Chimeric antigen receptors based on the hinge and / or transmembrane domains of CD30

Abstract

Aspects of the present disclosure include improvements in chimeric antigen receptor (CAR) constructs, CARs that constitute immune cells, and methods for making such constructs and / or cells, and methods for their use in treating diseases (e.g., cancer). Disclosed are immune cells that constitute CARs that contain hinges and / or transmembrane bodies derived from CD30, and methods for using such cells in treating malignancies (e.g., B-cell malignancies). Also disclosed are polynucleotides that encode CARs that contain hinges and / or transmembrane domains derived from CD30, as well as cells that contain such polynucleotides, and pharmaceutical compositions that contain such cells.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 335,560, filed April 27, 2022, which is incorporated by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing submitted in ST26 format, which is incorporated by reference in its entirety. The ST26 copy, created on April 3, 2023, is named MDAC_1327WO_Sequence_Listing.xml and is 53,662 bytes in size.

[0003] Technical Field The present disclosure relates generally to at least the fields of cancer biology, immunology and medicine. [Background technology]

[0004] Chimeric antigen receptor (CAR) cell therapy is generally considered an effective solution for relapsed or refractory tumors, especially hematological malignancies. Although commercially available anti-CD19 CAR-T therapies have achieved remarkable success, setbacks such as high relapse rates and resistance are common. There is a need to discover more effective engineered CAR components to enhance the therapeutic efficacy of effector cells and further improve patient outcomes. Summary of the Invention [Means for solving the problem]

[0005] Described herein are novel functional components of chimeric antigen receptors (CARs), CARs comprising one or more functional components, engineered cells comprising one or more functional components, compositions comprising one or more functional components, and methods of using the above.

[0006] Embodiments of the present disclosure include nucleic acids, polynucleotides, polypeptides, proteins, peptides, constructs, vectors, cells, therapeutic cells, immune cells, engineered cells, methods for generating engineered cells, methods for detecting engineered cells, methods for isolating engineered cells, methods for depleting engineered cells, and methods for purifying engineered cells. Nucleic acids of the present disclosure may encode one or more polypeptides of the present disclosure, including one or more functional components of a chimeric polypeptide. In some embodiments, a nucleic acid molecule of the present disclosure encodes a chimeric polypeptide. In some embodiments, a nucleic acid molecule of the present disclosure encodes two or more chimeric polypeptides. A chimeric polypeptide of the present disclosure may comprise at least one, two, three or more of the following regions or domains: a signal peptide, an extracellular domain, a hinge region, a transmembrane domain, and an intracellular region. An engineered cell of the present disclosure may comprise one, two, three, four or more polynucleotides and / or polypeptides of the present disclosure. The methods of the disclosure may include at least one, two, three, four or more of the following steps: introducing a polynucleotide into a cell, introducing a vector into a cell, introducing a polypeptide into a cell, expressing the polypeptide in the cell, expanding a population of cells, contacting the cell with an antigen binding protein, contacting the cell with an antibody drug conjugate, and detecting the cell using an imaging agent.

[0007] In certain embodiments, the present disclosure provides a functional component of a chimeric antigen receptor (CAR), the functional component comprising CD30 (also known as TNFRSF8, D1S166E, and Ki-1), a hinge domain, and / or a CD30 transmembrane domain. In certain embodiments, the present disclosure provides a CAR comprising: i) an antigen binding domain; ii) a CD30 hinge domain; iii) a CD30 transmembrane domain; iv) at least one intracellular costimulatory domain; and v) an intracellular stimulatory domain. In certain embodiments, the CD30 hinge domain comprises less than 51 contiguous amino acids and at least 7 contiguous amino acids of the extracellular domain of CD30. In certain embodiments, the CD30 transmembrane domain comprises no more than 27 contiguous amino acids of CD30. In certain embodiments, the CD30 hinge domain comprises no more than 51 contiguous amino acids and at least 7 contiguous amino acids of the extracellular domain of CD30, and the CD30 transmembrane domain comprises no more than 27 contiguous amino acids of CD30.

[0008] In certain embodiments, the CD30 hinge and / or transmembrane domain does not contain a cysteine. In certain embodiments, the CD30 hinge is at least 80%, 85%, 90%, 95%, or 98% identical to SEQ ID NO:3. In certain embodiments, the CD30 transmembrane domain is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:4. In certain embodiments, the CD30 hinge domain and / or transmembrane domain lacks three or more consecutive amino acids set forth in SEQ ID NO:6. In certain embodiments, the CD30 hinge domain comprises SEQ ID NO:3. In certain embodiments, the CD30 transmembrane domain comprises SEQ ID NO:4. In certain embodiments, the CD30 domain and transmembrane domain are encoded by a nucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, or 98% identical to SEQ ID NO:39.

[0009] In some certain embodiments, the present disclosure provides a method for the production and / or storage of a CD30 hinge and / or transmembrane domain, as well as CD4, CD5, CD7, CD10, CD19, CD20, CD22, CD30, CD79a, CD79b, SLAM-F7, CD123, CD70, CD72, CD33, CD38, CD80, CD86, CD138, CLL-1, FLT3, ROR-1, TACI, TRBC1, MUC1, PD-L1, CD117, FR, LeY , HER2, IL13Rα2, DLL3, DR5, FAP, LMP1, MAGE-A1, MAGE-A4, MG7, MUC16, PMEL, ROR2, VEGFR2, AFP, EphA2, PSCA, EPCAM, EGFR, PSMA, EGFRvIII, GPC3, CEA, GD2, NY-ESO-1, TCL1, mesothelin, and / or BAFF-R. In certain embodiments, the present disclosure provides CARs comprising a CD30 hinge and / or transmembrane domain and at least one antigen binding domain targeted to CD19, CD20, CD22, CD70, CD79B, CD79A, ROR1, BCMA, BAFF receptor, GD2, and / or claudin 18.2. In certain embodiments, the antigen binding domain is targeted to CD19, CD79B, and / or CD70.

[0010] In certain embodiments, the present disclosure provides CARs comprising a CD30 hinge and / or transmembrane domain and at least one intracellular costimulatory domain comprising CD8, 4-1BB (CD137), CD27, CD28, CD30, OX-40 (CD134), CD3ε, CD3ζ, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, or CD154, an intracellular costimulatory domain. In certain embodiments, the at least one intracellular costimulatory domain comprises CD28.

[0011] In certain embodiments, the present disclosure provides CARs comprising a CD30 hinge and / or transmembrane domain and at least one intracellular stimulatory domain comprising a DAP12, DAP10, FCER1G (Fc epsilon receptor I gamma chain), CD3δ (CD3 delta), CD3ε (CD3 epsilon), CD3γ (CD3 gamma), CD3ζ (CD3 zeta), or CD79A, intracellular stimulatory domain. In certain embodiments, the at least one intracellular stimulatory domain comprises a CD3ζ (CD3 zeta) intracellular stimulatory domain.

[0012] In certain embodiments, the present disclosure provides a CAR comprising a CD30 hinge and / or transmembrane domain, at least one intracellular costimulatory domain, and at least one intracellular stimulatory domain, wherein the polypeptide sequence comprising it is at least 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:2.

[0013] In certain embodiments, the present disclosure provides a cell comprising a CAR comprising a CD30 hinge and / or transmembrane domain. In certain embodiments, the cell is an immune cell. In certain embodiments, the cell is a T cell or a NK cell. In certain embodiments, the cell is derived from a healthy donor. In certain embodiments, the cell is derived from a patient. In certain embodiments, the T cell comprises a CD4+ T cell, a CD8+ T cell, an iNKT cell, a NKT cell, a γδ T cell, a regulatory T cell, an innate lymphocyte, or a combination thereof. In certain embodiments, the T cell is a γδ T cell. In certain embodiments, the immune cell is an immune cell described in International Publication No. WO2021034982A1, the entirety of which is incorporated herein by reference for purposes.

