CD28 activator and incubated for 24, 48 or 72 hours. The numbers on the X-axis represent mesothelin concentration.

Fig. 11A-11C show relative IRF activity in THP-1 monocytes treated with Cell Turnover Factor (CTF) collected from Jurkat T cell lines containing anti-mesothelin CAR and/or inducible miniTRIF constructs. Cells were treated with various concentrations of mesothelin or CD3/CD28 activator and incubated for 24, 48 or 72 hours prior to collection of CTF. The numbers on the X-axis represent mesothelin concentration.

Disclosure of Invention

In certain aspects, the disclosure relates to an immune cell that has been engineered to include one or more heterologous polynucleotides that promote sanoχ transfer of the immune cell.

In certain aspects, the disclosure relates to a pharmaceutical composition comprising: immune cells that have been engineered to contain one or more heterologous polynucleotides that promote the delivery of sanos to the immune cells; and a pharmaceutically acceptable carrier.

In one embodiment, the composition comprises an amount of immune cells sufficient to induce a biological response in the target cells. In one embodiment, the immune cell comprises a heterologous targeting domain. In one embodiment, the heterologous targeting domain is an antigen binding domain. In one embodiment, the immune cell comprises a heterologous signal transduction domain that triggers cell turnover. In one embodiment, the heterologous signal transduction domain is an intracellular signal transduction domain. In one embodiment, the targeting domain is operably linked to the signal transduction domain. In one embodiment, the polynucleotide that promotes saenox transfer in an immune cell is operably linked to a heterologous promoter that induces expression of the gene upon activation of the signal transduction domain. In one embodiment, the heterologous promoter is selected from the group consisting of: activating T cell Nuclear Factor (NFAT) promoter, STAT promoter, AP-1 promoter, NF-. Kappa.B promoter and IRF4 promoter. In one embodiment, the immune cell comprises a Chimeric Antigen Receptor (CAR) comprising the antigen binding domain and the intracellular signaling domain.

In certain aspects, the disclosure relates to an immune cell comprising:

(a) One or more nucleic acid sequences encoding a Chimeric Antigen Receptor (CAR), wherein the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular signaling domain; and

(b) A polynucleotide that promotes sanoχ transfer to an immune cell, operably linked to a heterologous promoter that induces expression of the polynucleotide upon activation of the immune cell,

wherein the immune cell is a T cell, natural Killer (NK) cell or macrophage.

In one embodiment, the intracellular signaling domain comprises at least one TCR-type signaling domain. In one embodiment, the intracellular signaling domain further comprises at least one costimulatory signaling domain. In one embodiment, the CAR further comprises a hinge domain. In one embodiment, the promoter induces expression of the polynucleotide upon binding of the antigen binding domain to an antigen. In one embodiment, the promoter is an activated T cell Nuclear Factor (NFAT) promoter, STAT promoter, NF-. Kappa.B promoter, AP-1 promoter or IRF4 promoter. In one embodiment, the TCR-type signaling domain comprises an intracellular domain of cd3ζ. In one embodiment, the intracellular domain of cd3ζ comprises a mutation of one or more tyrosine residues in one or more immunoreceptor tyrosine-based activation motifs (ITAMs).

In one embodiment, the intracellular signaling domain comprises a combination of domains selected from the group consisting of: (a) A costimulatory signaling domain of CD28 and an intracellular domain of cd3ζ; (b) 4-1BB costimulatory signaling domain and intracellular domain of cd3ζ; and (c) a costimulatory signaling domain of CD28, a costimulatory signaling domain of 4-1BB, a costimulatory signaling domain of CD27 or CD134, and an intracellular domain of CD3 ζ. In one embodiment, the intracellular signaling domain comprises the costimulatory signaling domain of CD28 and the intracellular domain of cd3ζ. In one embodiment, the intracellular signaling domain comprises a costimulatory signaling domain of 4-1BB and an intracellular domain of CD3 zeta.

In one embodiment, the antigen binding domain binds to a protein that is preferentially expressed on the surface of cancer cells. In one embodiment, the cancer cell is a solid cancer. In one embodiment, the cancer is a non-solid cancer. In one embodiment, the antigen binding domain binds to a protein selected from the group consisting of: CD19, CD20, CD22, CD23, kappa light chain, CD5, CD30, CD70, CD38, CD138, BCMA, CD33, CD123, CD44v6, CS1 and ROR1. In one embodiment, the antigen binding domain binds to a protein selected from the group consisting of: CD44v6, CAIX (carbonic anhydrase IX), CEA (carcinoembryonic antigen), CD133, C-Met (hepatocyte growth factor receptor), EGFR (epidermal growth factor receptor), EGFRvIII (type III epidermal growth factor receptor), epcam (epithelial adhesion molecule), ephA2 (erythropoietin-producing hepatocellular carcinoma A2), fetal acetylcholine receptor, FRa (folate receptor alpha), GD2 (ganglioside GD 2), GPC3 (glypican-3), GUCY2C (guanylate cyclase C), HER1 (human epidermal growth factor receptor 1), HER2 (human epidermal growth factor receptor 2) (ERBB 2), ephA2 (erythropoietin-producing hepatocellular carcinoma A2) ICAM-1 (intercellular adhesion molecule 1), IL13Ra2 (interleukin 13 receptor A2), IL11Ra (interleukin 11 receptor a), kras (Kirsten rat sarcoma virus oncogene homolog), kras G12D, L1CAM (L1-cell adhesion molecule), MAGE, MET, mesothelin, MUC1 (mucin 1), MUC16 ecto (mucin 16), NKG2D (natural killer group 2 member D), NY-ESO-1, PSCA (prostate Stem cell antigen), WT-1 (Wilms tumor 1), PSMA1, LAP3, ANXA3, breast mitoxin, olfactory protein 4, CD11b, integrin alpha-2, FAP (fibroblast activation protein), lewis-Y and TAG72. In one embodiment, the antigen binding domain binds mesothelin.

In one embodiment, the polynucleotide that promotes sano delivery encodes a death fold domain. In one embodiment, the death fold domain is selected from the group consisting of: death domain, thermo protein domain (pyrin domain), death Effector Domain (DED), C-terminal caspase recruitment domain (CARD), and variants thereof. In one embodiment, the death domain is from a protein selected from the group consisting of: fas-associated protein with death domain (FADD), fas, tumor necrosis factor receptor type 1-associated death domain (TRADD), tumor necrosis factor receptor type 1 (TNFR 1), and variants thereof. In one embodiment, the thermal protein domain is from a protein selected from the group consisting of: the NLR family contains the thermal protein domain protein 3 (NLRP 3) and apoptosis-related speckle-like proteins (ASCs). In one embodiment, the Death Effector Domain (DED) is from a protein selected from the group consisting of: fas-associated protein with death domain (FADD), caspase-8 and caspase-10. In one embodiment, the CARD is from a protein selected from the group consisting of: RIP related ICH1/CED3 homologous protein (RAIDD), apoptosis related spotting protein (ASC), mitochondrial antiviral signaling protein (MAVS), caspase-1, and variants thereof.

In one embodiment, the polynucleotide that facilitates sano delivery encodes a Toll/interleukin-1 receptor (TIR) domain. In one embodiment, the TIR domain is from a protein selected from the group consisting of: myeloid differentiation primary response protein 88 (MyD 88), toll/interleukin-1 receptor (TIR) domain containing adaptor-induced interferon- β (tif), toll-like receptor 3 (TLR 3), toll-like receptor 4 (TLR 4), TIR domain containing adaptor protein (tirp), translocation chain related membrane protein (TRAM) and variants thereof. In one embodiment, the polynucleotide that facilitates sano delivery encodes a protein comprising a TIR domain. In one embodiment, the protein comprising a TIR domain is selected from the group consisting of: myeloid differentiation primary response protein 88 (MyD 88), toll/interleukin-1 receptor (TIR) domain containing adaptor-induced interferon- β (tif), toll-like receptor 3 (TLR 3), toll-like receptor 4 (TLR 4), TIR domain containing adaptor protein (tirp), translocation chain related membrane protein (TRAM) and variants thereof.

In one embodiment, the polynucleotide that promotes sano delivery encodes a tri or a tri variant. In one embodiment, the TRIF variant is a TRIF variant listed in Table 2. In one embodiment, the TRIF variant comprises an amino acid sequence set forth in Table 2. In one embodiment, the polynucleotide that promotes saenox transfer encodes a polypeptide selected from the group consisting of: cellularFLICE(FADD-likeIL-1betaconvertingenzyme)-inhibitorprotein(c-FLIP),receptorinteractingserine/threonineproteinkinase1(RIPK1),receptorinteractingserine/threonineproteinkinase3(RIPK3),Z-DNAbindingprotein1(ZBP1),mixedlineagekinasedomain-likepseudokinase(MLKL),N-terminaltruncationsofthetifcomprisingonlytheTIRdomainandRHIMdomain,dominantnegativemutantsofFas-relatedproteinswithdeathdomain(FADD-DD),myr-FADD-DD,inhibitorkBalphasuper-repressor(IkBalpha-SR),interleukin-1receptor-relatedkinase1(IRAK1),tumornecrosisfactorreceptortype1relateddeathdomain(TRADD),dominantnegativemutantsofcaspase-8,interferonregulator3(IRF3),desetin-a(GSDM-a),desetin-b(GSDM-b),desetin-c(dm-c),desetin-d(GSDM-d),desetin-e(GSDM-e),asbestein-c(asc-c),andvariantswithapoptosis-relatedprotein(asc-c)withtheirapoptosisdomain. In some embodiments, the N-terminal truncations of the tri comprising only the TIR domain and the RHIM domain comprise deletions of amino acid residues 1-311 of human tri. In some embodiments, the N-terminal truncate of TRIF comprising only the TIR domain and the RHIM domain comprises or consists of SEQ ID NO. 12.

In one embodiment, cflup is human cflup. In one embodiment, cflup is caspase-8 and FADD-like apoptosis modulator (cfar). In one embodiment, ZBP1 comprises a deletion of the receptor-interacting protein homotypic interaction motif (RHIM) C, a deletion of RHIM D, and a deletion at the N-terminus of the Za1 domain. In one embodiment, ZBP1 is a ZBP1-Za1/RHIM A truncate.

In one embodiment, the polynucleotide that promotes sano delivery is a viral gene. In one embodiment, the viral gene encodes a polypeptide selected from the group consisting of: vFLIP (ORF 71/K13) from Kaposi sarcoma-associated herpesvirus (KSHV), MC159L from molluscum contagiosum virus, E8 from equine herpesvirus 2, vICA from Human Cytomegalovirus (HCMV) or Murine Cytomegalovirus (MCMV), crmA from vaccinia virus and P35 from Medicago sativa spodoptera nuclear polyhedrosis virus (AcMNPV).

In some embodiments, the one or more polynucleotides that promote saenox transfer encode two or more different saenox transfer polypeptides, wherein the two or more saenox transfer polypeptides are selected from the group consisting of: TRADD, TRAF2, TRAF6, ciaP1, ciaP2, XIAP, NOD2, myD88, TRAM, HOIL, HOIP, sharpin, IKKg, IKKa, IKKb, relA, MAVS, RIGI, MDA, tak1, TBK1, IKKe, IRF3, IRF7, IRF1, TRAF3, caspase, FADD, TNFR1, TRAILR2, FAS, bax, bak, bim, bid, noxa, puma, TRIF, ZBP1, RIPK3, MLKL, destina B, destina C, destina D, destina E, tumor necrosis factor receptor superfamily (TNFSF) proteins, and variants thereof. In some embodiments, at least one of the polynucleotides encodes a chimeric protein comprising at least two of the saenopassing polypeptides. In some embodiments, at least one of these polynucleotides is transcribed as a single transcript encoding two or more different saenox transfer polypeptides.

In some embodiments, at least two of the saenox polypeptides encoded by the one or more polynucleotides activate NF-kB. In some embodiments, at least two of the saenox polypeptides encoded by the one or more polynucleotides activate IRF3 and/or IRF7. In some embodiments, at least two of the saenopassing polypeptides encoded by the one or more polynucleotides promote extrinsic apoptosis. In some embodiments, at least two of the saenox polypeptides encoded by the one or more polynucleotides promote programmed necrosis. In some embodiments, at least one of the saenox polypeptides encoded by the one or more saenox polynucleotides activates NF-kB and at least one of the saenox polypeptides encoded by the one or more polynucleotides activates IRF3 and/or IRF7. In some embodiments, at least one of the saenox polypeptides encoded by the one or more polynucleotides activates NF-kB and at least one of the saenox polypeptides encoded by the one or more polynucleotides promotes extrinsic apoptosis. In some embodiments, at least one of the saenox polypeptides encoded by the one or more polynucleotides activates NF-kB and at least one of the saenox polypeptides encoded by the one or more polynucleotides promotes procedural necrosis.

In some embodiments, at least one of the saenox polypeptides encoded by the one or more polynucleotides activates IRF3 and/or IRF7, and at least one of the saenox polypeptides encoded by the one or more polynucleotides promotes extrinsic apoptosis. In some embodiments, at least one of the saenox polypeptides encoded by the one or more polynucleotides activates IRF3 and/or IRF7, and at least one of the saenox polypeptides encoded by the one or more polynucleotides promotes procedural necrosis. In some embodiments, at least one of the saenox polypeptides encoded by the one or more polynucleotides promotes apoptosis, and at least one of the saenox polypeptides encoded by the one or more saenox polynucleotides promotes programmed necrosis. In some embodiments, the programmed necrosis comprises necrotic apoptosis. In some embodiments, the programmed necrosis comprises apoptosis.

In some embodiments, the saenox transfer polypeptide that activates NF-kB is selected from the group consisting of: TRIF, TRADD, TRAF2, TRAF6, cIAP1, cIAP2, XIAP, NOD2, myD88, TRAM, HOIL, HOIP, sharpin, IKKg, IKKa, IKKb, relA, MAVS, RIGI, MDA5, tak1, TNFSF proteins and variants thereof. In some embodiments, the saenox transfer polypeptide that activates IRF3 and/or IRF7 is selected from the group consisting of: TRIF, myD88, MAVS, TBK1, IKKe, IRF3, IRF7, IRF1, TRAF3, and variants thereof. In some embodiments, the saenox transfer polypeptide that promotes extrinsic apoptosis is selected from the group consisting of: TRIF, RIPK1, caspase, FADD, TRADD, TNFR1, TRAILR2, FAS, bax, bak, bim, bid, noxa, puma, and variants thereof. In some embodiments, the saenox transfer polypeptide that promotes procedural necrosis is selected from the group consisting of: ZBP1, RIPK3, MLKL, desetin and variants thereof.

In some embodiments, at least one of the saenox polypeptides comprises tri or a variant thereof. In some embodiments, at least one of the saenox polypeptides comprises RIPK3 or a variant thereof. In some embodiments, at least one of the saenox polypeptides encoded by the one or more saenox polynucleotides comprises a tri or variant thereof, and at least one of the saenox polypeptides encoded by the one or more polynucleotides comprises RIPK3 or a variant thereof. In some embodiments, at least one of the saenox polypeptides comprises MAVS or a variant thereof, and at least one of the saenox polypeptides comprises RIPK3 or a variant thereof.

In some embodiments, the TRIF variant is a TRIF variant listed in Table 2. In some embodiments, the TRIF variant comprises an amino acid sequence set forth in Table 2. In some embodiments, the variant of TRIF is an N-terminal truncate of TRIF comprising only a TIR domain and a RHIM domain. In some embodiments, the TRIF variant comprises a deletion of amino acid residues 1-311 of human TRIF. In some embodiments, the N-terminal truncate of TRIF comprising only the TIR domain and the RHIM domain comprises or consists of SEQ ID NO. 12. In some embodiments, the one or more polynucleotides further encode a polypeptide that inhibits caspase activity. In some embodiments, the polypeptide that inhibits caspase activity is selected from the group consisting of: FADD dominant negative mutant (FADD-DN), cFLIP, vcca, caspase 8 dominant negative mutant (Casp 8-DN), cIAP1, cIAP2, tak1, IKK, and variants thereof. In some embodiments, the polypeptide that inhibits caspase activity is FADD-DN. In some embodiments, the polypeptide that inhibits caspase activity is cflup. In some embodiments, the polypeptide that inhibits caspase activity is vcica.

In some embodiments, the one or more polynucleotides encode at least one mesothelin or variant thereof. In some embodiments, at least one of the saenox polypeptides comprises TRIF or a variant thereof, and at least one of the saenox polypeptides comprises RIPK3 or a variant thereof, and at least one of the saenox polypeptides comprises mesothelin or a variant thereof. In some embodiments, at least one of the saenox polypeptides comprises MAVS or a variant thereof, and at least one of the saenox polypeptides comprises RIPK3 or a variant thereof, and at least one of the saenox polypeptides comprises mesothelin or a variant thereof. In some embodiments, the mesothelin is mesothelin E or a variant thereof. In some embodiments, the variant is a functional fragment of a saenox transfer polypeptide.

In some embodiments, the cell further comprises at least one heterologous polynucleotide encoding a dimerization domain. In some embodiments, at least one of these saenox polypeptides is contained within a fusion protein further comprising a dimerization domain. In some embodiments, the dimerization domain is heterologous to the saenox transfer polypeptide.

In certain aspects, the disclosure relates to a method of promoting saenox transfer in a subject, the method comprising administering an immune cell described herein in an amount and for a duration sufficient to promote saenox transfer in the subject.

In certain aspects, the disclosure relates to a method of promoting saenox transfer to a target cell, the method comprising contacting the target cell or tissue comprising the target cell with an immune cell described herein in an amount and for a duration sufficient to promote saenox transfer to the target cell.

In certain aspects, the disclosure relates to a method of promoting an immune response in a subject in need thereof, the method comprising administering to the subject an immune cell described herein in an amount and for a duration sufficient to promote saenox transfer of the immune cell, thereby promoting the immune response in the subject.

In one embodiment, the immune cells are administered to the subject in an amount and for a duration sufficient to promote saenox delivery in the target cells. In one embodiment, the target cell is selected from the group consisting of: cancer cells, immune cells, endothelial cells, and fibroblasts. In one embodiment, the subject has an infection. In one embodiment, the target cell is infected with a pathogen. In one embodiment, the infection is a viral infection. In one embodiment, the infection is a chronic infection. In one embodiment, the chronic infection is selected from the group consisting of HIV infection, HCV infection, HBV infection, HPV infection, hepatitis b infection, hepatitis c infection, EBV infection, CMV infection, TB infection, and parasitic infection.

In certain aspects, the disclosure relates to a method of treating cancer in a subject in need thereof, the method comprising administering to the subject an immune cell described herein, thereby treating the cancer in the subject. In one embodiment, administering immune cells to a subject reduces proliferation of cancer cells in the subject. In one embodiment, the proliferation of the cancer cells is a hyperproliferative of the cancer cells resulting from a cancer therapy administered to the subject. In one embodiment, administering immune cells to a subject reduces metastasis of cancer cells in the subject. In one embodiment, administering immune cells to a subject reduces neovascularization of a tumor in the subject.

In one embodiment, treating the cancer comprises any one or more of: a decrease in tumor burden, a decrease in tumor size, inhibition of tumor growth, achieving stable cancer in a subject with advanced cancer prior to treatment, a delay in cancer progression time, and an increase in survival time. In one embodiment, an immunostimulatory cell turnover pathway is induced in the cancer. In one embodiment, the cancer lacks an immunostimulatory cell turnover pathway. In one embodiment, the immunostimulatory cell turnover pathway is selected from the group consisting of: necrotic apoptosis, extrinsic apoptosis, iron death, and apoptosis of the cell coke. In one embodiment, the cancer is a cancer responsive to immune checkpoint therapy. In one embodiment, the cancer is selected from the group consisting of carcinoma, sarcoma, lymphoma, melanoma, and leukemia. In one embodiment, the cancer is a metastatic cancer. In one embodiment, the cancer is a solid tumor.

In one embodiment, the solid tumor is selected from the group consisting of: colon cancer, soft Tissue Sarcoma (STS), metastatic clear cell renal cell carcinoma (ccRCC), ovarian cancer, gastrointestinal cancer, colorectal cancer, hepatocellular carcinoma (HCC), glioblastoma (GBM), breast cancer, melanoma, non-small cell lung cancer (NSCLC), sarcoma, malignant pleura, mesothelioma (MPM), retinoblastoma, glioma, medulloblastoma, osteosarcoma, ewing's sarcoma, pancreatic cancer, lung cancer, stomach cancer (gastric cancer), gastric cancer (stomach cancer), esophageal cancer, liver cancer, prostate cancer, gynecological cancer, nasopharyngeal cancer, osteosarcoma, rhabdomyosarcoma, urothelial bladder cancer, neuroblastoma, and cervical cancer.

In one embodiment, the cancer is selected from the group consisting of: melanoma, cervical cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, urothelial cancer, bladder cancer, non-small cell lung cancer, sarcoma, colorectal adenocarcinoma, gastrointestinal stromal tumor, gastroesophageal cancer, colorectal cancer, pancreatic cancer, renal cancer, hepatocellular carcinoma, malignant mesothelioma, leukemia, lymphoma, myelodysplastic syndrome, multiple myeloma, transitional cell carcinoma, neuroblastoma, plasmacytoma, wilms' tumor, and hepatocellular carcinoma. In one embodiment, the cancer is not a solid tumor. In one embodiment, the cancer is selected from the group consisting of: leukemia, lymphoma, B-cell malignancy, T-cell malignancy, multiple myeloma, myeloid malignancy, and hematological malignancy.

In one embodiment, the immune cells are administered intravenously to the subject. In one embodiment, the immune cell is intratumorally administered to the subject.

In one embodiment, the method further comprises administering an anti-tumor agent to the subject. In one embodiment, the antineoplastic agent is a chemotherapeutic agent. In one embodiment, the antineoplastic agent is a biologic agent. In one embodiment, the biologic agent is an antigen binding protein. In one embodiment, the biologic agent is an oncolytic virus.

In one embodiment, the anti-neoplastic agent is an immunotherapeutic agent. In one embodiment, the immunotherapeutic agent is selected from the group consisting of: toll-like receptor (TLR) agonists, cell-based therapies, cytokines, cancer vaccines and immune checkpoint modulators of immune checkpoint molecules. In one embodiment, the TLR agonist is selected from the group consisting of Coley's toxin (BCG) and bacillus Calmette-guerin (BCG). In one embodiment, the cell-based therapy is chimeric antigen receptor T cell (CAR-T cell) therapy.

In one embodiment, the immune checkpoint molecule is selected from the group consisting of CD27, CD28, CD40, OX40, GITR, ICOS, 4-1BB, ADORA2A, B-H3, B7-H4, BTLA, CTLA-4, KIR, LAG-3, PD-1, PD-L2, TIM-3 and VISTA. In one embodiment, the immune checkpoint molecule is a stimulatory immune checkpoint molecule and the immune checkpoint modulator is an agonist of the stimulatory immune checkpoint molecule. In one embodiment, the immune checkpoint molecule is an inhibitory immune checkpoint molecule and the immune checkpoint modulator is an antagonist of the inhibitory immune checkpoint molecule. In one embodiment, the immune checkpoint modulator is selected from the group consisting of a small molecule, an inhibitory RNA, an antisense molecule, and an immune checkpoint molecule binding protein.

In one embodiment, the immune checkpoint molecule is PD-1 and the immune checkpoint modulator is a PD-1 inhibitor. In one embodiment, the PD-1 inhibitor is selected from the group consisting of pembrolizumab, nivolumab, pirimab, SHR-1210, MEDI0680R01, BBg-A317, TSR-042, REGN2810 and PF-06801591. In one embodiment, the immune checkpoint molecule is PD-L1 and the immune checkpoint modulator is a PD-L1 inhibitor. In one embodiment, the PD-L1 inhibitor is selected from the group consisting of dimaruzumab, alemtuzumab, avermectin, MDX-1105, AMP-224, and LY3300054. In one embodiment, the immune checkpoint molecule is CTLA-4 and the immune checkpoint modulator is a CTLA-4 inhibitor. In one embodiment, the CTLA-4 inhibitor is selected from Ai Pili mab, trimelimab, JMW-3B3 and AGEN1884. In one embodiment, the antineoplastic agent is a histone deacetylase inhibitor. In one embodiment, the histone deacetylase inhibitor is a hydroxamic acid, a benzamide, a cyclic tetrapeptide, a depsipeptide, an electrophilic ketone, or an aliphatic compound. In one embodiment, the hydroxamic acid is vorinostat (SAHA), belinostat (PXD 101), LAQ824, trichostatin a, or panobinostat (LBH 589).

In one embodiment, the benzamide is entinostat (MS-275), 01994, or motitinostat (MGCD 0103). In one embodiment, the cyclic tetrapeptide is Qu Puxin (trapoxin) B. In one embodiment, the fatty acid is phenyl butyrate or valproic acid.

Detailed Description

The present invention relates to an immune cell that has been engineered to contain one or more heterologous polynucleotides that promote saenox delivery of the immune cell. Sano delivery is a communication process between cells, for example, between signaling cells (e.g., engineered immune cells as described herein) and responding cells, which is the result of activation of a cell turnover pathway (e.g., programmed cell death) in the signaling cells that signals the responding cells to conduct a biological response. Sano delivery may be induced in signaling cells by expression of a cell turnover pathway gene (e.g., a programmed cell death pathway gene). Signaling cells whose cell turnover pathway has been activated may signal the responding cells by factors actively released by the signaling cells, or by intracellular factors of signaling cells exposed to the responding cells during signaling cell turnover (e.g., cell death). In various embodiments of the invention, the one or more polynucleotides expressed by the engineered immune cells promote saenox transfer in the immune cells by increasing the expression or activity of one or more polypeptides that promote saenox transfer and/or by decreasing the expression or activity of one or more polypeptides that suppress saenox transfer in the immune cells. Accordingly, in certain aspects, the invention relates to a method of promoting saenox delivery in a subject, the method comprising administering an engineered immune cell described herein.

In some embodiments, signaling cells (e.g., engineered immune cells described herein) can further promote saenox transfer in a subject by: contact with or in proximity to the target cell to promote saenox transfer to the target cell (e.g., cancer cell). For example, factors released by engineered immune cells during cell turnover may also initiate cell turnover (e.g., programmed cell death) in target cells, thereby promoting sanoχ transfer to target cells. Accordingly, the present invention also relates to a method of promoting the delivery of saxophone to a target cell, the method comprising contacting the target cell or a tissue comprising the target cell with an engineered immune cell as described herein.

In some embodiments, the engineered immune cells additionally comprise a heterologous signal transduction domain that triggers cell turnover (e.g., programmed cell death). The signaling domain may be, for example, a Chimeric Antigen Receptor (CAR) intracellular signaling domain. In some embodiments, the polynucleotide that promotes sano delivery is under the transcriptional control of a promoter that induces expression of the polynucleotide upon activation of the signal transduction domain.

I. Definition of the definition

As used herein, the term "administering" includes any method of delivering a pharmaceutical composition or agent to a system of a subject or to a specific area in or on a subject.

As used herein, "combination administration," "co-administration," or "combination therapy" is understood to mean administration of two or more active agents using separate formulations or a single pharmaceutical formulation, or continuous administration in any order such that there is a period of time for the two (or all) active agents to overlap in exerting their biological activity. It is contemplated herein that one active agent (e.g., an immune cell that has been engineered to undergo cell turnover or programmed cell death and initiate sano delivery) can improve the activity of a second therapeutic agent (e.g., an immunotherapeutic agent), e.g., can sensitize a target cell (e.g., a cancer cell) to or can have a synergistic effect with the activity of the second therapeutic agent. "combination administration" does not require administration of agents simultaneously, at the same frequency, or through the same administration route. As used herein, "combination administration," "co-administration," or "combination therapy" includes administration of immune cells engineered to undergo cell turnover and initiate sano transfer with one or more additional therapeutic agents, such as an immunotherapeutic agent (e.g., an immune checkpoint modulator). Examples of immunotherapeutic agents are provided herein.

As used herein, the term "antigen binding domain" refers to a protein that binds to another protein on the surface of a target cell or target pathogen (e.g., fungus, bacteria, or virus). In some embodiments, the antigen binding domain is an immunoglobulin chain or fragment thereof, comprising at least one immunoglobulin variable domain sequence. The term "antigen binding domain" encompasses, but is not limited to, antibodies and antibody fragments.

As used herein, the term "antibody fragment" refers to at least one portion of an antibody that retains the ability to specifically interact (e.g., by binding, steric hindrance, stabilization/destabilization, spatial distribution) with an epitope of an antigen. Examples of antibody fragments include, but are not limited to, fab ', F (ab') ₂ Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFv), fd fragments consisting of VH and CH1 domains, linear antibodies, single domain antibodies such as sdabs (VL or VH), camelidae VHH domains, multispecific antibodies formed from antibody fragments (such as bivalent fragments comprising two Fab fragments linked by a disulfide bond of a hinge region), and isolated CDRs or other epitope-binding fragments of antibodies. Antigen binding fragments may also be incorporated into single domain antibodies, large antibodies, minibodies, nanobodies, intracellular antibodies, diabodies, triabodies, tetrabodies, v-NARs and bis-scFv (see, e.g., hollinger and Hudson, nature Biotechnology [ Nature Biotechnology ]]23:1126-1136,2005). Antigen binding fragments may also be based on grafting of polypeptides such as fibronectin type III (Fn 3) into a scaffold (see us patent No. 6,703,199, which describes a fibronectin polypeptide miniantibody).

The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light chain variable region and the heavy chain variable region are continuously linked, e.g., by a synthetic linker (e.g., a short flexible polypeptide linker) and are capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. As used herein, an scFv may have VL and VH variable regions in any order, e.g., an scFv may comprise a VL-linker-VH or may comprise a VH-linker-VL, relative to the N-terminus and C-terminus of a polypeptide, unless otherwise indicated.

As used herein, the term "chimeric antigen receptor" or "CAR" refers to a set of polypeptides that, when expressed in immune cells, provide the cells with specificity for target cells (e.g., cancer cells) and intracellular signaling. In some embodiments, the CAR comprises at least an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain. The intracellular signaling domain comprises at least one immune receptor tyrosine based activation motif (ITAM). In some embodiments, the intracellular signaling domain comprises at least one TCR-type signaling domain. In some embodiments, the intracellular signaling domain further comprises at least one costimulatory signaling domain, as defined below. In some embodiments, the set of polypeptides comprising the CAR are located in the same polypeptide chain (e.g., the CAR is a chimeric fusion protein comprising an antigen binding domain, a transmembrane domain, and an intracellular signaling domain). In some embodiments, the set of polypeptides that make up the CAR are not adjacent to each other, e.g., are in different polypeptide chains. In some embodiments, the set of polypeptides comprising the CAR comprises a dimerization switch that can couple the polypeptides to each other in the presence of the dimerization molecule, e.g., can couple an antigen binding domain to an intracellular signaling domain. In one embodiment, the TCR-type signaling domain of the CAR is a CD3 zeta chain associated with the T cell receptor complex.

As used herein, the term "T Cell Receptor (TCR) -type signaling domain" or "TCR-type signaling domain" refers to a component of the intracellular signaling domain of a CAR that initiates antigen-dependent primary activation by a T Cell Receptor (TCR).

As used herein, the term "costimulatory signaling domain" refers to a domain from a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response of the T cell, such as, but not limited to, proliferation. The costimulatory signaling domain may be derived from a cell surface molecule other than an antigen receptor or ligand thereof required for an effective immune response. For example, the costimulatory signaling domain may be derived from ase:Sub>A protein, including but not limited to MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activating molecules (SLAM proteins), activating NK cell receptors, BTLA, toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD 11 ase:Sub>A/CD 18), 4-1BB (CD 137), B7-H3, CDS, ICAM-1, ICOS (CD 278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), CD kirs 2, SLAMF7, NKp80 (KLRF 1), NKp44, NKp30, NKp46, CD19, CD4, CD8 ase:Sub>A, CD8 betase:Sub>A, IL2rβ, IL2rγ, IL7rα, ITGA4, VLA1, CD49 ase:Sub>A, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11D, ITGAE, CD103, ITGAL, CD11 ase:Sub>A, LFA-1, ITGAM, CD11B, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG C, TNFR2, TRANCE/RANKL, DNAM1 (CD 226), SLAMF4 (CD 244,2B 4), CD84, CD96 (Tactile), CEACAM1, CRTAM, ly9 (CD 229), CD160 (BY 55), PSGL1, CD100 (SEMA 4D), CD69, SLAMF6 (NTB-ase:Sub>A, ly 108), SLAM (SLAMF 1, CD150, IPO-3), BLAME (SLAMF 8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19 ase:Sub>A, and ligands that specifically bind to CD 83.

As used herein, the term "signal transduction domain" refers to a functional portion of a protein that functions by transmitting information within a cell to modulate cellular activity through a defined signaling pathway by generating a second messenger or by functioning as an effector in response to such a messenger.

The term "variant" as used herein with respect to a polypeptide refers to a polypeptide that differs from the corresponding wild-type polypeptide by at least one amino acid residue. In some embodiments, the variant polypeptide has at least one activity that differs from the corresponding naturally occurring polypeptide. The term "variant" as used herein with respect to a polynucleotide refers to a polynucleotide that differs from the corresponding wild-type polynucleotide by at least one nucleotide. In some embodiments, the variant polypeptide or variant polynucleotide has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the corresponding wild-type polypeptide or polynucleotide. In some embodiments, the variant is a functional fragment of a polypeptide.

The term "functional fragment" as used herein with respect to a polypeptide refers to a portion of the polypeptide that retains at least one biological activity of the polypeptide (e.g., the ability to promote saenox transfer). In some embodiments, the functional fragment is a domain of a polypeptide, such as a RHIM domain, death fold domain, or TIR domain of a polypeptide. In some embodiments, the functional fragment of the polypeptide is a portion of the domain that retains at least one biological activity of the domain.

As used herein, the term "death folding domain" refers to a structurally defined motif characterized by six to seven tightly-coiled alpha-helices found on proteins involved in apoptosis, inflammation, and other cellular signaling processes. Death fold domains bind to each other through homotypic protein-protein interactions, resulting in the formation of large functional complexes that are involved in initiating cell turnover and other cell signaling pathways. Examples of death folding domains include Death Domain (DD), death Effector Domain (DED), caspase recruitment domain (CARD), thermo protein domain (PYD), fas-associated protein with death domain (FADD), fas death domain, tumor necrosis factor receptor type 1-associated death domain (TRADD), and tumor necrosis factor receptor type 1 (TNFR 1). See Lahm et al, 2003,Cell Death&Differentiation [ cell death and differentiation ]10:10-12, the entire contents of which are incorporated herein by reference.

As used herein, the term "linker" refers to a flexible peptide consisting of amino acids such as glycine and/or serine residues, used alone or in combination, to join two polypeptide sequences together. In one embodiment, the linker is a Gly/Ser linker and comprises the amino acid sequence (Gly-Gly-Gly-Gly-Ser) _n Wherein n is a positive integer equal to or greater than 1. For example, n=1, n=2, n=3, n=4, n=5, n=6, n=7, n=8, n=9, n=10, n=11, n=12, n=13, n=14, or n=15. In some embodiments, the linker includes, but is not limited to (Gly) ₄ Ser) ₄ Or (Gly) ₄ Ser) ₃ . In another embodiment, the linker comprises (Gly ₂ Ser), (GlySer) or (Gly) ₃ Ser). Also included are linkers described in WO 2012/138475 (which is incorporated herein by reference in its entirety). In some embodiments, the linker is GSTSGSGKPGSGEGSTKG (SEQ ID NO: 26), e.g., whitlow et alHuman, protein Eng [ Protein engineering ]](1993) 6 (8) 989-895. In some embodiments, the linker comprises at least 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acid residues. In some embodiments, the linker comprises fewer than 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acid residues. The linker may in turn be modified to perform additional functions, such as attaching a drug or attaching to a solid support. As used herein, the terms "increase" and "decrease" refer to modulating a greater or lesser amount, function, or activity, respectively, of a production parameter relative to a reference. For example, after administration of a composition described herein, a parameter (e.g., activation of IRF, activation of NFkB, activation of macrophages, size of tumor, or growth) can be increased or decreased in a subject by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% or more relative to the amount of the parameter prior to administration. Typically, the post-administration indicator is measured at a time when the effect has been achieved by administration, e.g., at least one day, one week, one month, 3 months, 6 months after initiation of the treatment regimen. Similarly, a preclinical parameter (activation of NFkB or IRF by a composition described herein, such as an in vitro cell, and/or reduction in tumor burden in a test mammal) can be increased or decreased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% or more relative to the amount of the preclinical parameter.

As used herein, "antineoplastic agent" refers to a drug used to treat cancer. Antineoplastic agents include chemotherapeutic agents (e.g., alkylating agents, antimetabolites, antitumor antibiotics, topoisomerase inhibitors, mitotic inhibitor corticosteroids and enzymes), biological anticancer agents, and immune checkpoint modulators.

A "cancer treatment regimen" or "anti-tumor regimen" is a clinically acceptable dosing regimen for treating cancer that includes administering one or more anti-tumor agents to a subject in specific amounts on a specific schedule.

The term "heterologous" as used herein refers to a combination of elements that do not occur in a natural combination. For example, a polynucleotide heterologous to an immune cell refers to a polynucleotide that does not naturally occur in the immune cell, or a polynucleotide that occurs at a location in the immune cell that is different from the location in which it naturally occurs. For example, the 5 'and 3' ends of the heterologous polynucleotides may be bound to nucleic acid sequences to which they are not bound in nature. A polypeptide heterologous to an immune cell refers to a polypeptide that does not naturally occur in an immune cell.

As used herein, an "immune checkpoint" or "immune checkpoint molecule" is a molecule that modulates a signal in the immune system. The immune checkpoint molecule can be a stimulatory checkpoint molecule, i.e. increasing signal, or an inhibitory checkpoint molecule, i.e. decreasing signal. As used herein, a "stimulatory checkpoint molecule" is a molecule that increases a signal or has costimulatory properties in the immune system. As used herein, an "inhibitory checkpoint molecule" is a molecule that reduces a signal or has co-suppression in the immune system.

As used herein, an "immune checkpoint modulator" is an agent that is capable of altering the activity of an immune checkpoint in a subject. In certain embodiments, the immune checkpoint modulator alters the function of one or more immune checkpoint molecules (including, but not limited to, CD27, CD28, CD40, OX40, GITR, ICOS, 4-1BB, ADORA2A, B-H3, B7-H4, BTLA, CTLA-4, KIR, LAG-3, PD-1, PD-L2, TIM-3, and VISTA). The immune checkpoint modulator may be an agonist or antagonist of an immune checkpoint. In some embodiments, the immune checkpoint modulator is an immune checkpoint binding protein (e.g., an antibody Fab fragment, a bivalent antibody, an antibody drug conjugate, an scFv, a fusion protein, a bivalent antibody, or a tetravalent antibody). In other embodiments, the immune checkpoint modulator is a small molecule. In particular embodiments, the immune checkpoint modulator is an anti-PD 1, anti-PD-L1 or anti-CTLA-4 binding protein, such as an antibody or antibody fragment.

As used herein, "immunotherapy" refers to a pharmaceutically acceptable compound, composition, or therapy that induces or enhances an immune response. Immunotherapeutic agents include, but are not limited to, immune checkpoint modulators, toll-like receptor (TLR) agonists, cell-based therapies, cytokines, and cancer vaccines.

As used herein, "oncological disorder" or "cancer" or "neoplasm" refers to all types of cancer or neoplasm found in a human, including, but not limited to: leukemia, lymphoma, melanoma, epithelial carcinoma, and sarcoma. As used herein, the terms "oncological disorder," "cancer," and "neoplasm" are used interchangeably and refer to cells in the singular or plural that have undergone malignant transformation that renders the cells pathological to a host organism. Primary cancer cells (i.e., cells obtained from the vicinity of the malignant transformation site) can be readily distinguished from non-cancerous cells by established techniques, particularly histological examination. As used herein, the definition of cancer cells includes not only primary cancer cells, but also cancer stem cells, as well as cancer progenitor cells or any cells derived from a cancer cell ancestor. This includes metastatic cancer cells, in vitro cultures and cell lines derived from cancer cells.

Based on tumor size, histological features, tumor markers, and other criteria known to those of skill in the art, the particular criteria for cancer staging will depend on the particular cancer type. In general, the stage of cancer can be described as follows: (i) stage 0, carcinoma in situ; (ii) Stage I, stage II and stage III, wherein higher numbers indicate that the disease is more extensive, including larger tumor size and/or spread of the cancer beyond its originally developed organ to nearby lymph nodes and/or tissues or organs adjacent to the primary tumor site; and (iii) stage IV, wherein the cancer has spread to distant tissues or organs.

"solid tumor" is a tumor mass; for example, a tumor detectable by a procedure such as CAT scan, MR imaging, X-ray, ultrasound, or palpation, and/or a tumor detectable due to the expression of one or more cancer specific antigens in a sample obtainable from a patient. The tumor need not have a measurable size.

The "subject" treated by the methods of the invention may refer to a human or non-human animal, preferably a mammal, more preferably a human. In certain embodiments, the subject has a detectable or diagnosed cancer prior to starting treatment using the methods of the invention. In certain embodiments, the subject has a detectable or diagnosed infection, e.g., a chronic infection, prior to starting treatment using the methods of the invention.

As used herein, "suicide gene" refers to a gene encoding a protein (e.g., an enzyme) that converts a non-toxic precursor of a drug into a cytotoxic compound.

As used herein, "cell turnover" refers to a dynamic process of rearranging and spreading intracellular material and possibly ultimately leading to cell death. Cell turnover involves the production and release of cell turnover factors from cells.

As used herein, a "cell turnover factor" is a molecule and cell fragment produced by a cell undergoing cell turnover that is ultimately released from the cell and affects the biological activity of other cells. Cell turnover factors can include proteins, peptides, carbohydrates, lipids, nucleic acids, small molecules, and cell fragments (e.g., vesicles and cell membrane fragments).

As used herein, "cell turnover pathway gene" refers to a gene encoding a polypeptide that promotes, induces, or otherwise contributes to a cell turnover pathway.