[0014] In some embodiments, the present disclosure provides a cell comprising a CAR and at least one additional transgene. In certain embodiments, the at least one additional transgene encodes an immunomodulatory gene. In some embodiments, the immunomodulatory gene is a pro-survival gene. In some embodiments, the immunomodulatory gene is BCL6. In some embodiments, the immunomodulatory gene is an anti-apoptotic B cell lymphoma 2 (BCL-2) family gene. In certain embodiments, the anti-apoptotic BCL-2 family gene is BCL2L1 (Bcl-xL), BCL-2, MCL1, BCL2L2 (Bcl-w), BCL2A1 (Bfl-1), BCL2L10 (BCL-B), or a combination thereof. In certain embodiments, the anti-apoptotic BCL-2 family gene is Bcl-xL.

[0015] In some embodiments, the cell comprises at least one artificial mutation in an endogenous gene, at least one heterologous nucleic acid that can modify the expression of at least one endogenous gene, and / or a nucleic acid that can exert an anti-apoptotic function. In certain embodiments, the endogenous gene is an immunomodulatory gene. In certain embodiments, the endogenous gene is an apoptotic protein (e.g., caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14, etc.). In certain embodiments, the endogenous gene is a pro-apoptotic gene (e.g., BCL2L11 (BIM), BBC3 (PUMA), PMAIP1 (NOXA), BIK, BMF, BAD, HRK, BID, BAX, BAK1, BOK, etc.). In certain embodiments, nucleic acids capable of exerting anti-apoptotic functions include sequences encoding IGF1, HSPA4 (Hsp70), HSPB1 (Hsp27), CLAR (cFLIP), BNIP3, FADD, AKT, and NF-κB, RAF1, MAP2K1 (MEK1), RPS6KA1 (p90Rsk), JUN, C-Jun, BNIP2, BAG1, HSPA9, HSP90B1, miRNA21, miR-106b-25, miR-206, miR-221 / 222, miR-17-92, miR-133, miR-143, miR-145, miR-155, miR-330, and / or any combination thereof.

[0016] In certain embodiments, the cell comprises at least one safety switch. In some embodiments, the safety switch is a truncated EGFR (e.g., EGFR lacking domains 1 and 2). In certain embodiments, the cell is an immune cell (e.g., a T cell, an innate lymphoid cell, and / or an NK cell) that expresses IL-2, IL-15, other growth or differentiation factors, or a combination thereof.

[0017] In certain embodiments, the cells maintain a proliferation rate for at least 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or any range therebetween. In certain embodiments, the cells have enhanced anti-tumor cytotoxicity, in vivo proliferation, in vivo persistence, and / or improved function.

[0018] In certain embodiments, provided herein are compositions comprising at least 50 million, 100 million, 200 million, 500 million, 750 million, 1 billion, 2 billion, 3 billion, 4 billion, 5 billion, 6 billion, 7 billion, 8 billion, 9 billion, or 10 billion immune cells, including T cells, innate lymphoid cells, NK cells, or mixtures thereof.

[0019] In certain embodiments, a vector is provided herein. In certain embodiments, the vector is a lentiviral vector. In certain embodiments, introducing the vector into the cell comprises transducing the cell with the lentiviral vector in the presence of IL-2 and / or other growth factors. In certain embodiments, the IL-2 is at a concentration of 10 IU / mL to 1000 IU / mL, such as 10 to 50 IU / mL, 50 to 75 IU / mL, 75 to 100 IU / mL, 100 to 250 IU / mL, 250 to 500 IU / mL, 500 to 750 IU / mL, or 750 to 1000 IU / mL. In certain embodiments, the IL-2 is at a concentration of 100, 200, 300, 400, or 500 IU / mL.

[0020] In certain embodiments, provided herein are methods for the treatment of immune-related disorders, infectious diseases, and / or cancer, comprising treatment with at least one cell disclosed herein. In some embodiments, the disease or disorder is an infectious disease, a cancer, and / or an immune-related disorder. In certain embodiments, the immune-related disorder is an autoimmune disorder, graft-versus-host disease, allograft rejection, or other inflammatory conditions. In some embodiments, the immune cells are allogeneic. In some embodiments, the immune-related disorder is cancer. In some embodiments, the cancer is a solid cancer or a hematological malignancy. In some embodiments, the cancer is a hematological malignancy.

[0021] In some embodiments, the treatment method comprises treatment with at least one cell disclosed herein and further comprises treatment with at least one additional therapeutic agent. In some embodiments, the at least one additional therapeutic agent comprises chemotherapy, immunotherapy, surgery, radiation therapy, drug therapy, hormone therapy, biological therapy, or a combination thereof.

[0022] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error in the measuring or quantitating method.

[0023] The use of the word "a" or "an" used in conjunction with the term "comprising" may mean "one," but is also consistent with the meaning of "one or more," "at least one," or "one or more."

[0024] "And / or" means "and" or "or." For example, A, B, and / or C includes: A alone, B alone, C alone, A and B in combination, A and C in combination, B and C in combination, or A, B and C in combination.

[0025] The words "comprising" (and any form of including, such as "comprises" or "comprises"), "having" (and any form of having, such as "have" or "has"), "including" (and any form of including, such as "includes" or "include"), or "containing" (and any form of including, such as "contains" or "contain") are inclusive or open-ended and do not exclude additional, unrepresented elements or method steps.

[0026] Compositions and methods of use thereof may "comprise," "comprise essentially," or "comprise" any of the components or steps disclosed throughout this specification. Compositions and methods "consisting essentially" of any of the disclosed components or steps limit the scope of the claim to those specified materials or steps that do not materially affect the basic and novel characteristics of the claimed invention.

[0027] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the invention, and vice versa. Further, a composition of the invention can be used to achieve a method of the invention.

[0028] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating specific embodiments of the present invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

[0029] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Brief description of the drawings]

[0030] [Figure 1] Figure 1 depicts an exemplary lentiviral vector map used to select different hinge, transmembrane, and / or costimulatory domain combinations. Different hinge, transmembrane, and / or costimulatory domain combinations were cloned between FMC63 scFv and CD3ζ (CD3 zeta) domain to construct different CARs. Plasmids expressing different CARs were transfected into 293T cells using Lipofectamine 3000 for screening of CARs with acceptable CD19 antigen binding ability. LTR = long terminal repeat; HTM = hinge and transmembrane domain; costim = costimulatory domain.

[0031] [Diagram 2] Figure 2 depicts an exemplary lentiviral vector map of a CD19-targeted CAR used to transduce T cells (e.g., infidel γδ T cells) generated from healthy donor T cells. sEF1a = short (weak) EF1 alpha promoter; tPGK = TetO-PGK promoter; HTM = hinge and transmembrane domain; costim = costimulatory domain; tCD34 = cytoplasmic tail truncated CD34.

[0032] [Figure 3-1]Figures 3A-3B depict the expression of anti-CD19 CARs with different hinge and transmembrane domains in T cells (e.g., infinite γδ T cells). Figure 3A) T cells (e.g., infinite γδ T cells) generated from healthy donor T cells were transduced with lentiviral vectors expressing anti-CD19 CARs with CD28 hTM-CD28 costim, PDL1hTM-CD28 costim, and CD30hTM-CD28 costim. The CAR expression cassettes were driven by weak (short EF1a promoter) or strong promoters (PDL1hTM CAR used the MSCV promoter and CD30hTM CAR used the composite PGK promoter). Anti-CD19 CAR expression was determined by staining with FITC-conjugated CD19 antigen. All cells were sorted using FITC-conjugated CD19 antigen. Flow cytometry data were analyzed using FlowJo software. All constructs showed clear CAR-positive populations before and after sorting, but the CD30hTMCD28 costim CAR with the TRE-PGK promoter showed the highest expression. Figure 3B) Median fluorescence intensity (MFI) of the CAR-positive population derived from transduced cells (quantification of Figure 3A). For the CARs driven by the two weak promoters, the MFI of the CD30hTM-CD28 costim CAR population was higher than that of the CD28hTM-CD28 costim CAR before and after sorting. For the CARs driven by the two strong promoters, the MFI of the CD30hTM-CD28 costim CAR population was higher than that of the PDL1hTM-CD28 costim CAR before and after sorting. Data were representative of one of three independent experiments. HTM = hinge and transmembrane domain; costim = costimulatory domain. [Figure 3-2] Same as above.