As used herein, "programmed cell death" refers to the important terminal pathways of cells of a multicellular organism and involves a variety of biological events including morphogenesis, maintenance of tissue homeostasis, and elimination of deleterious cells.

As used herein, "programmed cell death gene" refers to a gene encoding a polypeptide that promotes, induces, or otherwise contributes to the programmed cell death pathway.

As used herein, "sano transfer" is communication between cells that is the result of activation of a cell turnover pathway (e.g., programmed cell death) in a signaling cell (e.g., an engineered immune cell as described herein) that signals a response to a target cell for a biological response. Sano transfer can be induced in signaling cells by: modulating cellular turnover pathway genes in the cells, such as by expressing heterologous genes that promote such pathways. Tables 1 through 6 describe exemplary genes and polypeptides capable of promoting various cell turnover pathways. Thus, a signaling cell in which a cell turnover pathway has been activated may signal a responsive target cell by a factor that is actively released by the signaling cell, or by an intracellular factor that is exposed to a signaling cell that responds to the target cell during cell turnover (e.g., cell death) of the signaling cell. In certain embodiments, activated signaling cells promote an immunostimulatory response (e.g., a pro-inflammatory response) in a responsive target cell (e.g., an immune cell).

As used herein, a "polynucleotide that promotes saenox transfer" refers to a polynucleotide whose expression in a signaling cell (e.g., an engineered immune cell as described herein) results in increased saenox transfer by the signaling cell. In some embodiments, the polynucleotide that promotes saenox transfer encodes a polypeptide that promotes saenox transfer, i.e., a polypeptide whose expression in a signaling cell increases saenox transfer in a target cell. In other embodiments, the polynucleotide that promotes saxored reduces expression and/or activity of a polypeptide that inhibits saxored in a signaling cell. For example, a polynucleotide that promotes saenox transfer may encode an RNA molecule that reduces the expression and/or activity of a polypeptide that inhibits saenox transfer in a signaling cell.

"therapeutically effective amount" refers to an amount of a compound or composition that, when administered to a patient to treat a disease, is sufficient to effect such treatment of the disease. When administered for the prevention of a disease, the amount is sufficient to avoid or delay the onset of the disease. The "therapeutically effective amount" will vary depending on the compound or composition, the disease and its severity, the age, weight, etc., of the patient to be treated. The therapeutically effective amount need not be curative. A therapeutically effective amount is not required to prevent the disease or condition from ever occurring. In contrast, a therapeutically effective amount is an amount that will at least delay or reduce the onset, severity, or progression of a disease or disorder.

As used herein, "treatment" and their cognate words refer to the medical management of a subject intended to improve, ameliorate, stabilize, prevent or cure a disease, pathological condition or disorder. The term includes active therapy (therapy aimed at ameliorating a disease, pathological condition or disorder), causal therapy (therapy aimed at the cause of the associated disease, pathological condition or disorder), palliative therapy (therapy aimed at alleviating symptoms), prophylactic therapy (therapy aimed at minimizing or partially or completely inhibiting the development of the associated disease, pathological condition or disorder); and supportive treatment (treatment for supplementing another therapy).

II cell turnover pathway

The immune cells described herein can be engineered to modulate cell turnover pathways in immune cells, thereby initiating saenox transfer in immune cells. In some embodiments, the immune cells are engineered to induce an immunostimulatory cell turnover pathway in the immune cells by expression of one or more polynucleotides that promote saenox transfer.

The immunostimulatory cell turnover pathway is a cell turnover pathway that, when activated in a cell, promotes an immunostimulatory response in a responsive cell (such as another immune cell). Immunostimulatory cell turnover pathways include, but are not limited to, programmed necrosis (e.g., necrotic apoptosis, pyro-apoptosis), apoptosis (e.g., extrinsic and/or intrinsic apoptosis), autophagy, iron death, and combinations thereof.

Programmed necrosis

As used herein, "programmed necrosis" refers to genetically controlled cell death with morphological features such as cell swelling (distension, death), membrane rupture, and release of cellular content, as opposed to maintenance of membrane integrity that occurs during apoptosis. In some embodiments, the programmed necrosis is apoptosis. In some embodiments, the programmed necrosis is necrotic apoptosis.

Apoptosis of cell coke

As used herein, "apoptosis" refers to the intrinsic inflammatory process of caspase 1, caspase 4, or caspase 5-dependent programmed cell death. The most prominent biochemical feature of cell apoptosis is early-induced proximity-mediated activation of caspase-1. The focal activation of caspase-1, 4 or 5 can occur in the context of a multiprotein platform called the inflammasome, which involves NOD-like receptors (NLR) or other receptors, such as cytoplasmic DNA receptor melanoma deficiency factor 2 (AIM 2), which recruits the adapter protein ASC that promotes caspase-1 activation. Caspase-4/5 may be activated directly by LPS. In both cases, active caspase-1 catalyzes the proteolytic maturation and release of interleukin-1 beta (IL-1 beta) and IL-18. Furthermore, in some (but not all) cases caspase activation targets GSDM-D to drive membrane rupture and cell death. See Galluzzi et al, 2018,Cell Death Differ, [ cell death and differentiation ] for 3 months; 25 (3):486-541.

In the methods of the present disclosure, apoptosis in immune cells may be induced by expressing one or more heterologous polynucleotides encoding polypeptides that induce apoptosis in immune cells. Polypeptides that can induce apoptosis in immune cells include, but are not limited to NLR, ASC, GSDM-D, AIM2 and BIRC1.

Several methods are known in the art and can be used to identify cells that undergo cell apoptosis and distinguish from other types of cell disassembly and/or cell death by detecting specific markers. Cell apoptosis requires caspase-1, caspase-4 or caspase-5 activity and is typically accompanied by processing of pre-IL-1 b and/or pre-IL-18, release of these mature cytokines and membrane permeabilization of the caspase-1/4/5 cleavage fragments of GSDM-D. Other mesothelins are also involved in apoptosis, including GSDM-B and GSDM-E, and can be used as markers of apoptosis.

Necrotic apoptosis

Necrotic apoptosis is the major type of programmed cell death pathway. Necrotic apoptosis involves cell swelling, organelle dysfunction and cytolysis (Wu W et al, (2012) crit.rev.oncol.Hematol. [ reviews of oncology and hematology ]82, 249-258). Unlike necrosis, which typically occurs unexpectedly or unregulated, necrotic apoptosis is a regulated process that can be induced by cellular metabolism and genotoxic stress or various anticancer agents. Necrotic apoptosis plays an indispensable role in the normal developmental process. Furthermore, it is involved in the pathogenesis of a variety of human diseases, including Cancer (Fulda S, (2013), cancer Biol Ther. [ Cancer biology and treatment ]14 (11): 999-1004). In some embodiments, necrotic apoptosis refers to receptor interacting protein kinase 3 (RIP 1-and/or RIPK 3)/mixed lineage kinase-like (MLKL) dependent necrosis. Several triggers can induce necrotic apoptosis, including alkylated DNA damage, excitotoxic, and ligation of death receptors. For example, when caspases (and in particular caspase-8 or caspase-10) are blocked by genetic manipulation (e.g., by gene knockout or RNA interference (RNAi)) or by pharmacological agents (e.g., chemical caspase inhibitors), RIPK3 phosphorylates MLKL, resulting in the assembly of MLKL into membrane pores, which ultimately activates the execution of necrotic cell death. See Galluzzi et al, 2018,Cell Death Differ, [ cell death and differentiation ] for 3 months; 25 (3) 486-541, which is incorporated herein by reference in its entirety.

In the methods of the present disclosure, necrotic apoptosis in immune cells may be induced by expression of one or more heterologous polynucleotides encoding polypeptides that induce necrotic apoptosis in immune cells. Polypeptides that can induce necrotic apoptosis in immune cells include, but are not limited to, toll-like receptor 3 (TLR 3), TLR4, TIRAP containing TIR domain, interferon- β (TRIF) containing Toll/interleukin-1 receptor (TIR) domain, Z-DNA binding protein 1 (ZBP 1), receptor interacting serine/threonine protein kinase 1 (RIPK 1), receptor interacting serine/threonine protein kinase 3 (RIPK 3), mixed lineage kinase domain-like pseudokinase (MLKL), tumor Necrosis Factor Receptor (TNFR), FS-7 related surface antigen (FAS), TNF-related apoptosis-inducing ligand receptor (TRAILR), and tumor necrosis factor receptor type 1 related death domain protein (TRADD).

Several methods are known in the art and can be used to identify cells that undergo necrotic apoptosis and distinguish from other types of cell disassembly and/or cell death by detecting specific markers. These include phosphorylation of RIPK1, RIPK3 and MLKL, which detects these post-translational modifications by antibodies, typically by immunoblotting or immunostaining of cells. Necrotic apoptosis can be distinguished from apoptosis and cell scorch by the absence of caspase activation, rapid membrane permeabilization, repositioning of MLKL on the membrane, accumulation of RIPK3 and MLKL into detergent insoluble fractions, RIPK3/MLKL complex formation, and MLKL oligomerization. Necrotic apoptosis can be defined both genetically and pharmacologically by the requirements for both RIPK3 and MLKL and their activation.

Apoptosis of

Apoptosis, as used herein, refers to a type of programmed cell death characterized by specific morphological and biochemical changes in dying cells, including cell shrinkage, nuclear concentration and rupture, dynamic membrane foaming and loss of adhesion to neighbors or to extracellular matrix (Nishida K et al, (2008) circ. Res. [ cycling research ]103, 343-351). There are two basic apoptosis signaling pathways: extrinsic and intrinsic pathways (Verbruge I et al, (2010) Cell [ cells ]. 143:1192-2). Intrinsic apoptotic pathways are activated by a variety of intracellular stimuli including DNA damage, growth factor deprivation, and oxidative stress. The extrinsic pathway of apoptosis is initiated by binding of death ligands to death receptors, followed by assembly of death-inducing signaling complexes that activate downstream effector caspases to directly induce Cell death or activate the mitochondrial-mediated intrinsic apoptosis pathway (verbruge I et al, (2010) Cell. [ Cell ] 143:1192-2).

Extrinsic apoptosis

The term "extrinsic apoptosis" as used herein refers to an example of apoptotic cell death induced by extracellular stress signals sensed and transmitted by specific transmembrane receptors. In addition apoptosis may be initiated by binding of ligands such as FAS/CD95 ligand (FASL/CD 95L), tumor necrosis factor alpha (tnfα) and TNF (ligand) superfamily member 10 (TNFSF 10, the most widely known TNF-related apoptosis-inducing ligand, TRAIL) to various death receptors (i.e., FAS/CD95, tnfα receptor 1 (TNFR 1) and TRAIL receptor (TRAIL r) 1-2, respectively). Alternatively, extrinsic pro-apoptotic signals may be emitted by so-called "dependent receptors", including netrin receptors (e.g. ans-D and deletions in colorectal cancer, DCC), which only play a lethal function when the concentration of their specific ligand falls below a critical threshold level. See Galluzzi et al, 2018,Cell Death Differ, [ cell death and differentiation ] for 3 months; 25 (3) 486-541, which is incorporated herein by reference in its entirety.

In the methods of the present disclosure, extrinsic apoptosis in immune cells may be induced by expression of one or more heterologous polynucleotides encoding polypeptides that induce extrinsic apoptosis in target cells. Polypeptides that induce extrinsic apoptosis in target cells include, but are not limited to, TNF, fas ligand (FasL), TRAIL (and cognate receptors thereof), TRADD, fas-related protein with death domain (FADD), transforming growth factor beta-activated kinase 1 (Tak 1), caspase-8, XIAP, BID, caspase-9, APAF-1, cytoC, caspase-3 and caspase-7. Polypeptides that can inhibit extrinsic apoptosis in target cells include apoptosis protein inhibitor 1 (cIAP 1), cIAP2, ikka, and Ikkb. Several methods are known in the art and can be used to identify cells that undergo apoptosis and distinguish from other types of cell disassembly and/or cell death by detecting specific markers. Apoptosis requires caspase activation and can be inhibited by inhibitors of caspase activation and/or prevented from death by the absence of caspases such as caspase-8 or caspase-9. Caspase activation systematically breaks down cells by cleaving specific substrates such as PARP and DFF45 and over 600 additional proteins. With the externalization of phosphatidylserine and concomitant membrane blebbing, apoptotic cell membranes initially remain intact. The mitochondrial outer membrane is typically destroyed and released into cytosolic proteins such as CytoC and HTRA 2. The nuclear DNA is cleaved into discrete fragments, which can be detected by assays known in the art.

Autophagy

As used herein, the term "autophagy" refers to an evolutionarily conserved catabolic process, starting from the formation of autophagosomes, i.e., double membrane-bound structures surrounding cytoplasmic macromolecules and organelles, intended for recycling (Liu JJ et al, (2011) Cancer Lett [ Cancer communication ]300, 105-114). Autophagy is a cellular strategy and mechanism that survives under stress conditions. When overactivated in some cases, excessive autophagy results in Cell death (Boya P et al, (2013) Nat Cell Biol [ Nature-Cell Biol ]15 (7): 713-20).

In the methods of the present disclosure, autophagy may be induced in immune cells by expression of one or more heterologous polynucleotides encoding polypeptides that induce autophagy in immune cells.

Iron death

As used herein, the term "iron death" refers to a cell death process that is subject to regulation, is iron dependent and involves the production of reactive oxygen species. In some embodiments, iron death involves iron-dependent accumulation of lipid hydroperoxides to lethal levels. Susceptibility to iron death is closely related to many biological processes, including amino acid, iron and polyunsaturated fatty acid metabolism, and biosynthesis of glutathione, phospholipids, NADPH and coenzyme Q10. Iron death involves metabolic dysfunction that results in the production of both cytosolic ROS and lipid ROS that are independent of mitochondria, but in some cellular environments depend on NADPH oxidase (see, e.g., dixon et al 2012, cell [ cell ]149 (5): 1060-72, which is incorporated herein by reference in its entirety).

In the methods of the present disclosure, iron death may be induced in immune cells by expression of one or more heterologous polynucleotides that, when expressed in immune cells, reduce expression or activity of a protein endogenous to immune cells that inhibits iron death. Proteins that inhibit iron death include, but are not limited to, FSP1, GPX4, and glutamate/cystine antiporter (System XC).

Several methods are known in the art and can be used to identify cells that undergo iron death and distinguish from other types of cell disassembly and/or cell death by detecting specific markers. (see, e.g., stockwell et al, 2017, cell [ cell ]171:273-285, which is incorporated herein by reference in its entirety). For example, since iron death may be caused by fatal lipid peroxidation, measuring lipid peroxidation provides a method of identifying cells that undergo iron-dependent cell disassembly. C11-BODIPY and Liperfluo are lipophilic ROS sensors that provide a rapid, indirect means to detect lipid ROS (Dixon et al 2012, cell [ cell ] 149:1060-1072). Liquid Chromatography (LC)/tandem Mass Spectrometry (MS) analysis can also be used to directly detect specific oxidized lipids (Friedmann Angeli et al 2014,Nat.Cell Biol [ Nature cell biology ]16:1180-1191; kagan et al 2017, nat. Chem. Biol. [ Nature chemical biology ] 13:81-90). Isoprost-and Malondialdehyde (MDA) can also be used to measure lipid peroxidation (Milne et al, 2007, nat. Protoc. [ Nature laboratory Manual ]2:221-226; wang et al, 2017, hepatology ]66 (2): 449-465). Kits for measuring MDA are commercially available (Beyotime, haimen, china).

Other useful assays for studying iron death include measuring iron abundance and GPX4 activity. Iron abundance can be measured using inductively coupled plasma-MS or calcein AM quenching, as well as other specific iron probes (Hirayama and Nagasawa,2017, J. Clin. Biochem. Nutr. [ J. Biochem. Biol. 60:39-48; spanger et al 2016, nat. Chem. Biol. [ Nature Biol. ] 12:680-685), whereas GPX4 activity can be detected using LC-MS using phosphatidylcholine hydrogen peroxide reduction in cell lysates (Yang et al, 2014, cell [ cell ] 156:317-331). In addition, iron death can be assessed by measuring Glutathione (GSH) content. GSH may be measured, for example, by using a commercially available GSH-Glo glutathione assay (Promega, madison (Madison), wisconsin).

Iron death may also be assessed by measuring the expression of one or more marker proteins. Suitable marker proteins include, but are not limited to, glutathione peroxidase 4 (GPX 4), prostaglandin endoperoxide synthase 2 (PTGS 2), and cyclooxygenase 2 (COX-2). The expression level of a marker protein or nucleic acid encoding a marker protein may be determined using suitable techniques known in the art, including, but not limited to, polymerase Chain Reaction (PCR) amplification reactions, reverse transcriptase PCR assays, quantitative real-time PCR, single strand conformational polymorphism assays (SSCP), mismatch cleavage detection, heteroduplex assays, northern blot analysis, western blot analysis, in situ hybridization, array analysis, deoxyribonucleic acid sequencing, restriction fragment length polymorphism assays, and combinations or sub-combinations thereof.

The engineered immune cells of the invention

The immune cells of the invention are engineered to comprise one or more polynucleotides that promote saenox transfer. In some embodiments, the engineered immune cell comprises at least one heterologous polynucleotide encoding a polypeptide that promotes saenox transfer of the immune cell. In other embodiments, the engineered immune cell comprises at least one heterologous polynucleotide encoding a polypeptide that promotes saenox transfer in the target cell.

In some embodiments, a polynucleotide that promotes sano delivery may be under the transcriptional control of a heterologous promoter, e.g., operably linked to a heterologous promoter that induces expression of the polynucleotide upon immune cell activation. In some embodiments, an immune cell is activated when a signal transduction domain contained in the immune cell is activated and/or binds to a target antigen. Suitable promoters include, but are not limited to, the activated T Nuclear Factor (NFAT) promoter, STAT promoter, AP-1 promoter, NF-. Kappa.B promoter, and IRF4 promoter.

Expression of one or more polynucleotides or polypeptides that promote saenox transfer in immune cells can alter cell turnover pathways in immune cells. For example, expression of one or more polynucleotides or polypeptides in an immune cell can alter the normal cell turnover pathway of the immune cell to a cell turnover pathway that promotes the delivery of sano, such as necrotic apoptosis, autophagy, iron death, or apoptosis.

In some embodiments, the engineered immune cell comprises at least 2, 3, 4, or 5 polynucleotides each encoding a polypeptide that promotes saenox transfer. Exemplary polypeptides that promote sanoχ transfer are provided in table 1 below. In some embodiments, the polynucleotide that promotes sano delivery encodes a wild-type protein. In some embodiments, the polynucleotide that promotes sano delivery encodes a biologically active fragment of a wild-type protein, e.g., an N-terminal or C-terminal truncate of the wild-type protein. In some embodiments, the polynucleotide that promotes sano delivery encodes a protein comprising one or more mutations. In some embodiments, the polynucleotide that promotes sano delivery encodes a human protein, e.g., a human wild-type protein.

TABLE 1 exemplary polypeptides that facilitate Sano delivery

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In some embodiments, the one or more polynucleotides that promote saenox transfer encode any one or more of the following: receptor interacting serine/threonine protein kinase 3 (RIPK 3), Z-DNA binding protein 1 (ZBP 1), mixed lineage kinase domain-like pseudokinase (MLKL), adaptor-induced interferon- β (TRIF) containing Toll/interleukin-1 receptor (TIR) domain, N-terminal truncations of TRIF containing only TIR domain and RHIM domain, interferon regulatory factor 3 (IRF 3), truncated Fas-associated protein with death domain (FADD) and cellular FLICE (FADD-like IL-1 β convertase) inhibitor protein (c-FLIP). In some embodiments, the one or more polynucleotides that promote saenox transfer encode a polypeptide selected from the group consisting of: desetin-A(GSDM-A),desetin-B(GSDM-B),desetin-C(GSDM-C),desetin-D(GSDM-D),desetin-E(GSDM-E),apoptosis-relatedspeckle-likeproteins(ASC-card)containingaC-terminalcaspaserecruitingdomainwithadimerizationdomain,andmutantsthereof. In some embodiments, the N-terminal truncations of the tri comprising only the TIR domain and the RHIM domain comprise a deletion of amino acid residues 1-311 of human tri (e.g., mini-tri).

In some embodiments, the one or more polynucleotides that promote saenox transfer encode a variant of TRIF, e.g., a variant of wild-type human TRIF protein. Exemplary human TRIF variants are provided in Table 2 below.

TABLE 2 human TRIF and variants thereof

In some embodiments, the one or more polynucleotides that promote sano delivery encode a polypeptide selected from the group consisting of cIAP1, cIAP2, IKKa, IKKb, XIAP, and Nemo. Although these polypeptides may inhibit cell death, they may promote sanoχ transfer, for example, by promoting NF-kB activation. Thus, in some embodiments, increasing expression of cIAP1, cIAP2, ikkα, ikkβ, XIAP, and/or Nemo in an immune cell promotes saenox transfer in the immune cell. In other embodiments, decreasing expression of cIAP1, cIAP2, ikkα, ikkβ, XIAP, and/or Nemo in immune cells promotes sano delivery to immune cells, e.g., by attenuating inhibition of cell death by these proteins, thereby promoting cell turnover.

In some embodiments, the polynucleotide that promotes sano delivery encodes a death fold domain. Examples of death folding domains include, but are not limited to, death domains, hot protein domains, death Effector Domains (DED), C-terminal caspase recruitment domains (CARD), and variants thereof.

In some embodiments, the death domain is selected from the death domain of Fas-associated protein (FADD) having a death domain, the death domain of Fas receptor, the death domain of tumor necrosis factor receptor type 1-associated death domain protein (TRADD), the death domain of tumor necrosis factor receptor type 1 (TNFR 1), and variants thereof. FADD is a 23kDa protein consisting of 208 amino acids. It contains two main domains: a C-terminal Death Domain (DD) and an N-terminal Death Effector Domain (DED). These domains are similar in structure to each other, each consisting of 6 alpha-helices. The DDs of FADD bind to receptors such as Fas receptor at plasma membrane via their DDs. The DED of FADD binds to the DED of intracellular molecules such as procaspase 8. In some embodiments, the FADD-DD is a dominant negative mutant of the FADD-DD, or myristoylated FADD-DD (myr-FADD-DD).

In some embodiments, the thermal protein domain is from a protein selected from the group consisting of NLR family thermal protein domain-containing protein 3 (NLRP 3) and apoptosis-related spot-like protein (ASC).

In some embodiments, the Death Effector Domain (DED) is from a protein selected from the group consisting of Fas-related protein (FADD), caspase-8, and caspase-10 having a death domain.

In some embodiments, the CARD is from a protein selected from the group consisting of RIP related ICH1/CED3 homologous protein (RAIDD), apoptosis related spotting protein (ASC), mitochondrial antiviral signaling protein (MAVS), caspase-1, and variants thereof.

In some embodiments, the polynucleotide that facilitates sano delivery encodes a protein comprising a TIR domain. The TIR domains may be derived from proteins including, but not limited to, myeloid differentiation primary response protein 88 (MyD 88), toll/interleukin-1 receptor (TIR) domain-containing adaptor-induced interferon- β (tif), toll-like receptor 3 (TLR 3), toll-like receptor 4 (TLR 4), TIR domain-containing adaptor protein (TIRAP), and position chain-related membrane protein (TRAM).

In some embodiments, the polynucleotide that promotes saenox transfer encodes a polypeptide selected from the group consisting of: cellularFLICE(FADD-likeIL-1βconvertingenzyme)-inhibitorprotein(c-FLIP),receptorinteractingserine/threonineproteinkinase1(RIPK1),receptorinteractingserine/threonineproteinkinase3(RIPK3),Z-DNAbindingprotein1(ZBP1),mixedlineagekinasedomain-likepseudokinase(MLKL),N-terminaltruncationsoftifcomprisingonlyTIRdomainandRHIMdomain,FADD,inhibitorkBalphasuper-repressor(ikbα-SR),interleukin-1receptorassociatedkinase1(IRAK1),tumornecrosisfactorreceptortype1associateddeathdomain(TRADD),dominantnegativemutantsofcaspase-8,interferonregulatoryfactor3(IRF3),destin-a(GSDM-a)andmutantsthereof,destin-b(GSDM-b)andmutantsthereof,destin-c(GSDM-c)andmutantsthereof,destin-d(GSDM-d)andmutantsthereof,destin-e(dm-e)andmutantsthereof,apoptosis-relatedproteins(asc-a)andmutantsthereofwithanapoptosis-relatedparticle-domainandapoptosis-likereceptor-relateddomain(asc-containingreceptor-end-domain).

In some embodiments, cflup is human cflup. In some embodiments, cflup is caspase-8 and FADD-like apoptosis modulator (cfar).

In some embodiments, the polynucleotide that promotes sano delivery is a viral gene. In some embodiments, the viral gene encodes a polypeptide selected from the group consisting of vfip (ORF 71/K13) of kaposi's sarcoma-associated herpesvirus (KSHV), MC159L from molluscum contagiosum virus, E8 from equine herpesvirus 2, vcica from Human Cytomegalovirus (HCMV) or Murine Cytomegalovirus (MCMV), crmA from vaccinia virus, and P35 from alfalfa y spodoptera frugiperda nucleopolyhedrovirus (AcMNPV).

It is understood that any of the polypeptides described herein that promote saenox transfer to immune cells may be mutated to further enhance their ability to promote saenox transfer. For example, in some embodiments, the polynucleotide encoding ZBP1 comprises any one or any combination of the following: deletion of the receptor interacting protein homotypic interacting motif (RHIM) C, deletion of RHIM D, deletion of RHIM B and deletion in the region encoding the N-terminal end of the zα1 domain. In some embodiments, ZBP1 is a ZBP1 Za1/RHIM a truncate.

In some embodiments, one or more polynucleotides that promote sano delivery inhibit expression or activity of receptor-interacting serine/threonine protein kinase 1 (RIPK 1). RIPK1 may promote sano delivery by driving necrotic apoptosis downstream of death receptors such as TNF and Fas. However, the RHIM domain in RIPK1 can also inhibit TRIF and ZBP 1-mediated necrotic apoptosis by preventing aberrant RHIM oligomerization, such that necrotic apoptosis can also be enhanced in the absence of RIPK 1. Thus, in some embodiments, RIPK1 may inhibit sano delivery by preventing TRIF and ZBP 1-mediated necrotic apoptosis.

Fusion proteins that promote saenox delivery

In some embodiments, a polynucleotide that promotes sano delivery may encode a fusion protein. The fusion protein may comprise two or more domains listed in table 3 below, for example 2, 3, 4 or 5 domains listed in table 3. For example, in some embodiments, the polynucleotide that facilitates sano delivery encodes a fusion protein comprising a TRIF TIR domain, a TRIF RHIM domain, and an ASC-CARD. This fusion protein will recruit caspase-1 and activate cell apoptosis. In some embodiments, the fusion protein comprises a ZBP1 Za2 domain and an ASC-CARD. Such fusion proteins activate cell apoptosis. In some embodiments, the fusion protein comprises a RIPK3 RHIM domain and a caspase large subunit/small subunit (L/S) domain. Such fusion proteins will drive constitutive activation of caspases, resulting in different types of cell death, depending on the caspase L/S domain selected, as shown in table 3. In some embodiments, the fusion protein comprises a TRIF TIR domain, a TRIF RHIM domain, and a FADD death domain (FADD-DD). Such fusion proteins block apoptosis but induce necrotic apoptosis. In some embodiments, the fusion protein comprises an inhibitor of a kbαsuper repressor (ikbαsr) and a caspase-8 DED domain. This fusion protein inhibits NF-kB and induces apoptosis.

TABLE 3 polypeptide domains that promote Sano delivery. The abbreviations shown are Death Domain (DD), death Effector Domain (DED), caspase recruitment domain (CARD) and large subunit/small subunit (L/S). The approximate size of the polynucleotide encoding the polypeptide domain is indicated.

Domain	Size (-bp)	Expected results
			ZBP1-RHIMA	100	Necrotic apoptosis
TRIF-RHIM	100	Necrotic apoptosis
			RIPK3-RHIM	100	Necrotic apoptosis
M45-RHIM	100	Inhibition of necrotic apoptosis
			ICP6-RHIM	100	Inhibition of necrotic apoptosis
MyD88-DD	300	Inhibition of IL-1R/TLR
			MyD88-TIR	300	Inhibition of IL-1R/TLR
IRAK4-DD	300	Inhibition of IL-1R/TLR
			ASC-CARD	300	Apoptosis of cell coke
ASC-thermal proteins	300	Apoptosis of cell coke
			MAVS-CARD	300	Blocking RLR
FADD-DD	300	Blocking extrinsic apoptosis
			FADD-DED	300	Induction of extrinsic apoptosis
TRADD-DD	300	Inhibition/induction of extrinsic apoptosis
			FAS-DD	300	Induction of extrinsic apoptosis
TNFR-DD	300	Induction of extrinsic apoptosis
			caspase-8-CARD	300	Induction of extrinsic apoptosis
caspase-8-L/S	600	Induction of extrinsic apoptosis
			caspase-1-CARD	300	Apoptosis of cell coke
Caspase-1-L/S	300	Apoptosis of cell coke
			caspase-9-CARD	300	Intrinsic apoptosis
Caspase 9-L/S	300	Intrinsic apoptosis

In some embodiments, the immune cells are engineered to contain only one polynucleotide that promotes saenox transfer. In some embodiments, such a single polynucleotide that facilitates saxophone delivery encodes only one saxophone delivery polypeptide. In some embodiments, such a single polynucleotide encodes two or more saenox transfer polypeptides, e.g., two or more saenox transfer polypeptides selected from the group consisting of: TRADD, TRAF2, TRAF6, ciaP1, ciaP2, XIAP, NOD2, myD88, TRAM, HOIL, HOIP, sharpin, IKKg, IKKa, IKKb, relA, MAVS, RIGI, MDA, tak1, TBK1, IKKe, IRF3, IRF7, IRF1, TRAF3, caspase, FADD, TNFR1, TRAILR2, FAS, bax, bak, bim, bid, noxa, puma, TRIF, ZBP1, RIPK3, MLKL, destina, destinin B, destinin C, destinin D, destinin E, tumor necrosis factor receptor superfamily (TNFSF) proteins, variants thereof, and functional fragments thereof.

In other embodiments, the immune cell is engineered to comprise two or more polynucleotides that promote saenox transfer, wherein each polynucleotide encodes a different saenox transfer polypeptide, e.g., wherein the different saenox transfer polypeptides are selected from the group consisting of: TRADD, TRAF2, TRAF6, ciaP1, ciaP2, XIAP, NOD2, myD88, TRAM, HOIL, HOIP, sharpin, IKKg, IKKa, IKKb, relA, MAVS, RIGI, MDA, tak1, TBK1, IKKe, IRF3, IRF7, IRF1, TRAF3, caspase, FADD, TNFR1, TRAILR2, FAS, bax, bak, bim, bid, noxa, puma, TRIF, ZBP1, RIPK3, MLKL, destina, destinin B, destinin C, destinin D, destinin E, tumor necrosis factor receptor superfamily (TNFSF) proteins, variants thereof, and functional fragments thereof.

Suitable caspases include caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, and caspase-12.

Exemplary TNFSF proteins are provided in Table 4 below.

Table 4. Exemplary TNFSF proteins were adapted from Locksley et al, 2001, cell [ cell ].104 (4): 487-501, which is incorporated herein by reference in its entirety.

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Polynucleotide sequences encoding the saenox transfer polypeptides are provided in table 5 below. Any other polynucleotide sequence encoding a saenox transfer polypeptide of table 5 (or encoding a polypeptide that is at least 85%, 87%, 90%, 95%, 97%, 98% or 99% identical thereto) may also be used in the methods and compositions described herein.

TABLE 5 polynucleotides encoding Sano delivery polypeptides

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The two or more saenox polypeptides may be expressed as separate polypeptides or they may be comprised in a chimeric protein. In some embodiments, at least one of these polynucleotides that promote saenox transfer is transcribed as a single transcript encoding two or more saenox transfer polypeptides.

The saenox delivery polypeptides described herein may facilitate saenox delivery by a variety of mechanisms, including, but not limited to, activation of NF-kB, activation of IRF3 and/or IRF7, promotion of apoptosis, and promotion of programmed necrosis (e.g., necrotic apoptosis or cell pyrosis). When a combination of two or more saenox transfer polypeptides is used, each of the two or more saenox transfer polypeptides may promote saenox transfer by a similar mechanism or by a different mechanism. For example, in some embodiments, at least two of the saenox polypeptides encoded by the one or more polynucleotides activate NF-kB. In some embodiments, at least two of the saenox polypeptides encoded by the one or more polynucleotides activate IRF3 and/or IRF7. In some embodiments, at least two of the saenopassing polypeptides encoded by the one or more polynucleotides promote apoptosis. In some embodiments, at least two of the saenopassing polypeptides encoded by the one or more polynucleotides promote programmed necrosis (e.g., necrotic apoptosis or cell pyrosis).

When the two or more saenox transfer polypeptides facilitate saenox transfer by different mechanisms, a combination of the various mechanisms may be used. For example, in some embodiments, at least one of the saenox polypeptides encoded by the one or more saenox polynucleotides activates NF-kB and at least one of the saenox polypeptides encoded by the one or more polynucleotides activates IRF3 and/or IRF7. In some embodiments, at least one of the saenox polypeptides encoded by the one or more polynucleotides activates NF-kB and at least one of the saenox polypeptides encoded by the one or more polynucleotides promotes apoptosis. In some embodiments, at least one of the saenox polypeptides encoded by the one or more polynucleotides activates NF-kB, and at least one of the saenox polypeptides encoded by the one or more polynucleotides promotes programmed necrosis (e.g., necrotic apoptosis or cell apoptosis). In some embodiments, at least one of the saenox polypeptides encoded by the one or more polynucleotides activates IRF3 and/or IRF7, and at least one of the saenox polypeptides encoded by the one or more polynucleotides promotes apoptosis. In some embodiments, at least one of the saenox polypeptides encoded by the one or more saenox polynucleotides activates IRF3 and/or IRF7, and at least one of the saenox polypeptides encoded by the one or more polynucleotides promotes programmed necrosis (e.g., necrotic apoptosis or cell apoptosis). In some embodiments, at least one of the saenox polypeptides encoded by the one or more polynucleotides promotes apoptosis, and at least one of the saenox polypeptides encoded by the one or more saenox polynucleotides promotes programmed necrosis (e.g., necrotic apoptosis or cell pyrosis).

In some embodiments, the saenox transfer polypeptide that activates NF-kB is selected from the group consisting of: TRIF, TRADD, TRAF2, TRAF6, cIAP1, cIAP2, XIAP, NOD2, myD88, TRAM, HOIL, HOIP, sharpin, IKKg, IKKa, IKKb, relA, MAVS, RIGI, MDA, tak1, TNFSF proteins, and functional fragments and variants thereof. In some embodiments, the saenox transfer polypeptide that activates IRF3 and/or IRF7 is selected from the group consisting of: TRIF, myD88, MAVS, TBK1, IKKe, IRF3, IRF7, IRF1, TRAF3, and functional fragments and variants thereof. In some embodiments, the apoptosis-promoting saenox transfer polypeptide is selected from the group consisting of: TRIF, RIPK1, caspase, FADD, TRADD, TNFR1, TRAILR2, FAS, bax, bak, bim, bid, noxa, puma, and functional fragments and variants thereof. In some embodiments, the saenox delivery polypeptide that promotes programmed necrosis (e.g., necrotic apoptosis or cell pyrosis) is selected from the group consisting of: ZBP1, RIPK3, MLKL, desetin, and functional fragments and variants thereof.

In some embodiments, the combination of the saenox transfer polypeptides is selected from: TRADD and TRAF2, TRADD and TRAF6, TRADD and cIAP1, TRADD and cIAP2, TRADD and XIAP, TRADD and NOD2, TRADD and MyD88, TRADD and TRAM, TRADD and HOIL, TRADD and HOIP, TRADD and Sharpin, TRADD and IKKKKg, TRADD and IKKa, TRADD and IKKKb, TRADD and RelA, TRADD and MAVS, TRADD and RIGI, TRADD and MDA5, TRADD and Tak1, TRADD and TBK1, TRADD and IKKe, TRADD and IRF3, TRADD and IRF7, TRADD and IRF1, TRADD and TRAF3, TRADD and caspase, TRADD and FADD, TRADD and TNFR1, TRADD and TRAILR2, TRADD and FAS TRADD and Bax, TRADD and Bak, TRADD and Bim, TRADD and Bid, TRADD and Noxa, TRADD and Puma, TRADD and TRIF, TRADD and ZBP1, TRADD and RIPK3, TRADD and MLKL, TRADD and Xiaoretin A, TRADD and Xiaoretin B, TRADD and Deuterin C, TRADD and Deuterin D, TRADD and Deuterin E, TRAF2 and TRAF6, TRAF2 and cIAP1, TRAF2 and cIAP2, TRAF2 and XIAP, TRAF2 and NOD2, TRAF2 and MyD88, TRAF2 and TRAM, TRAF2 and HOIL, TRAF2 and HOIP, TRAF2 and Sharpin, TRAF2 and IKKKg, TRAF2 and Ka, TRAF2 and IKKKKb, TRAF2 and RelA, TRAF2 and IKKKKG TRAF2 and MAVS, TRAF2 and RIGI, TRAF2 and MDA5, TRAF2 and Tak1, TRAF2 and TBK1, TRAF2 and IKKe, TRAF2 and IRF3, TRAF2 and IRF7, TRAF2 and IRF1, TRAF2 and TRAF3, TRAF2 and caspase, TRAF2 and FADD, TRAF2 and TNFR1, TRAF2 and TRAILR2, TRAF2 and FAS, TRAF2 and Bax, TRAF2 and Bak, TRAF2 and Bim, TRAF2 and Bid, TRAF2 and Noxa, TRAF2 and Puma, TRAF2 and TRIF, TRAF2 and ZBP1, TRAF2 and RIPK3, TRAF2 and KL, TRAF2 and ZBP1, TRAF2 and RIPK3, TRAF2 and KL, and TRAF2 and BIM TRAF2 and Desertraline A, TRAF and Desertraline B, TRAF2 and Desertraline C, TRAF2 and Desertraline D, TRAF2 and Desertraline E, TRAF6 and cIAP1, TRAF6 and cIAP2, TRAF6 and XIAP, TRAF6 and NOD2, TRAF6 and MyD88, TRAF6 and TRAM, TRAF6 and HOIL, TRAF6 and HOIP, TRAF6 and Sharpin, TRAF6 and IKKKg, TRAF6 and IKKa, TRAF6 and IKKb, TRAF6 and RelA, TRAF6 and MAVS, TRAF6 and RIGI, TRAF6 and MDA5, TRAF6 and Tak1, TRAF6 and IKKe, TRAF6 and IRF3, TRAF6 and IRF7, TRAF6 and IRF1, TRAF6 and TRAF3, TRAF6 and cystease, radix asparagines and Fadd, the method comprises the steps of (1) mixing a first component with a second component, and (b) heating the mixture to obtain a mixture of the first component and the second component, the method comprises the steps of The method comprises the following steps of Sharp and IKKKKKg, sharp and IKKKb, sharp and RelA, sharp and MAVS, sharp and RIGI, sharp and MDA5, sharp and Tak1, sharp and TBK1, sharp and IKKe, sharp and IRF3, sharp and IRF7, sharp and IRF1, sharp and TRAF3, sharp and caspase, sharp and FADD, sharp and TNFR1, sharp and TRAILR2, sharp and FAS, sharp and Bax, sharp and Bak, sharp and Bim, sharp and Bid, sharp and Noxa, sharp and TRA Sharpin and TRIF, sharpin and ZBP1, sharpin and RIPK3, sharpin and MLKL, sharpin and destin A, sharpin and destin B, sharpin and destin C, sharpin and destin D, sharpin and destin E, IKKg and IKKa, IKKKKg and IKKb, IKKKg and RelA, IKKKg and MAVS, IKKg and RIGI, IKKKg and MDA5, IKKKg and Tak1, IKKg and TBK1, IKKKKe, IKKKand IRF3, IKKg and IRF7, IKKKg and IRF1, IKKKg and TRAF3, IKKKKand caspase, IKKKKKKKand FADD, IKKKg and TNFR1, IKKKKKKg and TRAILR1, IKKKKKg and TRAILR 2; IKKg and FAS, IKKg and Bax, IKKg and Bak, IKKg and Bim, IKKg and Bid, IKKg and Noxa, IKKg and Puma, IKKg and TRIF, IKKg and ZBP1, IKKg and RIPK3, IKKg and MLKL, IKKg and degranulation 823 and degranulation B, IKKg and degranulation C, IKKg and degranulation D, IKKg and degranulation E, IKKa and IKKb, ik and RelA, IKKa and MAVS, IKKa and RIGI, IKKa and MDA5, IKKa and Tak1, IKKa and TBK1, IKKa and ikkek, IKKa and IRF3, IKKa and IRF7, IKKa and IRF1, IKKa and TRAF3, IKKa and caspase, IKKa and tbf 1 IKKa and FADD, IKKa and TNFR1, IKKa and TRAILR2, IKKa and FAS, IKKa and Bax, IKKa and Bak, IKKa and Bim, IKKa and Bid, IKKa and Noxa, IKKa and Puma, IKKa and TRIF, IKKa and ZBP1, IKKa and RIPK3, IKKa and MLKL, IKKa and degummed A, IKKa and degummed B, IKKa and degummed C, IKKa and degummed D, IKKa and degummed E, IKKb and RelA, IKKb and MAVS, IKKb and RIGI, IKKb and MDA5, IKKb and Tak1, IKKb and TBK1, IKKb and IKKe, IKKb and IRF3, IKKb and IRF7 IKKb and IRF1, IKKb and TRAF3, IKKb and caspase, IKKb and FADD, IKKb and TNFR1, IKKb and TRAILR2, IKKb and FAS, IKKb and Bax, IKKb and Bak, IKKb and Bim, IKKb and Bid, IKKb and Noxa, IKKb and Puma, IKKb and tri, IKKb and ZBP1, IKKb and RIPK3, IKKb and MLKL, IKKb and degumptin B, IKKb and degumptin C, IKKb and degumptin E, IKKb and RelA, IKKb and MAVS, IKKb and RIGI, IKKb and MDA5, IKKb and Tak1, IKKb and ZBP1, IKKb and RIPK3, IKKb and MLKL, IKKb and degumptin 6324 and degumptin 7452 and degumptin E, IKKb, IKKb and MAVS; IKKb and TBK1, IKKb and IKKe, IKKb and IRF3, IKKb and IRF7, IKKb and IRF1, IKKb and TRAF3, IKKb and caspase, IKKb and FADD, IKKb and TNFR1, IKKb and TRAILR2, IKKb and FAS, IKKb and Bax, IKKb and Bak, IKKb and Bim, IKKb and Bid, IKKb and Noxa, IKKb and Puma, IKKb and TRIF, IKKb and ZBP1, IKKb and RIPK3, IKKb and MLKL, IKKb and decetin A, IKKb and decetin 45B, IKKb and decetin C, IKKb and decetin 25 and decetin 54 and MAVS, and MAVS RelA and RIGI, relA and MDA5, relA and Tak1, relA and TBK1, relA and IKKe, relA and IRF3, relA and IRF7, relA and IRF1, relA and TRAF3, relA and caspase, relA and FADD, relA and TNFR1, relA and TRAILR2, relA and FAS, relA and Bax, relA and Bak, relA and Bim, relA and Bid, relA and Noxa, relA and Puma, relA and TRIF, relA and ZBP1, relA and RIPK3, relA and MLKL RelA and desetin A, relA and desetin B, relA and desetin C, relA and desetin D, relA and desetin E, MAVS and RIGI, MAVS and MDA5, MAVS and Tak1, MAVS and TBK1, MAVS and IKKe, MAVS and IRF3, MAVS and IRF7, MAVS and IRF1, MAVS and TRAF3, MAVS and caspase, MAVS and FADD, MAVS and TNFR1, MAVS and TRAILR2, MAVS and FAS, MAVS and Bax, MAVS and Bak, MAVS and Bim, MAVS and Bid, MAVS and Noxa, MAVS and Puma, MAVS and TRIF, MAVS and ZBP1, MAVS and RIPK1, MAVS and RIPK3, MAVS and MLKL, MAVS and xigenin A, MAVS and xigenin B, MAVS and xigenin C, MAVS and xigenin D, MAVS and xigenin E, RIGI and MDA5, RIGI and Tak1, RIGI and TBK1, RIGI and IKKe, RIGI and IRF3, RIGI and IRF7, RIGI and IRF1, RIGI and TRAF3, RIGI and caspase, RIGI and FADD, RIGI and TNFR1, RIGI and TRAILR2, RIGI and FAS, RIGI and Bax, RIGI and Bak, RIGI and rim, RIGI and Bid, RIGI and Noxa, RIGI and Puma, RIGI and TRIF, RIGI and ZBP1 RIGI and RIPK1, RIGI and RIPK3, RIGI and MLKL, RIGI and RIILL A, RIGI and RIILR 2, MDA5 and FAS, MDA5 and Bax, MDA5 and Bak, MDA5 and Bim, MDA5 and Bid, MDA5 and Noxa, MDA5 and Puma, MDA5 and TRAF3, MDA5 and caspase, MDA5 and FADD, MDA5 and TNFR1, MDA5 and TRAILR2, MDA5 and FAS, MDA5 and Bax, MDA5 and Bak, MDA5 and Bim, MDA5 and Bid, MDA5 and Noxa, MDA5 and Puma MDA5 and TRIF, MDA5 and ZBP1, MDA5 and RIPK3, MDA5 and MLKL, MDA5 and desetin A, MDA and desetin B, MDA5 and desetin C, MDA5 and desetin D, MDA5 and desetin E, tak1 and TBK1, tak1 and IKKe, tak1 and IRF3, tak1 and IRF7, tak1 and IRF1, tak1 and TRAF3, tak1 and caspase, tak1 and FADD, tak1 and TNFR1, tak1 and TRAILR2, tak1 and FAS, tak1 and Bax, tak1 and Bak, tak1 and Bim, tak1 and Bid, tak1 and Noxa Tak1 and Puma, tak1 and TRIF, tak1 and ZBP1, tak1 and RIPK3, tak1 and MLKL, tak1 and Deamin A, tak1 and Deamin B, tak1 and Deamin C, tak1 and Deamin D, tak1 and Deamin E, TBK1 and IKKe, TBK1 and IRF3, TBK1 and IRF7, TBK1 and IRF1, TBK1 and TRAF3, TBK1 and caspase, TBK1 and FADD, TBK1 and TNFR1, TBK1 and TRAILR2, TBK1 and FAS, TBK1 and Bax, TBK1 and Bak, TBK1 and Bim, TBK1 and Bid, TBK1 and Noxa, noxa, TBK1 and Puma, TBK1 and TRIF, TBK1 and ZBP1, TBK1 and RIPK3, TBK1 and MLKL, TBK1 and desetin A, TBK1 and desetin B, TBK1 and desetin C, TBK1 and desetin D, TBK1 and desetin E, IKKe and IRF3, IKKe and IRF7, IKKe and IRF1, IKKe and TRAF3, IKKe and caspase, IKKe and FADD, IKKe and TNFR1, IKKe and TRAILR1, IKKe and trair 2, IKKe and FAS, IKKe and Bax, IKKe and Bak, IKKe and Bim, IKKe and Bid, IKKe and Noxa, IKKe and Puma, IKKe and TRIF, IKKe and FADD IKKe and ZBP1, IKKe and RIPK3, IKKe and MLKL, IKKe and degerming A, IKKe and degerming B, IKKe and degerming C, IKKe and degerming D, IKKe and degerming E, IRF and IRF7, IRF3 and IRF1, IRF3 and TRAF3, IRF3 and caspase, IRF3 and FADD, IRF3 and TNFR1, IRF3 and TRAILR2, IRF3 and FAS, IRF3 and Bax, IRF3 and Bak, IRF3 and Bim, IRF3 and Bid, IRF3 and Noxa, IRF3 and Puma, IRF3 and TRIF, IRF3 and ZBP1, IRF3 and RIPK1 IRF3 and RIPK3, IRF3 and MLKL, IRF3 and degerming A, IRF3 and degerming B, IRF and degerming C, IRF3 and degerming D, IRF3 and degerming E, IRF7 and IRF1, IRF7 and TRAF3, IRF7 and caspase, IRF7 and FADD, IRF7 and TNFR1, IRF7 and TRAILR2, IRF7 and FAS, IRF7 and Bax, IRF7 and Bak, IRF7 and Bim, IRF7 and Bid, IRF7 and Noxa, IRF7 and Puma, IRF7 and ZBP1, IRF7 and RIPK3, IRF7 and MLKL IRF7 and desugarin A, IRF and desugarin B, IRF and desugarin C, IRF and desugarin D, IRF and desugarin E, IRF1 and TRAF3, IRF1 and caspase, IRF1 and FADD, IRF1 and TNFR1, IRF1 and TRAILR1, IRF1 and FAS, IRF1 and Bax, IRF1 and Bak, IRF1 and Bim, IRF1 and Bid, IRF1 and Noxa, IRF1 and Puma, IRF1 and TRIF, IRF1 and ZBP1, IRF1 and RIPK3, IRF1 and MLKL, IRF1 and desugarin A, IRF1 and desugarin B, IRF1 and desugarin C, IRF1 and desquamation D, IRF1 and desquamation E, TRAF and caspase, TRAF3 and FADD, TRAF3 and TNFR1, TRAF3 and TRAILR2, TRAF3 and FAS, TRAF3 and Bax, TRAF3 and Bak, TRAF3 and Bim, TRAF3 and Bid, TRAF3 and Noxa, TRAF3 and Puma, TRAF3 and tri, TRAF3 and ZBP1, TRAF3 and RIPK3, TRAF3 and MLKL, TRAF3 and desquamation A, TRAF3 and desquamation B, TRAF3 and desquamation C, TRAF and desquamation D, TRAF3 and desquamation E, cystein and FADD caspases and TNFR1, caspases and TRAILR2, caspases and FAS, caspases and Bax, caspases and Bak, caspases and Bim, caspases and Bid, caspases and Noxa, caspases and Puma, caspases and TRIF, caspases and ZBP1, caspases and RIPK3, caspases and MLKL, caspases and xifolin A, caspase and caspase and desgenin B, caspase and desgenin C, caspase and desgenin D, caspase and desgenin E, FADD and TNFR1, FADD and TRAILR2, FADD and FAS, FADD and Bax, FADD and Bak, FADD and Bim, FADD and Bid, FADD and Noxa, FADD and Puma, FADD and TRIF, FADD and ZBP1, FADD and RIPK3, FADD and MLKL, FADD and desgenin A, FADD and desgenin B, FADD and desgenin C, FADD and desgenin 3723 and desgenin E, TNFR1 and TRAILR1 TNFR1 and TRAILR2, TNFR1 and FAS, TNFR1 and Bax, TNFR1 and Bak, TNFR1 and Bim, TNFR1 and Bid, TNFR1 and Noxa, TNFR1 and Puma, TNFR1 and TRIF, TNFR1 and ZBP1, TNFR1 and RIPK3, TNFR1 and MLKL, TNFR1 and Xiaofitin A, TNFR1 and Xiaofitin B, TNFR1 and Xiaofitin C, TNFR and Xiaofitin D, TNFR1 and Xiaofitin E, TRAILR1 and TRAILR2, TRAILR1 and FAs, TRAILR1 and Bax, TRAILR1 and Bak, TRAILR1 and Bim, TRAILR1 and Bid, TRAILR1 and Noxa, TRAILR1 and Puma, TRAILR1 and TRIF, TRAILR1 and ZBP1, TRAILR1 and RIPK3, TRAILR1 and MLKL, TRAILR1 and xiaosu 2 and FAS, TRAILR2 and Bax, TRAILR2 and Bak, TRAILR2 and Bim, TRAILR2 and Bid, TRAILR2 and Noxa, TRAILR2 and Puma, TRAILR2 and TRIF, TRAILR2 and ZBP1, TRAILR2 and RIPK1 TRAILR2 and RIPK3, TRAILR2 and MLKL, TRAILR2 and Xiao Su and Bax, FAS and Bak, FAS and Bim, FAS and Bid, FAS and Noxa, FAS and Puma FAS and TRIF, FAS and ZBP1, FAS and RIPK3, FAS and MLKL, FAS and desetin and Bak, bax and Bim, bax and Bid, A and B Bax and Noxa, bax and Puma, bax and TRIF, bax and ZBP1, bax and RIPK3, bax and MLKL, bax and degerming and Bim, bak and Bid, and Bak and Noxa, bak and Puma, bak and TRIF, bak and ZBP1, bak and RIPK3, bak and MLKL, bak and degerming and bir, a. The composition of Bak and Noxa, bak and Puma, bak and TRIF, bak and ZBP1, bak and RIPK3 Bak and MLKL, bak and degerming and Bid, ZBP1 and RIPK3, ZBP1 and MLKL, ZBP1 and xiaosu 1 and RIPK3 RIPK1 and MLKL, RIPK1 and desetin 3 and MLKL ZBP1 and RIPK3, ZBP1 and MLKL, ZBP1 and xiaosu 1 and RIPK3, RIPK1 and MLKL, RIPK1 and xiaosu 3 and MLKL RIPK3 and xiaosu E the compounds are selected from the group consisting of desetin A and B, desetin A and C, desetin A and D, desetin A and E, desetin B and C, desetin B and D, desetin B and E, desetin C and D, desetin C and E, desetin D and TRADD, TNFSF protein and TRAF2, TNFSF protein and TRAF6, TNFSF protein and cIAP1, TNFSF protein and cIAP2, TNFSF protein and XIAP, TNFSF protein and NOD2, TNFSF protein and MyD88, TNFSF protein and TRAM, TNFSF protein and HOIL, TNFSF protein and HOIP, TNFSF protein and IKKF protein and IKKa, TNFSF protein and TNFSKb, TNFSF protein and RelA, TNFSF protein and RelKg, TNFSF protein and MAVS, TNFSF protein and RIGI, TNFSF protein and MDA5, TNFSF protein and Tak1, TNFSF protein and TBK1, TNFSF protein and IKKe, TNFSF protein and IRF3, TNFSF protein and IRF7, TNFSF protein and IRF1, TNFSF protein and TRAF3, TNFSF protein and caspase, TNFSF protein and FADD, TNFSF protein and TNFR1, TNFSF protein and TRAILR2, TNFSF protein and FAS TNFSF protein and Bax, TNFSF protein and Bak, TNFSF protein and Bim, TNFSF protein and Bid, TNFSF protein and Noxa, TNFSF protein and Puma, TNFSF protein and TRIF, TNFSF protein and ZBP1, TNFSF protein and RIPK3, TNFSF protein and MLKL, TNFSF protein and desetin A, TNFSF protein and desetin B, TNFSF protein and desetin C, TNFSF protein and desetin D, TNFSF protein and desetin E, as well as variants thereof and functional fragments thereof.

In particular embodiments, at least one of the saenox polypeptides is a TRIF or a functional fragment or variant thereof.

In particular embodiments, at least one of these saenox polypeptides is RIPK3 or a functional fragment or variant thereof.

In particular embodiments, at least one of the saenox polypeptides encoded by the one or more saenox polynucleotides comprises a TRIF or functional fragment thereof, and at least one of the saenox polypeptides encoded by the one or more polynucleotides comprises a RIPK3 or functional fragment thereof.

In particular embodiments, at least one of the saenox polypeptides is MAVS or a functional fragment or variant thereof, and at least one of the saenox polypeptides is RIPK3 or a functional fragment or variant thereof.

In particular embodiments, at least one of the saenox polypeptides is MAVS or a functional fragment or variant thereof, and at least one of the saenox polypeptides is MLKL or a functional fragment or variant thereof.

In some embodiments, the functional fragment of Bid is a truncated Bid (tBID). TNFR1/Fas conjugation results in cleavage of cytoplasmic Bid into truncated tBIDs, which translocate to mitochondria. the tBID polypeptide functions as a membrane-targeted death ligand. Bak-deficient mitochondria and blocking antibodies reveal that tBID binds to its mitochondrial partner Bak to release cytochrome c. The activated tBID results in allosteric activation of BAK, inducing its intramembranous oligomerization into proposed pores of cytochrome c outflow, integrating the pathway from death receptor to cell death. See Wei et al, 2000, genes & Dev. [ Gene and development ]14:2060-2071.

In particular embodiments, at least one of the saenox polypeptides is MAVS or a functional fragment or variant thereof, and at least one of the saenox polypeptides is tBID or a functional fragment or variant thereof.

In some embodiments, a virus engineered to include one or more polynucleotides that promote sanoχ transfer does not include a polynucleotide encoding a tri.

Additional polynucleotides included in the engineered viruses are described below.

Caspase inhibitors

The engineered virus may further comprise one or more polynucleotides that inhibit caspase activity in the target cell. In some embodiments, the polynucleotide that inhibits caspase activity in the target cell reduces the expression or activity of one or more caspases that are endogenous to the target cell. Polynucleotides that reduce caspase expression may include, but are not limited to, antisense DNA molecules, antisense RNA molecules, double stranded RNAs, sirnas, or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) -CRISPR associated (Cas) (CRISPR-Cas) system guide RNAs.

In some embodiments, the polynucleotide that inhibits caspase activity in the target cell encodes a polypeptide that inhibits caspase activity. In some embodiments, the polypeptide that inhibits caspase activity is a viral protein or variant or functional fragment thereof. Exemplary viral protein caspase inhibitors are provided in table 6 below. In some embodiments, the polypeptide that inhibits caspase activity is a human protein or variant or functional fragment thereof. In some embodiments, the polypeptide that inhibits caspase activity inhibits one or more caspases selected from the group consisting of: caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9 and caspase 10. In certain embodiments, the polypeptide that inhibits caspase activity inhibits caspase 8. In certain embodiments, the polypeptide that inhibits caspase activity inhibits caspase 10. In certain embodiments, the polypeptide that inhibits caspase activity inhibits caspase 8 and caspase 10.

Table 6. Exemplary viral protein caspase inhibitors adapted from Mocarski et al, 2011,Nat Rev Immunol [ Natural comment immunology ]12 month 23 day; 12 (2) 79-88.Doi:10.1038/nri3131, which is incorporated herein by reference in its entirety. BHV-4, bovine herpes virus 4; CMV, cytomegalovirus; DAI, a DNA-dependent activator of interferon regulatory factors; EHV-1, equine herpesvirus 1; FADD, FAS-related death domain proteins; HPV-16, human papillomavirus 16; HSV, herpes simplex virus; KSHV, kaposi's sarcoma-associated herpesvirus; MCMV, murine cytomegalovirus; MCV, molluscum contagiosum virus; RHIM, RIP homotypic interaction motif; RIP, receptor-interacting proteins; TRIF, an adapter protein containing a TIR domain that induces IFN beta; vICA, caspase 8 activates viral inhibitors; vIRA, RIP activates viral inhibitors.

In some embodiments, the polypeptide that inhibits caspase activity is selected from the group consisting of: fas-related death domain protein (FADD-DN), caspase 8-activating viral inhibitor (vICA), cellular FLICE (FADD-like IL-1β converting enzyme) inhibitor protein (cFLIP), caspase 8-dominant negative mutant (Casp 8-DN), apoptosis protein inhibitor-1 (cIAP 1), apoptosis protein inhibitor-1 (cIAP 2), X-linked apoptosis inhibitor (XIAP), TGF-activating kinase 1 (Tak 1), IκB kinase (IKK) and functional fragments thereof.

In a particular embodiment, the polypeptide that inhibits caspase activity is FADD-DN. Death-inducing signaling complex (DISC) recruits adaptor proteins including FADD and an initiating caspase such as caspase 8. See Morgan et al, 2001,Cell Death&Differentiation [ cell death and differentiation ] Vol.8, pages 696-705. Aggregation of caspase 8 in DISC results in activation and apoptosis of the caspase cascade. FADD consists of two protein interaction domains: death domain and death effector domain. Because FADD is an essential component of DISC, a dominant negative mutant containing a death domain but no death effector domain (FADD-DN) has been widely used in death receptor-induced apoptosis studies. FADD-DN acts as a dominant negative inhibitor because it binds to the receptor but is unable to recruit caspase 8.

In a particular embodiment, the polypeptide that inhibits caspase activity is vcica. The vcica protein is a human Cytomegalovirus (CMV) protein encoded by the UL36 gene. See Skaletskaya et al, PNAS [ journal of the national academy of sciences of the united states ]7 month 3 day, 2001 98 (14) 7829-7834, which is incorporated herein by reference in its entirety. The ica protein inhibits Fas-mediated apoptosis by binding to and preventing activation of the prodomain of caspase-8.

In a particular embodiment, the polypeptide that inhibits caspase activity is cflup. The cFLIP protein is the primary anti-apoptosis regulator and resistance factor that inhibits tumor necrosis factor-alpha (TNF-alpha), fas-L, and TNF-related apoptosis-inducing ligand (TRAIL) -induced apoptosis. See Safa,2012, exp Oncol [ experimental oncology ] for 10 months; 34 (3) 176-84, which is incorporated herein by reference in its entirety. The cflup protein is expressed in human cells as long (cflup (L)), short (cflup (S)) and cflup (R) splice variants. The cFLIP protein binds FADD and/or caspase-8 or-10 and TRAIL receptor 5 (DR 5) in a ligand-dependent and non-dependent manner and forms an apoptosis-inhibiting complex (AIC). This interaction in turn prevents the formation of death-inducing signaling complexes (DISC) and subsequent activation of the caspase cascade. It is also known that c-FLIP (L) and c-FLIP (S) have a multifunctional role in various signaling pathways. In a particular embodiment, cFLIP is cFLIP (L). In a particular embodiment, cFLIP is cFLIP (S).

In some embodiments, at least one of the saenox polypeptides is a TRIF or a functional fragment or variant thereof, at least one of the saenox polypeptides is a RIPK3 or a functional fragment or variant thereof, and at least one of the saenox polypeptides is a FADD-DN or a functional fragment or variant thereof.

In some embodiments, at least one of the saenox polypeptides is a tri or functional fragment or variant thereof, at least one of the saenox polypeptides is a RIPK3 or functional fragment or variant thereof, and at least one of the saenox polypeptides is a vca or functional fragment or variant thereof.

In some embodiments, at least one of the saenox polypeptides is a TRIF or a functional fragment or variant thereof, at least one of the saenox polypeptides is a RIPK3 or a functional fragment or variant thereof, and at least one of the saenox polypeptides is a cFLIP or a functional fragment or variant thereof.

In some embodiments, at least one of the saenox polypeptides is MAVS or a functional fragment or variant thereof, at least one of the saenox polypeptides is RIPK3 or a functional fragment or variant thereof, and at least one of the saenox polypeptides is FADD-DN or a functional fragment or variant thereof.

Xiao Su (Xiao Su)

Desertraline is a family of pore-forming effector proteins that cause membrane permeabilization and cell apoptosis. The mesothelin protein comprises mesothelin A, mesothelin B, mesothelin C, mesothelin D and mesothelin E. The degerming contains a cytotoxic N-terminal domain and a C-terminal repressor domain linked by a flexible linker. Proteolytic cleavage between these two domains releases intramolecular inhibition of the cytotoxic domain, allowing it to intercalate into the cell membrane and form large oligomeric pores, which disrupt ion homeostasis and induce cell death. See Broz et al, 2020,Nature Reviews Immunology [ Nature review-immunology ]20:143-157, which is incorporated herein by reference in its entirety. For example, the desetin E (GSDME, also known as DFNA 5) can be cleaved by caspase 3, thereby converting non-inflammatory apoptosis into apoptosis in GSDME-expressing cells. Similarly, caspases 1, 4 and 5 cleave and activate desetin D.

A nucleic acid molecule, or vector (e.g., virus, plasmid, or transposon), cell, or pharmaceutical composition encoding two or more saenox polypeptides may comprise at least one polynucleotide encoding a mesothelin or a functional fragment or variant thereof. In some embodiments, the functional fragment of a mesothelin is the N-terminal domain of mesothelin a, mesothelin B, mesothelin C, mesothelin D, or mesothelin E.

In some embodiments, at least one of the saenox polypeptides is a TRIF or a functional fragment or variant thereof, at least one of the saenox polypeptides is a RIPK3 or a functional fragment or variant thereof, and at least one of the saenox polypeptides is a mesothelin or a functional fragment or variant thereof.

In some embodiments, at least one of the saenox polypeptides is a TRIF or a functional fragment or variant thereof, at least one of the saenox polypeptides is a RIPK3 or a functional fragment or variant thereof, and at least one of the saenox polypeptides is a mesothelin E or a functional fragment or variant thereof.

In some embodiments, at least one of the saenox polypeptides is MAVS or a functional fragment or variant thereof, and at least one of the saenox polypeptides is a mesothelin D N-terminal domain or a functional fragment or variant thereof.

In some embodiments, at least one of the saenox polypeptides is MAVS or a functional fragment or variant thereof, and at least one of the saenox polypeptides is a mesothelin E N-terminal domain or a functional fragment or variant thereof.

In some embodiments, at least one of the saenox transfer polypeptides is MAVS or a functional fragment or variant thereof, at least one of the saenox transfer polypeptides is tBID or a functional fragment or variant thereof, and at least one of the saenox transfer polypeptides is mesothelin E or a functional fragment or variant thereof.

Additional proteins for expression in engineered immune cells

In addition to one or more polynucleotides encoding polypeptides that promote sanoχ transfer, such as those provided in table 5 above, the engineered immune cells disclosed herein may further comprise one or more polynucleotides encoding immunostimulatory proteins. In one embodiment, the immunostimulatory protein is an antagonist of transforming growth factor beta (TGF-beta), a colony stimulating factor, a cytokine, an immune checkpoint modulator, a flt3 ligand, or an antibody agonist of flt 3.

The colony stimulating factor may be granulocyte-macrophage colony stimulating factor (GM-CSF). In one embodiment, the polynucleotide encoding GM-CSF is inserted into the ICP34.5 locus.

The cytokine may be an interleukin. In some embodiments, the interleukin is selected from the group consisting of: IL-1α, IL-1β, IL-2, IL-4, IL-12, IL-15, IL-18, IL-21, IL-24, IL-33, IL-36α, IL-36β, and IL-36γ. Other suitable cytokines include type I interferons, gamma interferons, type III interferons, and tnfα.

In some embodiments, the immune checkpoint modulator is an antagonist of an inhibitory immune checkpoint protein. Examples of inhibitory immune checkpoint proteins include, but are not limited to, ADORA2A, B7-H3, B7-H4, KIR, VISTA, PD-1, PD-L2, LAG3, tim3, BTLA and CTLA4. In some embodiments, the immune checkpoint modulator is an agonist of a stimulatory immune checkpoint protein. Examples of stimulatory immune checkpoint proteins include, but are not limited to, CD27, CD28, CD40, OX40, GITR, ICOS, and 4-1BB. In some embodiments, the agonist of the stimulatory immune checkpoint protein is selected from the group consisting of a CD40 ligand (CD 40L), ICOS ligand, GITR ligand, 4-1-BB ligand, 0X40 ligand, and modified forms of any of them. In some embodiments, the agonist of the stimulatory immune checkpoint protein is an antibody agonist of a protein selected from the group consisting of CD40, ICOS, GITR, 4-1-BB and 0X 40.

In addition to one or more polynucleotides encoding polypeptides that promote saenox transfer, such as those provided in table 5 above, the engineered immune cells disclosed herein may further comprise a suicide gene. The term "suicide gene" refers to a gene encoding a protein (e.g., an enzyme) that converts a non-toxic precursor of a drug into a cytotoxic compound. In some embodiments, the suicide gene encodes a polypeptide selected from the group consisting of: FK506 binding protein (FKBP) -FAS, FKBP-caspase-8, FKBP-caspase-9, polypeptides having cytosine deaminase (CDase) activity, polypeptides having thymidine kinase activity, polypeptides having uracil phosphoribosyl transferase (UPRTase) activity, and polypeptides having purine nucleoside phosphorylase activity.

In some embodiments, the polypeptide having CDase activity is FCY1, FCA1, or CodA.

In some embodiments, the polypeptide having UPRTase activity is FUR1 or a variant thereof, e.g., fur1Δ105.Fur1Δ105 is a FUR1 gene lacking the first 105 nucleotides in the 5' region of the coding region, allowing the synthesis of UPRTase, wherein the first 35 amino acid residues have been deleted at the N-terminus. Fur1Δ105 starts with methionine at position 36 of the native protein.

Suicide genes may encode fusion proteins, for example fusion proteins having CDase and UPRTase activity. In some embodiments, the fusion protein is selected from the group consisting of codA:: upp, FCY1:: FUR1, FCYI:: FUR1Δ105 (FCU 1), and FCU1-8 polypeptides.

Polypeptides inhibiting saenox transmission

In some embodiments, the polynucleotide that promotes saxotransmission is a polynucleotide (e.g., a polynucleotide encoding an siRNA) that reduces expression or activity in an immune cell of a polypeptide that inhibits saxotransmission that is endogenous to the immune cell. Exemplary polypeptides that can inhibit saenox transfer that are endogenous to immune cells are provided in table 7 below.

Table 7. Exemplary polypeptides that inhibit saenox transfer in immune cells.

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Polynucleotides that reduce expression of genes that inhibit sanoχ delivery may include, but are not limited to, antisense DNA molecules, antisense RNA molecules, double stranded RNAs, sirnas, or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) -CRISPR associated (Cas) (CRISPR-Cas) system guide RNAs.

Signal transduction and targeting domains

The engineered immune cells of the invention can comprise a heterologous signal transduction domain (e.g., an intracellular signaling domain) that triggers cell turnover and/or a heterologous targeting domain (e.g., an antigen binding domain) that directs the engineered immune cells to a target cell. The signal transduction domain is not necessarily required to be operably linked to a targeting domain. For example, in some embodiments, the signal transduction domain and the targeting domain are assembled only in the presence of a heterodimeric small molecule, such that the engineered immune cell is activated only when the target antigen is engaged and the small molecule aggregates the signal transduction domain and the targeting domain together. See Wu et al 2015 science [ science ]10 month 16 day; 350 (6258) aab4077.

In some embodiments, the heterologous signal transduction domain comprises an intracellular signal transduction domain described herein, e.g., a signal transduction domain comprising ITAM. However, other types of heterologous signal transduction domains besides the intracellular signal transduction domains described herein are also suitable for engineering immune cells. For example, in some embodiments, the heterologous signal transduction domain comprises a synthetic Notch (synNotch) receptor signaling system. The synNotch receptor signaling system contains a core regulatory domain from the cell-cell signaling receptor Notch, but has a synthetic antigen binding domain (e.g., a single chain antibody) and a synthetic intracellular transcription domain (Gordon et al 2015; morput et al 2016). When the synNotch receptor binds to a cognate antigen, the synNotch receptor undergoes induced transmembrane cleavage, similar to natural Notch activation, releasing the intracellular transcription domain to enter the nucleus and activate expression of the target gene regulated by the cognate upstream cis-activating promoter. Thus, the synNotch signaling system can be used to generate engineered immune cells in which a tailored antigen recognition event can drive expression of a heterologous polynucleotide (e.g., a polynucleotide that facilitates sanoχ transfer). The synNotch signaling system is described in the following documents: for example, U.S. patent No. 9,670,281; roybal et al 2016, cell [ cell ]167:419-432; and Morput et al 2016, cell [ cell ]164 (4): 780-91.

In some embodiments, the heterologous targeting domain is an antigen binding domain. However, other types of heterologous targeting domains besides antigen binding domains are also suitable for engineering immune cells. For example, in some embodiments, the heterologous targeting domain is an oxygen-sensitive subdomain of hif1α. This oxygen-sensitive subdomain is responsive to the hypoxic environment, which is a hallmark of certain tumors. See juillerate et al 2017, sci.rep. [ science report ]7:39833.

In some embodiments, the targeting domain (e.g., antigen binding domain) is operably linked to a signaling domain (e.g., intracellular signaling domain). In some embodiments, the targeting domain and the signal transduction domain are contained within a Chimeric Antigen Receptor (CAR).

Chimeric Antigen Receptor (CAR)

The engineered immune cells of the invention may further comprise a heterologous polynucleotide encoding a Chimeric Antigen Receptor (CAR). Chimeric Antigen Receptors (CARs) are molecules that combine antibody-based specificity for a desired antigen (e.g., a tumor antigen) with a T cell receptor activating intracellular domain to generate a chimeric protein that exhibits specific immune activity.

In some embodiments, the CAR comprises a signaling domain (e.g., an intracellular signaling domain). In some embodiments, the CAR comprises a targeting domain (e.g., an antigen binding domain) that directs the engineered immune cell to the target cell. In some embodiments, the CAR comprises an antigen binding domain, a transmembrane domain, and a signaling domain. The CAR may further comprise a hinge domain between the antigen binding domain and the transmembrane domain.

In some embodiments, the polynucleotide that facilitates sano delivery can be under transcriptional control of a heterologous promoter, e.g., operably linked to a heterologous promoter that induces expression of the polynucleotide upon activation of the immune cell, e.g., upon activation of the antigen binding domain of the CAR with a target antigen and/or the signaling domain (e.g., intracellular signaling domain of the CAR).

Expression of one or more polynucleotides or polypeptides that promote saenox transfer in immune cells can alter cell turnover pathways in immune cells. For example, expression of one or more polynucleotides or polypeptides in an immune cell can alter the normal cell turnover pathway of the immune cell to a cell turnover pathway that promotes the delivery of sano, such as necrotic apoptosis, autophagy, iron death, or apoptosis.

A. Antigen binding domains

An antigen binding domain is a target-specific binding element on the surface of an engineered immune cell that recognizes a surface marker on a target cell or pathogen (e.g., a cancer cell, a fungal cell, a bacterial cell, or a virus). The choice of antigen binding domain of a CAR depends on the type of target protein found on the surface of the target cell or pathogen. For example, in some embodiments, the antigen binding domain specifically binds to a target protein on the surface of a cancer cell (e.g., a solid tumor cell or a non-solid cancer cell). In some embodiments, the antigen binding domain specifically binds to a target protein on the surface of a fungal cell, such as aspergillus fumigatus (Aspergillus fumigatus). In some embodiments, the antigen binding domain specifically binds to a target protein on the surface of a bacterial cell. In some embodiments, the antigen binding domain specifically binds to a target protein on the surface of a virus (e.g., HIV, HBV, HCV or CMV). In some embodiments, the antigen binding domain specifically binds to a target protein on the surface of a host cell infected with a pathogen.

When the target cell is a tumor cell, the antigen binding domain target protein may be a Tumor Specific Antigen (TSA) or a Tumor Associated Antigen (TAA). TSA is specific to tumor cells and does not occur on other cells in the body. TAA is not specific for tumor cells and, conversely, it is also expressed on normal (e.g., non-cancerous) cells under conditions that do not induce an immune tolerance state against the antigen. TAAs may be antigens expressed on normal cells during fetal development when the immune system is immature and unable to respond, or may be antigens that are normally present at very low levels on normal cells but are expressed at much higher levels on tumor cells.

The choice of antigen binding domain and corresponding target protein on a cancer cell will depend on the particular type of cancer to be treated. In some embodiments, the cancer cell is a solid tumor cell. Proteins found on the surface of solid tumor cells are known in the art and are described in the following documents: for example, martinez et al 2019,Front Immunol [ immunological front ]2 months 5 days; 10:128; and Fesnak et al 2016,Nat Rev Cancer [ cancer natural review ] 8, 23, 2016; 16 (9):566-81. Non-limiting exemplary proteins on the surface of solid tumor cells that can be targeted by the antigen binding domain are provided in table 8 below. In certain embodiments, the antigen binding domain binds mesothelin.

TABLE 8 exemplary antigen binding domain target proteins for solid tumors

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In some embodiments, the target cell is a non-solid tumor cell, i.e., a non-solid cancer cell. Proteins found on the surface of non-solid tumor cells are known in the art and are described in the following documents: for example, fesnak et al 2016,Nat Rev Cancer [ cancer natural review ] 8, 23, 2016; 16 (9):566-81. Non-limiting exemplary proteins on the surface of non-solid tumor cells that can be targeted by the antigen binding domains are provided in table 9 below.

TABLE 9 exemplary antigen binding domain target proteins for non-solid tumors

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TABLE 10 exemplary antigen binding domain target proteins for pathogens

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In some embodiments, the antigen binding domain binds to a target protein on a Human Immunodeficiency Virus (HIV), such as gp120. In particular embodiments, the antigen binding domain comprises or consists of a bispecific molecule in which the CD4 segment is linked to a single chain variable fragment of a 17b human monoclonal antibody that recognizes a highly conserved CD 4-induced epitope on gp120 involved in co-receptor binding. See Liu et al, 2015, J Virol J Virol 89 (13): 6685-6694, which is incorporated herein by reference in its entirety. In another specific embodiment, the antigen binding domain comprises or consists of a bispecific molecule comprising a human CD4 segment linked to a carbohydrate recognition domain of a human C-type lectin. These antigen binding domains target two independent regions on HIV-1gp120, which presumably must be conserved across clinically significant viral variants (i.e., primary receptor binding sites and compact oligomannose plaques). See Ghanem et al 2018, cytotherapy [ cytotherapy ]2018;20 (3):407-419.

In some embodiments, the engineered immune cell comprises more than one antigen binding domain (e.g., 2, 3, 4, or 5 antigen binding domains), each targeting a different protein. For example, in some embodiments, the engineered immune cell comprises a first antigen binding domain operably linked to a TCR-type signaling domain and a second antigen binding domain operably linked to a costimulatory signaling domain.

B. Hinge domain

The extracellular region of the CAR, i.e., the antigen binding domain of the CAR, can be linked to the transmembrane domain by a hinge domain (e.g., a hinge from a human protein). For example, in some embodiments, the hinge may be a human Ig (immunoglobulin) hinge, e.g., an IgG4 hinge or a CD8a hinge. In some embodiments, the hinge domain comprises or consists of an IgD hinge. In some embodiments, the hinge domain comprises an inhibitory killer cell Ig-like receptor (KIR) 2DS2 hinge. Suitable amino acid sequences for the hinge domain and nucleic acid sequences encoding the hinge domain are known in the art and are described, for example, in U.S. patent No. 8,911,993 and U.S. patent No. 10,273,300, each of which is incorporated herein by reference in its entirety.

C. Transmembrane domain

The CAR may comprise a transmembrane domain, optionally fused to an antigen binding domain of the CAR via a hinge domain. In some embodiments, the transmembrane domain is naturally associated with one of the other domains in the CAR, e.g., derived from the same protein as one of the other domains in the CAR. In some embodiments, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of the transmembrane domain to the transmembrane domain of the same or a different surface membrane protein, thereby minimizing interactions with other members of the receptor complex. The transmembrane domain may be derived from a naturally occurring protein or may be engineered. Where the source is natural, the transmembrane domain may be derived from any membrane-bound protein or transmembrane protein. The transmembrane region of particular interest may be derived from (e.g., comprise at least the transmembrane region of) the alpha, beta or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD 154.

Alternatively, the transmembrane domain may be engineered, in which case it will predominantly comprise hydrophobic residues such as leucine and valine. In some embodiments, the engineered transmembrane comprises a triplet of phenylalanine, tryptophan, and valine at each end of the transmembrane domain. Optionally, a short polypeptide linker (e.g., between 2 and 10 amino acids in length) can form a linkage between the transmembrane domain and the intracellular signaling domain of the CAR. Glycine-serine duplex is one example of a suitable linker. In a particular embodiment, the transmembrane domain is a CD8 transmembrane domain.

D. Intracellular signaling domains

The intracellular signaling domain of the CAR is responsible for activating at least one effector function of the CAR-expressing immune cell. The term "effector function" refers to a specific function of a cell. For example, the effector function of a T cell may be cytolytic activity or helper activity, including secretion of cytokines. Although the entire intracellular signaling domain of a protein may be employed, in many cases the entire chain need not be used. To the extent that truncated portions of the intracellular signaling domain are used, such truncated portions can be used instead of the complete chain, so long as they transduce effector function signals. Thus, the term intracellular signaling domain is intended to include any truncated portion of the intracellular signaling domain of a protein sufficient to transduce an effector function signal.

Various intracellular signaling domains suitable for use in CARs are known in the art and are described in the following documents: for example, fesnak et al 2016,Nat Rev Cancer [ cancer natural review ] 8, 23, 2016; 16 566-81; and Tokarew et al, 2019, br J Cancer [ journal of Cancer England ]1 month; 120 (1):26-37. Examples of components of intracellular signaling domains for CARs include cytoplasmic sequences of T Cell Receptors (TCRs) and co-receptors that cooperate to initiate signal transduction upon antigen receptor engagement, as well as any derivatives or variants of these sequences and any synthetic sequences with the same functional capabilities. It is known that the signal generated by TCR alone is not sufficient to fully activate T cells, and that a secondary signal or co-stimulatory signal is also required. Thus, T cell activation can be said to be mediated by two different classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation by TCRs (i.e., TCR-type signaling domains) and those that act in an antigen-independent manner to provide secondary or costimulatory signals (i.e., costimulatory signaling domains).

The TCR-type signaling domain modulates primary activation of the TCR complex in a stimulatory manner or in an inhibitory manner. The stimulatory acting TCR-type signaling domain may contain signaling motifs known as immune receptor tyrosine-based activation motifs or ITAMs. Examples of ITAMs containing primary cytoplasmic signaling sequences include those derived from TCR ζ, fcrγ, fcrβ, cd3γ, cd3δ, cd3ε, CD5, CD22, CD79a, CD79b and CD66 d.

In particular embodiments, the intracellular signaling domain of the CAR comprises a cytoplasmic signaling sequence from cd3ζ. The human CD3 zeta protein amino acid sequence is provided, for example, in Uniprot accession number P20963 (incorporated herein by reference in its entirety). The cytoplasmic signaling sequence of cd3ζ consists of amino acid residues 52-164 of the cd3ζ protein. The cytoplasmic signaling sequence of CD3 zeta contains three ITAMs at amino acid residues 61-89, 100-128 and 131-159 of the CD3 zeta protein. In some embodiments, one or more tyrosine residues in one or more cd3ζitams are mutated. It is believed that redundancy in signaling in CARs incorporating all three CD3 zeta ITAMs promotes counterproductive T cell differentiation and depletion. Thus, mutating one or more tyrosine residues in one or more cd3ζitams can block their phosphorylation and downstream signaling, thereby yielding CARs with enhanced therapeutic properties. See Feucht et al, 2019,Nature Medicine [ Nature medical ]1 month; 25 82-88, the entire contents of which are incorporated herein by reference. In some embodiments, the tyrosine residue is mutated by substitution with another amino acid (e.g., phenylalanine).

The intracellular signaling domain further comprises one or more costimulatory signaling domains from a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands, which are necessary for the effective response of lymphocytes to antigens. Examples of such molecules include CD27, CD28, 4-1BB (CD 137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD 83. For example, in particular embodiments, the intracellular signaling domain of the CAR can comprise a cd3ζ chain portion and one or more costimulatory signaling domains. In some embodiments, the costimulatory signaling domain is from an immunostimulatory molecule, e.g., from one or more of the following: CD27, CD28, 4-1BB (CD 137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C and a ligand that specifically binds to CD 83. In some embodiments, the costimulatory signaling domain is from an inhibitory immune checkpoint protein, such as PD-1 or B7-H3.

The TCR-type signaling domain and the co-stimulatory signaling domain within the intracellular signaling domain of the CAR may be linked to each other in a random or specific order. Optionally, a short polypeptide linker (e.g., between 2 and 10 amino acids in length) may form a linkage. Glycine-serine duplex is one example of a suitable linker.