[0033] [Figure 4]Figure 4 depicts the cytotoxic activity of T cells containing anti-CD19 CAR with CD30hTM-CD28-CD3z against cancer cells. T cells (e.g., infinite γδ T cells) generated from healthy donor T cells were transduced with lentiviral vectors expressing anti-CD19 CAR based on CD30hTM-CD28 costim (tPGK promoter). The CAR expression cassette was driven by a strong composite PGK promoter. After expansion of the transduced T cells, the percentage of CAR positivity was about 20% before sorting. These CAR T cells (e.g., CAR infinite γδ T cells) were co-cultured with Nalm6 tumor cells with 200 IU of exogenous IL-2 in the medium, and the percentage change of Nalm6 cells was monitored over 3 days. The results showed that the percentage of live Nalm6 tumor cells rapidly decreased over 3 days. HTM = hinge and transmembrane domain; costim = costimulatory domain. tPGK = TetO-PGK promoter.

[0034] [Figure 5-1]Figures 5A-5C depict a comparison of the cytotoxic activity of anti-CD19 CARs with different hinge and transmembrane domains. T cells (e.g., infinite γδ T cells) generated from healthy donor T cells were transduced with lentiviral vectors expressing anti-CD19 CARs with CD8hTM-CD28 costim, CD28hTM-CD28 costim, PDL1hTM-CD28 costim, and CD30hTM-CD28 costim (see Figure 2 for vector map). After sorting, CAR-positive cells were co-cultured with Nalm6 tumor cells expressing RFP-luciferase in duplicate wells. The absolute number of live Nalm6 cells was calculated using CountBright™ absolute counting beads on days 0, 1, and 2. Figure 5A shows that CD28hTM-CD28 costim-based anti-CD19 CAR inhibited the proliferation of Nalm6 cells better than CD8hTM-CD28 costim-based anti-CD19 CAR when co-cultured without exogenous IL-2 in the medium. For the co-culture experiment with 200 IU / mL IL-2 in the medium, Figure 5B shows that CD30hTM-CD28 costim-based anti-CD19 CAR had stronger cytotoxicity than CD28hTM-CD28 costim CAR. Figure 5C showed that CD30hTM-CD28 costim-based anti-CD19 CAR had stronger cytotoxicity than PDL1hTM-CD28 costim CAR. Data were representative of one of three independent experiments. Statistical significance (P<0.05) between experimental conditions was determined by paired t-test. HTM=hinge and transmembrane domain; costim=co-stimulatory domain. [Figure 5-2] Same as above. [Figure 5-3] Same as above.

[0035] [Figure 6]Figure 6 depicts the expression of anti-CD79B CAR with CD30 hinge and transmembrane domain. 293T cells were transfected with lentiviral plasmids expressing CD30hTM-CD28 costim anti-CD79B CARs (one containing scFv derived from SN8 clone of CD79B antibody, the other containing scFv derived from 2F2 clone of CD79B antibody). All CAR expression cassettes were driven by composite human PGK promoter. 24 hours after transfection, anti-79B CAR expression was determined by staining with APC-conjugated CD79B antigen. Flow cytometry data was analyzed using FlowJo software. The results showed that CD30 hinge and transmembrane domain also worked well with scFv derived from other antibodies (e.g., not only those targeting CD19 using FMC63 scFv antibody). HTM = hinge and transmembrane domain; costim = costimulatory domain.

[0036] [Figure 7]Figure 7 depicts the expression of anti-CD19 CARs with different hinge and transmembrane domains in 293T cells. 293T cells were transfected with lentiviral plasmids expressing anti-CD19 CARs with different hinge, transmembrane, and / or costimulatory domains. CAR expression cassettes were all driven by MSCV promoter (see Figure 1 for vector map). Transfection efficiency was determined by AF647-conjugated anti-EGFR antibody, and anti-CD19 CAR expression was determined by staining transduced 293T cells with FITC-conjugated CD19 antigen. Data showed that hinge and transmembrane domains derived from the same transmembrane receptor did not support optimal CAR expression (e.g., CD79AhTM-CD28 costim, Long CTLA4hTM-CD28 costim, TIM3hTM-CD28 costim). Furthermore, the data suggested that the entire construct with the hinge and transmembrane domains and the costimulatory domain together was important for optimal CAR expression. This was based on the observation that combining the CD28 costimulatory domain with PD1 or PDL1 hTM resulted in good CAR expression on the cell surface, but not with the hTM domains derived from CTLA4, TIM3, and CD79A. HTM = hinge and transmembrane domain; costim = costimulatory domain.

[0037] [Figure 8]Figure 8 depicts the signaling ability of anti-CD19 CARs with different hinge and transmembrane domains. CAR plasmids that showed permissive expression in 293T cells were used to produce lentiviral vectors and transduced into Jurkat-Lucia™ NFAT reporter cell line (InvivoGen), which were used to quantify CAR-induced signaling by measuring luciferase activity. After sorting the CAR-positive population, each population was co-cultured with Raji lymphoma cells at an effector:target (E:T) ratio of 1:1. After 24 hours, luciferase activity was measured in the supernatant according to the manufacturer's instructions. The results showed that all CARs could be specifically activated by Raji cells, but the CD30hTM-CD28 CAR induced the highest activity. Furthermore, the PD1hTM-CD28 CAR had higher tonic signaling than the other CARs based on the observed activity in the absence of Raji cells. Data were representative of one of two independent experiments. HTM=hinge and transmembrane domain; costi=costimulatory domain.

[0038] [Figure 9-1]Figures 9A-9B depict the expression of anti-CD19 CARs with different hinge and transmembrane domains in infinite αβ T cells. Figure 9A) αβ T cells generated from healthy donor T cells were transduced with lentiviral vectors expressing anti-CD19 CARs containing different hinge and transmembrane domains as well as costimulatory domains. All CAR expression cassettes were driven by the MSCV promoter. Anti-CD19 CAR expression was determined by staining the transduced T cells with FITC-conjugated CD19 antigen. The CD30hTM-OX40 costim CAR showed a superior CAR-positive population, as did the CD28HTM-CD28 costim and CD8HTM-BAFF-R costim CARs. For the other constructs, CAR expression was low or absent. Figure 9B) Anti-CD19 CAR expression on the transduced cells in Figure 9A was determined by staining with FITC-conjugated CD19 antigen. The MFI of the CD30HTM-OX40 costim CAR+ population was the highest among all constructs tested in this experiment. Together, these results suggested that all constructs with hinge and transmembrane domains and costimulatory domains are together important for optimal CAR folding and surface expression. HTM = hinge and transmembrane domains; costim = costimulatory domain. [Figure 9-2] Same as above.

[0039] [Figure 10-1]Figures 10A-10E depict the signaling capabilities of different CARs with CD30 hinge and transmembrane domains (HTM) and CD28-CD3z signaling domains. Lentiviral vectors expressing different CAR constructs were transduced into Jurkat-Lucia™ NFAT reporter cell line, CAR+ cells were sorted, and CAR-induced signaling was quantified by measuring luciferase activity with or without co-culture with Daudi lymphoma cells at a 1:1 effector:target ratio for 24 hours. Figures 10A, 10B, and 10D showed that both FMC63 scFv-CD30HTM-CD28costim (CD19-CD30HTM-CD28 CAR) and SN8 scFv-CD30HTM-CD28costim (CD79b-CD30HTM-CD28 CAR) signal only in the presence of cells expressing CD19 and / or CD79b (e.g., Daudi tumor cells). Figure 10C) Jurkat-Lucia™ NFAT reporter cells were also transduced with Fc receptor CAR (FcR CAR - CD16V-CD30HTM-CD28). The data showed that FcR CAR signals only in the presence of both rituximab (anti-CD20 antibody) and Daudi tumor cells, but not when co-cultured with Daudi tumor cells alone. FIG. 10E) scFv-CD30HTM-41BB CAR (CD19-CD30HTM-41BB CAR) was lentivirally transduced into Jurkat-Lucia™ NFAT reporter cell line, CAR+ cells were sorted, and CAR-induced signaling was quantified by measuring luciferase activity with or without co-culture with PDX203 lymphoma cells, a high-grade B-cell lymphoma cell line developed in our laboratory derived from a patient-derived xenograft. After 24 hours, luciferase activity was measured in the supernatant. The results showed that CD19-CD30HTM-41BB CAR only signals in the presence of PDX-203 tumor cells. Data were representative of one of two or three independent experiments.Overall, these results indicated that the CD30 HTM domain functions as an efficient HTM component with multiple CAR designs and / or different costimulatory domains targeting different antigens on tumor cells. [Figure 10-2] Same as above. [Figure 10-3] Same as above. [Figure 10-4] Same as above. [Figure 10-5] Same as above.