In some embodiments, the intracellular signaling domain comprises an intracellular domain of cd3ζ. Various combinations of signaling domains may also be used. For example, the CAR intracellular signaling domain can comprise signaling domains from at least 2, 3, 4, or 5 different proteins. For example, in some embodiments, the CAR intracellular signaling domain comprises the intracellular domain of cd3ζ and the signaling domain of CD 28. In some embodiments, the CAR intracellular signaling domain comprises an intracellular domain of cd3ζ and a signaling domain of 4-1 BB. In some embodiments, the CAR intracellular signaling domain comprises the intracellular domain of cd3ζ and the signaling domains of CD28 and 4-1 BB. In some embodiments, the CAR intracellular signaling domain comprises the intracellular domain of cd3ζ, the signaling domains of CD28 and 4-1BB, and the signaling domain of CD27 or CD 134. Amino acid sequences for various signaling domains, and nucleic acid sequences encoding them, suitable for use in the intracellular signaling domain of a CAR are known in the art and are described, for example, in U.S. patent No. 8,911,993, which is incorporated herein by reference in its entirety.

For example, in some embodiments, the intracellular signaling domain comprises a combination of signaling domains selected from the group consisting of: (a) A costimulatory signaling domain of CD28 and an intracellular domain of cd3ζ; (b) 4-1BB costimulatory signaling domain and intracellular domain of cd3ζ; and (c) a costimulatory signaling domain of CD28, a costimulatory signaling domain of 4-1BB, a costimulatory signaling domain of CD27 or CD134, and an intracellular domain of CD3 ζ.

The engineered immune cells can further comprise a protein that is constitutively or inductively expressed upon CAR activation, such as a cytokine (e.g., interleukin 12 (IL-12)). T cells transduced with these CARs are known as T cells (TRUCK) redirected for universal cytokine killing. Activation of these CARs promotes production and secretion of the required cytokines to promote tumor killing by several synergistic mechanisms such as exocytosis (perforins, granzymes) or death ligand-death receptor (Fas-FasL, TRAIL) systems. See Tokarew et al, 2019, br J Cancer journal, 1 month; 120 (1):26-37.

In some embodiments, the CAR intracellular signaling domain may comprise a domain that drives IL-12 activation or transcription. For example, the CAR intracellular signaling domain may further comprise a domain that drives IL-12 activation or IL-12 transcription, such as an IL-2rβ truncated intracellular interleukin-2β chain receptor with a STAT3/5 binding motif. Antigen-specific activation of this receptor triggers TCR (e.g., via the cd3ζ domain), co-stimulation (e.g., the CD28 domain), and cytokine (JAK-STAT 3/5) signaling simultaneously, which effectively provides all three synergistic signals required to physiologically drive complete T cell activation and proliferation. See Tokarew et al, 2019, br J Cancer journal, 1 month; 120 (1):26-37.

Non-limiting exemplary combinations of signaling domains that may be present in the CAR intracellular signaling domains are provided in table 11 below.

Table 11. Exemplary CAR intracellular signaling domains.

Reference to the literature

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193.Finney HM,Akbar AN,Lawson AD.Activation of resting human primary T cells with chimeric receptors:costimulation from CD28,inducible costimulator,CD134,and CD137 in series with signals from the TCR zeta chain.J Immunol.2004；172:104–113.[PubMed:14688315]

194.Imai C,et al.Chimeric receptors with 4-1BB signaling capacity provoke potent cytotoxicity against acute lymphoblastic leukemia.Leukemia.2004；18:676–684.DOI:10.1038/sj.leu.2403302[PubMed:14961035]

197.Wang J,et al.Optimizing adoptive polyclonal T cell immunotherapy of lymphomas,using a chimeric T cell receptor possessing CD28 and CD137 costimulatory domains.Hum Gene Ther.2007；18:712–725.DOI:10.1089/hum.2007.028[PubMed:17685852]

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CAR encoding vectors

CARs for expression in engineered immune cells can be encoded by a DNA construct comprising a nucleic acid sequence encoding a domain of the CAR, e.g., a nucleic acid sequence encoding an antigen binding domain, a nucleic acid sequence encoding a transmembrane domain, and a nucleic acid sequence encoding an intracellular signaling domain, all of which are operably linked. The DNA construct encoding the CAR may further comprise a nucleic acid sequence encoding a hinge domain. Nucleic acid sequences encoding these domains can be obtained using recombinant methods known in the art, such as by screening libraries from cells expressing the gene, by obtaining the gene from vectors known to contain the gene, or by direct isolation from cells and tissues containing the gene, using standard techniques. Alternatively, the gene of interest may be synthetically produced, rather than cloned.

The DNA construct encoding the CAR is inserted into a vector for transfer into immune cells. Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer, as they allow long-term stable integration of transgenes and their propagation in daughter cells. Lentiviral vectors have additional advantages over vectors derived from tumor retroviruses such as murine leukemia virus, because lentiviral vectors can transduce non-proliferating cells such as hepatocytes. It also has the additional advantage of low immunogenicity.

Expression of the natural or synthetic nucleic acid encoding the CAR is typically achieved by operably linking the nucleic acid encoding the CAR polypeptide or portion thereof to a promoter, and incorporating the construct into an expression vector. Vectors may be suitable for replication and integration in eukaryotes. Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters for regulating expression of the desired nucleic acid sequence. Methods for gene delivery are known in the art. See, for example, U.S. Pat. nos. 5,399,346, 5,580,859, 5,589,466, which are incorporated herein by reference in their entirety. In some embodiments, the vector is a gene therapy vector. The nucleic acid sequences may be cloned into various types of vectors, such as plasmids, phagemids, phage derivatives, animal viruses and cosmids. Vectors of particular interest include expression vectors and replication vectors. The expression vector may be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al (2001,Molecular Cloning:ALaboratory Manual: molecular cloning: A laboratory Manual ], cold Spring Harbor Laboratory, new York [ Cold spring harbor laboratory, new York ]), and other virology and molecular biology handbooks. Viruses that may be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses (AAV), herpesviruses, and lentiviruses.

In general, suitable vectors contain an origin of replication that is functional in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selectable markers (see, e.g., WO 01/96584, WO 01/29058, and U.S. Pat. No. 6,326,193). A variety of virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene may be inserted into a vector and packaged into retroviral particles using techniques known in the art.

The recombinant virus may then be isolated and delivered to cells of the subject in vivo or ex vivo. Many retroviral systems are known in the art. In some embodiments, an adenovirus vector is used. Many adenoviral vectors are known in the art. In one embodiment, lentiviral vectors are used. Additional promoter elements (e.g., enhancers) regulate the frequency of transcription initiation. One example of a suitable promoter is the immediate early Cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence to which it is operably linked. Other examples of suitable promoters include the elongation growth factor-lα (EF-1α), phosphoglycerate kinase 1 (PGK), or fragments thereof that retain the ability to drive gene expression. However, other constitutive promoter sequences may also be used, including but not limited to simian virus 40 (SV 40) early promoter, mouse Mammary Tumor Virus (MMTV), human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, moMuLV promoter, avian leukemia virus promoter, epstein-Barr virus (Epstein-Barr virus) immediate early promoter, rous sarcoma virus (Rous sarcoma virus) promoter, and human gene promoters such as but not limited to actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Inducible promoters may also be used. The use of an inducible promoter provides a molecular switch that can switch on expression of a polynucleotide sequence to which it is operably linked when such expression is desired or switch off expression when it is not desired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

To assess expression of the CAR polypeptide or portion thereof, the expression vector to be introduced into the immune cell may also contain a selectable marker gene or a reporter gene or both to facilitate identification and selection of the expressing cells from a population of cells that are attempted to be transfected or infected by the viral vector. In other aspects, the selectable marker may be performed on a single piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes, such as neo and the like. The reporter gene is used to identify potentially transfected immune cells and evaluate the function of the regulatory sequences. Suitable reporter genes may include genes encoding luciferases, beta-galactosidases, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., ui-Tei et al, 2000FEBS Letters [ European society of Biochemical Association ] 479:79-82).

The engineered immune cells may further comprise a cetuximab epitope or a rituximab epitope as a safety switch, allowing the engineered immune cells to be killed by administration of cetuximab or rituximab when necessary. See Wang et al 2011, blood [ hematology ]118:1255-63; and Sommer et al 2019 mol Ther [ molecular therapy ]27 (6): 1126-1138.

Methods for producing cells comprising vectors and/or exogenous nucleic acids are well known in the art. See, e.g., sambrook et al ((2001,Molecular Cloning:A Laboratory Manual: molecular cloning: A laboratory Manual), cold Spring Harbor Laboratory, new York [ Cold spring harbor laboratory, N.Y.).

Type of immune cells to be engineered

Immune cells that can be engineered to include one or more polynucleotides that promote sano delivery and/or polynucleotides encoding Chimeric Antigen Receptors (CARs) that include a signaling domain that triggers cell turnover (e.g., an intracellular signaling domain) and a targeting domain that directs the engineered immune cells to target cells (e.g., an antigen binding domain) include, but are not limited to, T lymphocytes (T cells), macrophages, natural Killer (NK) cells, and dendritic cells.

T lymphocyte (T cell)

In some embodiments, the engineered immune cell is a T lymphocyte (T cell). T cells mediate a wide range of immune functions including assisting B cells in the ability to develop into antibody-producing cells, enhancing the ability of monocytes/macrophages to act microbiocidally, suppressing certain types of immune responses, directly killing target cells, and mobilizing inflammatory responses. (Paul, W.E. "Chapter 1:The immune system:an introduction [ Chapter 1: immune System: introduction) ]", fundamental Immunology,4th Edition [ basic immunology,4th Edition ]]Paul, W.E. editions, lippicott-Raven Publishers, philadelphia [ Lippicott-Raven Press, philadelphia)], (1999)). T cells are divided according to their expressed cell surface receptorsTwo different categories. Most T cells express T Cell Receptors (TCRs) consisting of alpha and beta chains. A panel of T cells express receptors consisting of gamma and delta chains. There are two sub-lineages of alpha/beta T cells: those cells expressing the co-receptor molecule CD4 (CD 4 ⁺ T cells); and those expressing CD8 (CD 8) ⁺ T cells). These cells differ in the way they recognize antigens and their effector and regulatory functions. In some embodiments, the T cells engineered to comprise one or more heterologous polynucleotides that promote sanoχ transfer are α/β T cells. In some embodiments, the T cells engineered to comprise one or more heterologous polynucleotides that promote sanoχ transfer are gamma/delta T cells.

CD4 ⁺ T cells are the primary regulatory cells of the immune system. Their regulatory function depends on the expression of their cell surface molecules (e.g., CD40 ligand, whose expression is induced when T cells are activated), and on the various cytokines they secrete upon activation. T cells also mediate important effector functions, some of which are determined by the pattern of cytokines they secrete. Cytokines can be directly toxic to target cells and can mobilize potent inflammatory mechanisms. Furthermore, T cells, in particular CD8 ⁺ T cells, which can develop into Cytotoxic T Lymphocytes (CTLs), which can effectively lyse target cells expressing the antigen recognized by the CTLs (Paul, W.E. "Chapter 1:The immune system:an introduction [ Chapter 1: immune system: introduction]", fundamental Immunology,4th Edition [ basic immunology,4th Edition ]]Paul, W.E. editions, lippicott-Raven Publishers, philadelphia [ Lippicott-Raven Press, philadelphia)], (1999)). T cells can also be classified according to their function as helper T cells; t cells involved in inducing cellular immunity; regulatory T (Treg) cells; and cytotoxic T cells.

T cells may be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. T cells may be obtained from a unit of blood taken from a subject using various techniques known to the skilled artisan, such as ficoll (tm) isolation. T cells can also be collected by apheresis, a process in which whole blood is removed from an individual, separated into selected components, and the remainder returned to the circulation. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In one aspect, cells collected by apheresis can be washed to remove plasma fractions, and optionally, the cells are placed in an appropriate buffer or medium for subsequent processing steps. In one embodiment, the cells are washed with Phosphate Buffered Saline (PBS). Methods for isolating T cells from blood samples are known in the art and are described, for example, in U.S. patent nos. 8,911,993 and 10,273,300, each of which is incorporated herein by reference in its entirety. In addition, various T cell lines available in the art may be used. Before or after genetic modification of T cells (e.g., to express a desired CAR and/or polynucleotide that promotes sanoχ transfer), T cells can generally be activated and expanded using methods described in the following documents: for example, U.S. patent nos. 6,352,694, 6,534,055, 6,905,680, 6,692,964, 5,858,358, 6,887,466, 6,905,681, 7,144,575, 7,067,318, 7,172,869, 7,232,566, 7,175,843, 5,883,223, 6,905,874, 6,797,514, 6,867,041 and U.S. patent application publication No. 20060121005. In some embodiments of the methods of the present disclosure, the T cells are autologous to the subject to be treated. In some embodiments, the T cells are allogeneic to the subject to be treated.

Methods of engineering T cells to express Chimeric Antigen Receptors (CARs) are described herein and in the art, for example, in U.S. patent No. 8,911,993 and U.S. patent No. 10,273,300, each of which is incorporated herein by reference in its entirety.

Macrophages with a function of promoting the growth of human body

In some embodiments, the engineered immune cell is a macrophage. Macrophages engulf and digest substances such as cell debris, foreign substances, microorganisms, and cancer cells in a process called phagocytosis. In addition to phagocytosis, macrophages play a critical role in nonspecific defenses (innate immunity) and also help initiate specific defenses mechanisms (adaptive immunity) by recruiting other immune cells (e.g., lymphocytes). For example, macrophages are important as antigen presenters for T cells.

Macrophages useful in the compositions and methods described herein can be prepared, for example, from proliferative, conditionally-stunted primary macrophage progenitor cells. Non-transformed self-renewing progenitor cells are established by overexpressing the transcription factor Hoxb8 in myeloid progenitor cells in medium supplemented with GM-CSF or Flt 3L. Hoxb8 activity results in a block of progenitor cell differentiation. This results in a rapidly proliferating clonable cell. Removal of Hoxb8 activity allows the progenitor cells to resume differentiation and produce differentiated macrophages. See Lee et al, 2016,J Control Release [ journal of controlled release ] 10, 28, 2016; 240:527-540. In some embodiments of the methods of the present disclosure, the macrophage is autologous to the subject to be treated. In some embodiments, the macrophage is allogeneic to the subject to be treated.

Methods of engineering macrophages to express Chimeric Antigen Receptors (CARs) are described herein and in the art, e.g., klichinsky et al, 2020,Nat Biotechnol [ Nature Biotechnology ]]. For example, CD34 from human umbilical cord blood ⁺ Hematopoietic stem cells/precursor cells (HS/PC) can be transduced and grown in a clonogenic assay. Heterologous polynucleotides that facilitate sanoχ transfer may be expressed under the control of the hTIE2 promoter. The TIE2 promoter is expressed upon macrophage differentiation at the tumor site. See Escobar et al, 2014,Sci Transl Med [ science conversion medicine ]]6,217a3. TIE2 has a unique extracellular region that contains two immunoglobulin-like domains, three Epidermal Growth Factor (EGF) -like domains, and three fibronectin type III repeats. It is widely expressed in human tumors and plays an important role in the progression of many cancers by stimulating the secretion of Matrix Metalloproteinases (MMPs) and cytokines.

In some embodiments, the engineered macrophage expresses a chimeric antigen receptor that comprises the intracellular domain of a CD147 molecule. CD147 is a member of the immunoglobulin superfamily in humans and is widely expressed in human tumors and plays an important role in the progression of many cancers by stimulating the secretion of Matrix Metalloproteinases (MMPs) and cytokines. CD147 is essential for extracellular matrix (ECM) remodeling by the expression of MMPs, which are responsible for the degradation of ECM. Degradation of ECM improves access to target cells (e.g., tumor cells). For example, degradation of ECM can improve tumor infiltration of immune cells. See Caruana et al, 2015,Nature Medicine [ Nature medical ] for 5 months; 21 (5):524-529. Heterologous polynucleotides that promote sanoχ transfer may be expressed under the control of a promoter activated by CD 147. Examples of promoters activated by CD147 include, but are not limited to, NF-. Kappa.B promoters, AP1 binding and cyclic AMP response element binding protein promoters, and activating transcription factor-2 promoters. See Xiong et al, 2014,Int J Mol Sci [ J.International molecular science ] for 10 months; 15 (10):17411-17441. In some embodiments, the engineered macrophage expresses a Chimeric Antigen Receptor (CAR) that is activated upon recognition of tumor antigen HER2 to trigger internal signaling of CD147 and increase expression of MMP. For example, in some embodiments, the CAR comprises a single chain antibody fragment that targets human HER 2. In particular embodiments, the CAR comprises a single chain antibody fragment that targets human HER2, a hinge region of mouse IghG1, and a transmembrane region and an intracellular region of CD147 (e.g., a mouse CD147 molecule). See Zhang et al, 2019,British Journal of Cancer, J.England cancer, volume 121, pages 837-845.

Natural Killer (NK) cells

In some embodiments, the engineered immune cell is a Natural Killer (NK) cell. NK cells are cytotoxic lymphocytes that lyse certain tumor cells and virus-infected cells without any prior stimulation or immunization. NK cells can be isolated from peripheral blood or can be derived in vitro from cord blood, bone marrow, human embryonic stem cells and induced pluripotent stem cell production. NK cell lines (such as NK-92, NKL and YTS) can be stably transfected to express any of the chimeric antigen receptor constructs described herein. In some embodiments, the NK cells comprise a heterologous signal transduction domain. In some embodiments, the heterologous signal transduction domain comprises a YINM domain from the cytoplasmic domain of DAP10, and/or a tyrosine-based motif TIYXX (V/I) from the cytoplasmic domain of CD 244. See Lanier,2008, nat Immunol 9 (5): 495-502. In some embodiments, the NK cells comprise a heterologous targeting domain, such as an antigen binding domain. In some embodiments, the antigen binding domain recognizes bissialoganglioside GD2. For example, in some embodiments, the antigen binding domain consists of or comprises an anti-GD 2 ch14.18 single chain Fv antibody fusion protein. In some embodiments, NK cells may express a chimeric antigen receptor. In some embodiments, the chimeric antigen receptor comprises an antigen binding domain that recognizes bissialoganglioside GD2, e.g., comprises an antigen binding domain that is an anti-GD 2 ch14.18 single chain Fv antibody fusion protein or consists of an anti-GD 2 ch14.18 single chain Fv antibody fusion protein. The chimeric antigen receptor can further comprise a CD3 chain as a signaling moiety. See ess et al 2012, j.cell.mol.med. [ journal of cell and molecular medicine ]16:569-581.

Methods of engineering NK cells to express Chimeric Antigen Receptors (CARs) are described herein and in the art, for example, in us patent No. 10,273,300. In some embodiments of the methods of the present disclosure, the NK cells are autologous to the subject to be treated. In some embodiments, the NK cells are allogeneic to the subject to be treated.

Dendritic cells

In some embodiments, the engineered immune cell is a dendritic cell. Dendritic Cells (DCs) play a key role in stimulating immune responses against pathogens and maintaining immune homeostasis against innocuous antigens. DCs represent a heterogeneous group of specialized antigen sensing and Antigen Presenting Cells (APCs) that are essential for inducing and modulating immune responses. In peripheral blood, human DCs are characterized by the absence of T cell markers (CD 3, CD4, CD 8), B cell markers (CD 19, CD 20) and monocyte markers (CD 14, CD 16), but high expression of HLA-DR and other DC lineage markers (e.g., CD1a, CD1 c). See Murphy et al, janeway's immunobiology.8th ed. [ Zhan Wei immunobiology, 8th edition ] Garland Science [ Galand Science Press ]; new York, NY, USA [ New York, U.S. No.: 2012, page 868. Methods for preparing and engineering dendritic cells are known in the art and are described in the following documents: for example, osada et al, 2015,J Immunotherapy [ J.Immunotherapy ] for 5 months; 38 (4):155-64.

Target cells for engineered immune cells

The engineered immune cells of the invention comprising one or more polynucleotides that promote sano delivery and/or polynucleotides encoding Chimeric Antigen Receptors (CARs) comprising a signaling domain that triggers cell turnover (e.g., an intracellular signaling domain) and a targeting domain (e.g., an antigen binding domain) that directs the engineered immune cells to the target cells can induce a biological response in a range of different target cells.

Types of target cells include, but are not limited to, cancer cells, immune cells, endothelial cells, and fibroblasts. In some embodiments, the saenox transfer of the engineered immune cells induces or increases the immune activity of endogenous immune cells in the subject, thereby promoting an immunostimulatory response in the subject. For example, in some embodiments, the sanoχ transfer of an engineered immune cell can alter the phenotype of an endogenous immune cell (such as a tumor-associated macrophage) and make it more inflammatory. Other biological responses that can be modulated in target cells by engineered immune cells include, for example, promotion of cancer cell growth and angiogenesis. For example, in some embodiments, the sanoχ transfer of the engineered immune cells can alter the phenotype of endogenous cancer-associated fibroblasts to deviate from the cancer-promoting phenotype. In some embodiments, the sanoχ delivery of the engineered immune cells can inhibit angiogenesis of endogenous endothelial cells.

The biological response induced by the engineered immune cells in the target cells may also be to promote the delivery of sano by the target cells. For example, engineering immune cells to produce cell turnover factors can in turn induce cell turnover in target cells, thereby promoting saenox transfer in target cells. The engineered immune cells may have more than one type of target cell. For example, in some embodiments, the engineered immune cells increase the immune activity of endogenous immune cells and also promote sanoχ transfer of another type of target cell (such as a cancer cell). Promoting sanoχ transfer of cancer cells may induce the cancer cells to produce additional cell turnover factors that increase the immune activity of endogenous immune cells, thereby further amplifying the immune stimulatory response of the subject. Cell turnover factors that can promote the sano delivery of target cells include, but are not limited to, cytokines (e.g., inflammatory cytokines such as IL6 and IL 1), immunomodulatory proteins (e.g., IFN), growth factors (e.g., FGF VEGF), chemokines, ATP, histones, nucleic acids (e.g., DNA, RNA), phosphatidylserine, heat Shock Proteins (HSP), high mobility group box protein 1 (HMGB 1), and calreticulin.

Engineered immune cells can promote sanoχ transfer in target cells (e.g., cancer cells) by altering the type of cell turnover that the target cells undergo. For example, in some embodiments, engineering immune cells to produce a cell turnover factor can change a cell turnover pathway in a target cell from a non-immunostimulatory cell turnover pathway to an immunostimulatory cell turnover pathway, such as necrotic apoptosis, extrinsic apoptosis, iron death, cell scorch, and combinations thereof.

A range of different cell turnover factors produced by engineered immune cells can promote the delivery of sano to target cells, for example, by inducing immunostimulatory cell turnover pathways in the target cells. For example, in some embodiments, the engineered immune cells can produce a granzyme (e.g., granzyme a) that promotes sanoχ transfer in the target cell. Granzymes are a family of serine proteases that can be delivered to target cells through perforin-mediated pores. Granzyme a from cytotoxic lymphocytes has been shown to cleave GSDM-B to trigger apoptosis in target cancer cells. See Zhou et al 2020, science 368 (6494). Thus, delivery of granzyme a produced by engineered immune cells to target cancer cells can promote sanoχ transfer in cancer cells by inducing apoptosis in the cell coke in the cancer cells. In some embodiments, the cell turnover factor produced by the engineered immune cell is a cytokine that induces an immunostimulatory cell turnover pathway in the target cell to facilitate sanopassing by the target cell. Other cell turnover factors include FasL or other TNF family members that engage partners on cancer cells and induce cell turnover in cancer cells, thereby promoting sanoχ transfer.

Any of the cancer cells described herein may be suitable as target cells for an engineered immune cell. In some embodiments, the target cell is a metastatic cancer cell. In some embodiments, the target cell is a cell in a Tumor Microenvironment (TME), such as a tumor-associated macrophage (TAM), a cancer-associated fibroblast (CAF), or a tumor-associated endothelial cell.

In some embodiments, the target cell is an immune cell selected from the group consisting of a mast cell, a Natural Killer (NK) cell, a monocyte, a macrophage, a dendritic cell, a lymphocyte (e.g., a B cell and a T cell), and any other immune cell described herein.

The target cells are sufficiently close to the engineered immune cells to be contacted with the cell turnover factors produced by the engineered immune cells.

VII method for promoting Sano delivery

The engineered immune cells of the invention are useful for promoting saenox delivery in a subject. For example, in certain aspects, the invention relates to a method of promoting saenox transfer in a subject, the method comprising administering an engineered immune cell described herein in an amount and for a duration sufficient to promote saenox transfer in the subject. Expression of a polynucleotide that promotes sano delivery in an engineered immune cell induces the engineered immune cell to produce cell turnover factors that are actively released by the immune cell or exposed during immune cell turnover (e.g., death). These factors signal that the responding cell (e.g., immune cell) experiences a biological response (e.g., an increase in immune activity).

In some embodiments, the engineered immune cells can further promote sanoχ transfer to target cells, such as cancer cells. For example, exposing the target cell to a cell turnover factor produced by the engineered immune cell can in turn initiate production of the cell turnover factor in the target cell, thereby promoting sanoχ transfer to the target cell. Thus, in certain aspects, the disclosure relates to a method of promoting saenox transfer to a target cell, the method comprising contacting the target cell or tissue comprising the target cell with an engineered immune cell described herein in an amount and for a duration sufficient to promote saenox transfer to the target cell.

Methods of increasing immune activity

In one aspect, the engineered immune cells of the invention can be used to increase immune activity in a subject (e.g., a subject who would benefit from increased immune activity). In certain aspects, the disclosure relates to a method of promoting an immune response in a subject in need thereof, the method comprising administering to the subject an engineered immune cell described herein in an amount and for a duration sufficient to promote saenox transfer of the immune cell, thereby promoting the immune response in the subject. For example, factors produced by an engineered immune cell when expressing one or more polynucleotides that promote sanoχ transfer may induce an immunostimulatory response (e.g., a pro-inflammatory response) in a responsive cell (e.g., an immune cell). In one embodiment, the immune response is an anti-cancer response.

According to the methods of the present disclosure, immune activity may be modulated by engineering the interaction of immune cells, including, for example, any one or more of mast cells, natural Killer (NK) cells, basophils, neutrophils, monocytes, macrophages, dendritic cells, eosinophils, lymphocytes (e.g., B-lymphocytes (B-cells)) and T-lymphocytes (T-cells), with a wide range of immune cells endogenous to a subject.

Immune cell type

Mast cells are a granulocyte that contains granules rich in histamine and heparin (an anticoagulant). Upon activation, the mast cells release the inflammatory compounds from the granules into the local microenvironment. Mast cells play a role in allergies, allergic reactions, wound healing, angiogenesis, immune tolerance, pathogen defense and blood brain barrier function.

Natural Killer (NK) cells are cytotoxic lymphocytes that lyse certain tumor cells and virus-infected cells without any prior stimulation or immunization. NK cells are also effective producers of various cytokines, such as IFN-gamma (IFNgamma), TNF-alpha (TNF alpha), GM-CSF, and IL-3. Thus, NK cells are also thought to function as regulatory cells in the immune system, affecting other cells and responses. In humans, NK cells are broadly defined as CD56+CD3-lymphocytes. The cytotoxic activity of NK cells is tightly controlled by a balance between cell surface receptor activation and inhibition signals. The major group of receptors that inhibit NK cell activation is the inhibitory killer immunoglobulin-like receptor (KIR). Upon recognition of self MHC class I molecules on target cells, these receptors will deliver inhibitory signals that cease activating the signaling cascade, thereby protecting cells with normal MHC class I expression from NK cell lysis. Activating receptors include the Natural Cytotoxic Receptors (NCR) and NKG2D, which push equilibrium towards cytolysis by binding to different ligands on the target cell surface. Thus, NK cell recognition by target cells is tightly controlled by processes involving integration of multiple activating receptors and signals inhibiting receptor delivery.

Monocytes are bone marrow derived mononuclear phagocytes that circulate in the blood for several hours/day before being recruited to the tissue. See Wacleche et al 2018, viruses [ virus ] (10) 2:65. Expression of various chemokine receptors and cell adhesion molecules on their surfaces enables them to enter the blood from the bone marrow and subsequently be recruited from the blood into the tissue. Monocytes belong to the innate arms of the immune system, providing a response to viral, bacterial, fungal or parasitic infections. Their functions include killing pathogens by phagocytosis, production of Reactive Oxygen Species (ROS), nitric Oxide (NO), myeloperoxidase and inflammatory cytokines. Under certain conditions, monocytes can stimulate or inhibit T cell responses in cancer, infectious diseases and autoimmune diseases. They are also involved in tissue repair and neovascularization.

Macrophages engulf and digest substances such as cell debris, foreign substances, microorganisms, and cancer cells in a process called phagocytosis. In addition to phagocytosis, macrophages play a critical role in nonspecific defenses (innate immunity) and also help initiate specific defenses mechanisms (adaptive immunity) by recruiting other immune cells (e.g., lymphocytes). For example, macrophages are important as antigen presenters for T cells. In addition to increasing inflammation and stimulating the immune system, macrophages also play an important anti-inflammatory role and can reduce the immune response by releasing cytokines. Macrophages that promote inflammation are called M1 macrophages, while macrophages that reduce inflammation and promote tissue repair are called M2 macrophages. In some embodiments, the macrophage is a tumor-associated macrophage.

Dendritic Cells (DCs) play a key role in stimulating immune responses against pathogens and maintaining immune homeostasis against innocuous antigens. DCs represent a heterogeneous group of specialized antigen sensing and Antigen Presenting Cells (APCs) that are essential for inducing and modulating immune responses. In peripheral blood, human DCs are characterized by the absence of T cell markers (CD 3, CD4, CD 8), B cell markers (CD 19, CD 20) and monocyte markers (CD 14, CD 16), but high expression of HLA-DR and other DC lineage markers (e.g., CD1a, CD1 c). See Murphy et al, janeway's immunobiology.8th ed. [ Zhan Wei immunobiology, 8th edition]Garland Science [ Galland Science Press ]]The method comprises the steps of carrying out a first treatment on the surface of the New York, NY, USA [ New York, U.S. ] and the like]2012. Page 868. In some embodiments, the dendritic cell is CD103 ⁺ Dendritic cells.

The term "lymphocytes" refers to small white blood cells formed in whole-body lymphoid tissues and in normal adults, accounting for about 22% -28% of the total number of white blood cells in circulating blood, and play an important role in protecting the body against disease. Individual lymphocytes are specialized in that they are dedicated to responding to a limited set of structurally related antigens by recombination of their genetic material (e.g., production of T cell receptors and B cell receptors). This behavior occurs prior to the first contact of the immune system with a given antigen, expressed by the presence of specific receptors for determinants (epitopes) on the antigen on the lymphocyte surface membrane. Each lymphocyte has a unique receptor population, all receptors of which have the same binding site. One group or clone of lymphocytes differs from another clone in the structure of the binding region of its receptor and thus also differs in the epitope that it can recognize. Lymphocytes differ from each other not only in the specificity of the receptor, but also in their function. (Paul, W.E. "," Chapter 1:The immune system:an introduction [ Chapter 1: immune system: introduction ] ", fundamental Immunology,4th Edition [ basic immunology,4th Edition ], paul, W.E. editions, lippicott-Raven Publishers, philadelphia [ Lippicott-Raven Press, philadelphia ], (1999), page 102).

Lymphocytes include B lymphocytes (B cells) and T lymphocytes (T cells) that are precursors of antibody secreting cells.

B lymphocyte (B cell)

B lymphocytes are derived from hematopoietic cells of the bone marrow. Mature B cells can be activated with antigens expressing epitopes recognized by their cell surfaces. The activation process may be direct (which depends on the cross-linking of the membrane Ig molecules by the antigen (cross-linked dependent B cell activation) or indirect (through interactions with helper T cells in a process called homology-assisted). Under many physiological conditions, receptor cross-linking stimulation and homologs help synergistically produce a more potent B-cell response (Paul, W.E. "Chapter 1:The immune system:an introduction [ Chapter 1: immune System: instruction ]", fundamental Immunology,4th Edition [ basic immunology,4th Edition ], paul, W.E. editions, lippicott-Raven Publishers, philadelphia [ Lippicott-Raven Press, philadelphia ], (1999)).

Cross-linking dependent B cell activation requires multiple copies of an epitope that is expressed by the antigen that is complementary to the binding site of the cell surface receptor, as each B cell expresses an Ig molecule having the same variable region. Other antigens with repetitive epitopes, such as capsular polysaccharides or viral envelope proteins of microorganisms, may fulfill this requirement. Cross-linked dependent B-cell activation is the primary protective immune response against these microorganisms (Paul, W.E. "Chapter 1:The immune system:an introduction [ Chapter 1: immune System: introduction ]", fundamental Immunology,4th Edition [ basic immunology,4th Edition ], paul, W.E. editions, lippicott-Raven Publishers, philadelphia [ Lippicott-Raven publishing, philadelphia ], (1999)).

Homology-assisted B-cell responses to antigens that fail to crosslink receptors while providing co-cell responses when B-cells are stimulated by weak crosslinking eventsStimulation signals rescue B cells from inactivation. Homology assistance relies on the binding of B-cell membrane immunoglobulins (Ig) to antigens, endocytosis of antigens, and their disruption into peptides at the endosomal/lysosomal compartments of the cell. Some of the resulting peptides are loaded into grooves of a specialized set of cell surface proteins called class II Major Histocompatibility Complex (MHC) molecules. The resulting class II/peptide complex is expressed on the cell surface and serves as a set of T cells (called CD4 ⁺ T cells) are described. CD4 ⁺ T cells have receptors on their surface specific for class II/peptide complexes of B cells. B cell activation not only relies on the binding of T cells through their T Cell Receptor (TCR), but this interaction also allows the activating ligand (CD 40 ligand) on the T cells to bind to their receptor (CD 40) on the B cells, thereby signaling B cell activation. In addition, T helper cells secrete several cytokines that regulate the growth and differentiation of stimulated B cells by binding to cytokine receptors on B cells (Paul, W.E. "Chapter1: the immune system:an interaction [ Chapter1: immune system: introduction) ]", fundamental Immunology,4th Edition [ basic immunology,4th Edition ]]Paul, W.E. editions, lippicott-Raven Publishers, philadelphia [ Lippicott-Raven Press, philadelphia)],(1999))。

In a homologous helper process for antibody production, the CD40 ligand is in activated CD4 ⁺ Transient expression on T helper cells and it binds to CD40 on antigen specific B cells, thereby transducing the second costimulatory signal. The second co-stimulatory signal is necessary for the growth and differentiation of B cells and for the production of memory B cells (by preventing apoptosis of germinal center B cells that have encountered the antigen). Over-expression of CD40 ligand in B and T cells is associated with the production of pathogenic autoantibodies in human SLE patients (Desai-Mehta, A. Et al, J.Clin.Invest. [ J.clinical study journal)]Vol.97 (9), 2063-2073, (1996)).

T lymphocyte (T cell)

T lymphocytes derived from hematopoietic tissue precursors differentiate in the thymus and are then seeded into peripheral lymphoid tissues and lymphocyte recirculation pools. T lymphocytes or T cells mediate a wide range of immunological functions. These include the ability to assist B cells in developing into antibody-producing cells, to enhance the ability of monocytes/macrophages to act microbiocidally, to suppress certain types of immune responses, to directly kill target cells, and to mobilize inflammatory responses. These effects depend on T cell expression of specific cell surface molecules and secretion of cytokines (Paul, W.E. "Chapter 1:The immune system:an introduction [ Chapter 1: immune System: introduction ]", fundamental Immunology,4th Edition [ basic immunology,4th Edition ], paul, W.E. editions, lippicott-Raven Publishers, philadelphia [ Lippicott-Raven publishing, philadelphia ], (1999)).

T cells differ from B cells in their antigen recognition mechanisms. Immunoglobulins (B-cell receptor) bind to unique epitopes on the surface of soluble molecules or particles. B cell receptors are directed against epitopes expressed on the surface of natural molecules. When antibodies and B cell receptors are advanced to bind and protect against microorganisms in the extracellular fluid, T cells recognize antigens on the surface of other cells and mediate their function by interacting with and altering the behavior of these Antigen Presenting Cells (APCs). There are three major APC types in peripheral lymphoid organs that can activate T cells: dendritic cells, macrophages and B cells. Of these, the most potent ones are dendritic cells, whose only function is to present foreign antigens to T cells. Immature dendritic cells are located in tissues throughout the body, including the skin, gut and respiratory tract. When they encounter invading microorganisms at these sites, they engulf pathogens and their products and carry them via lymph to regional lymph nodes or to gut-associated lymphoid organs. Encounter with pathogens induces dendritic cells to mature from antigen capturing cells into APCs that activate T cells. APCs display three types of protein molecules on their surface that play a role in activating T cells into effector cells: (1) MHC proteins which present foreign antigens to T cell receptors; (2) A costimulatory protein that binds to a T cell surface complementary receptor; and (3) cell-cell adhesion molecules that are capable of binding T cells to APCs for a sufficient period of time to become activated ("Chapter 24: the adaptive immune system [ Chapter24: adaptive immune system ]" Molecular Biology of the Cell [ molecular biology of cells ], alberts, B.et al, garland Science, NY [ Galand Science, new York ], (2002)).

T cells are classified into two different classes according to the cell surface receptors they express. Most T cells express T Cell Receptors (TCRs) consisting of alpha and beta chains. A panel of T cells express receptors consisting of gamma and delta chains. There are two sub-lineages of alpha/beta T cells: those cells expressing the co-receptor molecule CD4 (CD 4 ⁺ T cells); and those expressing CD8 (CD 8) ⁺ T cells). These cells differ in the way they recognize antigens and their effector and regulatory functions. CD4 ⁺ T cells are key cells of the adaptive immune system that use T cell antigen receptors to recognize peptides that are produced in endosomes or phagosomes and displayed on the surface of host cells that bind to the major histocompatibility complex molecule. Their regulatory function depends on the expression of their cell surface molecules (such as CD40 ligands whose expression is induced when T cells are activated), and the large number of cytokines they secrete when activated.

T cells also mediate important effector functions, some of which are determined by the pattern of cytokines they secrete. Cytokines can be directly toxic to target cells and can mobilize potent inflammatory mechanisms.

Furthermore, T cells, in particular CD8 ⁺ T cells, which can develop into Cytotoxic T Lymphocytes (CTLs), which can effectively lyse target cells expressing the antigen recognized by the CTLs (Paul, W.E. "Chapter 1:The immune system:an introduction [ Chapter 1: immune system: introduction]", fundamental Immunology,4th Edition [ basic immunology,4th Edition ]]Paul, W.E. editions, lippicott-Raven Publishers, philadelphia [ Lippicott-Raven Press, philadelphia)],(1999))。

T Cell Receptors (TCRs) recognize a complex from the group consisting of: peptides of proteolytic origin of antigens binding to the specialised grooves of class II or class I MHC proteins. CD4 ⁺ T cells recognize only peptide/class II complexes, while CD8 ⁺ T cell recognition peptide/class I complex (Paul, W.E. "Chapter 1:The immune system:a)n introduction [ chapter 1: the immune system: introduction to the invention]", fundamental Immunology,4th Edition [ basic immunology,4th Edition ]]Paul, W.E. editions, lippicott-Raven Publishers, philadelphia [ Lippicott-Raven Press, philadelphia)],(1999))。

Ligands for TCRs (i.e., peptide/MHC protein complexes) are produced within APCs. Typically, class II MHC molecules bind peptides derived from proteins that are taken up by APCs through an endocytic process. These peptide-loaded class II molecules are then expressed on the cell surface where they can be bound by CD4 ⁺ T cells (which have TCRs capable of recognizing the expressed cell surface complex) bind. Thus, CD4 ⁺ T cells react exclusively with antigens derived from extracellular sources (Paul, W.E. "Chapter1: the immune system:an interaction [ Chapter 1:immune system: introduction)]", fundamental Immunology,4th Edition [ basic immunology,4th Edition ]]Paul, W.E. editions, lippicott-Raven Publishers, philadelphia [ Lippicott-Raven Press, philadelphia)],(1999))。

In contrast, class I MHC molecules are predominantly loaded with peptides derived from internal synthetic proteins (e.g. viral proteins). These peptides are produced from cytosolic proteins by proteolysis of the proteasome and transported into the rough endoplasmic reticulum. Such peptides, typically consisting of nine amino acids in length, bind to class I MHC molecules and are brought to the cell surface where they can be expressed by CD8, a suitable receptor ⁺ T cell recognition. This allows for a T cell system (in particular CD8 ⁺ T cells) are able to detect cells expressing proteins that are different from or produced in much greater amounts than those of cells of the rest of the organism (e.g., viral antigens) or mutant antigens (such as active oncogene products), even though these proteins are neither expressed nor secreted on the cell surface in their intact form (Paul, w.e. "Chapter1:The immune system:an introduction [ Chapter1: the immune system: introduction to the invention ]", fundamental Immunology,4th Edition [ basic immunology,4th Edition ]]Paul, W.E. editions, lippicott-Raven Publishers, philadelphia [ Lippicott-Raven Press, philadelphia)],(1999))。

T cells can also be classified according to their function as helper T cells; t cells involved in inducing cellular immunity; a suppressor T cell; and cytotoxic T cells.

Helper T cell

Helper T cells are T cells that stimulate B cells to produce an antibody response to proteins and other T cell-dependent antigens. T cell-dependent antigens are immunogens in which unique epitopes are present only once or a limited number of times, so they are unable to cross-link membrane immunoglobulins (Ig) or inefficiency of B cells. B cells bind antigen through their membrane Ig and the complex undergoes endocytosis. In the endosomal and lysosomal compartments, the antigen is fragmented into peptides by proteolytic enzymes, and one or more of the resulting peptides are loaded into MHC class II molecules, which are transported through the vesicle compartment. The resulting peptide/MHC class II complex is then exported to a B cell surface membrane. T cells with peptide/class II molecule complex specific receptors recognize this complex on the surface of B cells. (Paul, W.E., "Chapter 1:The immune system:an introduction [ Chapter 1: immune System: instructions ]", fundamental Immunology,4th Edition [ basic immunology,4th Edition ], paul, W.E. editions, lippicott-Raven Publishers, philadelphia [ Lippicott-Raven Press, philadelphia ], (1999)).

B cell activation depends on both: t cells interact with CD40 on B cells through the binding of their TCR, the T cell CD40 ligand (CD 40L). T cells do not constitutively express CD40L. Instead, the result of the interaction with APCs that express the cognate antigen recognized by the TCR of the T cell and CD80 or CD86 is to induce CD40L expression. CD80/CD86 is normally expressed by activated, rather than resting, B cells, and thus helper interactions involving activated B cells and T cells can lead to efficient antibody production. However, in many cases, the initial induction of CD40L on T cells depends on their recognition of surface antigens by APC (e.g., dendritic cells) that constitutively express CD 80/86. Such activated helper T cells may then effectively interact with and assist B cells. Even though inefficient, crosslinking of membrane Ig on B cells can cooperate with CD40L/CD40 interactions to produce strong B cell activation. Subsequent events in B-cell responses, including proliferation, ig secretion and class switching of expressed Ig classes, depend on or are enhanced by the action of T-cell derived cytokines (Paul, W.E. "Chapter 1:The immune system:an introduction [ Chapter 1: immune System: introduction ]", fundamental Immunology,4th Edition [ basic immunology,4th Edition ], paul, W.E. editions, lippicott-Raven Publishers, philadelphia [ Lippicott-Raven publication, philadelphia ], (1999)).

CD4 ⁺ T cells tend to differentiate into cells that secrete the major cytokines IL-4, IL-5, IL-6, and IL-10 (T _H 2 cells) or differentiation into a major IL-2, IFN-gamma and lymphotoxin (T) _H 1 cells). T (T) _H 2 cells are very effective in aiding B cell development into antibody-producing cells, while T _H 1 cells are then potent inducers of cellular immune responses, involving enhancement of the microbiocidal activity of monocytes and macrophages, and thus increasing the efficiency of lysis of microorganisms in intracellular vesicle compartments. Although having T _H CD4 of the 2 cell phenotype ⁺ T cells (i.e., IL-4, IL-5, IL-6 and IL-10) are potent helper cells, but T _H 1 cells also have the capacity as helper cells (Paul, W.E. "Chapter 1:The immune system:an introduction [ Chapter 1: immune System: introduction)]", fundamental Immunology,4th Edition [ basic immunology,4th Edition ]]Paul, W.E. editions, lippicott-Raven Publishers, philadelphia [ Lippicott-Raven Press, philadelphia)],(1999))。

T cells involved in cellular immune induction

T cells can also function to enhance the ability of monocytes and macrophages to destroy intracellular microorganisms. In particular, interferon-gamma (IFN-gamma) produced by helper T cells enhances several mechanisms by which mononuclear phagocytes destroy intracellular bacteria and parasites, including the production of nitric oxide and the induction of Tumor Necrosis Factor (TNF) production. T (T) _H1 Cells are effective in enhancing microbiocidal action because they produce IFN-gamma. In contrast, by T _H2 The two major cytokines IL-4 and IL-10 produced by cells block these activities (Paul, W.E. "Chapter 1:The immune system:an introduction [ Chapter 1: immune system: introduction)]", fundamental Immunology,4th Edition [ basic immunology,4th Edition ]],Paul,W.EEditing, lippicott-Raven Publishers, philadelphia [ Lippicott-Raven Press, philadelphia)],(1999))。

Regulatory T (Treg) cells

Immune homeostasis is maintained by a controlled balance between initiation and downregulation of the immune response. Mechanism of apoptosis and T cell anergy (a tolerating mechanism in which T cells are inherently functionally inactive following antigen encounter (Schwartz, r.h. "T cell anergy [ T cell anergy ]]", annu.Rev.Immunol" [ review of immunological years ]]Volume 21: 305-334 (2003))) to facilitate down-regulation of immune responses. The third mechanism is through repressive or regulatory CD4 ⁺ T (Treg) cells actively suppress activated T cells (provided in Kronenberg, m. et al, nature]Reviewed in volume 435, 598-604 (2005). CD4 constitutively expressing IL-2 receptor alpha (IL-2 Ralpha) chain ⁺ Treg(CD4 ⁺ CD25 ⁺ ) Are naturally occurring T cell subsets that are anergic and inhibitory (Taams, l.s. Et al, eur.J.immunol. [ journal of European immunology.) ]31:1122-1131 (2001)). Human CD4 ⁺ CD25 ⁺ Tregs, like their murine counterparts, are produced in the thymus and are characterized by the ability to suppress proliferation of responsive T cells, the inability to produce IL-2, and an in vitro anergy phenotype through cell-cell contact dependent mechanisms. Human CD4 based on the expression level of CD25 ⁺ CD25 ⁺ T cells can be classified as repressible (CD 25 ^{High height} ) And non-repressible (CD 25) ^{Low and low} ) And (3) cells. The member FOXP3 of the fork-shaped transcription factor family has been shown in murine and human CD4 ⁺ CD25 ⁺ Treg expression and appears to control CD4 ⁺ CD25 ⁺ Major genes for Treg development (Battaglia, m. et al, j. Immunol. [ journal of immunology ]]Volumes 177:8338-8347, (2006)). Thus, in some embodiments, an increase in immune response may be associated with the activation or proliferation of T cells lacking regulation.

Cytotoxic T lymphocytes

CD8 recognizing peptide from protein produced in target cell ⁺ T cells are cytotoxic in that they cause target cells to lyse. The mechanism of CTL-induced lysis involves the production of perforin by CTLs, which is an insertable targetA molecule in the membrane of a cell that promotes lysis of the cell. Perforin-mediated cleavage is enhanced by granzyme, a series of enzymes produced by activated CTLs. Many active CTLs also express a large number of fas ligands on their surface. Interaction of fas ligands on the CTL surface with fas on the target cell surface initiates apoptosis of the target cells, resulting in death of these cells. CTL mediated lysis appears to be the primary mechanism to destroy virus-infected cells.

Lymphocyte activation

The term "activation" or "lymphocyte activation" refers to the stimulation of lymphocytes by specific antigens, non-specific mitogens or allogeneic cells, resulting in the synthesis of RNA, proteins and DNA and the production of lymphokines; followed by proliferation and differentiation of various effector cells and memory cells. T cell activation is dependent on the interaction of the TCR/CD3 complex with its cognate ligand, which is a peptide that binds in a groove in an MHC class I or class II molecule. Molecular events mobilized by receptor binding are complex. One of the earliest steps appeared to be the activation of tyrosine kinases, resulting in tyrosine phosphorylation of a group of substrates that control several signaling pathways. These include a group of adaptor proteins that link TCRs to the ras pathway, phospholipase cγ1, whose tyrosine phosphorylation increases its catalytic activity and participates in the inositol phospholipid metabolic pathway, leading to an increase in intracellular free calcium concentration and protein kinase C activation, as well as a series of other enzyme activations that control cell growth and differentiation. Complete responsiveness of T cells, in addition to receptor engagement, requires co-stimulatory activity delivered by the helper cell, e.g., engagement of CD28 on the T cells with CD80 and/or CD86 on the APC.

T memory cell

After recognition and eradication of pathogens by an adaptive immune response, most (90% -95%) T cells undergo apoptosis, with the remaining cells forming a pool of memory T cells, known as central memory T Cells (TCM), effector memory T cells (TEM) and resident memory T cells (TRM) (Clark, r.a. "Resident memory T cells in human health and disease [ resident memory T cells in human health and disease ]", sci.Transl.med. [ science of transformation ],7,269rv1, (2015)).

These memory T cells have a long life span compared to standard T cells, with different phenotypes, such as expression of specific surface markers, rapid production of different cytokine profiles, the ability to direct effector cell function, and unique homing distribution patterns. Memory T cells exhibit a rapid response upon re-exposure to their respective antigens, thereby eliminating re-infection by offenders and thereby rapidly restoring balance of the immune system. There is growing evidence that autoimmune memory T cells hamper most attempts to treat or cure autoimmune diseases (Clark, r.a., "Resident memory T cells in human health and disease [ resident memory T cells in human health and disease ]", sci.Transl.med. [ science conversion medicine ], volume 7,269rv1, (2015)).

Increasing immune activity

An engineered immune cell comprising one or more polynucleotides that promote the delivery of a sano described herein and/or a polynucleotide encoding a Chimeric Antigen Receptor (CAR) can increase immune activity in a tissue or subject by increasing the level or activity of any one or more of the immune cells described herein (e.g., macrophages, monocytes, dendritic cells, B cells, T cells, and cd4+, cd8+ or cd3+ cells (e.g., cd4+, cd8+ or cd3+ T cells)) in the tissue or subject. For example, in one embodiment, the engineered immune cells are administered in an amount sufficient to increase one or more of the following in a tissue or subject: macrophage level or activity, monocyte level or activity, dendritic cell level or activity, T cell level or activity, B cell level or activity, and cd4+, cd8+ or cd3+ cell (e.g., cd4+, cd8+ or cd3+ T cells) level or activity.

In some aspects, the disclosure relates to a method of increasing the level or activity of macrophages, monocytes, B cells, T cells and/or dendritic cells in a tissue or subject comprising administering to the tissue or subject an immune cell engineered to comprise one or more polynucleotides that promote sano delivery and/or polynucleotides encoding Chimeric Antigen Receptors (CARs), wherein the immune cell is administered in an amount sufficient to increase the level or activity of macrophages, monocytes, B cells, T cells and/or dendritic cells relative to the tissue or subject not treated with the engineered immune cell. In one embodiment, the subject is in need of increased levels or activity of macrophages, monocytes, dendritic cells, B cells and/or T cells. In one embodiment, the level or activity of macrophages, monocytes, B-cells, T-cells or dendritic cells is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%, or at least 2-fold, 4-fold, 6-fold, 8-fold or 10-fold relative to the tissue or subject not treated with the engineered immune cells.

In some aspects, the disclosure relates to a method of increasing the level or activity of cd4+, cd8+, or cd3+ cells in a tissue or subject, the method comprising administering to the subject immune cells engineered to comprise one or more polynucleotides that promote sano delivery in an amount sufficient to increase the level or activity of cd4+, cd8+, or cd3+ cells relative to the tissue or subject not treated with the engineered immune cells. In one embodiment, the subject is in need of increased levels or activity of cd4+, cd8+ or cd3+ cells. In one embodiment, the level or activity of cd4+, cd8+ or cd3+ cells is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%, or at least 2-fold, 4-fold, 6-fold, 8-fold or 10-fold relative to tissue or subject not treated with engineered immune cells.

The engineered immune cells of the invention can also increase immune activity in a cell, tissue, or subject by increasing the level or activity of a pro-immune cytokine produced by the immune cell. For example, in some embodiments, the engineered immune cells are administered in an amount sufficient to increase the level or activity of a pro-immune cytokine produced by the immune cells in the cell, tissue, or subject. In one embodiment, the pro-immune cytokine is selected from IFN- α, IL-1, IL-12, IL-18, IL-2, IL-15, IL-4, IL-6, TNF- α, IL-17, and GMCSF.

In some aspects, the disclosure relates to a method of inducing a pro-inflammatory transcriptional response in immune cells endogenous to a tissue or subject, e.g., inducing NFkB pathway, interferon IRF signaling, and/or STAT signaling in immune cells of a tissue or subject, the method comprising administering to the tissue or subject an immune cell engineered to comprise one or more polynucleotides that promote sano signaling and/or Chimeric Antigen Receptors (CARs) comprising a signaling domain and/or a targeting domain in an amount sufficient to induce a pro-inflammatory transcriptional response, interferon IRF signaling, and/or STAT signaling in NFkB pathway of the endogenous immune cell.

The engineered immune cells of the invention can also increase immune activity in a cell, tissue, or subject by modulating signaling through intracellular receptors of the nucleic acid, such as an interferon gene stimulating factor (STING). Thus, in some aspects, the disclosure relates to a method of increasing immune activity in a cell, tissue, or subject by modulating signaling through intracellular receptors of a nucleic acid, such as an interferon gene Stimulus (STING), the method comprising administering to the cell, tissue, or subject an immune cell engineered to comprise one or more polynucleotides that promote sanoχ transfer and/or polynucleotides encoding Chimeric Antigen Receptors (CARs) in an amount sufficient to increase immune activity in the cell, tissue, or subject by modulating signaling through intracellular receptors of a nucleic acid, such as an interferon gene Stimulus (STING).

The engineered immune cells of the invention can also increase immune activity in a tissue or subject by inducing or modulating an antibody response. For example, in some embodiments, immune cells engineered to include one or more polynucleotides that promote sano delivery and/or polynucleotides encoding Chimeric Antigen Receptors (CARs) are administered in an amount sufficient to modulate an antibody response in a tissue or subject.

Thus, in some aspects, the disclosure relates to a method of increasing immune activity in a tissue or subject by inducing or modulating an antibody response of an immune cell in the tissue or subject, the method comprising administering to the tissue or subject an immune cell engineered to comprise one or more polynucleotides that promote sano delivery and/or polynucleotides encoding a Chimeric Antigen Receptor (CAR), wherein the immune cell is administered in an amount sufficient to increase immune activity in the tissue or subject relative to the tissue or subject not treated with the engineered immune cell.

In some aspects, the disclosure relates to a method of increasing the level or activity of an pro-immune cytokine in a cell, tissue, or subject, the method comprising administering to the cell, tissue, or subject an immune cell engineered to comprise one or more polynucleotides that promote sano delivery and/or polynucleotides encoding a Chimeric Antigen Receptor (CAR), wherein the immune cell is administered in an amount sufficient to increase the level or activity of the pro-immune cytokine relative to a cell, tissue, or subject not treated with the engineered immune cell. In one embodiment, the pro-immune cytokine is selected from IFN- α, IL-1, IL-12, IL-18, IL-2, IL-15, IL-4, IL-6, TNF- α, IL-17, and GMCSF. In one embodiment, the subject is in need of increased levels or activity of an immunocytokine. In one embodiment, the level or activity of the pro-immune cytokine is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%, or at least 2-fold, 4-fold, 6-fold, 8-fold or 10-fold relative to a cell, tissue or subject not treated with the engineered immune cell.

In some embodiments, the methods disclosed herein further comprise evaluating one or more of the following of the tissue or subject prior to administering the engineered immune cells: macrophage level or activity; level or activity of monocytes; the level or activity of dendritic cells; levels or activities of cd4+ cells, cd8+ cells, or cd3+ cells; level or activity of T cells; b cell level or activity, and level or activity of an immunocytokine.

In one embodiment, the method of the invention further comprises, after administration of the engineered immune cells, evaluating one or more of the following of the tissue or subject: levels or activity of NFkB, IRF or STING; macrophage level or activity; level or activity of monocytes; the level or activity of dendritic cells; levels or activities of cd4+ cells, cd8+ cells, or cd3+ cells; level or activity of T cells; and the level or activity of an immunocytokine.

Measuring the level or activity of NFkB, IRF or STING; macrophage level or activity; level or activity of monocytes; the level or activity of dendritic cells; levels or activities of cd4+ cells, cd8+ cells, or cd3+ cells; level or activity of T cells; and methods of stimulating the level or activity of immune cytokines are known in the art.

For example, protein levels or activity of NFkB, IRF or STING can be measured by suitable techniques known in the art, including ELISA, western blot or in situ hybridization. The level of nucleic acid (e.g., mRNA) encoding NFkB, IRF, or STING can be measured using suitable techniques known in the art, including Polymerase Chain Reaction (PCR) amplification reactions, reverse transcriptase PCR assays, quantitative real-time PCR, single strand conformational polymorphism assays (SSCP), mismatch cleavage detection, heteroduplex assays, northern blot analysis, in situ hybridization, array analysis, deoxyribonucleic acid sequencing, restriction fragment length polymorphism assays, and combinations or sub-combinations thereof.

Methods for measuring macrophage levels and activity are described, for example, in Chitu et al, 2011,Curr Protoc Immunol [ current protocol for immunology ] 14:1-33. The level and activity of monocytes can be measured by flow cytometry, as described, for example, in Henning et al, 2015,Journal of Immunological Methods J immunology methods 423:78-84. The level and activity of dendritic cells can be measured by flow cytometry, as described, for example, in Dixon et al, 2001,Infect Immun [ infection and immunization ]69 (7): 4351-4357. Each of these references is incorporated by reference herein in its entirety.

Proliferation assays based on human cd4+ T cells can be used to assess the level or activity of T cells. For example, cells are labeled with the fluorescent dye 5, 6-carboxyfluorescein diacetate succinimidyl ester (CFSE). Those proliferating cells showed a decrease in CFSE fluorescence intensity, which can be measured directly by flow cytometry. Alternatively, radioactive thymidine incorporation can be used to assess the growth rate of T cells.

In some embodiments, an increase in immune response may be associated with a decrease in activation of regulatory T cells (tregs). Functionally active T regs can be assessed using an in vitro Treg suppression assay. Such assays are described in Collison and Vignali (Methods Mol Biol. [ method molecular biology ]2011;707:21-37, which is incorporated herein by reference in its entirety).

The level or activity of the pro-immune cytokine may be quantified, for example, in cd8+ T cells. In embodiments, the pro-immune cytokine is selected from the group consisting of interferon alpha (IFN-alpha), interleukin-1 (IL-1), IL-12, IL-18, IL-2, IL-15, IL-4, IL-6, tumor necrosis factor alpha (TNF-alpha), IL-17, and granulocyte-macrophage colony stimulating factor (GMCSF). Quantification can be performed using the ELISPOT technique, which detects T cells that secrete a given cytokine (e.g., IFN- α) in response to antigenic stimulation. T cells are cultured with antigen presenting cells in wells that have been coated with, for example, anti-IFN- α antibodies. Secreted IFN- α is captured by the coated antibody and then displayed with a secondary antibody coupled to a chromogenic substrate. Thus, locally secreted cytokine molecules form spots, each spot corresponding to one IFN- α secreting cell. The number of spots allows one to determine the frequency of IFN- α secreting cells in the sample analyzed that are specific for a given antigen. ELISPOT assays are also described for detecting TNF- α, interleukin-4 (IL-4), IL-6, IL-12 and GMCSF.

Methods of treating disorders

The engineered immune cells of the invention comprising a polynucleotide that promotes sanoχ transfer and/or a polynucleotide that encodes a Chimeric Antigen Receptor (CAR) can be used to increase immune activity in a cell or subject. Thus, the engineered immune cells of the invention are useful in the treatment of disorders that may benefit from increased immune activity, such as cancer and infectious diseases and disorders.

A. Cancer of the human body

As provided herein, immune cells engineered to include one or more heterologous polynucleotides that promote sano delivery and/or polynucleotides encoding Chimeric Antigen Receptors (CARs) can promote or induce immune activity of endogenous immune cells (e.g., T cells, B cells, NK cells, etc.), and thus can enhance immune cell function, such as those involved in immunotherapy for treating cancer. Thus, in certain aspects, the disclosure relates to a method of treating cancer in a subject in need thereof, the method comprising administering to the subject an immune cell engineered to comprise one or more heterologous polynucleotides that promote sano delivery and/or polynucleotides encoding Chimeric Antigen Receptors (CARs), thereby treating cancer in the subject.

The ability of cancer cells to prevent the immune system from distinguishing itself from non-itself using a complex series of overlapping mechanisms represents the fundamental mechanism by which cancer evades immune surveillance. One or more mechanisms include disruption of antigen presentation, disruption of regulatory pathways that control T cell activation or inhibition (immune checkpoint modulation), recruitment of cells that contribute to immune suppression (tregs, MDSCs) or release of factors that affect immune activity (IDO, PGE 2). ( See Harris et al, 2013,J Immunotherapy Cancer [ journal of cancer immunotherapy ]1:12; chen et al 2013, immunity [ immunity ]39:1; pardoll et al, 2012,Nature Reviews:Cancer [ natural review: cancer ]12:252; and Shalma et al, 2015, cell [ cells ]161:205, each of which is incorporated herein by reference in its entirety. )

Cancers treated using the methods described herein include, for example, all types of cancers or neoplasms or malignant tumors found in mammals, including, but not limited to: sarcomas, melanomas, epithelial cancers, leukemias and lymphomas.

The term "sarcoma" generally refers to a tumor composed of a substance such as embryonic connective tissue, and is generally composed of closely packed cells embedded in a fibrous or homogeneous substance. Examples of sarcomas that can be treated with the methods of the invention include, for example, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanomas, myxosarcoma, osteosarcoma, abemetic sarcoma (Abemethyl's sarcomas), liposarcoma, acinoid soft tissue sarcoma, enameloblastoma, botryoma, green sarcoma, choriocarcinoma, embryonal sarcoma, wilms ' turmor sarcoma, endometrial sarcoma, mesenchymal sarcoma, ewing's sarcomas, fascia sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytosarcoma, hodgkin's sarcoma, idiopathic multiple pigment hemangiosarcoma, B cell immunoblastic sarcoma, lymphomas, T cell immunoblastic sarcoma, zhan Enxun's sarcomas, kaposi's sarcoma, koependymoma, vascular sarcoma, leukemia, mesophylloma, endometrial sarcoma, reticuloendoma, rous's sarcoma, sarcomas, capillary sarcoma, and myxogenic sarcoma (osteosarcoma, hemangiosarcoma, myxoma, and myxoma (osteosarcoma).

The term "melanoma" refers to a tumor derived from the melanocyte system of the skin and other organs. Melanomas that can be treated with the methods of the invention include, for example, acral lentigo melanoma (acral-lentiginous melanoma), melanomas, benign adolescent melanoma, cloudman melanoma, S91 melanoma, harding-Passey melanoma, juvenile melanoma, malignant lentigo melanoma, malignant melanoma, nodular melanoma, sublingual melanoma, and superficial diffuse melanoma.

The term "epithelial cancer" refers to a malignant new growth consisting of epithelial cells that tends to infiltrate surrounding tissue and cause metastasis. As described herein, epithelial cancers that may be treated with the methods of the invention include, for example, acinar, adenocarcinoma, adenocyst, adenoid cystic, adenoma, adrenocortical, alveolar cell, basal epithelial cell, basal-like, basal squamous cell, bronchoalveolar, bronchus, bronchiogenic, brain-like, cholangiocellular, chorioallantoic, colloid, colonic adenocarcinomas of the colon, acne-like, uterine, screen, armor, skin, columnar, catheter, dura, embryonal, medullary, epithelioid, adenoid, explanted, ulcerative gastric (carcinoma ex ulcere), fibroma, colloid, colloidal, giant cell (giant cell carcinoma), giant cell (carcinoma gigantocellulare), hyaline carcinoma 6), granulosa, hair-mother, blood, hepatocellular, xu Teer, clear (Hurthle cell carcinoma), adrenal-like cancer, naive embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, krompcher's cancer, large cell carcinoma, bean-like carcinoma (lenticular carcinoma), bean-like carcinoma (carcinoma lenticulare), lipoma carcinoma, lymphatic epithelial carcinoma, medullary carcinoma (carcinoma medullare), medullary carcinoma (medullary carcinoma), melanosquamous carcinoma, soft carcinoma (carpinoma molle), mercker cell carcinoma (merkel cell carcinoma), mucinous carcinoma (mucinous carcinoma), mucinous cell carcinoma, mucinous epidermoid carcinoma, mucinous carcinoma (carcinoma mucosum), mucinous cancer (mucoid), myxomatoid cancer (carcinoma myxomatode), nasopharyngeal cancer, oat cell cancer, ossified cancer (carcinoma ossifican), bone-like cancer, papillary carcinoma, periportal cancer, pre-invasive cancer, acanthocellular cancer, brain-like cancer, renal cell carcinoma of the kidney, reserve cell carcinoma, sarcoidosis, schneider's cancer (schneiderian carcinoma), hard cancer, scrotum cancer (carpinoma sciti), ring cell carcinoma, simple cancer, small cell carcinoma, eggplant cancer (solanoid carcinoma), globular cell carcinoma, spindle cell carcinoma, medullary cancer (carcinoma spongiosum), squamous cell carcinoma (squamous carcinoma), squamous cell carcinoma (squamous cell carcinoma), string-bound carcinoma (stringing carbioma), vasodilating cancer (carcinoma telangiectaticum), vasodilating cancer (carcinoma telangiectodes), transitional cell carcinoma, nodular skin carcinoma (carcinoma tuberosum), nodular skin carcinoma (tuberous carcinoma), wart cancer (verrucous carcinoma), squamous cell carcinoma, tonsil cell carcinoma, and villous cancer. In particular embodiments, the cancer is renal cell carcinoma.

The term "leukemia" refers to a type of cancer of the blood or bone marrow characterized by an abnormal increase in immature leukocytes (referred to as "blasts"). Leukemia is a broad term covering a wide range of diseases. In turn, it is part of a broader group of diseases affecting the blood, bone marrow and lymphatic systems, all of which are known as hematological neoplasms. Leukemia can be divided into four main categories: acute Lymphoblastic Leukemia (ALL), acute myelogenous (or myeloid or non-lymphocytic) leukemia (AML), chronic Lymphocytic Leukemia (CLL), and Chronic Myelogenous Leukemia (CML). Other types of leukemia include Hairy Cell Leukemia (HCL), T-cell lymphocytic leukemia (T-PLL), large granular lymphocytic leukemia, and adult T-cell leukemia. In certain embodiments, the leukemia comprises acute leukemia. In certain embodiments, the leukemia comprises chronic leukemia.

The term "lymphoma" refers to a group of blood cell tumors that develop from lymphocytes. Two major categories of lymphomas are Hodgkin's Lymphoma (HL) and non-hodgkin's lymphoma (NHL). Lymphomas include any neoplasm of lymphoid tissue. The main category is cancer of lymphocytes, a type of white blood cell that belongs to both the lymph fluid and the blood and extends throughout both.

In some embodiments, the engineered immune cells and compositions comprising the engineered immune cells of the disclosure are used to treat various types of solid tumors, such as breast cancer (e.g., triple negative breast cancer), bladder cancer, genitourinary tract cancer, colon cancer, rectal cancer, endometrial cancer, kidney (renal cell) cancer, pancreatic cancer, prostate cancer, thyroid cancer (e.g., papillary thyroid cancer), skin cancer, bone cancer, brain cancer, cervical cancer, liver cancer, stomach cancer, oral and buccal cancer, esophageal cancer, adenoid cystic cancer, neuroblastoma, testicular cancer, uterine cancer, thyroid cancer, head and neck cancer, kidney cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer), mesothelioma, ovarian cancer, sarcoma, stomach cancer, uterine cancer, cervical cancer, medulloblastoma, and vulval cancer. In certain embodiments, skin cancers include melanoma, squamous cell carcinoma, and Cutaneous T Cell Lymphoma (CTCL).

In some embodiments, the solid tumor is selected from the group consisting of: colon cancer, soft Tissue Sarcoma (STS), metastatic clear cell renal cell carcinoma (ccRCC), ovarian cancer, gastrointestinal cancer, colorectal cancer, hepatocellular carcinoma (HCC), glioblastoma (GBM), breast cancer, melanoma, non-small cell lung cancer (NSCLC), sarcoma, malignant pleura, mesothelioma (MPM), retinoblastoma, glioma, medulloblastoma, osteosarcoma, ewing's sarcoma, pancreatic cancer, lung cancer, stomach cancer, gastric cancer, esophageal cancer, liver cancer, prostate cancer, gynaecological cancer, nasopharyngeal cancer, osteosarcoma, rhabdomyosarcoma, urothelial bladder cancer, neuroblastoma, and cervical cancer. Exemplary antigen binding domain target proteins for targeting these solid tumors are provided in table 8.

In particular embodiments, the cancer may be an "immunocompromised" cancer, such as a tumor containing small numbers of infiltrating T cells, or a cancer that is not recognized by the immune system and does not elicit a strong response, which makes it difficult to treat with current immunotherapy. For example, in one embodiment, the cancer is selected from the group consisting of: melanoma, cervical cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, urothelial cancer, bladder cancer, non-small cell lung cancer, sarcoma, colorectal adenocarcinoma, gastrointestinal stromal tumor, gastroesophageal cancer, colorectal cancer, pancreatic cancer, renal cancer, malignant mesothelioma, leukemia, lymphoma, myelodysplastic syndrome, multiple myeloma, transitional cell carcinoma, neuroblastoma, plasmacytoma, wilms' tumor, and hepatocellular carcinoma (e.g., hepatocellular carcinoma).

In some embodiments, the therapies described herein can be administered to a subject who has previously failed to treat cancer with another anti-tumor (e.g., immunotherapy) regimen. A "subject who fails an anti-tumor regimen" is a subject with cancer who does not respond or ceases to respond to treatment with an anti-tumor regimen according to RECIST 1.1 criteria, i.e., does not reach a complete response, partial response or disease stabilization in the target lesion; or during or after completion of an anti-tumor treatment regimen (alone or in combination with surgery and/or radiation therapy (possibly often clinically indicated in combination with anti-tumor therapy), does not achieve a complete response to non-target lesions or non-CR/non-PD. The RECIST 1.1 standard is described, for example, in Eisenhauer et al 2009, eur j. Cancer [ journal of european cancer ]45:228-24 (which is incorporated herein by reference in its entirety) and discussed in more detail below. Failure of anti-tumor therapy results in, for example, tumor growth, increased tumor burden, and/or tumor metastasis. As used herein, a failed anti-tumor regimen includes a treatment regimen that is terminated due to dose-limiting toxicity (e.g., grade III or IV toxicity that cannot be resolved to allow continued or resumption of treatment with the toxic anti-tumor agent or regimen). In one embodiment, the subject fails to be treated with an anti-tumor regimen comprising administration of one or more anti-angiogenic agents.

Failed anti-tumor regimens include treatment regimens that do not result in at least stable disease for an extended period of time for all target lesions and non-target lesions, e.g., at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 12 months, at least 18 months, or less than any period of clinically defined cure. Failed anti-tumor regimens include treatment regimens that result in progressive disease of at least one target lesion during treatment with an anti-tumor agent, or less than 2 weeks, less than 1 month, less than two months, less than 3 months, less than 4 months, less than 5 months, less than 6 months, less than 12 months, or less than 18 months, or less than any time period less than a clinically defined cure after the treatment regimen is completed.

Failed anti-tumor regimens do not include those in which the subject treating the cancer reaches a clinically defined cure (e.g., a 5 year complete response) after the treatment regimen is completed, and in which the subject is subsequently diagnosed with a different cancer after the treatment regimen is completed, e.g., more than 5 years, more than 6 years, more than 7 years, more than 8 years, more than 9 years, more than 10 years, more than 11 years, more than 12 years, more than 13 years, more than 14 years, or more than 15 years.

RECIST criteria are clinically accepted evaluation criteria, standard methods for providing a measure of solid tumors, and definitions for objective assessment of tumor size changes in clinical trials. Such criteria may also be used to monitor the response of an individual undergoing treatment for a solid tumor. The RECIST 1.1 standard is discussed in detail in, for example, eisenhauer et al 2009, eur j. Cancer journal 45:228-24, which is incorporated herein by reference. The response criteria for target lesions included:

complete Response (CR): all target lesions disappeared. Any pathological lymph node (whether targeted or non-targeted) must have its minor axis reduced to <10mm.

Partial Response (PR): the sum of target lesion diameters is reduced by at least 30% with reference to the sum of baseline diameters.

Progressive Disease (PD): the sum of diameters of the target lesions is increased by at least 20% with the minimum sum in the study as a reference (including the baseline sum if the baseline sum in the study is at a minimum). In addition to a relative increase of 20%, the sum must also show an absolute increase of at least 5 mm. ( And (3) injection: the appearance of one or more new lesions is also considered progress. )

Stable Disease (SD): taking the smallest sum of diameters at the time of investigation as a reference, there is neither sufficient shrinkage to meet PR nor sufficient increase to meet PD.

RECIST 1.1 criteria also contemplates non-target lesions, which are defined as lesions that are measurable but do not require measurement, and should only be qualitatively assessed at the desired time points. Response criteria for non-target lesions included:

complete Response (CR): all non-target lesions disappeared and tumor marker levels normalized. All lymph node sizes must be non-pathological (short axis <10 mm).

non-CR/non-PD: the persistence of one or more non-target lesions and/or tumor marker levels are maintained above normal limits.

Progressive Disease (PD): clear progress in existing non-target lesions. The appearance of one or more new lesions is also considered to progress. In order to achieve a "clear progression" on the basis of non-target disease, the overall level of non-target disease must be severely worsened, so that the overall tumor burden has increased sufficiently to merit discontinuation of therapy even in the presence of SD or PR in the target disease. A modest "increase" in the size of one or more non-target lesions is often insufficient to justify a clear progression state. Therefore, it is very rare to specify overall progression based only on changes in non-target disease facing SD or PR in target disease.

In some embodiments, the pharmaceutical compositions and combination therapies described herein can be administered to a subject having refractory cancer. A "refractory cancer" is a surgically ineffective malignancy that is initially unresponsive to chemotherapy or radiation therapy, or that is unresponsive to chemotherapy or radiation therapy over time.

The disclosure further provides a method of inhibiting tumor cell growth in a subject, the method comprising administering an engineered immune cell described herein such that tumor cell growth is inhibited. In certain embodiments, treating cancer comprises extending survival or extending tumor progression time as compared to a control (e.g., a subject not treated with engineered immune cells). In certain embodiments, the subject is a human subject. In some embodiments, the subject is identified as having cancer (e.g., tumor) prior to administration of the first dose of engineered immune cells. In certain embodiments, the subject has cancer (e.g., tumor) when the engineered immune cells are first administered.

In one embodiment, administering the engineered immune cells results in one or more of the following: reducing proliferation of cancer cells, reducing metastasis of cancer cells, reducing neovascularization of a tumor, reducing tumor burden, reducing tumor size, weight, or volume, inhibiting tumor growth, increasing the time to progression of a cancer, and/or extending survival time of a subject with a tumor disorder. In certain embodiments, administration of the engineered immune cells reduces proliferation of cancer cells, reduces metastasis of cancer cells, reduces neovascularization of a tumor, reduces tumor burden, reduces tumor size, weight or volume, increases time to progression, inhibits tumor growth, and/or increases survival time of the subject by at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, or 500% relative to a corresponding control subject to which the engineered immune cells were not administered. In certain embodiments, administration of the engineered immune cells reduces proliferation of cancer cells, reduces metastasis of cancer cells, reduces neovascularization of a tumor, reduces tumor burden, reduces tumor size, weight or volume, increases time of progression, inhibits tumor growth, and/or increases survival time of a population of subjects with a tumor disorder by at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, or 500% relative to a corresponding population of control subjects with a tumor disorder without administration of the engineered immune cells. In some embodiments, the proliferation of cancer cells is hyperproliferative of cancer cells caused by cancer therapies administered to the subject. In some embodiments, administration of the engineered immune cells stabilizes the tumor disorder in a subject having a progressive tumor disorder prior to treatment.

Combination therapy of the engineered immune cells of the invention with one or more additional therapeutic agents

The terms "combination administration," "combination therapy," "co-administration," or "co-administration" may refer to administration of an engineered immune cell of the invention in combination with one or more additional therapeutic agents, i.e., an immune cell engineered to comprise one or more heterologous polynucleotides that promote saenox transfer and/or polynucleotides encoding Chimeric Antigen Receptors (CARs). The one or more additional therapeutic agents may be administered prior to, simultaneously with, or substantially simultaneously with, subsequent to, or intermittently with the administration of the engineered immune cells of the invention. In certain embodiments, the one or more additional therapeutic agents are administered prior to administration of the engineered immune cells. In certain embodiments, the one or more additional therapeutic agents are administered concurrently with the engineered immune cells. In certain embodiments, the one or more additional therapeutic agents are administered after the engineered immune cells are administered.

One or more additional therapeutic agents act additively or synergistically with the engineered immune cells of the invention. In one embodiment, the one or more additional therapeutic agents and the engineered immune cells act synergistically. In some embodiments, the synergy is used to treat a neoplastic disorder or infection. For example, in one embodiment, the combination of one or more additional therapeutic agents and the engineered immune cells improves the persistence of the immune response against the cancer, i.e., extends the duration. In some embodiments, the one or more additional therapeutic agents and the engineered immune cells act additively.

1.Immune checkpoint modulators

In some embodiments, the additional therapeutic agent administered in combination with the engineered immune cells of the invention is an immune checkpoint modulator of an immune checkpoint molecule. Examples of immune checkpoint molecules include LAG-3 (Triebel et al, 1990, J.Exp.Med. [ J.Endoc. Endoc. Sci. ] 171:1393-1405), TIM-3 (Sakuishi et al, 2010, J.Exp.Med. [ J.Endoc. Sci. ] 207:2187-2194), VISTA (Wang et al, 2011, J.Exp.Med. [ J.Endoc. Sci. ] 208:577-592), ICOS (Fan et al, 2014, J.Exp.Med. [ J.Endoc. Sci. ] 211:715-725), OX40 (Curti et al, 2013, cancer Res. [ J.Sci. Sci. 73:7189-7198), and 4-1BB (Melaro et al, 1997, nat. Sci. 3:682-685).

The immune checkpoint may be a stimulatory immune checkpoint (i.e. a molecule that stimulates an immune response) or an inhibitory immune checkpoint (i.e. a molecule that inhibits an immune response). In some embodiments, the immune checkpoint modulator is an antagonist of an inhibitory immune checkpoint. In some embodiments, the immune checkpoint modulator is an agonist of a stimulatory immune checkpoint. In some embodiments, the immune checkpoint modulator is an immune checkpoint binding protein (e.g., an antibody Fab fragment, a bivalent antibody, an antibody drug conjugate, an scFv, a fusion protein, a bivalent antibody, or a tetravalent antibody). In certain embodiments, the immune checkpoint modulator is capable of binding or modulating the activity of more than one immune checkpoint. Examples of stimulatory and inhibitory immune checkpoints and molecules that modulate these immune checkpoints that can be used in the methods of the invention are provided below.

i. Stimulatory immune checkpoint molecules

CD27 supports antigen-specific expansion of naive T cells and is critical for the generation of T cell memory (see, e.g., hendriks et al (2000) Nat. Immunol. [ Nature immunology ]171 (5): 433-40). CD27 is also a memory marker for B cells (see, e.g., agematsu et al (2000) Histol. Histopath [ histology and histology ]15 (2): 573-6). CD27 activity is governed by the transient availability of its ligand CD70 on lymphocytes and dendritic cells (see, e.g., borst et al (2005) Curr. Opin. Immunol. [ recent immunology perspective ]17 (3): 275-81). A variety of immune checkpoint modulators have been developed that are specific for CD27 and can be used as disclosed herein. In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and/or expression of CD 27. In some embodiments, the immune checkpoint modulator is an agent that binds to CD27 (e.g., an anti-CD 27 antibody). In some embodiments, the checkpoint modulator is a CD27 agonist. In some embodiments, the checkpoint modulator is a CD27 antagonist. In some embodiments, the immune checkpoint modulator is a CD27 binding protein (e.g., an antibody). In some embodiments, the immune checkpoint modulator is valirumumab (hild administration therapeutics (Celldex Therapeutics). Additional CD27 binding proteins (e.g., antibodies) are known in the art and are disclosed, for example, in U.S. patent nos. 9,248,183, 9,102,737, 9,169,325, 9,023,999, 8,481,029; U.S. patent application publication nos. 2016/0185870, 2015/0337047, 2015/0299330, 2014/012942, 2013/0336976, 2013/0243995, 2013/0183316, 2012/0213771, 2012/0093805, 2011/0274685, 2010/0173324; and PCT publication nos. WO 2015/016718, WO 2014/140374, WO 2013/138586, WO/004367, WO 2011/130434, WO 2010/001908, and WO 2008/051424, each of which is incorporated herein by reference in its entirety.

Cd28. cluster of differentiation 28 (CD 28) is one of the proteins expressed on T cells that provide the costimulatory signals required for T cell activation and survival. In addition to the T Cell Receptor (TCR), stimulation of T cells by CD28 provides an effective signal to produce various interleukins (especially IL-6). Binding to its two ligands CD80 and CD86 expressed on dendritic cells promotes T cell expansion (see, e.g., prasad et al (1994) Proc.Nat' l. Acad. Sci. USA [ Proc. Natl. Acad. Sci. USA [ 91 (7) of the national academy of sciences USA ]): 2834-8). A variety of immune checkpoint modulators have been developed that are specific for CD28 and can be used as disclosed herein. In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and/or expression of CD28. In some embodiments, the immune checkpoint modulator is an agent that binds to CD28 (e.g., an anti-CD 28 antibody). In some embodiments, the checkpoint modulator is a CD28 agonist. In some embodiments, the checkpoint modulator is a CD28 antagonist. In some embodiments, the immune checkpoint modulator is a CD28 binding protein (e.g., an antibody). In some embodiments, the immune checkpoint modulator is selected from the group consisting of: TAB08 (TheraMab LLC), lu Lizhu mab (also known as BMS-931699, bristol-Myers Squibb), and FR104 (OSE immunotherapy Co (OSE Immunotherapeutics)). Additional CD28 binding proteins (e.g., antibodies) are known in the art and are described, for example, in U.S. patent nos. 9,119,840, 8,709,414, 9,085,629, 8,034,585, 7,939,638, 8,389,016, 7,585,960, 8,454,959, 8,168,759, 8,785,604, 7,723,482; U.S. patent application publication nos. 2016/0017039, 2015/0299321, 2015/0150968, 2015/007496, 2015/0376278, 2013/0078557, 2013/023040, 2013/0078136, 2013/0109846, 2013/0266577, 2012/0201814, 2012/0082683, 2012/0219553, 2011/0189735, 2011/0097339, 2010/0266605, 2010/0168400, 2009/02024404, 2008/0038273; and PCT publications WO 2015198147, WO 2016/05421, WO 2014/1209168, WO 2011/101791, WO 2010/007476, WO 2010/009391, WO 2004/004768, WO 2002/030459, WO 2002/051871 and WO 2002/047721, each of which is incorporated herein by reference in its entirety.