[0040] [Figure 11-1] Figures 11A-11C depict a comparison of the cytotoxic activity of anti-CD19 CARs with CD28 or CD30 hinge and transmembrane (HTM) domains. T cells (e.g., infinite γδ T cells) generated from healthy donors M4 and M5 were transduced with lentiviral vectors expressing anti-CD19 CARs with CD28HTM-CD28 costimulatory domains or CD30HTM-CD28 costimulatory domains, respectively, and then the cells were sorted for CAR+ cells and co-cultured with luciferase-RFP-expressing Nalm6 acute lymphoblastic leukemia tumor cells in duplicate wells at an effector:target ratio of 5:1. The absolute number of live Nalm6 tumor cells was calculated using CountBright™ absolute counting beads on days 0 and 1. Figure 11A depicts the change in the absolute number of live Nalm6 cells, and Figures 11B and 11C depict the percentage change in live tumor cells. The results showed that CD19 CARs with CD30HTM domains had significantly more potent cytotoxicity than CD19 CARs with CD28HTM domains. P values ​​were calculated by unpaired t-test. [Figure 11-2] Same as above. [Figure 11-3] Same as above.

[0041] [Figure 12-1]Figures 12A-12B show the antitumor effect of infinity γδ CAR T cells transduced with scFv CD30HTM CAR in vivo. Luciferase-labeled Daudi Burkitt lymphoma tumor cells (2 × 104 tumor cells / mouse) were intravenously injected into three groups of human IL-15 transgenic NSG mice (secreting physiological levels of human IL-15) on day -2. Three fusions of infinity γδ T or infinity anti-CD19CD30HTM-CD28Cos CAR-γδ T were injected into mice on days 0, 3, and 8 at a dose of 8 × 106 T cells / mouse / injection. The results showed that CD30HTM-CD28Cos CAR-γδ T slowed lymphoma progression. Total tumor burden was assessed by bioluminescence imaging (Figure 12A) and the mean total flux in each group was quantified (Figure 12B). The data showed that the antitumor effect of infinite γδ CAR T cells was higher than that of infinite γδ T cells without CD30HTM-CAR. [Figure 12-2] Same as above.

[0042] [Figure 13-1]Figures 13A-13B show the transduction, signaling, and CD70 binding capacity of a truncated CD27 (tCD27) CAR with CD30 hinge and transmembrane domains. Figure 13A) A CD27-based anti-CD70 CAR was generated by fusing a truncated CD27 extracellular domain (SEQ ID NO: 48) with CD30 hinge and TM domains, CD28 costimulatory domain, and CD3z signaling domain. Signaling capacity was determined using Jurkat-Lucia™ NFAT reporter cell line (Invivogen). Lentiviral vectors expressing this CAR construct were transduced into Jurkat-Lucia™ NFAT reporter cell line, CAR+ cells were sorted, and CAR-induced signaling was quantified by measuring luciferase activity with or without co-culture with a CD70 positive T cell line at an effector:target ratio of 1:1. Luciferase activity was measured in the supernatant after 24 hours. As shown, the tCD27-CD30HTM-CD28cos-CD3z CAR only signaled in the presence of cells expressing CD70. The results indicated that the CD30HTM domain functions as an efficient HTM component with multiple CAR designs and / or different co-stimulatory domains targeting different antigens on tumor cells. RLU = relative light units. Figure 13B) shows the expression of the above lentiviral vector (tCD27-CD30HTM-CD28cos-CD3z CAR) transduced into primary human T cells. Ten days after transduction, cell surface expression of CD27 and CD70 on CAR-T cells was measured. The ability of the CAR to bind CD70 was also tested by staining with fluorescent dye-conjugated recombinant CD70 protein. Non-transduced T cells and a low affinity anti-CD70 scFv antibody (clone 1F6) were used as controls. Anti-CD27 antibody staining results showed that tCD27 was properly folded and expressed on the surface of primary T cells (top row). CD70 protein staining showed that tCD27 CAR bound to recombinant CD70 protein (middle row).Anti-CD70 antibody staining showed that CD70+ cells were nearly absent in T cells transduced with the tCD27 CAR, indicating that CD70-expressing cells were either efficiently deleted or CD70 on the cell surface was masked by cis binding (bottom row). [Figure 13-2] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] The inventors herein describe several studies that involve testing hinge and / or transmembrane domain scores for suitability in CAR construction. Surprisingly, the inventors show that functional and / or effective CAR molecules can be constructed using relatively few hinge and / or transmembrane domains. Among the example components tested, the hinge and / or transmembrane domain derived from CD30 was effective. Without being limited by theory, one reason for this observed effectiveness may be the primary amino acid sequence underlying the CD30 hinge and / or transmembrane domain. In some embodiments, the CD30-derived hinge and / or transmembrane domain has more amino acids (e.g., G or S) that provide flexibility than those derived from CD28 or CD8a, which are the most commonly used hinge and / or transmembrane domains in CARs. Furthermore, in some embodiments, the described CD30-derived hinge and / or transmembrane domain does not contain a cysteine ​​(C) in the hinge or transmembrane region. In contrast, the CD28 hinge and transmembrane domain have two cysteines, and the CD8a hinge and transmembrane domain have three cysteines. It is known that cysteine-containing proteins or peptides tend to form homodimers between themselves or heterodimers with other transmembrane proteins, which may lead to unpredictable results regarding CAR expression and folding of the antigen-binding domain of the CAR molecule (e.g., scFv). Thus, the unique sequence of the CD30-derived hinge and / or transmembrane domain allows for better folding of the antigen-binding domain of the CAR molecule (e.g., scFv), explaining the observed increase in cell surface expression of the CD30 hinge and / or transmembrane domain comprising the CAR described herein. Consistent with this, the results described herein show that anti-CD19 CARs comprising CD30h / TM-CD28-CD3z had strong surface expression in T cells, robust signaling capacity in Jurkat reporter cell lines (e.g., the highest signaling capacity of the vectors tested), and demonstrated potent cytotoxicity against B cell leukemia.

[0044] Since it is known that identical DNA sequences in lentiviral vectors can have detrimental effects on the stability of the underlying construct, the search for new effective hinge and / or transmembrane domains provides the field with additional tools that, in certain embodiments, can be used to construct dual, triple, quadruple, etc. CARs that target two or more different antigens. Thus, compared to the established CD8a or CD28 hinge and transmembrane domains, the significantly different DNA sequences encoding the described CD30 hinge and / or transmembrane domains facilitate the production of stable lentiviral vectors for generating bispecific, trispecific, etc. CARs that simultaneously target more than one antigen.

[0045] Aspects of the present disclosure relate to improvements in CAR construct components (e.g., domains and / or regions). In certain embodiments, the present disclosure provides novel hinge and / or transmembrane (TM) domains that can be used to construct chimeric antigen receptors (CARs). In certain embodiments, the hinge and / or TM domains are derived in part from the extracellular domain and complete transmembrane domain of the human CD30 molecule. In certain embodiments, the CD30 hinge and / or TM domains, when connected with costimulatory domains such as, but not limited to, OX40 or CD28, can support strong CAR expression and effector cell cytotoxicity against tumor cells.

[0046] In some embodiments, a CAR construct comprising CD30h / TM-CD28-CD3z can induce stronger downstream signaling of CD3z in the Jurkat-Lucia™ reporter cell line (as an example) than a comparable CAR construct comprising CD28h / TM-CD28-CD3z. In some embodiments, immune cells comprising a CAR construct comprising CD30h / TM-CD28-CD3z have better cytotoxicity against cancer cells when compared to immune cells comprising a CAR comprising CD28h / TM-CD28-CD3z.

[0047] I. Polypeptides As used herein, a "protein" or "polypeptide" refers to a molecule that comprises at least five amino acid residues. As used herein, the term "wild type" refers to the endogenous version of a molecule that naturally occurs in an organism. In some embodiments, a wild type version of a protein or polypeptide is used, while in many embodiments of the present disclosure, a modified protein or polypeptide is used to generate an immune response. The above terms can be used interchangeably. A "modified protein" or "modified polypeptide" or "variant" refers to a protein or polypeptide whose chemical structure, particularly its amino acid sequence, has been altered with respect to a wild type protein or polypeptide. In some embodiments, a modified / variant protein or polypeptide has at least one modified activity or function (recognizing that a protein or polypeptide may have multiple activities or functions). It is specifically contemplated that a modified / variant protein or polypeptide may be altered with respect to one activity or function but retain a wild type activity or function in other respects, such as immunogenicity.

[0048] When a protein is specifically described herein, it is generally a reference to a natural (wild type) or recombinant (modified) protein, or a protein with the signal sequence removed, if necessary. Proteins can be directly isolated from a natural organism, produced by recombinant DNA / exogenous expression methods, or produced by solid phase peptide synthesis (SPPS) or other in vitro methods. In certain embodiments, there are isolated nucleic acid segments and recombinant vectors that contain a nucleic acid sequence that encodes a polypeptide (e.g., an antibody or fragment thereof). The term "recombinant" may be used in connection with a polypeptide or the name of a particular polypeptide, and generally refers to a polypeptide that is produced from a nucleic acid molecule that has been manipulated in vitro, or is a product of replication of such a molecule.

[0049] In certain embodiments, the size of a protein or polypeptide (wild type or modified) is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 , 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725 , 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1100, 1200, 1300, 1400, 1500, 1750, 2000, 2250, 2500 amino acid residues or more, and any range derivable therefrom, or derivatives of the corresponding amino acid sequences described or referenced herein. It is contemplated that the polypeptides may be mutated by truncation, shortened from the corresponding wild type, and modified by fusing or joining heterologous protein or polypeptide sequences having specific functions (e.g., for targeting or localization, for enhancing immunogenicity, for purification purposes, etc.). As used herein, the term "domain" refers to any discrete functional or structural unit of a protein or polypeptide, generally a sequence of amino acids having a structure or function recognizable to one of skill in the art.

[0050] The polypeptides, proteins, or polynucleotides encoding such polypeptides or proteins of the present disclosure include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (or a range derivable therein) or more variant amino acid or nucleic acid substitutions for SEQ ID NOs: 1-52. or at least or at most 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82 , 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 1 18, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 9, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180 , 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211,212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 300, 400, 500, 550, 1000 or more consecutive amino acids or nucleic acids and at least is 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or a range derivable therein).

[0051] In some embodiments, the protein or polypeptide comprises amino acids 1-2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 20 7, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106 , 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 1 69, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 20 0, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231 , 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262,263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459、460、461、462、463、464、465、466、467、468、469、470、471、472、473、474、475、476、477、478、479、480、481、482、483、484、485、486、487、488、489、490、491、492、493、494、495、496、497、498、499、500、501、502、503、504、505、506、507、508、509、510、511、512、513、514、515、516、517、518、519、520、521、522、523、524、525、526、527、528、529、530、531、532、533、534、535、536、537、538、539、540、541、542、543、544、545、546、547、548、549、550、551、552、553、554、555、556、557、558、559、560、561、562、563、564、565、566、567、568、569、570、571、572、573、574、575、576、577、578、579、580、581、582、583、584、585、586、587、588、589、590、591、592、593、594、595、596、597、598、599、600、601、602、603、604、605、606、607、608、609、610、611、612、613、614、615、616、617、618、619、620、621、622、623、624、625、626、627、628、629、630、631、632、633、634、635、636、637、638、639、640、641、642、643、644、645、646、647、648、649、650、651、652、653、654、655、656、657、658、659、660、661、662、663、664、665、666、667、668、669、670、671、672、673、674、675、676、677、678、679、680、681、682、683、684、685、686、687、688、689、690、691、692、693、694、695、696、697、698、699、700、701、702、703、704、705、706、707、708、709、710、711、712、713、714、715、716、717、718、719、720、721、722、723、724、725、726、727、728、729、730、731、732、733、734、735、736、737、738、739、740、741、742、743、744、745、746、747、748、749、750、751、752、753、754、755、756、757、758、759、760、761、762、763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 79 3, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 8 24, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854 , 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 88 5, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946 , 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, or 1000 (or any range derivable therein).

[0052] In some embodiments, the protein or polypeptide comprises amino acids 1-2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 20 7, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106 , 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 1 69, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 20 0, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231 , 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262,263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459、460、461、462、463、464、465、466、467、468、469、470、471、472、473、474、475、476、477、478、479、480、481、482、483、484、485、486、487、488、489、490、491、492、493、494、495、496、497、498、499、500、501、502、503、504、505、506、507、508、509、510、511、512、513、514、515、516、517、518、519、520、521、522、523、524、525、526、527、528、529、530、531、532、533、534、535、536、537、538、539、540、541、542、543、544、545、546、547、548、549、550、551、552、553、554、555、556、557、558、559、560、561、562、563、564、565、566、567、568、569、570、571、572、573、574、575、576、577、578、579、580、581、582、583、584、585、586、587、588、589、590、591、592、593、594、595、596、597、598、599、600、601、602、603、604、605、606、607、608、609、610、611、612、613、614、615、616、617、618、619、620、621、622、623、624、625、626、627、628、629、630、631、632、633、634、635、636、637、638、639、640、641、642、643、644、645、646、647、648、649、650、651、652、653、654、655、656、657、658、659、660、661、662、663、664、665、666、667、668、669、670、671、672、673、674、675、676、677、678、679、680、681、682、683、684、685、686、687、688、689、690、691、692、693、694、695、696、697、698、699、700、701、702、703、704、705、706、707、708、709、710、711、712、713、714、715、716、717、718、719、720、721、722、723、724、725、726、727、728、729、730、731、732、733、734、735、736、737、738、739、740、741、742、743、744、745、746、747、748、749、750、751、752、753、754、755、756、757、758、759、760、761、762、763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793 , 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824 , 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855 , 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886 , 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 91 7, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 94 8, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, or 1000 (or any range derivable therein) consecutive amino acids.

[0053] In some embodiments, the polypeptide, protein, or nucleic acid is at least, at most, or exactly 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or a range derivable therein) of SEQ ID NOs: 1-52. 1 to 52, which are similar, identical, or homologous to at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 1 42, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 17 3, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204,205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223、224、225、226、227、228、229、230、231、232、233、234、235、236、237、238、239、240、241、242、243、244、245、246、247、248、249、250、251、252、253、254、255、256、257、258、259、260、261、262、263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459、460、461、462、463、464、465、466、467、468、469、470、471、472、473、474、475、476、477、478、479、480、481、482、483、484、485、486、487、488、489、490、491、492、493、494、495、496、497、498、499、500、501、502、503、504、505、506、507、508、509、510、511、512、513、514、515、516、517、518、519、520、521、522、523、524、525、526、527、528、529、530、531、532、533、534、535、536、537、538、539、540、541、542、543、544、545、546、547、548、549、550、551、552、553、554、555、556、557、558、559、560、561、562、563、564、565、566、567、568、569、570、571、572、573、574、575、576、577、578、579、580、581、582、583、584、585、586、587、588、589、590、591、592、593、594、595、596、597、598、599、600、601、602、603、604、605、606、607、608、609、610、611、612、613、614、615、616、617、618、619、620、621、622、623、624、625、626、627、628、629、630、631、632、633、634、635、636、637、638、639、640、641、642、643、644、645、646、647、648、649、650、651、652、653、654、655、656、657、658、659、660、661、662、663、664、665、666、667、668、669、670、671、672、673、674、675、676、677、678、679、680、681、682、683、684、685、686、687、688、689、690、691、692、693、694、695、696、697、698、699、700、701、702、703、704、705、706、707、708、709、710、711、712、713、714、715、716、717、718、719、720、721、722、723、724、725、726、727、728、729、730、731、732、733、734、735、736、737、738、739、740、741、742、743、744、745、746、747、748、749、750、751、752、753、754、755、756、757、758、759、760、761、762、763、764、765、766、767、768、769、770、771、772、773、774、775、776、777、778、779、780、781、782、783、784、785、786、787、788、789、790、791、792、793、794、795、796、797、798、799、800、801、802、803、804、805、806、807、808、809、810、811、812、813、814、815、816、817、818、819、820、821、822、823、824、825、826、827、828、829、830、831、832、833、834、835、836、837、838、839、840、841、842、843、844、845、846、847、848、849、850、851、852、853、854、855、856、857、858、859、860、861、862、863、864、865、866、867、868、869、870、871、872、873、874、875、876、877、878、879、880、881、882、883、884、885、886、887、888、889、890、891、892、893、894、895、896、897、898、899、900、901、902、903、904、905、906、907、908、909、910、911、912、913、914、915、916、917、918、919、920、921、922、923、924、925、926、927、928、929、930、931、932、933、934、935、936、937、938、939、940、941、942、943、944、945、946、947、948、949、950、951、952、953、954、955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, or 1000 (or any range derivable therein) consecutive amino acids or nucleotides.