CD40 cluster of differentiation 40 (CD 40, also known as TNFRSF 5) is found on a variety of immune system cells including antigen presenting cells. CD40L, also known as CD154, is a ligand for CD40 and is activated at CD4 ⁺ T cell surface transient expression. CD40 signaling is known to ' permit ' dendritic cell maturation and thereby trigger T cell activation and differentiation (see, e.g., O ' Sullivan et al (2003) crit.Rev.Immunol. [ immunology importance comment)]23 (1):83-107). A variety of immune checkpoint modulators have been developed that are specific for CD40 and can be used as disclosed herein. In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and/or expression of CD40. In some embodiments, the immune checkpoint modulator is an agent that binds to CD40 (e.g., an anti-CD 40 antibody). In some embodiments, the checkpoint modulator is a CD40 agonist. In some embodiments, the checkpoint modulator is a CD40 antagonist. In some embodiments, the immune checkpoint modulator is a CD40 binding protein selected from the group consisting of: darcy beadMonoclonal antibodies (Genntech/Seattle Genetics, inc. (Genntech/Seattle Genetics)), CP-870,893 (Pfizer), bruzumab (An Si Talars pharmaceutical, inc. (Astella Pharma)), lu Katuo Mumab (Novartis), CFZ533 (NoHua, inc.; see, e.g., cordoba et al (2015) am.J. Transplay. [ journal of American transplantation ] ]15 2825-36), RG7876 (Gene technologies Co., ltd.), FFP104 (Pan Gene Co., ltd. (PanGenetics, B.V.)), APX005 (Apexigen Co., ltd.), BI 655064 (Bolin and John Co., ltd. (Boehringer Ingelheim)), chi Lob 7/4 (British cancer research center (Cancer Research UK); see, e.g., johnson et al (2015) clin.cancer Res. [ clinical cancer research]21 1321-8), ADC-1013 (biological invention International company (BioInvent International)), SEA-CD40 (Seattle genetics), xmAb 5485 (Xencor company), PG120 (Pantoea. Co.), tenebxib (teneliximab) (Bai Meishi precious company); see, e.g., thompson et al (2011) am.j. Transfer [ journal of american transplantation ]]11 (5) 947-57), and AKH3 (Biogen); see, for example, international publication No. WO 2016/028810). Additional CD40 binding proteins (e.g., antibodies) are known in the art and are disclosed, for example, in U.S. patent nos. 9,234,044, 9,266,956, 9,109,011, 9,090,696, 9,023,360, 9,023,361, 9,221,913, 8,945,564, 8,926,979, 8,828,396, 8,637,032, 8,277,810, 8,088,383, 7,820,170, 7,790,166, 7,445,780, 7,361,345, 8,961,991, 8,669,352, 8,957,193, 8,778,345, 8,591,900, 8,551,485, 8,492,531, 8,362,210, 8,388,971; U.S. patent application publication nos. 2016/0045597, 2016/0152713, 2016/007592, 2015/0299329, 2015/0057437 2015/0315282, 2015/0307616, 2014/0099317, 2014/0179907, 2014/0349395, 2014/023444, 2014/0348836, 2014/0193405, 2014/01010103, 2014/0105907, 2014/024866, 2014/0093497, 2014/0010812, 2013/0024956, 2013/0023047, 2013/0315900, 2012/0087927, 2012/0263732, 2012/1488, 2011/0027276, 2011/0104182, 2010/02344578, 2010304687, 2009/0181015, 2009/013051254, 2008/0199471, 2008/00855531, 2014/0107721, 0111/0111, 2010111/0111, 2015/86991/2015, 2015/008602, 2015/2015,00864, 2015/2015,053/2015,059900, 2015/2015,035 11405. 2012/01010101011585, 2011/0033456, 2011/0002934, 2010/0172912, 2009/0081242, 2009/013095, 2008/0254026, 2008/007527, 2009/0304706, 2009/0202531, 2009/017111, 2009/0041773, 2008/027412, 2008/0057070, 2007/0098717, 2007/0218060, 2007/0098718, 2007/010754; and PCT publication nos. WO 2016/069919, WO 2016/023960, WO 2016/023875, WO 2016/028810, WO 2015/134988, WO 2015/091853, WO 2015/091655, WO 2014/065403, WO 2014/070934, WO 2014/065402, WO 2014/207064, WO 2013/034904, WO 2012/125569, WO 2012/14956, WO 2012/111762, WO 2012/145673, WO 2011/123489, WO 2010/123012, WO 2010/104761, WO 2009/094391, WO 2008/091954, WO 2007/129895, WO 2006/128103, WO 2005/0632289; in WO 2005/063981, WO 2003/040170, WO 2002/011763, WO 2000/075348, WO 2013/164789, WO 2012/075111, WO 2012/065950, WO 2009/062054, WO 2007/124299, WO 2007/053661, WO 2007/053767, WO 2005/044294, WO 2005/044304, WO 2005/044306, WO 2005/044855, WO 2005/044854, WO 2005/044305, WO 2003/045978, WO 2003/029296, WO 2002/028481, WO 2002/028480, WO 2002/028904, WO 2002/028905, WO 2002/088186, and WO 2001/02823, each of which is incorporated herein by reference.

The ox40.Ox40 receptor (also known as CD 134) promotes the expansion of effector T cells and memory T cells. OX40 also represses T-regulated cell differentiation and activity and regulates cytokine production (see, e.g., croft et al (2009) immunol. Rev. [ immunol comment ]229 (1): 173-91). A variety of immune checkpoint modulators have been developed that are specific for OX40 and can be used as disclosed herein. In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and/or expression of OX40. In some embodiments, the immune checkpoint modulator is an agent that binds to OX40 (e.g., an anti-OX 40 antibody). In some embodiments, the checkpoint modulator is an OX40 agonist. In some embodiments, the checkpoint modulator is an OX40 antagonist. In some embodiments, the immune checkpoint modulator is an OX40 binding protein (e.g., an antibody) selected from the group consisting of: MEDI6469 (AgonOx company/medical immune company (AgonOx/mediimune)), pongamuzumab (also known as MOXR0916 and RG7888; genentech inc.)), tavolizumab (also known as MEDI0562; medical immune company), and GSK3174998 (GlaxoSmithKline). Additional OX-40 binding proteins (e.g., antibodies) are known in the art and are described, for example, in U.S. patent nos. 9,163,085, 9,040,048, 9,006,396, 8,748,585, 8,614,295, 8,551,477, 8,283,450, 7,550,140; U.S. patent application publication nos. 2016/0068604, 2016/0031974, 2015/0315281, 2015/0132288, 2014/0308276, 2014/0377284, 2014/0044703, 2014/0294824, 2013/0330344, 2013/0280275, 2013/024372, 2013/0183315, 2012/0269825, 2012/024076, 2011/0008368, 2011/012352, 2010/0254978, 2010/0196359, 2006/0281072; and PCT publication nos. WO 2014/148895, WO 2013/068563, WO 2013/038191, WO 2013/028231, WO 2010/096418, WO 2007/062245, and WO 2003/106498, each of which is incorporated herein by reference in its entirety.

GITR glucocorticoid-induced TNFR family related Genes (GITR) are members of the Tumor Necrosis Factor Receptor (TNFR) superfamily, which are constitutively or conditionally expressed on Treg, CD4 and CD 8T cells. GITR was rapidly up-regulated on effector T cells following TCR ligation and activation. Human GITR ligand (GITRL) is constitutively expressed on APC in secondary lymphoid organs and some non-lymphoid tissues. Downstream effects of GITR: GITRL interactions induce reduced Treg activity and enhance CD4 ⁺ T cell activity, resulting in reversal of Treg-mediated immunosuppression and enhanced immune stimulation. A variety of immune checkpoint modulators have been developed that are specific for GITR and can be used as disclosed herein. In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and/or expression of GITR. In some embodiments, the immune checkpoint modulator is an agent that binds to GITR (e.g., an anti-GITR antibody). In some embodiments, the checkpoint modulator is a GITR agonist. In some embodiments, the checkpoint modulator is a GITR antagonist. In some embodiments, the immune checkpoint modulator is a GITR binding protein (e.g., antibody) selected from the group consisting of: TRX518 (flight therapy) Company (Leap Therapeutics)), MK-4166 (Merck company (Merck)&Co.)), MEDI-1873 (medical immunology), INCAGN1876 (An Dijun Sis/Inseide (Agenus/Incyte)), and FPA154 (five major therapies Co., ltd. (Five Prime Therapeutics)). Additional GITR binding proteins (e.g., antibodies) are known in the art and are described, for example, in U.S. patent nos. 9,309,321, 9,255,152, 9,255,151, 9,228,016, 9,028,823, 8,709,424, 8,388,967; U.S. patent application publication nos. 2016/0145342, 2015/0353637, 2015/0064204, 2014/0348841, 2014/0065152, 2014/00745566, 2014/00745565, 2013/0183321, 2013/0108641, 2012/0189639; and PCT publications WO 2016/054638, WO 2016/057841, WO 2016/057846, WO 2015/187835, WO 2015/184099, WO 2015/031667, WO 2011/028683, and WO 2004/107618, each of which is incorporated herein by reference.

ICOS inducible T cell costimulator (ICOS, also known as CD 278) is expressed on activated T cells. Its ligand is ICOSL expressed primarily on B cells and dendritic cells. ICOS is important in T cell effector functions. ICOS expression is up-regulated following T cell activation (see, e.g., fan et al (2014) J. Exp. Med. [ journal of Experimental medicine ]211 (4): 715-25). A variety of immune checkpoint modulators have been developed that are specific for ICOS and can be used as disclosed herein. In some embodiments, the immune checkpoint modulator is an agent that modulates ICOS activity and/or expression. In some embodiments, the immune checkpoint modulator is an agent that binds ICOS (e.g., an anti-ICOS antibody). In some embodiments, the checkpoint modulator is an ICOS agonist. In some embodiments, the checkpoint modulator is an ICOS antagonist. In some embodiments, the immune checkpoint modulator is an ICOS binding protein (e.g., antibody) selected from the group consisting of: MEDI-570 (also known as JMab-136, medical immune corporation), GSK3359609 (ghatti/insert) and JTX-2011 (bouncer therapy (Jounce Therapeutics)). Additional ICOS binding proteins (e.g., antibodies) are known in the art and are described, for example, in U.S. patent nos. 9,376,493, 7,998,478, 7,465,445, 7,465,444; U.S. patent application publication nos. 2015/0239978, 2012/0039874, 2008/0199466, 2008/0279851; and PCT publication No. WO 2001/087981, each of which is incorporated herein by reference.

4-1BB.4-1BB (also known as CD 137) is a member of the Tumor Necrosis Factor (TNF) receptor superfamily. 4-1BB (CD 137) is a type II transmembrane glycoprotein, CD4 which can be triggered ⁺ And CD8 ⁺ Expression is induced on T cells, activated NK cells, DCs and neutrophils and acts as a T cell costimulatory molecule when bound to 4-1BB ligand (4-1 BBL) found on activated macrophages, B cells and DCs. Ligation of the 4-1BB receptor results in activation of NF-B, c-Jun and p38 signaling pathways, and has been shown to promote CD8 by up-regulating expression of the anti-apoptotic genes Bcl-x (L) and Bfl-1 ⁺ T cell survival. In this way, 4-1BB acts to enhance or even rescue the sub-optimal immune response. A variety of immune checkpoint modulators have been developed that are specific for 4-1BB and can be used as disclosed herein. In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and/or expression of 4-1BB. In some embodiments, the immune checkpoint modulator is an agent that binds 4-1BB (e.g., an anti-4-1 BB antibody). In some embodiments, the checkpoint modulator is a 4-1BB agonist. In some embodiments, the checkpoint modulator is a 4-1BB antagonist. In some embodiments, the immune checkpoint modulator is a 4-1BB binding protein, which is Wu Ruilu mab (also known as BMS-663513; beatles Meissu precious Co.) or Wutuuzumab (pyroinc.). In some embodiments, the immune checkpoint modulator is a 4-1BB binding protein (e.g., an antibody). 4-1BB binding proteins (e.g., antibodies) are known in the art and are described, for example, in U.S. patent nos. 9,382,328, 8,716,452, 8,475,790, 8,137,667, 7,829,088, 7,659,384; U.S. patent application publication nos. 2016/0083474, 2016/0152722, 2014/0193422, 2014/0178368, 2013/0149301, 2012/0237498, 2012/0141494, 2012/0076122, 2011/0177104, 2011/0189189, 2010/0183621, 2009/0068192, 2009/0041763, 2008/0305113, 2008/0008716; and PCT publication nos. WO 2016/029073, WO 2015/188047, WO 2015/179236, WO 2015/119923, WO 2012/032333, WO 2012/145183, WO 2011/031063, WO 2010/132389, WO 2010/0424 33. WO 2006/126835, WO 2005/035584, WO 2004/010947; martinez-Forero et al (2013) J.Immunol. [ J.Immunol.]190 (12) 6694-706, and Dubrot et al (2010) Cancer immunol. Immunothers [ Cancer immunology and immunotherapy ]]59 1223-33, each of which is incorporated herein by reference in its entirety.

inhibitory immune checkpoint molecules

Adora2A adenosine A2A receptor (A2A 4) is a member of the G Protein Coupled Receptor (GPCR) family, which possesses seven transmembrane alpha helices and is considered an important checkpoint in cancer therapy. The A2A receptor can down-regulate an overreacting immune cell (see, e.g., ohta et al (2001) Nature [ Nature ]414 (6866): 916-20). A variety of immune checkpoint modulators have been developed that are specific for ADORA2A and can be used as disclosed herein. In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and/or expression of ADORA2A. In some embodiments, the immune checkpoint modulator is an agent that binds to ADORA2A (e.g., an anti-ADORA 2A antibody). In some embodiments, the immune checkpoint modulator is an ADORA2A binding protein (e.g., an antibody). In some embodiments, the checkpoint modulator is an ADORA2A agonist. In some embodiments, the checkpoint modulator is an ADORA2A antagonist. ADORA2A binding proteins (e.g., antibodies) are known in the art and are disclosed, for example, in U.S. patent application publication No. 2014/032366, which is incorporated herein by reference.

B7-H3.B7-H3 (also known as CD 276) belongs to the B7 superfamily, a group of molecules that co-stimulate or down-regulate T cell responses. B7-H3 efficiently and continuously down-regulates human T cell responses (see, e.g., leitner et al (2009) Eur. J. Immunol. European journal of immunology ]39 (7): 1754-64). A variety of immune checkpoint modulators have been developed that are specific for B7-H3 and can be used as disclosed herein. In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and/or expression of B7-H3. In some embodiments, the immune checkpoint modulator is an agent that binds to B7-H3 (e.g., an anti-B7-H3 antibody). In some embodiments, the checkpoint modulator is a B7-H3 agonist. In some embodiments, the checkpoint modulator is a B7-H3 antagonist. In some embodiments, the immune checkpoint modulator is an anti-B7-H3 binding protein selected from the group consisting of: DS-5573 (first co-product (Daiichi Sankyo, inc.)), enotuzumab (macrogenetics, inc.)) and 8H9 (snenkatelin cancer institute (Sloan Kettering Institute for Cancer Research)); see, for example, ahmed et al (2015) J.biol.chem. [ J.Biochem. ]290 (50): 30018-29). In some embodiments, the immune checkpoint modulator is a B7-H3 binding protein (e.g., an antibody). B7-H3-binding proteins (e.g., antibodies) are known in the art and are described, for example, in U.S. patent nos. 9,371,395, 9,150,656, 9,062,110, 8,802,091, 8,501,471, 8,414,892; U.S. patent application publication nos. 2015/0352224, 2015/0297748, 2015/0259434, 2015/0274838, 2014/032875, 2014/0161814, 2013/0287798, 2013/0078234, 2013/0149236, 2012/02947960, 2010/0143245, 2002/0102264; PCT publication nos. WO 2016/106004, WO 2016/033225, WO 2015/181267, WO 2014/057687, WO 2012/147713, WO 2011/109400, WO 2008/116219, WO 2003/075846, WO 2002/032375; and Shi et al (2016) mol. Med. Rep. [ molecular medical report ]14 (1): 943-8, each of which is incorporated herein by reference.

B7-H4.B7-H4 (also known as O8E, OV064 and V-set domain containing T cell activation inhibitor (VTCN 1)) belongs to the B7 superfamily. By preventing the cell cycle, the B7-H4 linkage of T cells has profound inhibitory effects on cell growth, cytokine secretion and development of cytotoxicity. Administration of B7-H4Ig to mice impairs antigen-specific T cell responses, whereas blocking endogenous B7-H4 by specific monoclonal antibodies promotes T cell responses (see, e.g., sica et al (2003) Immunity 18 (6): 849-61). A variety of immune checkpoint modulators have been developed that are specific for B7-H4 and can be used as disclosed herein. In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and/or expression of B7-H4. In some embodiments, the immune checkpoint modulator is an agent that binds to B7-H4 (e.g., an anti-B7-H4 antibody). In some embodiments, the immune checkpoint modulator is a B7-H4 binding protein (e.g., an antibody). In some embodiments, the checkpoint modulator is a B7-H4 agonist. In some embodiments, the checkpoint modulator is a B7-H4 antagonist. B7-H4 binding proteins (e.g., antibodies) are known in the art and are described, for example, in U.S. patent nos. 9,296,822, 8,609,816, 8,759,490, 8,323,645; U.S. patent application publication nos. 2016/0159910, 2016/0017040, 2016/0168249, 2015/0315275, 2014/013180, 2014/032969, 2014/0356364, 2014/0328751, 2014/0294861, 2014/0308259, 2013/0058864, 2011/0085970, 2009/007460, 2009/0208489; and PCT publication nos. WO 2016/040724, WO 2016/070001, WO 2014/159835, WO 2014/100483, WO 2014/100439, WO 2013/067492, WO 2013/025779, WO 2009/073533, WO 2007/067991, and WO 2006/104677, each of which is incorporated herein by reference.

Btla.b and T Lymphocyte Attenuators (BTLA), also known as CD272, have HVEM (herpes virus invasion medium) as their ligands. In human CD8 ⁺ Surface expression of BTLA is gradually down-regulated during the phenotypic differentiation of T cells from naive to effector cells, but tumor-specific human CD8 ⁺ T cells express high levels of BTLA (see, e.g., derre et al (2010) j.clin.invest. [ journal of clinical research ]]120 (1):157-67). A variety of immune checkpoint modulators have been developed that are specific for BTLA and can be used as disclosed herein. In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and/or expression of BTLA. In some embodiments, the immune checkpoint modulator is an agent that binds BTLA (e.g., an anti-BTLA antibody). In some embodiments, the immune checkpoint modulator is a BTLA binding protein (e.g., an antibody). In some embodiments, the checkpoint modulator is a BTLA agonist. In some embodiments, the checkpoint modulator is a BTLA antagonist. BTLA binding proteins (e.g., antibodies) are known in the art and are described, for example, in U.S. patent nos. 9,346,882, 8,580,259, 8,563,694, 8,247,537; U.S. patent application publication nos. 2014/0017255, 2012/0288500, 2012/0183565, 2010/0172900; and PCT publication nos. WO 2011/014438 and WO 2008/076560; each of which is incorporated herein by reference.

CTLA-4. Cytotoxic T lymphocyte antigen 4 (CTLA-4) is a member of the immunoregulatory CD28-B7 immunoglobulin superfamily and acts on naive and resting T lymphocytes to promote immunosuppression via B7-dependent and B7-independent pathways (see, e.g., kim et al (2016) J.Immunol.Res. [ J.Immunol. Study ], 14). CTLA-4 is also known as CD152.CTLA-4 modulates the threshold of T cell activation. See, e.g., gajewski et al (2001) J.Immunol. 166 (6): 3900-7. Various immune checkpoint modulators have been developed that are specific for CTLA-4 and can be used as disclosed herein. In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and/or expression of CTLA-4. In some embodiments, the immune checkpoint modulator is an agent that binds to CTLA-4 (e.g., an anti-CTLA-4 antibody). In some embodiments, the checkpoint modulator is a CTLA-4 agonist. In some embodiments, the checkpoint modulator is a CTLA-4 antagonist. In some embodiments, the immune checkpoint modulator is a CTLA-4 binding protein (e.g., an antibody) selected from the group consisting of: ai Pili Mumab (Yervoy; medary/Bai Meshi precious Co., medarex/Bristol-Myers Squibb)), qu Meili Mumab (formerly ticilimumab; pfizer/Astrazeneca), JMW-3B3 (Ab Di Ding university (University of Aberdeen)) and AGEN1884 (An Dijun S Co., agenus)). Additional CTLA-4 binding proteins (e.g., antibodies) are known in the art and are described, for example, in U.S. patent No. 8,697,845; U.S. patent application publication nos. 2014/0105914, 2013/0267688, 2012/0107320, 2009/0123777; and PCT publication nos. WO 2014/207064, WO 2012/120125, WO 2016/015675, WO 2010/097597, WO 2006/066568, and WO 2001/054732, each of which is incorporated herein by reference.

KIR killer cell immunoglobulin-like receptors (KIR) comprise a diverse pool of mhc i binding molecules that down-regulate Natural Killer (NK) cell function to protect cells from NK-mediated cell lysis. KIR is usually expressed on NK cells but has also been detected on tumor-specific CTLs. A variety of immune checkpoint modulators have been developed that are specific for KIR and can be used as disclosed herein. In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and/or expression of KIR. In some embodiments, the immune checkpoint modulator is an agent that binds to KIR (e.g., an anti-KIR antibody). In some embodiments, the immune checkpoint modulator is a KIR-binding protein (e.g., an antibody). In some embodiments, the checkpoint modulator is a KIR agonist. In some embodiments, the checkpoint modulator is a KIR antagonist. In some embodiments, the immune checkpoint modulator is Li Ruilu mab (also known as BMS-986015; BAIMEISHIGULAR). Additional KIR-binding proteins (e.g., antibodies) are known in the art and are described, for example, in U.S. patent nos. 8,981,065, 9,018,366, 9,067,997, 8,709,411, 8,637,258, 8,614,307, 8,551,483, 8,388,970, 8,119,775; U.S. patent application publication nos. 2015/0344576, 2015/0376275, 2016/0046712, 2015/0191547, 2015/0290316, 2015/0283234, 2015/0197569, 2014/0193430, 2013/0143269, 2013/0287770, 2012/0208237, 2011/0293627, 2009/0081240, 2010/0189723; and PCT publication nos. WO 2016/069589, WO 2015/069785, WO 2014/066532, WO 2014/055648, WO 2012/160448, WO 2012/071411, WO 2010/065939, WO 2008/084106, WO 2006/072625, WO 2006/072626, and WO 2006/003179, each of which is incorporated herein by reference.

LAG-3, lymphocyte activation gene 3 (LAG-3, also known as CD 223) is a CD 4-associated transmembrane protein that competitively binds to MHC II and serves as a co-inhibitory checkpoint for T cell activation (see, e.g., goldberg and Drake (2011) Curr. Top. Microbiol. Immunol. [ recent topics of microbiology ] 344:269-78). A variety of immune checkpoint modulators have been developed that are specific for LAG-3 and can be used as disclosed herein. In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and/or expression of LAG-3. In some embodiments, the immune checkpoint modulator is an agent that binds LAG-3 (e.g., an anti-PD-1 antibody). In some embodiments, the checkpoint modulator is a LAG-3 agonist. In some embodiments, the checkpoint modulator is a LAG-3 antagonist. In some embodiments, the immune checkpoint modulator is a LAG-3 binding protein (e.g., an antibody) selected from the group consisting of: pembrolizumab (Keytruda; formerly lanreoccur bead mab; merck corporation), nivolumab (Opdivo; bermexiv precious corporation), pildirizumab (CT-011, keri gene corporation), SHR-1210 (infledd/Jiangsu constant rayleigh pharmaceutical limited), MEDI0680 (also known as AMP-514; applied immune corporation/medical immune corporation), PDR001 (nohua corporation), BGB-a317 (berji shenzhou corporation), TSR-042 (also known as ANB011; an Pote biological corporation-t Sha Re corporation), REGN2810 (re-pharmaceutical corporation/cinofi-ambet corporation), and PF-06801591 (parecosystem). Additional PD-1-binding proteins (e.g., antibodies) are known in the art and are described, for example, in U.S. patent nos. 9,181,342, 8,927,697, 7,488,802, 7,029,674; U.S. patent application publication Nos. 2015/0152180, 2011/0171215, 2011/0171220; and PCT publication nos. WO 2004/056875, WO 2015/036394, WO 2010/029435, WO 2010/029434, WO 2014/194302, each of which is incorporated herein by reference.

PD-1 programmed cell death protein 1 (PD-1, also known as CD279 and PDCD 1) is an inhibitory receptor that down regulates the immune system. In contrast to CTLA-4, which affects primarily naive T cells, PD-1 is more widely expressed on immune cells and regulates mature T cell activity in surrounding tissues and tumor microenvironments. PD-1 inhibits T cell responses by interfering with T cell receptor signaling. PD-1 has two ligands, PD-L1 and PD-L2. A variety of immune checkpoint modulators have been developed that are specific for PD-1 and can be used as disclosed herein. In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and/or expression of PD-1. In some embodiments, the immune checkpoint modulator is an agent that binds to PD-1 (e.g., an anti-PD-1 antibody). In some embodiments, the checkpoint modulator is a PD-1 agonist. In some embodiments, the checkpoint modulator is a PD-1 antagonist. In some embodiments, the immune checkpoint modulator is a PD-1 binding protein (e.g., an antibody) selected from the group consisting of: pembrolizumab (Keytruda; formerly lanreoccur bead mab; merck corporation), nivolumab (Opdivo; bermexiv precious corporation), pildirizumab (CT-011, keri gene corporation), SHR-1210 (infledd/Jiangsu constant rayleigh pharmaceutical limited), MEDI0680 (also known as AMP-514; applied immune corporation/medical immune corporation), PDR001 (nohua corporation), BGB-a317 (berji shenzhou corporation), TSR-042 (also known as ANB011; an Pote biological corporation-t Sha Re corporation), REGN2810 (re-pharmaceutical corporation/cinofi-ambet corporation), and PF-06801591 (parecosystem). Additional PD-1-binding proteins (e.g., antibodies) are known in the art and are described, for example, in U.S. patent nos. 9,181,342, 8,927,697, 7,488,802, 7,029,674; U.S. patent application publication Nos. 2015/0152180, 2011/0171215, 2011/0171220; and PCT publication nos. WO 2004/056875, WO 2015/036394, WO 2010/029435, WO2010/029434, WO 2014/194302, each of which is incorporated herein by reference.

PD-L1/PD-L2.PD ligand 1 (PD-L1, also known as B7-H1) and PD ligand 2 (PD-L2, also known as PDCD1LG2, CD273 and B7-DC) bind to the PD-1 receptor. Both ligands belong to the same B7 family as the B7-1 and B7-2 proteins that interact with CD28 and CTLA-4. PD-L1 can be expressed on a number of cell types, including e.g., epithelial cells, endothelial cells, and immune cells. The ligation of PDL-1 reduces IFN, TNF, and IL-2 production and stimulates IL10 production, IL10 being an anti-inflammatory cytokine associated with reduced T cell reactivity and proliferation and antigen-specific T cell anergy. PDL-2 is expressed mainly on Antigen Presenting Cells (APCs). PDL2 ligation also results in T cell repression, but in the case where PDL-1-PD-1 interaction inhibits proliferation by cell cycle arrest at G1/G2 phase PDL2-PD-1 engagement has been shown to inhibit TCR-mediated signaling by blocking B7: CD28 signaling at low antigen concentrations and reducing cytokine production at high antigen concentrations. A variety of immune checkpoint modulators have been developed that are specific for PD-L1 and PD-L2 and can be used as disclosed herein.

In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and/or expression of PD-L1. In some embodiments, the immune checkpoint modulator is an agent that binds to PD-L1 (e.g., an anti-PD-L1 antibody). In some embodiments, the checkpoint modulator is a PD-L1 agonist. In some embodiments, the checkpoint modulator is a PD-L1 antagonist. In some embodiments, the immune checkpoint modulator is a PD-L1 binding protein (e.g., an antibody or Fc fusion protein) selected from the group consisting of: duvaluzumab (also known as MEDI-4736; aspirin/New Biopharmaceutical Co., ltd. (AstraZeneca/Celgene Corp.)), alelizumab (Tecentriq; also known as MPDL3280A and RG7446; gene technologies Co., ltd. (Genetech Inc.)), avlumab (also known as MSB0010718C; merck Serono/Aspirin Co., merck Serono/AstraZeneca)); MDX-1105 (Meddarey/Behcet Mitsui precious Co.), AMP-224 (applied immune Co., gelanin Smith), LY3300054 (Gift Lai Co., eli Lilly and Co.). Additional PD-L1 binding proteins are known in the art and are disclosed, for example, in U.S. patent application publication Nos. 2016/0084839, 2015/0355184, 2016/0175397, and PCT publication Nos. WO 2014/100079, WO 2016/030350, WO 2013181634, each of which is incorporated herein by reference.

In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and/or expression of PD-L2. In some embodiments, the immune checkpoint modulator is an agent that binds to PD-L2 (e.g., an anti-PD-L2 antibody). In some embodiments, the checkpoint modulator is a PD-L2 agonist. In some embodiments, the checkpoint modulator is a PD-L2 antagonist. PD-L2 binding proteins (e.g., antibodies) are known in the art and are described, for example, in U.S. patent nos. 9,255,147, 8,188,238; U.S. patent application publication nos. 2016/012431, 2013/0243152, 2010/0278816, 2016/0137731, 2015/0197571, 2013/0291136, 2011/0271358; and PCT publication nos. WO 2014/022758 and WO 2010/036959; each of which is incorporated herein by reference.

TIM-3.T cell immunoglobulin mucin 3 (TIM-3, also known as hepatitis a virus cell receptor (HAVCR 2)) is a type I glycoprotein receptor that binds to the S-type lectin galectin 9 (Gal-9). TIM-3 is a ligand widely expressed on lymphocytes, liver, small intestine, thymus, kidney, spleen, lung, muscle, reticulocytes, and brain tissue. Tim-3 was originally identified as selectively expressed on IFN-gamma secreting Th1 and Tc1 cells (Monney et al (2002) Nature [ Nature ] 415:536-41). TIM-3 receptor binding to Gal-9 triggers downstream signaling, thereby down regulating T cell survival and function. A variety of immune checkpoint modulators have been developed that are specific for TIM-3 and can be used as disclosed herein. For some embodiments, immune checkpoint modulators are agents that modulate the activity and/or expression of TIM-3. For some embodiments, the immune checkpoint modulator is an agent that binds to TIM-3 (e.g., an anti-TIM-3 antibody). For some embodiments, the checkpoint modulator is a TIM-3 agonist. For some embodiments, the checkpoint modulator is a TIM-3 antagonist. In some embodiments, the immune checkpoint modulator is an anti-TIM-3 antibody selected from the group consisting of: TSR-022 (An Pote Bio Inc. -Te Sha Re Co.) and MGB453 (North China Co.). Additional TIM-3 binding proteins (e.g., antibodies) are known in the art and are described, for example, in U.S. patent nos. 9,103,832, 8,552,156, 8,647,623, 8,841,418; U.S. patent application publication nos. 2016/0200815, 2015/0284468, 2014/0134539, 2014/0044728, 2012/0189617, 2015/0086574, 2013/0022623; and PCT publication nos. WO 2016/068802, WO 2016/068803, WO 2016/071448, WO2011/155607, and WO 2013/006490, each of which is incorporated herein by reference.

T cell activated V domain Ig repressors (VISTA, also known as platelet receptor Gi 24) are Ig superfamily ligands that down regulate T cell responses. See, e.g., wang et al, 2011, j.exp.med. [ journal of experimental medicine ]]208:577-92. VISTA expressed on APC directly represses CD4 ⁺ And CD8 ⁺ T cell proliferation and cytokine production (Wang et al (2010) J Exp Med journal of experimental medicine]208 (3):577-92). A variety of immune checkpoint modulators have been developed that are specific for VISTA and can be used as disclosed herein. In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and/or expression of VISTA. In some embodiments, the immune checkpoint modulator is an agent that binds to VISTA (e.g., an anti-VISTA antibody). In some embodiments, the checkpoint modulator is a VISTA agonist. In some embodiments, the checkpoint modulator is a VISTA antagonist. In some embodiments, the immune checkpoint modulator is a VISTA binding protein (e.g., an antibody) selected from the group consisting of:TSR-022 (An Pote Bio Inc. -Te Sha Re Co.) and MGB453 (North China Co.). VISTA binding proteins (e.g., antibodies) are known in the art and are described, for example, in U.S. patent application publication nos. 2016/0096891, 2016/0096891; and PCT publication nos. WO 2014/190356, WO 2014/197849, WO 2014/190356, and WO 2016/094837, each of which is incorporated herein by reference.

Methods of treating a tumor disorder by administering an engineered immune cell of the invention in combination with at least one immune checkpoint modulator to a subject are provided. In certain embodiments, the immune checkpoint modulator stimulates an immune response in the subject. For example, in some embodiments, an immune checkpoint modulator stimulates or increases the expression or activity of a stimulatory immune checkpoint (e.g., CD27, CD28, CD40, OX40, GITR, ICOS, or 4-1 BB). In some embodiments, the immune checkpoint modulator inhibits or reduces the expression or activity of an inhibitory immune checkpoint (e.g., A2A4, B7-H3, B7-H4, BTLA, CTLA-4, KIR, LAG3, PD-1, PD-L2, TIM-3, or VISTA).

In certain embodiments, the immune checkpoint modulator targets an immune checkpoint molecule selected from the group consisting of: CD27, CD28, CD40, OX40, GITR, ICOS, 4-1BB, A2A4, B7-H3, B7-H4, BTLA, CTLA-4, KIR, LAG3, PD-1, PD-L2, TIM-3 and VISTA. In certain embodiments, the immune checkpoint modulator targets an immune checkpoint molecule selected from the group consisting of: CD27, CD28, CD40, OX40, GITR, ICOS, 4-1BB, A2A4, B7-H3, B7-H4, BTLA, KIR, LAG3, PD-1, PD-L2, TIM-3 and VISTA. In a specific embodiment, the immune checkpoint modulator targets an immune checkpoint molecule selected from the group consisting of: CTLA-4, PD-L1 and PD-1. In another specific embodiment, the immune checkpoint modulator targets an immune checkpoint molecule selected from the group consisting of: PD-L1 and PD-1.

In some embodiments, more than one (e.g., 2, 3, 4, 5, or more) immune checkpoint modulator is administered to a subject. When more than one immune checkpoint modulator is administered, the modulator may each target a stimulatory immune checkpoint molecule, or each target an inhibitory immune checkpoint molecule. In other embodiments, the immune checkpoint modulator comprises at least one modulator that targets a stimulatory immune checkpoint and at least one immune checkpoint modulator that targets an inhibitory immune checkpoint molecule. In certain embodiments, the immune checkpoint modulator is a binding protein, such as an antibody. As used herein, the term "binding protein" refers to a protein or polypeptide that can specifically bind to a target molecule (e.g., an immune checkpoint molecule). In some embodiments, the binding protein is an antibody or antigen binding portion thereof, and the target molecule is an immune checkpoint molecule. In some embodiments, the binding protein is a protein or polypeptide that specifically binds to a target molecule (e.g., an immune checkpoint molecule). In some embodiments, the binding protein is a ligand. In some embodiments, the binding protein is a fusion protein. In some embodiments, the binding protein is a receptor. Examples of binding proteins useful in the methods of the invention include, but are not limited to, humanized antibodies, antibody Fab fragments, bivalent antibodies, antibody drug conjugates, scFv, fusion proteins, bivalent antibodies, and tetravalent antibodies.

As used herein, the term "antibody" refers to any immunoglobulin (Ig) molecule composed of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant or derivative thereof. Such mutant, variant or derived antibody forms are known in the art. In full length antibodies, each heavy chain consists of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH 3. Each light chain is composed of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is composed of one domain CL. The VH and VL regions can be further subdivided into regions of higher variability, termed Complementarity Determining Regions (CDRs), with more conserved regions, termed Framework Regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Immunoglobulin molecules may be of any type (e.g., igG, igE, igM, igD, igA and IgY), of any class (e.g., igG 1, igG2, igG 3, igG4, igA1, and IgA 2), or subclass. In some embodiments, the antibody is a full length antibody. In some embodiments, the antibody is a murine antibody. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is a humanized antibody. In other embodiments, the antibody is a chimeric antibody. Chimeric and humanized antibodies can be prepared by methods well known to those skilled in the art, including CDR grafting methods (see, e.g., U.S. Pat. nos. 5,843,708, 6,180,370, 5,693,762, 5,585,089 and 5,530,101), chain shuffling strategies (see, e.g., U.S. Pat. nos. 5,565,332; rader et al (1998) proc.nat' l.acad.sci.usa [ national academy of sciences of the united states of america ] 95:8910-8915), molecular modeling strategies (U.S. Pat. No. 5,639,641), and the like.

As used herein, the term "antigen-binding portion" of an antibody (or simply "antibody portion") refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen. It has been shown that the antigen binding function of antibodies can be performed by fragments of full length antibodies. Such antibody embodiments may also be in bispecific, bispecific or multispecific forms; for example, a multispecific form; specifically bind to two or more different antigens. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) A F (ab') 2 fragment, which is a bivalent fragment comprising two Fab fragments linked at the hinge region by a disulfide bridge; (iii) an Fd fragment consisting of VH and CH1 domains; (iv) Fv fragments consisting of the VL and VH domains of a single arm of an antibody; (v) dAb fragments (Ward et al (1989) NATURE [ Nature ]341:544-546; and WO 90/05144A1, the contents of which are incorporated herein by reference), which comprise a single variable domain; and (vi) an isolated Complementarity Determining Region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be formed as a single protein chain, in which the VL and VH regions pair to form a monovalent molecule (known as a single chain Fv (scFv); see, e.g., bird et al (1988) SCIENCE 242:423-426; and Huston et al (1988) PROC.NAT' L.ACAD.SCI.USA journal of national academy of SCIENCEs 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen binding portion" of the antibody. Other forms of single chain antibodies, such as diabodies, are also contemplated. Antigen binding moieties may also be incorporated into single domain antibodies, large antibodies (maxibodies), minibodies (minibodies), nanobodies, intracellular antibodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., hollinger and Hudson, nature Biotechnology [ Nature Biotechnology ]23:1126-1136,2005).

As used herein, the term "CDR" refers to a complementarity determining region within an antibody variable sequence. There are three CDRs in each of the variable regions of the heavy and light chains, designated CDR1, CDR2 and CDR3, respectively. As used herein, the term "set of CDRs" refers to a set of three CDRs present in a single variable region capable of binding an antigen. The exact boundaries of these CDRs have been defined differently for different systems. The system described by Kabat (Kabat et al National Institutes of Health, bethesda, md. [ national institutes of health, bessel da (1987) and (1991)) provides not only a well-defined residue numbering system for any variable region of an antibody, but also precise residue boundaries defining three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and its colleagues found that some of the sub-portions within the Kabat CDRs employed nearly identical peptide backbone conformations, although having great diversity at the amino acid sequence level (Chothia et al (1987) J.mol. BIOL. [ J.Molec. Biol. J. ]196:901-917, and Chothia et al (1989) NATURE [ Nature ] 342:877-883). These subfractions are designated as L1, L2 and L3 or H1, H2 and H3, where "L" and "H" represent the light and heavy chain regions, respectively. These regions may be referred to as Chothia CDRs, whose boundaries overlap with Kabat CDRs. Other boundaries defining CDRs overlapping Kabat CDRs have been described by the following documents: padlan et al (1995) FASEB J. [ journal of the American society of experimental biology ]9:133-139, and MacCallum et al (1996) J.mol.BIOL. [ journal of molecular biology ]262 (5): 732-45. Still other CDR boundary definitions may not strictly follow one of the above systems, but still overlap the Kabat CDRs, although they may be shortened or lengthened according to the following predictions or experiments: specific residues or groups of residues or even the entire CDR do not significantly affect antigen binding. Although the preferred embodiment uses CDRs defined by Kabat or Chothia, the methods used herein may utilize CDRs defined according to any of these systems.

As used herein, the term "humanized antibody" refers to a non-human (e.g., murine) antibody that is a chimeric immunoglobulin, immunoglobulin chain or fragment thereof (such as Fv, fab, fab ', F (ab') 2 or other antigen-binding subsequence of an antibody) that contains minimal sequence derived from a non-human immunoglobulin. In most cases, humanized antibodies and antibody fragments thereof are human immunoglobulins (recipient antibody or antibody fragment) in which residues from a Complementarity Determining Region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some cases, fv Framework Region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies/antibody fragments may comprise residues found in neither the recipient antibody nor the introduced CDR or framework sequences. These modifications may further improve and optimize the performance of the antibody or antibody fragment. Generally, a humanized antibody or antibody fragment thereof will comprise substantially all of at least one (typically two) variable domain, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or a substantial portion of the FR regions are those of a human immunoglobulin sequence. The humanized antibody or antibody fragment may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For more details, see Jones et al (1986) NATURE Nature 321:522-525; reichmann et al (1988) NATURE Nature 332:323-329; and Presta (1992) CURR.OP.STRUCT.BIOL [ recent views of structural biology ]2:593-596, each of which is incorporated herein by reference in its entirety.

As used herein, the term "immunoconjugate" or "antibody drug conjugate" refers to the attachment of an antibody or antigen binding fragment thereof to another agent, such as a chemotherapeutic agent, toxin, immunotherapeutic agent, imaging probe, or the like. The linkage may be covalent or non-covalent, such as by electrostatic forces. To form the immunoconjugate, various linkers known in the art may be used. In addition, the immunoconjugate may be provided in the form of a fusion protein that is expressible from a polynucleotide encoding the immunoconjugate. As used herein, "fusion protein" refers to a protein produced by the ligation of two or more genes or gene fragments that initially encode separate proteins (including peptides and polypeptides). Translation of the fusion gene produces a single protein with functional properties derived from each native protein.

"bivalent antibody" refers to an antibody or antigen-binding fragment thereof comprising two antigen-binding sites. The two antigen binding sites may bind the same antigen, or they may each bind different antigens, in which case the antibody or antigen binding fragment is characterized as "bispecific". "tetravalent antibody" refers to an antibody or antigen binding fragment thereof that comprises four antigen binding sites. In certain embodiments, the tetravalent antibody is bispecific. In certain embodiments, the tetravalent antibody is multispecific, i.e., binds to two or more different antigens.

Fab (fragment antigen binding) antibody fragments are immunoreactive polypeptides comprising a monovalent antigen binding domain of an antibody, which comprises a polypeptide consisting of a heavy chain variable region (V _H ) And heavy chain constant region 1 (C) _H1 ) Partially composed polypeptides and light chain variants (V _L ) Constant with light chain (C _L ) Partially composed polypeptides, wherein C _L And C _H1 The moieties are preferably bound together by disulfide bonds between Cys residues.

Immune checkpoint modulator antibodies include, but are not limited to, at least 4 major classes: i) Antibodies that block inhibitory pathways directly on T cells or Natural Killer (NK) cells (e.g., antibodies that target PD-1 such as nivolumab and pembrolizumab, antibodies that target TIM-3, and antibodies that target LAG-3, 2B4, CD160, A2aR, BTLA, CGEN-15049, and KIR), ii) antibodies that activate stimulatory pathways directly on T cells or NK cells (e.g., antibodies that target OX40, GITR, and 4-1 BB), iii) antibodies that block inhibitory pathways on immune cells or rely on antibody-dependent cytotoxicity to deplete inhibitory immune cell populations (e.g., antibodies that target CTLA-4 such as Ai Pili mab, antibodies that target PD-L2, gr1, and Ly 6G), and iv) antibodies that block inhibitory pathways directly on cancer cells or rely on antibody-dependent cytotoxicity to enhance cytotoxicity to cancer cells (e.g., rituximab, antibodies that target PD-L1, and antibodies that target B7-H7, B-4, and galsta 1 and Gal 9). Examples of checkpoint inhibitors include, for example, inhibitors of CTLA-4, such as Ai Pili mab or trimelimumab; inhibitors of the PD-1 pathway, such as anti-PD-1, anti-PD-L1 or anti-PD-L2 antibodies. Exemplary anti-PD-1 antibodies are described in WO 2006/121168, WO 2008/156712, WO2012/145493, WO 2009/014708, and WO 2009/114335. Exemplary anti-PD-L1 antibodies are described in WO 2007/005874, WO 2010/077634 and WO 2011/066389, and exemplary anti-PD-L2 antibodies are described in WO 2004/007679.

In particular embodiments, the immune checkpoint modulator is a fusion protein, e.g., a fusion protein that modulates the activity of the immune checkpoint modulator.

In one embodiment, the immune checkpoint modulator is a therapeutic nucleic acid molecule, e.g., a nucleic acid that modulates the expression of an immune checkpoint protein or mRNA. Nucleic acid therapeutics are well known in the art. Nucleic acid therapeutics include single-and double-stranded (i.e., nucleic acid therapeutics having a region of complementarity of at least 15 nucleotides in length) nucleic acids that are complementary to a target sequence in a cell. In certain embodiments, the nucleic acid therapeutic agent targets a nucleic acid sequence encoding an immune checkpoint protein.

Antisense nucleic acid therapeutics are single stranded nucleic acid therapeutics, typically about 16 to 30 nucleotides in length, and are complementary to a target nucleic acid sequence in a target cell in culture or organism.

In another aspect, the agent is a single stranded antisense RNA molecule. The antisense RNA molecule is complementary to a sequence within the target mRNA. Antisense RNA can inhibit translation in a stoichiometric manner by base pairing with mRNA and physically blocking the translation machinery, see Dias, N.et al, (2002) Mol Cancer Ther [ molecular Cancer therapy ]1:347-355. The antisense RNA molecule can have about 15-30 nucleotides complementary to the target mRNA. Patents relating to antisense nucleic acids, chemical modifications and therapeutic uses include, for example: us patent No. 5,898,031 relates to chemically modified RNA-containing therapeutic compounds; U.S. Pat. No. 6,107,094 relates to methods of using these compounds as therapeutic agents; us patent No. 7,432,250 relates to a method of treating a patient by administering a single-stranded chemically modified RNA-like compound; and us patent No. 7,432,249 relates to pharmaceutical compositions containing single-stranded chemically modified RNA-like compounds. U.S. patent No. 7,629,321 relates to a method of cleaving a target mRNA using a single stranded oligonucleotide having multiple RNA nucleosides and at least one chemical modification. The entire contents of each patent listed in this paragraph are incorporated herein by reference.

Nucleic acid therapeutics for use in the methods of the invention also include double-stranded nucleic acid therapeutics. As used interchangeably herein, "RNAi agent," "double stranded RNAi agent," double stranded RNA (dsRNA) molecules, also referred to as "dsRNA agent," "dsRNA," "siRNA," "iRNA agent," refers to a complex of ribonucleic acid molecules having a double stranded structure comprising two anti-parallel and substantially complementary (as defined below) nucleic acid strands. As used herein, RNAi agents can also include dsiRNA (see, e.g., U.S. patent publication 20070104688, which is incorporated herein by reference). Typically, the majority of the nucleotides of each strand are ribonucleotides, but as described herein, each or both strands may also include one or more non-ribonucleotides, such as deoxyribonucleotides and/or modified nucleotides. Furthermore, as used herein, an "RNAi agent" can include ribonucleotides with chemical modification; RNAi agents can include substantial modifications at multiple nucleotides. Such modifications may include all types of modifications disclosed herein or known in the art. For the purposes of the present specification and claims, any such modifications, as used in siRNA-type molecules, are encompassed by "RNAi agents". RNAi agents used in the methods of the invention include agents with chemical modifications, as disclosed, for example, in WO/2012/037254 and WO 2009/073809, the entire contents of each of which are incorporated herein by reference.

Immune checkpoint modulator may be administered at an appropriate dose to treat a neoplastic disorder, for example by using standard dosages. One skilled in the art will be able to determine, by routine experimentation, an effective non-toxic amount of immune checkpoint modulator for the purpose of treating a neoplastic disorder. Standard dosages of immune checkpoint modulator are known to those skilled in the art and may be obtained, for example, from product instructions provided by the manufacturer of the immune checkpoint modulator. Examples of standard doses of immune checkpoint modulator are provided in table 12 below. In other embodiments, the immune checkpoint modulator is administered at a dose that is different (e.g., lower) than the standard dose of the immune checkpoint modulator used to treat the tumor disorder at standard care for the particular tumor disorder.

TABLE 12 exemplary standard doses of immune checkpoint modulator

In certain embodiments, the dose of immune checkpoint modulator administered is 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% lower than the standard dose of immune checkpoint modulator for a particular tumor disorder. In certain embodiments, the dose of immune checkpoint modulator administered is 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of the standard dose of immune checkpoint modulator for a particular tumor disorder. In one embodiment, where a combination of immune checkpoint modulators is administered, at least one of the immune checkpoint modulators is administered at a dose that is lower than the standard dose of the immune checkpoint modulator for the particular tumor disorder. In one embodiment, where a combination of immune checkpoint modulator is administered, at least two of the immune checkpoint modulator are administered at a dose that is lower than the standard dose of immune checkpoint modulator for the particular tumor disorder. In one embodiment, where a combination of immune checkpoint modulators is administered, at least three of the immune checkpoint modulators are administered at a dose that is lower than the standard dose of immune checkpoint modulator for the particular tumor disorder. In one embodiment, where a combination of immune checkpoint modulators is administered, all of the immune checkpoint modulators are administered at a dose that is lower than the standard dose of immune checkpoint modulator for the particular tumor disorder.

Additional immunotherapeutic agents that may be administered in combination with the engineered immune cells of the invention include, but are not limited to, toll-like receptor (TLR) agonists, cell-based therapies, cytokines, and cancer vaccines.

2.TLR agonists

TLRs are single transmembrane, non-catalytic receptors that recognize structurally conserved molecules derived from microorganisms. The TLRs together with interleukin-1 receptors form a superfamily of receptors called the "interleukin-1 receptor/Toll-like receptor superfamily". Members of this family are structurally characterized by an extracellular leucine-rich repeat (LRR) domain, a conserved pattern of membrane-proximal cysteine residues, and an intracytoplasmic signaling domain that forms a platform for downstream signaling by recruiting TIR domain-containing adaptors (including MyD 88), TIR domain-containing adaptors (TRAP), and TIR domain-containing adaptors (induction of ifnβ) (tif) (O' Neill et al, 2007,Nat Rev Immunol [ natural comment immunology ]7,353).

TLRs include TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, and TLR10.TLR2 mediates cellular responses to a variety of microbial products including peptidoglycan, bacterial lipopeptides, lipoteichoic acid, mycobacterial lipoarabinomannan, and yeast cell wall components. TLR4 is a transmembrane protein that belongs to the family of Pattern Recognition Receptors (PRRs). Its activation leads to the intracellular signaling pathway NF- κb and inflammatory cytokine production, which are responsible for activating the innate immune system. TLR5 is known to recognize bacterial flagellin from invading mobile bacteria and has been shown to be involved in the onset of many diseases including inflammatory bowel disease.

TLR agonists are known in the art and described, for example, in US2014/0030294, which is incorporated herein by reference in its entirety. Exemplary TLR2 agonists include mycobacterial cell wall glycolipids, lipoarabinomannans (LAMs) and mannosylated Phosphatidylinositol (PIIM), MALP-2 and Pam3Cys, and synthetic variants thereof. Exemplary TLR4 agonists include lipopolysaccharide or synthetic variants thereof (e.g., MPL and RC 529) and lipid a or synthetic variants thereof (e.g., aminoalkyl glucosaminide 4-phosphate). See, e.g., cluff et al, 2005,Infection and Immunity [ infection and immunization ], pages 3044-3052:73; lembo et al, 2008,The Journal of Immunology [ J.Immunol. ]180,7574-7581; and Evans et al, 2003,Expert Rev Vaccines [ vaccine expert panel ]2:219-29. Exemplary TLR5 agonists include flagellin or synthetic variants thereof (e.g., pharmacologically optimized TLR5 agonists with reduced immunogenicity made by deleting a flagellin moiety not necessary for TLR5 activation, such as CBLB 502).

Other TLR agonists include the colestoxin and BCG. The collitoxin is a mixture of killed bacteria consisting of the species streptococcus pyogenes and serratia marcescens. See Taniguchi et al, 2006,Anticancer Res [ anti-cancer study ]26 (6A): 3997-4002.BCG was prepared from attenuated live Mycobacterium bovis, mycobacterium bovis (Mycobacterium bovis). See Venkataswamy et al 2012, vaccine [ vaccine ]30 (6): 1038-1049.

3.Cell-based therapies

Cell-based therapies for treating cancer include administering immune cells (e.g., T cells, tumor-infiltrating lymphocytes (TILs), natural killer cells, and dendritic cells) to a subject. In autologous cell-based therapies, the immune cells are derived from the same subject to which they are administered. In allogeneic cell-based therapies, immune cells are derived from one subject and administered to a different subject. The immune cells may be activated, for example, by treatment with cytokines prior to administration to a subject. In some embodiments, for example, in Chimeric Antigen Receptor (CAR) T cell immunotherapy, the immune cells are genetically modified prior to administration to a subject.

In some embodiments, the cell-based therapy comprises Adoptive Cell Transfer (ACT). ACT is generally composed of three parts: lymphocyte depletion, cell administration, and high dose IL-2 therapy. Cell types that can be administered in ACT include Tumor Infiltrating Lymphocytes (TILs), T Cell Receptor (TCR) transduced T cells, and Chimeric Antigen Receptor (CAR) T cells.

Tumor infiltrating lymphocytes are immune cells observed in many solid tumors, including breast cancer. They are cell populations comprising a mixture of cytotoxic T cells and helper T cells, B cells, macrophages, natural killer cells and dendritic cells. General procedure for autologous TIL therapy is as follows: (1) digesting the resected tumor into fragments; (2) Each fragment grows in IL-2 and lymphocyte proliferation destroys the tumor; (3) Amplifying the lymphocytes after the presence of the pure lymphocyte population; and (4) amplification to 10 ¹¹ After each cell, lymphocytes are injected into the patient. See Rosenberg et al 2015 science]348 (6230) 62-68, which is incorporated herein by reference in its entirety.

TCR-transduced T cells are produced by genetic induction of tumor-specific TCRs. This is typically accomplished by cloning a specific antigen-specific TCR into the retroviral backbone. Blood is drawn from the patient and Peripheral Blood Mononuclear Cells (PBMCs) are extracted. PBMCs were stimulated with CD3 in the presence of IL-2 and then transduced with retroviruses encoding antigen specific TCRs. These transduced PBMC were further expanded in vitro and infused back into the patient. See Robbins et al, 2015,Clinical Cancer Research [ clinical cancer Studies ]21 (5): 1019-1027, which is incorporated herein by reference in its entirety.

Chimeric Antigen Receptors (CARs) are recombinant receptors comprising an extracellular antigen recognition domain, a transmembrane domain, and a cytoplasmic signaling domain (e.g., cd3ζ, CD28, and 4-1 BB). The CAR has both antigen binding and T cell activation functions. Thus, T cells expressing CARs can recognize a variety of cell surface antigens, including glycolipids, carbohydrates, and proteins, and can attack malignant cells expressing these antigens by activating cytoplasmic co-stimulation. See Pang et al, 2018, mol Cancer 17:91, which is incorporated herein by reference in its entirety.

In some embodiments, the cell-based therapy is Natural Killer (NK) cell-based therapy. NK cells are large granular lymphocytes that have the ability to kill tumor cells without any prior sensitization or restriction of expression of Major Histocompatibility Complex (MHC) molecules. See Uppendahl et al, 2017,Frontiers in Immunology [ immunology preamble ]8:1825. Adoptive transfer of autologous lymphokine-activated killer (LAK) cells with high doses of IL-2 therapy has been evaluated in human clinical trials. Similar to LAK immunotherapy, cytokine Induced Killer (CIK) cells are generated from peripheral blood mononuclear cell cultures under stimulation with anti-CD 3 mAb, IFN- γ and IL-2. CIK cells are characterized by a mixed T-NK phenotype (cd3+cd56+), and show enhanced cytotoxic activity against ovarian and cervical cancers compared to LAK cells. Human clinical trials were also performed to investigate the adoptive transfer of autologous CIK cells following primary tumor mass reduction and adjuvant carboplatin/paclitaxel chemotherapy. See Liu et al, 2014,J Immunother J.Immunotherapy 37 (2): 116-122.

In some embodiments, the cell-based therapy is dendritic cell-based immunotherapy. Vaccination of Dendritic Cells (DCs) treated with tumor lysates has been shown to increase therapeutic anti-tumor immune responses in vitro and in vivo. See Jung et al 2018,Translational Oncology [ transforming oncology ]11 (3): 686-690. DCs capture and process antigens, migrate into lymphoid organs, express lymphocyte costimulatory molecules, and secrete cytokines that elicit immune responses. They also stimulate immune effector cells (T cells) expressing tumor-associated antigen-specific receptors and reduce the number of immunosuppressive factors (e.g., cd4+cd25+foxp3+ regulatory T (Treg) cells). For example, renal Cell Carcinoma (RCC) DC vaccination strategies based on tumor cell lysate-DC hybrids have shown therapeutic potential in preclinical and clinical trials. See Lim et al, 2007,Cancer Immunol Immunother [ cancer immunology and immunotherapy ]56:1817-1829.

4.Cytokines and methods of use

Several cytokines, including IL-2, IL-12, IL-15, IL-18 and IL-21 have been used in the treatment of cancer to activate immune cells, such as NK cells and T cells. IL-2 is one of the first cytokines used clinically, and it is desirable to induce antitumor immunity. IL-2 induces remission in certain Renal Cell Carcinoma (RCC) and metastatic melanoma patients as a single agent at high doses. Low doses of IL-2 have also been studied and the aim is to selectively link IL-2αβγ receptors (IL-2rαβγ) to reduce toxicity while maintaining biological activity. See Romee et al, 2014, scientific, volume 2014, article ID 205796, page 18, which is incorporated herein by reference in its entirety.

Interleukin 15 (IL-15) is a cytokine that is structurally similar to interleukin 2 (IL-2). Like IL-2, IL-15 binds and signals through a complex consisting of the IL-2/IL-15 receptor beta chain (CD 122) and a common gamma chain (gamma-C, CD 132). Recombinant IL-15 has been evaluated for the treatment of solid tumors (e.g., melanoma, renal cell carcinoma) and support of NK cells following adoptive transfer in cancer patients. See rome et al, as referenced above.

IL-12 is a heterodimeric cytokine composed of p35 and p40 subunits (IL-12α and β chains), and was originally identified as "NK cell stimulating factor (NKSF)" based on its ability to enhance NK cytotoxicity. Upon encountering pathogens, IL-12 is released by activated dendritic cells and macrophages and binds to its cognate receptor expressed primarily on activated T and NK cells. Many preclinical studies have shown that IL-12 has antitumor potential. See rome et al, as referenced above.

IL-18 is a member of the proinflammatory IL-1 family and, like IL-12, is secreted by activated phagocytes. IL-18 has shown significant anti-tumor activity in preclinical animal models and has been evaluated in human clinical trials. See Robertson et al, 2006,Clinical Cancer Research [ clinical cancer Studies ]12:4265-4273.

IL-21 has been used in anti-tumor immunotherapy because of its ability to stimulate NK cells and CD8+ T cells. For ex vivo NK cell expansion, membrane-bound IL-21 has been expressed in K562-stimulated cells with effective results. See Denman et al 2012, PLoS One [ public science library comprehensive ]7 (1) e30264. Recombinant human IL-21 has also been shown to increase soluble CD25 and induce perforin and granzyme B expression on cd8+ cells. IL-21 has been evaluated in several clinical trials for the treatment of solid tumors. See rome et al, as referenced above.

5.Cancer vaccine

Therapeutic cancer vaccines eliminate cancer cells by enhancing the patient's own immune response to cancer, particularly cd8+ T cell mediated responses, with the aid of a suitable adjuvant. The therapeutic efficacy of cancer vaccines depends on the differential expression of tumor-associated antigens (TAAs) by tumor cells relative to normal cells. TAAs are derived from cellular proteins and should be expressed predominantly or selectively on cancer cells to avoid immune tolerance or autoimmune effects. See Circelli et al 2015, vaccine [ vaccine ]3 (3): 544-555. Cancer vaccines include, for example, dendritic Cell (DC) based vaccines, peptide/protein vaccines, genetic vaccines and tumor cell vaccines. See Ye et al, 2018, J Cancer journal 9 (2): 263-268.

The combination therapy of the invention may be used to treat a neoplastic disorder. In some embodiments, the combination therapy of the engineered immune cells of the invention and additional therapeutic agents inhibits tumor cell growth. Accordingly, the invention further provides a method of inhibiting tumor cell growth in a subject, the method comprising administering to the subject an immune cell engineered to comprise one or more heterologous polynucleotides that promote saenox transfer and/or polynucleotides encoding Chimeric Antigen Receptors (CARs), and at least one additional therapeutic agent such that tumor cell growth is inhibited. In certain embodiments, treating the cancer comprises extending survival or extending time to tumor progression as compared to a control. In some embodiments, the control is a subject treated with an additional therapeutic agent but not with an engineered immune cell. In some embodiments, the control is a subject treated with the engineered immune cells but not with an additional therapeutic agent. In some embodiments, the control is a subject not treated with an additional therapeutic agent or an engineered immune cell. In certain embodiments, the subject is a human subject. In some embodiments, the subject is identified as having a tumor prior to administration of the first dose of the engineered immune cell or the first dose of the additional therapeutic agent. In certain embodiments, the subject has a tumor at the first administration of the engineered immune cells or at the first administration of the additional therapeutic agent.

In certain embodiments, a subject is administered at least 1, 2, 3, 4, or 5 cycles of a combination therapy comprising an engineered immune cell of the invention and one or more additional therapeutic agents. The subjects were evaluated for response criteria at the end of each cycle. Adverse events (e.g., coagulation, anemia, liver and kidney function, etc.) of the subject are also monitored throughout each cycle to ensure that the treatment regimen is adequately tolerated.

It should be noted that more than one additional therapeutic agent (e.g., 2, 3, 4, 5, or more additional therapeutic agents) may be administered in combination with the engineered immune cells of the invention.

In one embodiment, administration of the engineered immune cells of the invention and the additional therapeutic agents described herein results in one or more of the following: decreasing tumor size, weight, or volume, increasing progression time, inhibiting tumor growth, and/or extending survival time of a subject with a tumor disorder. In certain embodiments, administration of the engineered immune cells and the additional therapeutic agent reduces tumor size, weight, or volume, increases time of progression, inhibits tumor growth, and/or increases survival time of the subject by at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, or 500% relative to a corresponding control subject administered the engineered immune cells but not administered the additional therapeutic agent. In certain embodiments, administration of the engineered immune cells and the additional therapeutic agent reduces tumor size, weight or volume, increases time of progression, inhibits tumor growth, and/or increases survival time of a population of subjects with a tumor disorder by at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, or 500% relative to a corresponding population of control subjects with a tumor disorder to which the engineered immune cells were administered but to which the additional therapeutic agent was not administered. In other embodiments, administration of the engineered immune cells and the additional therapeutic agent stabilizes the tumor disorder in the subject having the progressive tumor disorder prior to treatment.

In certain embodiments, treatment with the engineered immune cells of the invention and an additional therapeutic agent (e.g., an immunotherapeutic agent) is combined with an additional anti-neoplastic agent, such as standard care for treating the particular cancer to be treated, e.g., by administering a standard dose of one or more anti-neoplastic agents (e.g., chemotherapeutic agents). Standard of care for a particular cancer type can be determined by one skilled in the art based on, for example, the type and severity of the cancer, the age, weight, sex, and/or medical history of the subject, and the success or failure of previous treatments. In certain embodiments of the invention, standard of care includes any one or combination of surgery, radiation, hormone therapy, antibody therapy, growth factor therapy, cytokines, and chemotherapy. In one embodiment, the additional anti-neoplastic agent is not an iron-dependent cell disassembly inducing agent and/or an immune checkpoint modulator.

Additional antineoplastic agents suitable for use in the methods disclosed herein include, but are not limited to, chemotherapeutic agents (e.g., alkylating agents such as altretamine, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, lomustine, melphalan, oxaliplatin, temozolomide, thiotepa); antimetabolites such as 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP); capecitabine Cytarabine->Fluorouridine, fludarabine, gemcitabineHydroxyurea, methotrexate, pemetrexed +.>Antitumor antibiotics, such as anthracyclines (e.g. daunorubicin, doxorubicin +.>Epirubicin, idarubicin), actinomycin-D, bleomycin, mitomycin-C, mitoxantrone (also used as topoisomerase II inhibitor); topoisomerase inhibitors such as topotecan, irinotecan (CPT-11), etoposide (VP-16), teniposide, mitoxantrone (also used as an antitumor antibiotic); mitotic inhibitors such as docetaxel, estramustine, ixabepilone, paclitaxel, vinblastine, vincristine, vinorelbine; corticosteroids, e.g. prednisone, methylprednisolone->DexamethasoneEnzymes, such as L-asparaginase and bortezomib +>Antitumor agents also include biological anticancer agents, such as anti-TNF antibodies, e.g., adalimumab or infliximab; anti-CD 20 antibodies, such as rituximab, anti-VEGF antibodies (e.g., bevacizumab); anti-HER 2 antibodies, such as trastuzumab; anti-RSV, such as palivizumab.

B. Infectious diseases

As provided herein, immune cells engineered to include polynucleotides that promote sano delivery can induce or increase immune activity in immune cells (e.g., T cells, B cells, NK cells, etc.) endogenous to a subject, and thus can enhance immune cell function, such as inhibiting bacterial and/or viral infection, and/or restoring immune surveillance and immune memory function to treat infection. Thus, in some embodiments, the engineered immune cells of the invention are used to treat an infection or an infectious disease, such as a chronic infection, in a subject.

As used herein, the term "infection" refers to any state in which a cell or tissue of an organism (i.e., a subject) is infected with an infectious agent (e.g., a subject has an intracellular pathogen infection, such as a chronic intracellular pathogen infection). As used herein, the term "infectious agent" refers to a foreign biological entity (i.e., pathogen) in at least one cell of an infected organism. For example, infectious agents include, but are not limited to, bacteria, viruses, protozoa, and fungi. Intracellular pathogens are of particular concern. Infectious diseases are disorders caused by infectious agents. Under certain conditions, some infectious agents do not cause identifiable symptoms or diseases, but under varying conditions may cause symptoms or diseases. These subject methods may be used to treat chronic pathogen infections including, but not limited to, viral infections such as retroviruses, lentiviruses, hepatitis viruses, herpes viruses, poxviruses, or human papillomaviruses; intracellular bacterial infections such as mycobacteria (Mycobacterium), chlamydophila (Chlamydophila), eirickettsia (Ehrlichia), rickettsia (Rickettsia), brucella (Brucella), legionella (Legionella), francissamella (francissela), listeria (Listeria), ke Kesi (Coxiella), neisseria (Neisseria), salmonella (Salmonella), yersinia species (Yersinia sp) or helicobacter pylori (Helicobacter pylori); and intracellular protozoan pathogens such as Plasmodium species (plasmmodium sp), trypanosoma species (Trypanosoma sp.), giardia species (Giardia sp.), toxoplasma species (Toxoplasma sp.) or Leishmania species (Leishmania sp.).

Infectious diseases that can be treated using the compositions described herein include, but are not limited to: HIV, influenza, herpes, giardia, malaria, leishmania, viral hepatitis (type A, type B or type C), herpes viruses (e.g., VZV, HSV-I, HAV-6, HSV-II and CMV, epstein Barr virus (Epstein Barr virus)), adenoviruses, influenza viruses, flaviviruses, echoviruses, rhinoviruses, coxsackieviruses, coronaviruses, respiratory syncytial viruses, mumps viruses, rotaviruses, measles viruses, rubella viruses, parvoviruses, vaccinia viruses, HTLV viruses, dengue viruses, papillomaviruses, mollusc viruses, polio viruses, rabies viruses, JC viruses and pathogen infections caused by arboencephalitis viruses, bacterial Chlamydia, rickettsia bacteria, mycobacteria, staphylococci, streptococci, pneumococci, meningococci and gonococci (conocci), klebsiella, protebuconas, serratia, pseudomonas, escherichia coli, legionella, diphtheria, salmonella, bacillus, cholera, tetanus, botulism, anthrax, plague, leptospirosis and infection with pathogens by Lyme disease bacteria, candida fungi (Candida albicans, candida krusei, candida glabrata, candida tropicalis, etc.), cryptococcus neoformans, aspergillus (Aspergillus fumigatus, aspergillus niger, etc.), mucor (Mucor, absidia, rhizopus), sporothecium, rhizopus dermatitis, paracoccus brasiliensis, coccidioides and histoplasma pathogen infections, proteus parasitic dysentery, tacrobag of colon, pathogen infection by naecognia flexneri, acanthamoeba species, giardia lamblia, cryptosporidium species, pneumosporidium californicum, plasmodium vivax, babesia tenuifolia, trypanosoma brucei, trypanosoma cruzi, leishmania donovani, toxoplasma gondii, and/or strongyloides brasiliensis.

The term "chronic infection" refers to an infection that lasts for about one month or more, for example, at least one month, two months, three months, four months, five months, or six months. In some embodiments, the chronic infection is associated with increased production of anti-inflammatory chemokines in and/or around the infected area or areas. Chronic infections include, but are not limited to, HIV infection, HCV infection, HBV infection, HPV infection, hepatitis b infection, hepatitis c infection, EBV infection, CMV infection, TB infection, and intracellular bacterial or parasitic infection. In some embodiments, the chronic infection is a bacterial infection. In some embodiments, the chronic infection is a viral infection.

IX. pharmaceutical compositions and modes of administration

In certain aspects, the disclosure relates to a pharmaceutical composition comprising an immune cell engineered to comprise one or more heterologous polynucleotides that promote sano delivery and/or polynucleotides encoding Chimeric Antigen Receptors (CARs). In some embodiments, the composition comprises an amount of immune cells sufficient to induce a biological response in the target cells. The pharmaceutical compositions described herein may be administered to a subject in any suitable formulation. The preferred form depends on the intended mode of administration and therapeutic application.

In certain embodiments, the pharmaceutical compositions are suitable for parenteral administration, including intravenous, intraperitoneal, intramuscular, and subcutaneous injection. In particular embodiments, the pharmaceutical composition is suitable for intravenous administration. In another particular embodiment, the pharmaceutical composition is suitable for intratumoral administration.

Pharmaceutical compositions for parenteral administration comprise aqueous solutions of the active compounds in water-soluble form. For intravenous administration, the formulation may be an aqueous solution. The aqueous solution may include Hank's solution, ringer's solution, phosphate Buffered Saline (PBS), physiological saline buffer, or other suitable salts or combinations to achieve the appropriate pH and osmotic pressure for the parenteral delivery formulation. The aqueous solution may be used to dilute the formulation to a desired concentration for administration. The aqueous solution may contain substances that increase the viscosity of the solution, such as sodium carboxymethyl cellulose, sorbitol, or dextran. In some embodiments, the formulation includes a phosphate buffered saline solution comprising disodium hydrogen phosphate, potassium dihydrogen phosphate, potassium chloride, sodium chloride, and water for injection.

It will be apparent to those skilled in the art that the useful in vivo dosage to be administered and the particular mode of administration will vary depending upon the age, weight, severity of the illness and the mammalian species being treated, the particular compounds employed, and the particular use for which these compounds are employed. The skilled artisan can determine effective dosage levels, i.e., the dosage levels required to achieve the desired result, using routine methods, such as human clinical trials, animal models, and in vitro studies.

In certain embodiments, the composition is administered parenterally. In certain embodiments, the composition is delivered by injection or infusion. In one embodiment, the compositions provided herein can be administered by direct injection into a tumor. In some embodiments, the composition may be administered by intravenous injection or intravenous infusion. In certain embodiments, the administration is systemic. In certain embodiments, the administration is topical.

Examples

The invention is further illustrated by the following examples, which should not be construed as limiting. The contents of all references, genBank accession numbers and gene numbers, and published patents and patent applications cited throughout this application are hereby incorporated by reference. Those skilled in the art will recognize that the invention can be practiced with modification of the disclosed structures, materials, compositions and methods, and that such modifications are considered to be within the scope of the invention.

Example 1. An engineered T cell comprising a polypeptide that promotes sano delivery and a CAR alone is prepared, wherein the CAR intracellular signaling domain comprises a costimulatory domain and an intracellular signaling domain of cd3ζ.

Production of the vector

The gene encoding the CAR construct was synthesized and cloned into a lentiviral expression vector (CAR vector). CAR consists of scFv against target antigen, hinge and transmembrane domains derived from human CD8a, costimulatory domains (e.g. CD28 or 4-1 BB) and intracellular signaling domains of human cd3ζ. In addition to CAR, the construct contains a fluorescent selectable marker (such as ZS-green) following the ribosome jump site (P2A, construct outlined in fig. 1A). Gene modules promoting Sano delivery, such as those described in examples 5-10 below, are synthesized and cloned into a separate lentiviral vector downstream of an activation-induced T cell promoter, such as NF-AT (Thano vector). In addition to the sano delivery module, the construct also contains a fluorescent selectable marker (such as DT-tomato red) following the ribosome jump site (F2A, construct outlined in fig. 1B).

Generating lentivirus stock solution

293T cells were plated the day prior to transfection. Lentiviral stocks for CAR generation were generated by transfecting 293T cells with CAR vector DNA along with lentiviral packaging mixtures. Separate lentiviral stocks for the Thano vector were prepared by transfecting 293T cells with the Thano vector DNA along with the lentiviral packaging mixture. Viral stocks were harvested 24 hours and 48 hours post-transfection and filtered through a 0.22uM filter to prepare lentiviral stocks.

Generating CAR T cells

Pan T cells were isolated from PBMCs from healthy human donors. The isolated T cells were activated with the activation beads for 2-3 days and the beads were removed. Lentiviral stock was added to activated T cells to transduce T cells. T cells were transduced with CAR vector or thano vector or both to prepare control CAR T cells, control thano T cells and thano CAR T cells, respectively. 1-3 days after transduction, transduced T cells are transferred to large scale expansion cultures in the presence of growth-supporting cytokines (e.g., human IL-2). Cells were harvested between day 8 and day 12 for the experiment. In some experiments, cells are purified based on the expression of fluorescent or selectable markers either before or after expansion.

Example 2 evaluation of in vitro engineered T cell function.

CAR T cell phenotype

The phenotype of CAR T cells generated as described in example 1 above was assessed by flow cytometry to detect markers of T cell senescence, depletion and function, e.g., CD4, CD8, CD25, 4-1BB, CD27, KLRG1, CD57. CAR T cells were also evaluated for metabolic fitness, for example using assays directed to mitochondrial function and redox status. The phenotype of CAR T cells is assessed in the presence or absence of their target antigen.

CAR T cell function and Sano delivery

Luciferase-labeled human tumor cells (e.g., BXPC-1, capan-2, HT-1080, HT-29) were plated in flat bottom 96-well plates at a density of 5000-10000 cells/well. CAR T cells were added at a T cell:target ratio in the range of 10:1 to 0.1:1. After 24 hours, supernatants from these co-cultures were harvested and incubated with reporter cell lines to measure the effect on NF-Kb and IRF activity. The levels of cytokines (e.g., IL-2, IFNγ) in the supernatants were also measured. The extent of target killing was determined by measuring the level of residual luciferase activity. In other experiments, the T cell killing kinetics of the target were determined by continuous live cell imaging of plates containing various ratios of target cells and T cells.

Example 3. CAR T cell function was evaluated in an immunodeficient mouse model of cancer.

Human tumor cells expressing different levels of antigen targets (e.g., BXPC-1, capan-2, ASC-1) were subcutaneously implanted into the flank of immunodeficient NSG mice. Once the tumor has formed, mice are treated once with different doses of CAR T ranging from 1 x 10 per mouse ⁵ Up to 1X 10 ⁷ . Tumor growth kinetics were monitored and the effect of different CAR T cells on tumor growth was assessed. In some experiments, mice were re-challenged with new tumors on the opposite flanks to evaluate the persistence of the anti-tumor CAR T cell response.

Example 4. Evaluation of murine CAR T cells in an immunocompetent mouse model of cancer.

For studies assessing the effect of CAR T cell activity on the intact immune system, the CAR constructs depicted in fig. 1A and 1B were murine. The construct is cloned into a retroviral backbone (e.g., SFG), where the VH and VL domains target human antigens, and the human is replaced with the mouse CD28 and mouse CD3 zeta signaling domains, thus stimulating mouse T cells. These constructs were used to generate stable packaging cell lines and primary murine T cells were genetically modified as described previously (Lee et al, cancer Res [ Cancer Industry ]2011,71 (8): 2871). The mouse CAR T cells are cultured with mouse tumor cells (e.g., B16 or CT 26) modified to express a human antigen target (e.g., mesothelin).

The B16 or CT26 tumors expressing the cognate antigen were subcutaneously implanted into WT mice. Once the tumor became palpable, mice were treated with murine CAR T and tumor growth kinetics were monitored. In addition, the host immune response to tumors in the presence of CAR T was monitored. In some experiments, checkpoint inhibitor antibodies (e.g., anti-mouse PD-1) and CAR T cells were also administered and the effect on tumor growth and host immune response was evaluated.

Example 5 cell death was induced in CT-26 mouse colon cancer cells expressing one or more Sanot delivery polypeptides.

CT-26 mouse colon cancer cells (ATCC; CRL-2638) were transduced with lentiviruses derived from pLVX-Tet3G vector (Takara; 631358) to establish stable Tet-On transactivator expression by the human PGK promoter. In the Tet-On system, gene expression may be induced by doxycycline. All lentiviral transduction was performed using 293T cells (ATCC; CRL-3216) and lentiviral packaging mixtures (Bosetta Corp. (Biosettia); pLV-PACK) using standard production protocols. CT-26-Tet3G cells were then transduced with lentiviruses expressing the human TRIF ORF (accession number NM-182919) in pLVX-TRE3G (Takara Shuzo Co., ltd.; 631193). Alternatively or additionally, CT-26-Tet3G cells were transduced with vectors expressing the mouse RIPK3ORF (accession number: NM-019955.2); RIPK3 expression was driven by a constitutive PGK promoter derivative of pLV-EF1a-MCS-IRES-Hyg (Bosita; cDNA-pLV 02). The two ORFs were modified by adding two DmrB domains (Takara Shuzo Co., ltd.; 635059) in tandem that oligomerize upon binding to the B-B ligand, to allow protein activation using the B/B homodimer (1. Mu.M) to promote oligomerization. After the initial test, dimerization with B/B had no substantial effect on the activity of the tif construct, but did promote the activity of the RIPK3 expressing construct. Thus, in all subsequent experiments, B/B-induced dimerization was not used to activate any construct including tif, but only a single construct expressing RIPK 3. Thus, B/B dimer was included in the experimental setup to ensure that all groups of experimental conditions were comparable, although it had no effect on the activity induced by tif. For example, as shown in FIG. 3B and described in example 6, the addition of dimer had little effect on IRF activity in macrophages treated with cell cultures from the engineered CT-26 cells described above.

CT26 mice colon cancer cells expressing the indicated Sano delivery module were inoculated and subsequently treated with doxycycline (1 mg/mL; sigma Aldrich, sigma Aldrich), 0219895525) and B/B homodimer (1. Mu.M) for 24 hours to promote expression and protein activation via oligomerization. Relative cell viability was determined 24 hours after treatment using the realtem-Glo MT cell viability assay kit (prolog, cat# G9712) according to the manufacturer's instructions and the plot shows relative viability measured by Relative Luminescence Units (RLUs).

As shown in FIG. 2A, induced expression and oligomerization of TRIF, RIPK3 or TRIF+RIPK3 induced a decrease in cell viability relative to the CT-26-Tet3G (Tet 3G) parental cell line. These results demonstrate that expression of one or more saenopassing polypeptides in cancer cells reduces the viability of the cancer cells.

In a separate experiment, the effect of the expression of the desetin E (GSDME) in the cancer cells expressing the TRIF, RIPK3 or both the TRIF and RIPK3 was examined. CT-26-Tet3G cells were transduced with human GSDME (NM-004403.3) cloned into pLV-EF1a-MCS-IRES-Puro vector (Bosita). GSDME was also transduced into CT-26-Tet3G-TRIF and CT26-Tet3G-TRIF-RIPK3 cells as described above. These cells were inoculated and subsequently treated with doxycycline (1 mg/mL; sigma aldrich, 0219895525) for 24 hours to promote expression. Relative cell viability was determined 24 hours after treatment using the realtem-Glo MT cell viability assay kit (prolog, cat# G9712) according to the manufacturer's instructions and the plot shows relative viability measured by Relative Luminescence Units (RLUs). B/B dimer was not used in these experiments.

As shown in FIG. 2B, expression of TRIF and TRIF+RIPK3 reduced cell viability relative to the CT-26-Tet3G parental cell line, confirming the results presented in FIG. 2A. In addition, induction of TRIF or TRIF+RIPK3 protein expression in GSDME-expressing cells also reduced cell viability compared to CT-26-Tet3G parent cells. Taken together, these results demonstrate that expression of one or more saenox transfer polypeptides (including TRIF, RIPK3, and GSDME) in cancer cells reduces the viability of the cancer cells.

Example 6 Effect of Cell Turnover Factor (CTF) from CT-26 mouse colon cancer cells expressing one or more Sanot delivering polypeptides on Interferon Stimulatory Gene (ISG) reporter genes in macrophages

J774-Dual ^TM Cells (Invivogen, J774-NFIS) were seeded at 100,000 cells/well in 96-well plates. J774-Dual ^TM Cells are induced by stable integration of twoIs derived from a mouse J774.1 macrophage-like cell line. These cells express a Secreted Embryonic Alkaline Phosphatase (SEAP) reporter gene under the control of an IFN- β minimal promoter fused to five copies of an NF- κb transcription response element and three copies of a c-Rel binding site. J774-Dual ^TM The cells also expressed the luciferases gene encoding secreted luciferases under the control of the ISG54 minimal promoter along with five Interferon Stimulated Response Elements (ISREs). As a result, J774-Dual ^TM Cells allow for the investigation of the NF- κB pathway by assessing SEAP activity and at the same time the Interferon Regulatory Factor (IRF) pathway by monitoring the activity of the luciferases.

As described in example 5 above, a medium containing Cell Turnover Factor (CTF) was produced from CT-26 mouse colon cancer cells. In addition to the sano delivery module described in example 5, additional RIPK3 constructs containing the full Tet-inducible promoter were also evaluated. This Tet-inducible RIPK3 is designated "RIPK3" in fig. 3A, and the RIPK3 construct containing the PGK promoter (described in example 5) is designated "pgk_ripk3" in fig. 3A.

Controls were also included that were predicted to induce cell death without immunostimulatory saenox delivery. These control constructs expressed i) the C-terminal caspase truncate of human Bid (nm_ 197966.3), ii) the N-terminal caspase truncate of human GSDMD (nm_ 001166237.1), iii) the synthetic dimerizable form of human caspase-8 (DmrB-caspase-8), or iv) both DmrB-caspase-8 and human GSDME (nm_ 004403.3). Then stimulate J774-Dual with the indicated CTF ^TM Cells were kept for 24 hours. Cell culture media was collected and luciferase activity was measured using a QUANTI-Luc (Injetty; rep-qlc 1) assay. Interferon Stimulated Response Element (ISRE) promoter activation was mapped relative to control cell line CT-26-Tet 3G.

As shown in FIG. 3A, in the examined CT-26 cell line, only the medium collected from cells expressing TRIF (alone or in combination with RIPK 3) was found in J774-Dual ^TM ISRE/IRF reporter activation is induced in the cells.