[0054] In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 0, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 11 6, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147 , 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 2 10, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 24 1, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272,273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459、460、461、462、463、464、465、466、467、468、469、470、471、472、473、474、475、476、477、478、479、480、481、482、483、484、485、486、487、488、489、490、491、492、493、494、495、496、497、498、499、500、501、502、503、504、505、506、507、508、509、510、511、512、513、514、515、516、517、518、519、520、521、522、523、524、525、526、527、528、529、530、531、532、533、534、535、536、537、538、539、540、541、542、543、544、545、546、547、548、549、550、551、552、553、554、555、556、557、558、559、560、561、562、563、564、565、566、567、568、569、570、571、572、573、574、575、576、577、578、579、580、581、582、583、584、585、586、587、588、589、590、591、592、593、594、595、596、597、598、599、600、601、602、603、604、605、606、607、608、609、610、611、612、613、614、615、616、617、618、619、620、621、622、623、624、625、626、627、628、629、630、631、632、633、634、635、636、637、638、639、640、641、642、643、644、645、646、647、648、649、650、651、652、653、654、655、656、657、658、659、660、661、662、663、664、665、666、667、668、669、670、671、672、673、674、675、676、677、678、679、680、681、682、683、684、685、686、687、688、689、690、691、692、693、694、695、696、697、698、699、700、701、702、703、704、705、706、707、708、709、710、711、712、713、714、715、716、717、718、719、720、721、722、723、724、725、726、727、728、729、730、731、732、733、734、735、736、737、738、739、740、741、742、743、744、745、746、747、748、749、750、751、752、753、754、755、756、757、758、759、760、761、762、763、764、765、766、767、768、769、770、771、772、773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 8 35, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 86 6, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897 , 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 9 99, 992, 993, 994, 995, 996, 997, 998, 999, or 1000 of SEQ ID NOs: 1-52, and wherein there is a nucleic acid molecule or polypeptide beginning at position 60, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, or 1000 of SEQ ID NOs: 1-52,15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223、224、225、226、227、228、229、230、231、232、233、234、235、236、237、238、239、240、241、242、243、244、245、246、247、248、249、250、251、252、253、254、255、256、257、258、259、260、261、262、263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、3、 16、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459、460、461、462、463、464、465、466、467、468、469、470、471、472、473、474、475、476、477、478、479、480、481、482、483、484、485、486、487、488、489、490、491、492、493、494、495、496、497、498、499、500、501、502、503、504、505、506、507、508、509、510、511、512、513、514、515、516、517、518、519、520、521、522、523、524、525、526、527、528、529、530、531、532、533、534、535、536、537、538、539、540、541、542、543、544、545、546、547、548、549、550、551、552、553、554、555、556、557、558、559、560、561、562、563、564、565、566、567、568、569、570、571、572、573、574、575、576、577、578、579、580、581、582、583、584、585、586、587、588、589、590、591、592、593、594、595、596、597、598、599、600、601、602、603、604、605、606、607、608、609、610、611、612、613、614、615、616、617、618、619、620、621、622、623、624、625、626、627、628、629、630、631、632、633、634、635、636、637、638、639、640、641、642、643、644、645、646、647、648、649、650、651、652、653、654、655、656、657、658、659、660、661、662、663、664、665、666、667、668、669、670、671、672、673、674、675、676、677、678、679、680、681、682、683、684、685、686、687、688、689、690、691、692、693、694、695、696、697、698、699、700、701、702、703、704、705、706、707、708、709、710、711、712、713、714、715、716、717、718、719、720、721、722、723、724、725、726、727、728、729、730、731、732、733、734、735、736、737、738、739、740、741、742、743、744、745、746、747、748、749、750、751、752、753、754、755、756、757、758、759、760、761、762、763、764、765、766、767、768、769、770、771、772、773、774、775、776、777、778、779、780、781、782、783、784、785、786、787、788、789、790、791、792、793、794、795、796、797、798、799、800、801、802、803、804、805、806、807、808、809、810、811、812、813、814、815、816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, or 1000 (or any range derivable therein) consecutive amino acids or nucleotides.

[0055] Nucleotide and protein, polypeptide and peptide sequences for various genes have been disclosed before and can be found in widely recognized computer databases.Two commonly used databases are National Center for Biotechnology Information's Genbank and GenPept databases (on the World Wide Web at ncbi.nlm.nih.gov / ) and The Universal Protein Resource (UniProt; on the World Wide Web at uniprot.org).The coding regions of these genes can be amplified and / or expressed using the techniques disclosed herein or as known to those skilled in the art.

[0056] It is contemplated that in certain embodiments of the compositions of the present disclosure, there is about 0.001 mg to about 10 mg of total polypeptide, peptide, and / or protein per ml. The concentration of protein in the composition may be about, at least about, or at most about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg / ml or more (or any range derivable therein).

[0057] A. Sequence The amino acid sequences of certain polypeptides, including chimeric antigen receptors and portions, regions, and / or domains thereof, are provided in Table 1. Table 1 - Amino acid sequences [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0058] B. Variant Polypeptides The following is a discussion of the change of amino acid subunits of proteins to create equivalent or even improved second generation variant polypeptides or peptides. For example, certain amino acids in a protein or polypeptide sequence can be replaced with other amino acids with or without appreciable loss of interactive binding ability with structures such as the antigen-binding region of an antibody or the binding site on a substrate molecule. Because it is the interacting ability and properties of a protein that define its functional activity, certain amino acid substitutions can be made in a protein sequence and its corresponding DNA coding sequence, but still produce a protein with similar or desirable properties. Thus, it is contemplated by the inventors that various changes can be made in the DNA sequence of the gene that codes for a protein without appreciable loss of the biological usefulness or activity of the protein.

[0059] As used herein, the term "functionally equivalent codons" refers to codons that code for the same amino acid, such as the six different codons for arginine. Also contemplated are "neutral substitutions" or "neutral mutations," which refer to changes in a codon or codons that code for biologically equivalent amino acids.

[0060] The amino acid sequence variant of the present disclosure may be a substitution, insertion, or deletion variant. Mutations in the polypeptide of the present disclosure may affect 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more non-contiguous or contiguous amino acids of a protein or polypeptide compared to wild type. Variants may include amino acid sequences that are at least 50%, 60%, 70%, 80%, or 90% identical (including all values ​​and ranges therebetween) to any of the sequences provided or referenced herein. The variant may contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more substituted amino acids.

[0061] It will also be understood that the amino acid and nucleic acid sequences may contain additional residues, such as additional N- or C-terminal amino acids, or 5' or 3' sequences, respectively, and may even be essentially identical as described in one of the sequences disclosed herein, so long as the sequence meets the above criteria, including the maintenance of biological protein activity where protein expression is concerned. The addition of terminal sequences applies particularly to nucleic acid sequences that may include, for example, various non-coding sequences flanking either the 5' or 3' portion of the coding region.

[0062] Typically, deletion variants lack one or more residues of the native or wild-type protein. Individual residues can be deleted, or several consecutive amino acids can be deleted. Stop codons can be introduced (by substitution or insertion) into the coding nucleic acid sequence to generate truncated proteins.

[0063] Insertional mutants typically involve the addition of amino acid residues at non-terminal points in the polypeptide. This may involve the insertion of one or more amino acid residues. Terminal additions may also be produced, and they may include fusion proteins that are multimers or concatamers of one or more peptides or polypeptides described or referenced herein.

[0064] Substitution variants typically contain the exchange of one amino acid for another at one or more sites within a protein or polypeptide, and can be designed to modulate one or more properties of the polypeptide, with or without the loss of other functions or properties. Substitutions can be conservative, i.e., an amino acid is replaced with one of similar chemical properties. A "conservative amino acid substitution" can include the exchange of a member of an amino acid class with another member of the same class. Conservative substitutions are well known in the art, and include, for example, alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartic acid to glutamic acid; cysteine ​​to serine; glutamine to asparagine; glutamic acid to aspartic acid; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. Conservative amino acid substitutions may include non-naturally occurring amino acid residues that are typically incorporated by chemical peptide synthesis rather than synthesis in living systems. These include peptide mimetics or other reverse or inverted amino acid moieties.

[0065] Alternatively, the substitutions may be "non-conservative" such that the function or activity of the polypeptide is affected. Non-conservative changes typically involve replacing an amino acid residue with a chemically dissimilar one, such as a polar or charged amino acid with a non-polar or charged amino acid, and vice versa. Non-conservative substitutions may involve the exchange of a member of one amino acid class for a member from another class.

[0066] Those skilled in the art can use well-known techniques to determine suitable variants of the polypeptides described herein.Those skilled in the art can identify suitable regions of the molecule that can be changed without destroying activity by targeting regions that are not considered important for activity.Those skilled in the art can also identify the amino acid residues and parts of the molecule that are conserved between similar proteins or polypeptides.In further embodiments, regions that may be important for biological activity or structure may undergo conservative amino acid substitutions that do not significantly change biological activity or have no adverse effect on protein or polypeptide structure.

[0067] In making such changes, the hydropathic index of the amino acids can be taken into consideration. The hydropathic profile of a protein is calculated by assigning a numerical value (the "hydropathy index") to each amino acid and then averaging these values ​​repeatedly along the peptide chain. Each amino acid is assigned a value based on its hydrophobicity and charge characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cysteine ​​(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte et al., J. Mol. Biol. 157:105-131 (1982)). It is accepted that the relative hydropathicity of amino acids contributes to the secondary structure of a resulting protein or polypeptide, which in turn dictates the interaction of the protein or polypeptide with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc. It is also known that certain amino acids can be substituted with other amino acids that have a similar hydropathic index or score and further retain similar biological activity. In making changes based on hydropathic index, certain embodiments include substitution of amino acids whose hydropathic index is within ±2. Some aspects of the disclosure include those within ±1, and other aspects of the disclosure include those within ±0.5.

[0068] It is also understood in the art that similar amino acid substitutions can be effectively made based on hydrophilicity.U.S. Patent No. 4,554,101, incorporated herein by reference, describes that the largest partial average hydrophilicity of a protein, governed by the hydrophilicity of adjacent amino acids, correlates with the biological properties of the protein.In certain embodiments, the largest partial average hydrophilicity of a protein, governed by the hydrophilicity of adjacent amino acids, correlates with its immunogenicity and antigen binding as biological properties of the protein. The following hydrophilicity values ​​have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0±1); glutamic acid (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). In making changes based on similar hydrophilicity values, certain embodiments include substitutions of amino acids whose hydrophilicity values ​​are within ±2, other embodiments include those that are within ±1, and still other embodiments include those that are within ±0.5. In some instances, one skilled in the art can also identify epitopes from primary amino acid sequences based on hydrophilicity. These regions are also referred to as "epitope core regions." It is understood that an amino acid can be substituted with another amino acid that has a similar hydrophilicity value and still results in biologically equivalent and immunologically equivalent proteins.

[0069] In addition, one skilled in the art can review structure-function studies that identify residues in similar polypeptides or proteins that are important for activity or structure. In light of such comparisons, one skilled in the art can predict the importance of amino acid residues in a protein that correspond to amino acid residues in the similar protein that are important for activity or structure. One skilled in the art can select substitutions of such predicted important amino acid residues with chemically similar amino acids.

[0070] Those skilled in the art can also analyze the three-dimensional structure and amino acid sequence with respect to its structure in a similar protein or polypeptide. In view of such information, those skilled in the art can predict the alignment of the amino acid residues of an antibody with respect to its three-dimensional structure. Those skilled in the art may choose not to make changes to amino acid residues predicted to be on the surface of a protein, since such residues may be involved in important interactions with other molecules. Furthermore, those skilled in the art can generate test variants containing single amino acid substitutions at each desired amino acid residue. These variants can then be screened using standard assays for binding and / or activity, thus obtaining information from such routine experiments and allowing those skilled in the art to determine amino acid positions where further substitutions should be avoided, either alone or in combination with other mutations. Various means available for determining secondary structure can be found on the World Wide Web at expasy.org / proteomics / protein_structure.

[0071] In some embodiments of the present disclosure, amino acid substitutions are made that (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) change binding affinity for forming protein complexes, (4) change ligand or antigen binding affinity, and / or (5) confer or modify other physicochemical or functional properties to such polypeptides. For example, single or multiple amino acid substitutions (in certain embodiments, conservative amino acid substitutions) can be made in naturally occurring sequences. Substitutions can be made in portions of antibodies outside of domains that form intermolecular contacts. In such embodiments, conservative amino acid substitutions that do not substantially change the structural features of proteins or polypeptides (e.g., one or more replacement amino acids that do not disrupt the secondary structure that characterizes natural antibodies) can be used.

[0072] C. Chimeric antigen receptor In some embodiments, chimeric antigen receptors (CARs) are disclosed. CARs generally comprise an extracellular antigen (or ligand) binding domain and / or a transmembrane domain linked, in some embodiments, by a linker to one or more intracellular signaling components. Such molecules typically mimic or approximate the signaling through a natural antigen receptor, the signaling through such receptor together with a costimulatory receptor, and / or the signaling through a costimulatory receptor alone.

[0073] It is contemplated that chimeric constructs can be introduced into any kind of immune cell as naked DNA or in a suitable vector.Methods for stably transfecting cells with naked DNA by electroporation are known in the art.See, for example, U.S. Patent No. 6,410,319.Naked DNA generally refers to the DNA encoding chimeric receptor contained in a plasmid expression vector in the proper orientation for expression.

[0074] Alternatively, a viral vector (such as a retroviral vector, an adenoviral vector, an adeno-associated viral vector, or a lentiviral vector) can be used to introduce the chimeric construct into immune cells.The vector suitable for use according to the method of the present disclosure is non-replicative in immune cells.A large number of vectors are known that are based on viruses, such as the vectors based on HIV, SV40, EBV, HSV, or BPV, and the copy number of the virus that is maintained in cells is low enough to maintain cell viability.

[0075] Certain embodiments of the present disclosure relate to the use of nucleic acids, including nucleic acids encoding cancer antigen-specific CAR polypeptides, including CARs (hCARs) that are humanized to reduce immunogenicity, in some cases comprising at least one intracellular signaling domain, a transmembrane domain, and an extracellular domain that comprises one or more signaling motifs. In certain embodiments, the binding region may comprise a complementarity determining region of a monoclonal antibody, a variable region of a monoclonal antibody, and / or an antigen-binding fragment thereof. In another embodiment, the specificity is derived from a peptide (e.g., a cytokine) that binds to a receptor.

[0076] It is contemplated that the CAR nucleic acid may be a human gene used to enhance cellular immunotherapy for human patients. In certain embodiments, the present disclosure includes a full-length CAR cDNA or coding region. The antigen-binding region or domain is a V domain of a single chain variable fragment (scFv) derived from a particular human monoclonal antibody (e.g., an anti-CD19 antibody such as FMC63.3 and / or an anti-CD79b antibody such as those described in PCT Patent Application Publication WO2021 / 222944). H and V LIn some embodiments, the fragment may comprise a fragment of a chain of a human antigen-specific antibody. In some embodiments, the fragment may be any number of different antigen-binding domains of a human antigen-specific antibody. In more particular embodiments, the fragment is a cancer antigen-specific scFv encoded by a sequence that is optimized for human codon usage for expression in human cells. In some embodiments, the antigen-binding region comprises a protein or polypeptide that acts as a ligand and / or receptor for another protein and / or polypeptide.