In a separate experiment, the combination of desetin E (GSDME) andeffect of combined expression of TRIF or TRIF+RIPK3. CTF-containing medium was produced from CT-26 cells expressing TRIF or TRIF+RIPK3 as described in example 5, and additionally from CT-26 cells expressing TRIF+Xiao-in-E or TRIF+RIPK3+Xiao-in-E. As shown in FIG. 3B, media from CT-26 cells expressing TRIF (iTRIF), TRIF+RIPK3 (iTRIF_c3), TRIF+Xiaogetin-E (iTRIF_ cGE) or TRIF+RIPK3+Xiaogetin-E (iTRIF_c3_ cGE) were each in J774-Dual ^TM ISRE/IRF reporter activation is induced in the cells. As discussed in example 5, the addition of dimer had little effect on ISRE/IRF reporter activation.

Taken together, these results demonstrate that CTF produced by cancer cells expressing one or more sanopassing polypeptides activates an immunostimulatory pathway (i.e., IRF pathway) in immune cells.

Example 7 Effect of Cell Turnover Factor (CTF) from CT-26 mouse colon cancer cells expressing one or more Sanodelivery polypeptides on bone marrow derived dendritic cells (BMDC)

Bone marrow cells were differentiated into dendritic cells using GM-CSF-rich RPMI medium for 8 days. 400,000 cells per 2ml were seeded in 6-well plates. On day 8, bone marrow-derived dendritic cells (BMDCs) were harvested and 100,000 cells/well were seeded in 96-well plates. BMDC were then stimulated with medium containing CTF derived from engineered CT-26 cells as described in example 5. At 24 hours, stimulated cells were harvested and expression of cell surface markers CD86, CD40 and PD-L1 was measured by flow cytometry and the Mean Fluorescence Intensity (MFI) was plotted against a Tet3G control. The sources of antibodies were as follows: CD86 (Biolegend, catalog number 105042); CD40 (bai sheng technology company, catalog number 102910); PD-L1 (Bai Sheng technology Co., catalog number 124312). Expression of the cell surface markers CD86, CD40 and PD-L1 are indicative of dendritic cell maturation.

As shown in FIG. 4, in the examined CT-26 cell line, only the medium collected from cells engineered to express TRIF (alone or in combination with RIPK 3) increased cell surface expression of CD86, CD40 or PD-L1. These results indicate that CTF from CT-26 cells engineered to express either TRIF or both TRIF and RIPK3 induces maturation of dendritic cells. Upregulation of CD86 and CD40 in dendritic cells indicates an increased ability to activate T cells. Thus, the results indicate that CTF from cancer cells engineered to express either tif or tif and RIPK3 will induce maturation of dendritic cells and increase their ability to activate T cells.

Example 8. Sano delivery polypeptide expression alone or in combination with anti-PD 1 antibodies affects tumor growth and survival in a mouse model of colon cancer.

CT-26 mouse colon cancer cells carrying the TRIF or TRIF+RIPK3 Sano delivery module as described in example 5 were trypsinized and treated at 1X 10 ⁶ Individual cells/mL were resuspended in serum-free medium. Cells (100 mL) were injected into the right subcutaneous flank of BALB/c mice. Conventional drinking water was supplemented with 2mg/ml doxycycline (sigma aldrich, cat# D9891) from day 11 to day 18 after CT-26 cell injection to induce the expression of the sanoxacin, and 2mg/kg B/B homodimer (bao biosystems, cat# 632622) was administered by daily IP injection from day 11 to day 18. anti-PD 1 antibodies (BioXcell, catalog No. BP 0273) and isotype controls were administered on days 14, 17 and 21. When the tumor reached 2000mm according to IACUC guidelines ³ Or at the end of the experiment, mice were euthanized.

As shown in FIG. 5A, expression of TRIF (CT 26-TF) alone increased survival compared to CT-26-Tet3G control (Tet 3G-isotype control) and CT26-RIPK3 cells (CT 26-P_R3), and even greater benefit was observed with the combination of TRIF and RIPK3 (Trif_RIPK3-isotype control). As shown in FIG. 5B, survival of mice injected with either TRIF-bearing CT-26 cells (CT 26-TF) or TRIF+RIPK3-bearing CT-26 cells (TRIF_RIPK3) was increased by treatment with anti-PD-1 antibodies, both treatment groups showed 100% survival (line overlap).

In a separate experiment, CT-26 mouse colon cancer cells carrying the TRIF+GSDME and TRIF+RIPK3+GSDME Sano delivery modules described in example 6 were trypsinized and treated with 1X 10 ⁶ Individual cells/mL were resuspended in serum-free medium. The B/B homodimer was not used in this experiment. Cells (100 mL) were injected into the right subcutaneous flank of BALB/c mice. From CT-26Mice were fed a Teklad basal diet supplemented with 625mg/kg doxycycline hydrochloride (envigor company td.01306) on days 15 to 21 after the cell injection. When the tumor reached 2000mm according to IACUC guidelines ³ Or at the end of the experiment, mice were euthanized.

As shown in fig. 5C, expression of GSDME in combination with either tif or tif+ripk3 further enhanced survival relative to mice implanted with tumors expressing either tif alone or tif-RIPK 3 alone.

Example 9 influence of chemical caspase inhibitors on U937 human myeloid leukemia cells expressing the Sanopassing polypeptide

U937 human myeloid leukemia cells and THP1-Dual cells were obtained from ATCC and England, respectively. U937 is a myeloid leukemia cell line. U937 cells expressing human Sano delivery polypeptides (tBId, caspase 8, RIPK3, or TRIF) were generated using the methods described in examples 5 and 6 and the doxycycline-inducible expression system described in example 5.

THP1-Dual cells are human monocyte lines that induce reporter proteins upon activation of NF-kB or IRF pathways. It expresses a Secreted Embryonic Alkaline Phosphatase (SEAP) reporter driven by an IFN- β minimal promoter fused to five copies of an NF- κb consensus transcriptional response element and three copies of a c-Rel binding site. THP1-Dual cells are also characterized by the Lucia gene, a secreted luciferase reporter under the control of the ISG54 minimal promoter along with five IFN stimulated response elements. As a result, THP1-Dual cells allowed the investigation of NF-kB pathways by monitoring the activity of SEAP, and at the same time, IRF pathways by assessing the activity of secreted luciferase (Lucia).

To generate conditioned medium, 5 million U937-tet3G, U937-tBId, U937-caspase 8, U937-RIPK3, or U937-TRIF cells were inoculated in RPMI in 10cm dishes, followed by treatment with doxycycline (1 μg/mL) for 24 hours to induce expression. B/B homodimers (100 nM) were added to U937-caspase 8, U937-RIPK3 and U937-TRIF cell cultures to promote expression and protein activation by oligomerization. In addition, U937-TRIF cells were additionally treated with 4. Mu. M Q-VD-Oph (ubiquitin caspase inhibitor), 10. Mu.M GSK872 (RIPK 3 inhibitor), or a combination of both. After 24 hours incubation of the cells, conditioned medium was harvested and sterile filtered.

To measure the effect of the sanoxatransfer polypeptides on NF-kB or IRF reporter gene expression, 100,000 THP1-Dual cells/well were seeded in 96-well flat-bottom plates at a volume of 100 μl. To each well 100 μl of conditioned medium generated by U937 cells expressing the sanoχ transfer module was added. After a 24 hour incubation period, 20 μl of THP1-Dual cell culture supernatant was transferred to flat bottom 96 well white (opaque) assay plates and 50 μl of QUANTI-Luc assay solution was added to each well, immediately followed by reading luminescence by a plate reader. To measure NF-kB activity, 20. Mu.l of THP1-Dual culture supernatant was transferred to a flat bottom 96 well transparent assay plate and 180. Mu.l of resuspended QUANTI-Blue solution was added to each well. Plates were incubated at 37℃for 1 hour and SEAP levels were measured at 655nm using a plate reader.

As shown in FIGS. 6A and 6B, treatment of THP-1Dual cells with cell cultures from U937-TRIF cells treated with caspase inhibitor alone (Q-VD-Oph) or in combination with RIPK3 inhibitor (Q-VD-Oph+GSK872) greatly increased NF-kB activation and IRF activity. (in FIGS. 6A to 6C, + + represents U937 cells treated with doxycycline, and++ represents U937 cells treated with doxycycline and B/B homodimer). Cell culture media from U937-TRIF cells treated with RIPK3 inhibitor alone had little effect on NF-kB activation of THP-1Dual cells, indicating that increased NF-kB activation was due to caspase inhibition. As shown in FIGS. 6B and 6C, treatment of THP-1Dual cells with cell culture media from U937-TRIF cells not treated with caspase inhibitors also increased IRF activity, although to a lesser extent than U937-TRIF cells treated with caspase inhibitors.

Taken together, these results demonstrate that CTF produced by human cancer cells expressing tif activates immunostimulatory pathways (i.e., NF-kB and IRF pathways) in immune cells, and caspase inhibition enhances this effect.

Example 10. Sano delivery in CT-26 murine colon cancer cells is modulated by expression of Sano delivery polypeptides comprising a combination of caspase inhibitor proteins.

The experiments described in this example test the effect of caspase inhibitor protein expression on saenox transmission in cancer cells expressing TRIF and RIPK 3.

CT26 mouse colon cancer cells expressing the sanoxatransfer polypeptides TRIF and RIPK3 were transduced with the genes encoding: (i) A dominant negative version of the human Fas-associated protein with death domain (FADD; accession NM-003824); (ii) Short forms of human cell FLICE-like inhibitor protein (cFLIPs; accession No. NM-001127184.4); or (iii) a viral inhibitor of caspase (vICA, HCMV gene UL36; accession NC-006273.2) to modulate sanoχ transfer by inhibiting caspase activity. FADD-DN, cFLIP, and vICA were each cloned into pLV-EF1a-MCS-IRES-Puro vector (Boseki Co.) and used to transduce CT26-TRIF-RIPK3 expressing cells.

These cells were inoculated and subsequently treated with doxycycline (1 mg/mL; sigma aldrich, 0219895525) for 24 hours to promote expression. B/B homodimers were not used in this experiment. Relative cell viability was determined 24 hours after treatment using the realtem-Glo MT cell viability assay kit (prolog, cat# G9712) according to the manufacturer's instructions and the plot shows relative viability measured by Relative Luminescence Units (RLUs).

As shown in FIG. 7A, expression of either FADD-DN, cFLIPs, or vICA in CT26-TRIF+RIPK3 cells attenuated the decrease in cancer cell viability induced by TRIF+RIPK3 expression. However, expression of cflup+trif+ripk3 or vcca+trif+ripk3 in CT26 cells still reduced cancer cell viability relative to the parental line CT26-Tet3G cell line, but to a lesser extent than TRIF-RIPK3 alone. See fig. 7A.

Next, a medium containing CTF was produced from CT-26 mouse colon cancer cells as described above. Then stimulate J774-Dual with the indicated CTF ^TM Cells were kept for 24 hours. Cell culture media was collected and luciferase activity was measured using a QUANTI-Luc (Injetty; rep-qlc 1) assay. Interferon Stimulated Response Element (ISRE) promoter activation was mapped relative to a control cell line, tet 3G. As shown in FIG. 7B, the medium collected from CT26 cell line expressing TRIF or TRIF+RIPK3 was fine in J774-Dual IRF reporter gene expression is induced in the cell. In addition, media from CT26 cells expressing FADD-DN, cFLIPs, or vICA in addition to TRIF+RIPK3 also induced IRF reporter activation in J774-Dual cells.

CT-26-TRIF+RIPK3 mouse colon cancer cells carrying the FADD-DN, cFLIPs or vICA Sano delivery module described above were trypsinized and treated with 1X 10 ⁶ Individual cells/mL were resuspended in serum-free medium. B/B homodimers were not used in this experiment. Cells (100. Mu.L) were injected into the right subcutaneous flank of immunocompetent BALB/c mice. From day 15 to day 21 after CT-26 cell injection, mice were fed a Teklad basal diet supplemented with 625mg/kg doxycycline hydrochloride (Envelo company TD.01306). When the tumor reached 2000mm according to IACUC guidelines ³ Or at the end of the experiment, mice were euthanized.

As shown in FIG. 7C, the growth of all tumors expressing the Sano delivery module (i.e., TRIF+RIPK3, TRIF+RIPK3+FADD-DN, TRIF+RIPK3+cFLIPS or TRIF+RIPK3+vICA) was reduced relative to control CT26-Tet3G cells. In particular, expression of FADD-DN or vcca in combination with tif+ripk3 further reduced tumor growth compared to parental CT 26-tif+ripk3 cells. Interestingly, although the sano delivery module comprising FADD-DN or vcca in addition to TRIF+RIPK3 was most effective in reducing tumor growth in vivo, FADD-dn+TRIF+RIPK3 had little effect on CT26 cancer cell viability in vitro relative to TRIF+RIPK3 cells, whereas vcca+TRIF+RIPK3 co-expression enhanced cell killing in vitro relative to TRIF+RIPK3. These results indicate that in addition to the magnitude of killing of cancer cells by the saenox transfer modules, accurate Cell Turnover Factor (CTF) profiles of cancer cells due to the expression of these modules can also contribute to the immune response to tumor cells in vivo.

Example 11 evaluation of cell death pathway and cytokine Activity in Jurkat T cells expressing anti-mesothelin CAR and/or inducible miniTRIF

The purpose of this experiment was to determine the effect of inducible miniTRIF expression on the pattern of cell death, and the immunostimulatory activity of the cell turnover factor produced by miniTRIF expressing cells.

Slow useViral transduction methods Jurkat T cells containing anti-mesothelin CAR and/or an inducible payload comprising minitri are prepared. The CAR contains an anti-mesothelin scFv (SS 1), a CD3z intracellular signaling domain, and a co-stimulatory domain comprising CD28 or 4-1 BB. miniTRIF is under the transcriptional control of the promoter NF-AT induced by T cell activation. Schematic diagrams of the CAR and miniTRIF constructs are provided in fig. 8. All lentiviruses were generated as described in example 5. Jurkat T cells were transduced with lentiviruses expressing NF-AT/miniTRIF, anti-mesothelin CAR containing the 4-1BB co-stimulatory domain, or anti-mesothelin CAR containing the CD28 co-stimulatory domain, using TransDux Max (systems bioscience Co (System Biosciences); LV 680A-1) as per the manufacturer's instructions. Selection of cells transduced with NF-AT/miniTRIF lentiviruses with puromycin to generate stable TS _miniTRIF Jurkat cells. To generate CAR _{Mesothelin-bbz} +TS _miniTRIF Jurkat cells and CARs _{Mesothelin-28 z} +TS _miniTRIF Jurkat cells transduced stable TS with lentiviruses expressing either a 4-1 BB-containing anti-mesothelin CAR or a CD 28-containing anti-mesothelin CAR _miniTRIF Jurkat cell line.

The following cell lines were evaluated.

Cells were seeded at 100,000 cells/well in 96-well flat bottom cell culture plates. Recombinant human mesothelin-Fc chimeric protein (0, 30, 62, 125, 250, 500ng/ml, BAOCHINE DEVICE CARRENT No. 593202) or human CD3/CD28 activator (25 ul/ml, stem cell technology Co (Stemcell Technologies) catalog No. 10971) was added to each well and the cells were incubated at 37℃with 5% CO ₂ Incubate for 24, 48 or 72 hours. CD3/CD28 activators are used to activate endogenous T Cell Receptors (TCRs), while recombinant mesothelin is used to activate CARs. At each time point, CTF-containing medium was collected from cell cultures for THP1-Dual assay, cells were harvested, and the fixable vital dye eFluor was used ^TM 780 (1:2000, invitrogen catalog number 65-0865-14) and annexin V (Invitrogen catalog number)88-8005-74) to assess cell viability. Fixable vital dye eFluor ^TM 780 marks dead cells. Thus, eFluor ^TM 780% labeled cells reflect the percentage of total cell death in culture. Annexin V staining indicates apoptotic cell death.

To examine the effect of CAR and miniTRIF on the pattern of cell death, annexin V staining was used to bind to the fixable vital dye eFluor ^TM 780 to analyze the ratio of necrotic to apoptotic cell populations. Necrotic cell population is defined as annexin V ^- And a vital dye eFluor780 ⁺ While the apoptotic cell population is defined as annexin V ⁺ And a vital dye eFluor780 ⁺ Is a cell of (a) a cell of (b).

CTF samples collected from different CAR Jurkat cells were used in THP1-Dual assays to check for immunogenicity of CTF. THP1-dual reporter cells were seeded at 100,000 cells/well in 96-well flat bottom plates. CTF samples were added to THP1-dual reporter cells at a 1:1 ratio and at 37℃with 5% CO ₂ Incubate for 24 hours. Cell culture media was then collected and IRF reporter gene expression (luciferase activity) was measured using a QUANTI-Luc assay (invitrogen).

Results

As shown in fig. 9A-9C, CAR-expressing cells died in a dose-dependent manner upon target engagement, and expression of the minitri payload in the cells did not alter the total amount of cell death.

As shown in fig. 10A-10C, the corresponding Cells (CAR) with no miniTRIF construct _{Mesothelin-bbz} Jurkat), CAR _{Mesothelin-bbz} +TS _miniTRIF The ratio of necrotic cells to apoptotic cells in Jurkat cells increased in a dose-dependent manner, indicating that inducible expression of miniTRIF promotes a change in the pattern of cell death from apoptosis to necrosis. Furthermore, relative to the corresponding Cells (CAR) without miniTRIF construct _{Mesothelin-28 z} Jurkat), at 48 hour and 72 hour time points, at CAR _{Mesothelin-28 z} +TS _miniTRIF A similar but smaller increase in necrotic cell death was observed in Jurkat cells. These results further demonstrate that minThe expression of itraf promotes a change from apoptotic cell death to necrotic cell death.

As shown in fig. 11A-11C, CTF from CAR Jurkat T cells expressing the ministif payload activated IRF reporter expression in THP1-Dual cells, whereas CTF from Jurkat T cells without the ministif construct resulted in only background levels of IRF reporter expression. These results demonstrate that expression of miniTRIF in Jurkat T cells increases the immunostimulatory activity of CTF produced by these cells. Furthermore, in CTF-treated THP1 cells harvested at the 72 hour time point, the effect on IRF activity was higher, indicating that the level of immunostimulatory CTF increased over time.

Conclusion(s)

Inducible expression of miniTRIF promotes a change in the death pattern of cells from apoptosis to necrosis and increases the immunostimulatory activity of CTF produced by these cells.

Sequences of the present disclosure

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Claims (117)

1. An immune cell, the immune cell comprising:

(a) One or more polynucleotides encoding a Chimeric Antigen Receptor (CAR), wherein the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular signaling domain; and

(b) One or more polynucleotides that promote the Sagnac transfer of the immune cell, operably linked to a heterologous promoter that induces expression of the polynucleotide upon activation of the immune cell,

wherein the immune cell is a T cell, natural Killer (NK) cell or macrophage.

2. The immune cell of claim 1, wherein the intracellular signaling domain comprises at least one TCR-type signaling domain.

3. The immune cell of claim 2, wherein the intracellular signaling domain further comprises at least one costimulatory signaling domain.

4. The immune cell of claim 3, wherein the CAR further comprises a hinge domain.

5. The immune cell of claim 1, wherein the promoter induces expression of the polynucleotide upon binding of the antigen binding domain to an antigen.

6. The immune cell of claim 1, wherein the heterologous promoter is selected from the group consisting of: activating T cell Nuclear Factor (NFAT) promoter, STAT promoter, AP-1 promoter, NF-. Kappa.B promoter and IRF4 promoter.

7. The immune cell of claim 2, wherein the TCR-type signaling domain comprises an intracellular domain of cd3ζ.

8. The immune cell of claim 7, wherein the intracellular domain of cd3ζ comprises a mutation of one or more tyrosine residues in one or more immune receptor tyrosine-based activation motifs (ITAMs).

9. The immune cell of claim 1, wherein the intracellular signaling domain comprises a combination of domains selected from the group consisting of:

(a) A costimulatory signaling domain of CD28 and an intracellular domain of cd3ζ;

(b) 4-1BB costimulatory signaling domain and intracellular domain of cd3ζ; and

(c) The costimulatory signaling domain of CD28, the costimulatory signaling domain of 4-1BB, the costimulatory signaling domain of CD27 or CD134, and the intracellular domain of CD3 ζ.

10. The immune cell of claim 1, wherein the intracellular signaling domain comprises a costimulatory signaling domain of CD28 and an intracellular domain of cd3ζ.

11. The immune cell of claim 1, wherein the intracellular signaling domain comprises a costimulatory signaling domain of 4-1BB and an intracellular domain of cd3ζ.

12. The immune cell of claim 1, wherein the antigen binding domain binds to a protein preferentially expressed on the surface of a cancer cell.

13. The immune cell of claim 12, wherein the cancer cell is a solid cancer.

14. The immune cell of claim 12, wherein the cancer cell is a non-solid cancer.

15. The immune cell of claim 1, wherein the antigen binding domain binds mesothelin.

16. The immune cell of claim 1, wherein the antigen binding domain binds to a protein selected from the group consisting of: CD19, CD20, CD22, CD23, kappa light chain, CD5, CD30, CD70, CD38, CD138, BCMA, CD33, CD123, CD44v6, CS1 and ROR1.

17. The immune cell of claim 1, wherein the antigen binding domain binds to a protein selected from the group consisting of: CD44v6, carbonic Anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CD133, hepatocyte growth factor receptor (C-Met), epidermal Growth Factor Receptor (EGFR), type III epidermal growth factor receptor (EGFRvIII), epithelial cell adhesion molecule (Epcam), erythropoietin-producing hepatocellular carcinoma A2 (EphA 2), fetal acetylcholine receptor, folate receptor alpha (Fra), ganglioside GD2 (GD 2), glypican-3 (GPC 3), guanylate cyclase C (GUCY 2C), human epidermal growth factor receptor 1 (HER 1), human epidermal growth factor receptor 2 (HER 2), intercellular adhesion molecule 1 (ICAM-1), interleukin 13 receptor A2 (IL 13Ra 2), interleukin 11 receptor a (IL 11 Ra), kirsten rat sarcoma virus oncogene homolog (Kras), kras G12D, L-cell adhesion molecule (L1 CAM), GE, MET, mesothelin, mucin 1 (MUC 1), mucin 16 (MUO 16), nd2_d2, human tumor cell activation factor receptor 2 (IL-11), human tumor cell growth factor receptor alpha (IL-2), human tumor cell signaling factor 1 (PSMA-3, PSP 2, PSP-3, and human tumor cell activation factor 1 (PSP-3, PSP-PSP 2, PSP-3, PSP-activated human tumor cell tissue factor 1 (PSP), human tumor cell antigen (hR 1 (hR) E), human tumor cell tumor antigen (hR 1).

18. The immune cell of any one of claims 1 to 17, wherein at least one polynucleotide of the one or more polynucleotides that promote sanoχ transfer encodes a tri or variant thereof.

19. The immune cell of claim 18, wherein the tri variant is a tri variant listed in table 2.

20. The immune cell of claim 18, wherein the tri variant comprises the amino acid sequences listed in table 2.

21. The immune cell of any one of claims 1 to 17, wherein at least one polynucleotide of the one or more polynucleotides that promote sanoχ transfer encodes a death folding domain.

22. The immune cell of claim 21, wherein the death folding domain is selected from the group consisting of: death domain, thermo protein domain, death Effector Domain (DED), C-terminal caspase recruitment domain (CARD), and variants thereof.

23. The immune cell of claim 22, wherein the death domain is from a protein selected from the group consisting of: fas-associated protein with death domain (FADD), fas, tumor necrosis factor receptor type 1-associated death domain (TRADD), tumor necrosis factor receptor type 1 (TNFR 1), and variants thereof.

24. The immune cell of claim 22, wherein the thermal protein domain is from a protein selected from the group consisting of: the NLR family contains the thermal protein domain protein 3 (NLRP 3) and apoptosis-related speckle-like proteins (ASCs).

25. The immune cell of claim 22, wherein the Death Effector Domain (DED) is from a protein selected from the group consisting of: fas-associated protein with death domain (FADD), caspase-8 and caspase-10.

26. The immune cell of claim 22, wherein the CARD is from a protein selected from the group consisting of: RIP related ICH1/CED3 homologous protein (RAIDD), apoptosis related spotting protein (ASC), mitochondrial antiviral signaling protein (MAVS), caspase-1, and variants thereof.

27. The immune cell of any one of claims 1 to 17, wherein at least one polynucleotide of the one or more polynucleotides that promote sanoχ transfer encodes a Toll/interleukin-1 receptor (TIR) domain.

28. The immune cell of claim 27, wherein the TIR domain is from a protein selected from the group consisting of: myeloid differentiation primary response protein 88 (MyD 88), toll/interleukin-1 receptor (TIR) domain containing adaptor-induced interferon- β (tif), toll-like receptor 3 (TLR 3), toll-like receptor 4 (TLR 4), TIR domain containing adaptor protein (tirp), translocation chain related membrane protein (TRAM) and variants thereof.

29. The immune cell of any one of claims 1 to 17, wherein at least one polynucleotide of the one or more polynucleotides that promote sanoχ transfer encodes a protein comprising a TIR domain.

30. The immune cell of claim 29, wherein the protein comprising a TIR domain is selected from the group consisting of: myeloid differentiation primary response protein 88 (MyD 88), toll/interleukin-1 receptor (TIR) domain containing adaptor-induced interferon- β (tif), toll-like receptor 3 (TLR 3), toll-like receptor 4 (TLR 4), TIR domain containing adaptor protein (tirp), translocation chain related membrane protein (TRAM) and variants thereof.

31. The immune cell of any one of claims 1 to 17, wherein at least one polynucleotide of the one or more polynucleotides that promote sanoχ transfer encodes a polypeptide selected from the group consisting of: cellularFLICE(FADD-likeIL-1betaconvertingenzyme)-inhibitorprotein(c-FLIP),receptorinteractingserine/threonineproteinkinase1(RIPK1),receptorinteractingserine/threonineproteinkinase3(RIPK3),Z-DNAbindingprotein1(ZBP1),mixedlineagekinasedomain-likepseudokinase(MLKL),N-terminaltruncationsofthetifcomprisingonlytheTIRdomainandRHIMdomain,dominantnegativemutantsofFas-relatedproteinswithdeathdomain(FADD-DD),myr-FADD-DD,inhibitorkBalphasuper-repressor(IkBalpha-SR),interleukin-1receptor-relatedkinase1(IRAK1),tumornecrosisfactorreceptortype1relateddeathdomain(TRADD),dominantnegativemutantsofcaspase-8,interferonregulator3(IRF3),desetin-a(GSDM-a),desetin-b(GSDM-b),desetin-c(dm-c),desetin-d(GSDM-d),desetin-e(GSDM-e),asbestein-c(asc-c),andvariantswithapoptosis-relatedprotein(asc-c)withtheirapoptosisdomain.

32. The immune cell of claim 31, wherein the N-terminal truncate of the TRIF comprising only a TIR domain and a RHIM domain comprises a deletion of amino acid residues 1-311 of human TRIF.

33. The immune cell of claim 31, wherein the N-terminal truncate of the tif comprising only a TIR domain and a RHIM domain comprises or consists of SEQ ID No. 12.

34. The immune cell of claim 31, wherein the cflup is human cflup.

35. The immune cell of claim 31, wherein the cflup is caspase-8 and FADD-like apoptosis modulator (cfar).

36. The immune cell of claim 31, wherein the ZBP1 comprises a deletion of a receptor-interacting protein homotypic interaction motif (RHIM) C, a deletion of RHIM D, and a deletion at the N-terminus of the Za1 domain.

37. The immune cell of claim 31, wherein the ZBP1 is a ZBP1-Za1/RHIM a truncate.

38. The immune cell of any one of claims 1 to 17, wherein at least one polynucleotide of the one or more polynucleotides that promote sanoχ transfer comprises a viral gene.

39. The immune cell of claim 38, wherein the viral gene encodes a polypeptide selected from the group consisting of: vFLIP (ORF 71/K13) from Kaposi sarcoma-associated herpesvirus (KSHV), MC159L from molluscum contagiosum virus, E8 from equine herpesvirus 2, vICA from Human Cytomegalovirus (HCMV) or Murine Cytomegalovirus (MCMV), crmA from vaccinia virus and P35 from Medicago sativa spodoptera nuclear polyhedrosis virus (AcMNPV).

40. The immune cell of any one of claims 1 to 17, wherein the one or more polynucleotides that promote saenox transfer encode two or more different saenox transfer polypeptides, wherein the two or more saenox transfer polypeptides are selected from the group consisting of: TRADD, TRAF2, TRAF6, ciaP1, ciaP2, XIAP, NOD2, myD88, TRAM, HOIL, HOIP, sharpin, IKKg, IKKa, IKKb, relA, MAVS, RIGI, MDA, tak1, TBK1, IKKe, IRF3, IRF7, IRF1, TRAF3, caspase, FADD, TNFR1, TRAILR2, FAS, bax, bak, bim, bid, noxa, puma, TRIF, ZBP1, RIPK3, MLKL, destina B, destina C, destina D, destina E, tumor necrosis factor receptor superfamily (TNFSF) proteins, and variants thereof.

41. The immune cell of claim 40, wherein the one or more polynucleotides that promote saenox transfer comprise at least two polynucleotides, wherein each polynucleotide encodes a different saenox transfer polypeptide selected from the group consisting of: TRADD, TRAF2, TRAF6, ciaP1, ciaP2, XIAP, NOD2, myD88, TRAM, HOIL, HOIP, sharpin, IKKg, IKKa, IKKb, relA, MAVS, RIGI, MDA, tak1, TBK1, IKKe, IRF3, IRF7, IRF1, TRAF3, caspase, FADD, TNFR1, TRAILR2, FAS, bax, bak, bim, bid, noxa, puma, TRIF, ZBP1, RIPK3, MLKL, destina B, destina C, destina D, destina E, tumor necrosis factor receptor superfamily (TNFSF) proteins, and variants thereof.

42. The immune cell of claim 40, wherein at least one of the polynucleotides encodes a chimeric protein comprising at least two of the saenopassing polypeptides.

43. The immune cell of claim 40, wherein at least one of the polynucleotides is transcribed as a single transcript encoding two or more different saenopassing polypeptides.

44. The immune cell of any one of claims 40 to 43, wherein at least one of the saenox polypeptides comprises tri or a variant thereof.

45. The immune cell of any one of claims 40 to 43, wherein at least one of the saenox polypeptides comprises RIPK3 or a variant thereof.

46. The immune cell of any one of claims 40 to 43, wherein at least one of the saenox polypeptides comprises tri or a variant thereof and at least one of the saenox polypeptides comprises RIPK3 or a variant thereof.

47. The immune cell of any one of claims 40 to 43, wherein at least one of the saenox polypeptides comprises MAVS or a variant thereof and at least one of the saenox polypeptides comprises RIPK3 or a variant thereof.

48. The immune cell of claim 44, wherein the TRIF variant is a TRIF variant listed in Table 2 or comprises an amino acid sequence listed in Table 2.

49. The immune cell of claim 44, wherein the TRIF variant is an N-terminal truncate of TRIF comprising only a TIR domain and a RHIM domain.

50. The immune cell of claim 44, wherein the TRIF variant comprises a deletion of amino acid residues 1-311 of human TRIF.

51. The immune cell of claim 49, wherein the N-terminal truncate of TRIF comprising only a TIR domain and a RHIM domain comprises or consists of SEQ ID NO. 12.

52. The immune cell of any one of claims 1 to 51, wherein the one or more polynucleotides that promote sanoχ transfer further encode a polypeptide that inhibits caspase activity.

53. The immune cell of claim 52, wherein the polypeptide that inhibits caspase activity is selected from the group consisting of: FADD dominant negative mutant (FADD-DN), cFLIP, vcca, caspase 8 dominant negative mutant (Casp 8-DN), cIAP1, cIAP2, tak1, IKK, and variants thereof.

54. The immune cell of claim 52, wherein the polypeptide that inhibits caspase activity is FADD-DN.

55. The immune cell of claim 52, wherein the polypeptide that inhibits caspase activity is cflup.

56. The immune cell of claim 52, wherein the polypeptide that inhibits caspase activity is vcica.

57. The immune cell of any one of claims 1 to 56, wherein at least one polynucleotide of the one or more polynucleotides that promote sanoχ transfer encodes a mesothelin or a variant thereof.

58. The immune cell of any one of claims 1 to 17, wherein at least one polynucleotide of the one or more polynucleotides that promote sanoχ transfer encodes a tri or variant thereof, and at least one polynucleotide of the one or more polynucleotides that promote sanoχ transfer encodes a RIPK3 or variant thereof, and at least one polynucleotide of the one or more polynucleotides that promote sanoχ transfer encodes an mesothelin or variant thereof.

59. The immune cell of any one of claims 1 to 17, wherein at least one polynucleotide of the one or more polynucleotides that promote sanoχ transfer encodes MAVS or a variant thereof, and at least one polynucleotide of the one or more polynucleotides that promote sanoχ transfer encodes RIPK3 or a variant thereof, and at least one polynucleotide of the one or more polynucleotides that promote sanoχ transfer encodes mesothelin or a variant thereof.

60. The immune cell of any one of claims 57 to 59, wherein the mesothelin is mesothelin E or variant thereof.

61. The immune cell of any one of claims 18 to 60, wherein the variant is a functional fragment of the saenox polypeptide.

62. The immune cell of any one of claims 1 to 61, wherein the immune cell further comprises at least one heterologous polynucleotide encoding a dimerization domain.

63. The immune cell of any one of claims 1 to 62, wherein at least one of the saenox polypeptides is contained within a fusion protein further comprising a dimerization domain.

64. The immune cell of claim 62 or 63, wherein the dimerization domain is heterologous to the saenox transfer polypeptide.

65. A method of promoting saenox transfer in a subject, the method comprising administering the immune cell of any one of claims 1 to 64 in an amount and for a duration sufficient to promote saenox transfer in the subject.

66. A method of promoting saenox transfer to a target cell, the method comprising contacting the target cell or tissue comprising the target cell with the immune cell of any one of claims 1 to 64 in an amount and for a duration sufficient to promote saenox transfer to the target cell.

67. A method of promoting an immune response in a subject in need thereof, the method comprising administering the immune cell of any one of claims 1 to 64 to the subject in an amount and for a duration sufficient to promote saenox transfer of the immune cell, thereby promoting an immune response in the subject.

68. The method of any one of claims 65-67, wherein the immune cell is administered to the subject in an amount and for a duration sufficient to promote saenox transfer of a target cell.

69. The method of claim 68, wherein the target cell is selected from the group consisting of: cancer cells, immune cells, endothelial cells, and fibroblasts.

70. The method of any one of claims 65-69, wherein the subject has an infection.

71. The method of any one of claims 66 and 68 to 70, wherein the target cell is infected with a pathogen.

72. The method of claim 70 or 71, wherein the infection is a viral infection.

73. The method of any one of claims 70-72, wherein the infection is a chronic infection.

74. The method of claim 73, wherein the chronic infection is selected from the group consisting of an HIV infection, an HCV infection, an HBV infection, an HPV infection, a hepatitis B infection, a hepatitis C infection, an EBV infection, a CMV infection, a TB infection, and a parasitic infection.

75. A method of treating cancer in a subject in need thereof, the method comprising administering the immune cell of any one of claims 1 to 64 to the subject, thereby treating the cancer in the subject.

76. The method of claim 75, wherein administering the immune cell to the subject reduces proliferation of cancer cells in the subject.

77. The method of claim 76, wherein the proliferation of the cancer cells is hyperproliferative of the cancer cells caused by cancer therapy administered to the subject.

78. The method of any one of claims 75 to 77, wherein administering the immune cell to the subject reduces metastasis of cancer cells in the subject.

79. The method of any one of claims 75-78, wherein administering the immune cells to the subject reduces neovascularization of a tumor in the subject.

80. The method of any one of claims 75 to 79, wherein treating the cancer comprises any one or more of: a decrease in tumor burden, a decrease in tumor size, inhibition of tumor growth, achieving stable cancer in a subject with advanced cancer prior to treatment, a delay in cancer progression time, and an increase in survival time.

81. The method of any one of claims 75 to 80, wherein an immunostimulatory cell turnover pathway is induced in the cancer.

82. The method of claim 81, wherein the cancer lacks the immunostimulatory cell turnover pathway.

83. The method of claim 81 or 82, wherein the immunostimulatory cell turnover pathway is selected from the group consisting of: necrotic apoptosis, extrinsic apoptosis, iron death, and apoptosis of the cell coke.

84. The method of any one of claims 75 to 83, wherein the cancer is a cancer responsive to immune checkpoint therapy.

85. The method of any one of claims 75 to 84, wherein the cancer is selected from the group consisting of an epithelial carcinoma, a sarcoma, a lymphoma, a melanoma, and a leukemia.

86. The method of any one of claims 75 to 85, wherein the cancer is a metastatic cancer.

87. The method of any one of claims 75 to 86, wherein the cancer is a solid tumor.

88. The method of claim 87, wherein the solid tumor is selected from the group consisting of: colon cancer, soft Tissue Sarcoma (STS), metastatic clear cell renal cell carcinoma (ccRCC), ovarian cancer, gastrointestinal cancer, colorectal cancer, hepatocellular carcinoma (HCC), glioblastoma (GBM), breast cancer, melanoma, non-small cell lung cancer (NSCLC), sarcoma, malignant pleura, mesothelioma (MPM), retinoblastoma, glioma, medulloblastoma, osteosarcoma, ewing's sarcoma, pancreatic cancer, lung cancer, stomach cancer, gastric cancer, esophageal cancer, liver cancer, prostate cancer, gynaecological cancer, nasopharyngeal cancer, osteosarcoma, rhabdomyosarcoma, urothelial bladder cancer, neuroblastoma, and cervical cancer.

89. The method of any one of claims 75 to 87, wherein the cancer is selected from the group consisting of: melanoma, cervical cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, urothelial cancer, bladder cancer, non-small cell lung cancer, sarcoma, colorectal adenocarcinoma, gastrointestinal stromal tumor, gastroesophageal cancer, colorectal cancer, pancreatic cancer, renal cancer, hepatocellular carcinoma, malignant mesothelioma, leukemia, lymphoma, myelodysplastic syndrome, multiple myeloma, transitional cell carcinoma, neuroblastoma, plasmacytoma, wilms' tumor, and hepatocellular carcinoma.

90. The method of any one of claims 75 to 86, wherein the cancer is not a solid tumor.

91. The method of claim 90, wherein the cancer is selected from the group consisting of: leukemia, lymphoma, B-cell malignancy, T-cell malignancy, multiple myeloma, myeloid malignancy, and hematological malignancy.

92. The method of any one of claims 65-91, wherein the immune cell is administered intravenously to the subject.

93. The method of any one of claims 65-91, wherein the immune cell is intratumorally administered to the subject.

94. The method of any one of claims 65-93, wherein the method further comprises administering an anti-tumor agent to the subject.

95. The method of claim 94, wherein the anti-tumor agent is a chemotherapeutic.

96. The method of claim 94, wherein the anti-tumor agent is a biologic agent.

97. The method of claim 96, wherein the biologic agent is an antigen binding protein.

98. The method of claim 96, wherein the biologic agent is an oncolytic virus.

99. The method of claim 94, wherein the anti-tumor agent is an immunotherapeutic agent.

100. The method of claim 99, wherein the immunotherapeutic agent is selected from the group consisting of: toll-like receptor (TLR) agonists, cell-based therapies, cytokines, cancer vaccines and immune checkpoint modulators of immune checkpoint molecules.

101. The method of claim 100, wherein the cell-based therapy is chimeric antigen receptor T cell (CAR-T cell) therapy.

102. The method of claim 100, wherein the immune checkpoint molecule is selected from the group consisting of CD27, CD28, CD40, OX40, GITR, ICOS, 4-1BB, ADORA2A, B-H3, B7-H4, BTLA, CTLA-4, KIR, LAG-3, PD-1, PD-L2, TIM-3, and VISTA.

103. The method of claim 100, wherein the immune checkpoint molecule is a stimulatory immune checkpoint molecule and the immune checkpoint modulator is an agonist of the stimulatory immune checkpoint molecule.

104. The method of claim 100, wherein the immune checkpoint molecule is an inhibitory immune checkpoint molecule and the immune checkpoint modulator is an antagonist of the inhibitory immune checkpoint molecule.

105. The method of any one of claims 100-104, wherein the immune checkpoint modulator is selected from the group consisting of a small molecule, an inhibitory RNA, an antisense molecule, and an immune checkpoint molecule binding protein.

106. The method of claim 100, wherein the immune checkpoint molecule is PD-1 and the immune checkpoint modulator is a PD-1 inhibitor.

107. The method of claim 106, wherein the PD-1 inhibitor is selected from the group consisting of pembrolizumab, nivolumab, pilizumab, SHR-1210, MEDI0680R01, BBg-a317, TSR-042, REGN2810, and PF-06801591.

108. The method of claim 100, wherein the immune checkpoint molecule is PD-L1 and the immune checkpoint modulator is a PD-L1 inhibitor.

109. The method of claim 108, wherein the PD-L1 inhibitor is selected from the group consisting of dimaruzumab, alemtuzumab, avermectin, MDX-1105, AMP-224, and LY3300054.

110. The method of claim 100, wherein the immune checkpoint molecule is CTLA-4 and the immune checkpoint modulator is a CTLA-4 inhibitor.

111. The method of claim 110, wherein the CTLA-4 inhibitor is selected from Ai Pili mumab, trimelimab, JMW-3B3 and AGEN1884.

112. The method of claim 94, wherein the anti-tumor agent is a histone deacetylase inhibitor.

113. The method of claim 112, wherein the histone deacetylase inhibitor is a hydroxamic acid, a benzamide, a cyclic tetrapeptide, a depsipeptide, an electrophilic ketone, or an aliphatic compound.

114. The method of claim 113, wherein the hydroxamic acid is vorinostat (SAHA), belicastat (PXD 101), LAQ824, trichostatin a, or panobinostat (LBH 589).

115. The method of claim 113, wherein the benzamide is entinostat (MS-275), 01994, or motiroxostat (MGCD 0103).

116. The method of claim 113, wherein the cyclic tetrapeptide is Qu Puxin B.

117. The method of claim 113, wherein the aliphatic compound is phenyl butyrate or valproic acid.