[0077] In some embodiments, the configuration may be multimeric, such as a diabody or multimer. Multimers are most likely formed by cross-pairing in the diabody of the variable portions of the light and heavy chains. The hinge portion of the construct may have multiple options, from being deleted entirely, to maintaining the first cysteine, to a proline rather than serine substitution, to being truncated to the first cysteine. In some embodiments, the Fc portion may be deleted. In some embodiments, any protein that is stable and / or dimerizes can serve this purpose. In some embodiments, only one Fc domain is used, for example, either the CH2 or CH3 domain from a human immunoglobulin. In some embodiments, the hinge, CH2 and CH3 regions of a human immunoglobulin that have been modified to improve dimerization can be used. In some embodiments, only the hinge portion of an immunoglobulin can be used.

[0078] The sequence of the open reading frame encoding the chimeric receptor can be obtained from genomic DNA sources, cDNA sources, or synthesized (e.g., by PCR), or a combination thereof. Depending on the size of the genomic DNA and the number of introns, it may be desirable to use cDNA or a combination thereof, since introns are known to stabilize mRNA. It may also be advantageous to use endogenous or exogenous non-coding regions to stabilize the mRNA.

[0079] In some aspects, an antigen-specific binding (e.g., anti-CD19, anti-CD79b, anti-CD70, etc.) or recognition component is linked to one or more transmembrane and intracellular signaling domains. In some embodiments, the CAR comprises a transmembrane domain fused to the extracellular domain of the CAR. In some embodiments, a transmembrane domain that is naturally associated with one of the domains in the CAR is used. In some embodiments, a transmembrane domain that is not naturally associated with one of the domains in the CAR is used. In some examples, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex. In some embodiments, the transmembrane domain is derived from a natural or synthetic source. In some embodiments, if the source is natural, the transmembrane domain is derived from any membrane-bound or transmembrane protein. In some embodiments, the transmembrane region includes those derived from (i.e., of; also includes at least a transmembrane region "derived from") the alpha, beta or zeta chain of the T cell receptor, CD28, DAP12, DAP10, NKG2D, CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD30, CD33, CD37, CD64, CD80, CD86, OX-40 (CD134), 4-1BB (CD137), CD154, ICOS / CD278, and the like. In some embodiments, the transmembrane domain of the present disclosure is a transmembrane domain derived from CD8α. In some embodiments, the transmembrane domain of the present disclosure is a transmembrane domain derived from CD30.

[0080] In some embodiments, the CAR nucleic acid includes sequences encoding other costimulatory receptors, such as transmembrane domains and one or more intracellular signaling domains. In addition to the primary T cell activation signal, such as may be initiated by CD3ζ and / or FcεRIγ, additional stimulatory signals for immune effector cell proliferation and effector function after engagement of the chimeric receptor with the target antigen can be used. For example, some or all of the human costimulatory receptors for enhanced activation of cells can be used, which may help improve in vivo persistence and improve the therapeutic success of adoptive immunotherapy. Examples include costimulatory domains derived from molecules such as DAP12, DAP10, NKG2D, CD2, CD28, CD27, 4-1BB (CD137), OX-40 (CD134), ICOS, (CD278), CD30, HVEM, CD40, LFA-1 (CD11a / CD18), and ICAM-1, although in certain alternative embodiments, any one of these enumerated ones may be excluded from use in the CAR.

[0081] In certain embodiments, certain CAR molecules are encompassed herein. In some cases, the antigen-binding domain of the CAR is an scFv, and any scFv that binds to a cancer antigen can be used herein. When an scFv is used in the extracellular domain of the CAR, the variable heavy and variable light chains of the scFv can be in any order from N-terminus to C-terminus. For example, the variable heavy chain can be N-terminal to the variable light chain, or vice versa. The scFv and / or ligand that binds to the antigen in the CAR can be codon-optimized or not. In some embodiments, the antigen-binding domain of the CAR is a ligand of another protein (e.g., a "bait" protein), such as the CD27 molecule that acts to target CD70. In some embodiments, the CD27 molecule is a truncated CD27 extracellular binding domain (e.g., tCD27).

[0082] In certain embodiments, the vector encodes a cancer antigen-specific CAR and also encodes one or more other molecules. For example, the vector may encode both a first CAR (e.g., an anti-CD19 CAR) and a second CAR (e.g., an anti-CD79b CAR, an anti-CD70 CAR, etc.).

[0083] In some embodiments, on the same molecule, a cancer antigen-specific CAR may include one or more antigen-specific extracellular domains, a specific hinge, a specific transmembrane domain, one or more specific costimulatory domains, and one or more specific activation signals. When more than one antigen-specific extracellular domain is used, such as to target two different antigens, a linker may be present between the two antigen-specific extracellular domains. Examples of CARs contemplated herein include, but are not limited to, CD19-specific (also "anti-CD19") CARs, anti-CD70 CARs (also CD70 CARs or tCD27-CARs), and CD79b-specific (also "anti-CD79b") CARs.

[0084] In certain embodiments of specific CAR molecules, the CAR may use CD28, DAP10, DAP12, 4-1BB, NKG2D, etc., or other costimulatory domains (also referred to herein as cytoplasmic domains). In some cases, CD3 zeta is used without any costimulatory domain. In certain embodiments of specific CAR molecules, the CAR may use any suitable transmembrane domain from CD30, DAP12, DAP10, 4-1BB, 2B4, OX40, CD27, NKG2D, CD8, CD28, IL12Rβ1, or IL12Rβ2, etc. In certain embodiments of specific CAR molecules, the CAR may use a transmembrane domain from CD30.

[0085] 1. Signal peptide The polypeptide of the present disclosure may include a signal peptide. "Signal peptide" refers to a peptide sequence that directs the transport and localization of a protein within a cell, for example, to a certain cell organelle (such as the endoplasmic reticulum) and / or to the cell surface. In some embodiments, the signal peptide directs the nascent protein to the endoplasmic reticulum. This is essential when the receptor is glycosylated and anchored to the cell membrane. In general, the signal peptide that naturally binds to the amino-terminal many components is used (e.g., in scFvs with a light chain-linker-heavy chain orientation, the natural signal of the light chain is used).

[0086] In some embodiments, the signal peptide is cleaved after passage through the endoplasmic reticulum (ER), i.e., it is a cleavable signal peptide, hi some embodiments, the restriction site is at the carboxy terminus of the signal peptide to facilitate cleavage.

[0087] 2. Antigen-binding domain The polypeptides of the present disclosure may include one or more antigen-binding domains. An "antigen-binding domain" describes a region of a polypeptide that can bind to an antigen under appropriate conditions. In some embodiments, the antigen-binding domain is a single-chain variable fragment (scFv) based on one or more antibodies (e.g., CD20 antibodies). In some embodiments, the antigen-binding domain includes a variable heavy (VH) region and a variable light (VL) region, where the VH and VL regions are on the same polypeptide. In some embodiments, the antigen-binding domain includes a linker between the VH and VL regions. The linker allows the antigen-binding domain to form the desired structure for antigen binding.

[0088] In certain embodiments of the chimeric antigen receptor, the antigen-specific portion of the receptor (which may be referred to as the extracellular domain containing the antigen-binding region) comprises a tumor-associated antigen or a pathogen-specific antigen-binding domain. The antigen comprises a carbohydrate antigen recognized by a pattern recognition receptor, such as Dectin-1. The tumor-associated antigen may be of any type, so long as it is expressed on the cell surface of the tumor cell. Exemplary embodiments of tumor-associated antigens include CD19, CD70, CD20, carcinoembryonic antigen, alphafetoprotein, CA-125, MUC-1, CD56, EGFR, c-Met, AKT, Her2, Her3, epithelial tumor antigen, melanoma-associated antigen, mutated p53, mutated ras, CD79a, CD79b, and the like. In certain embodiments, the tumor-associated antigen is CD19. In certain embodiments, the tumor-associated antigen is CD79b. In certain embodiments, the tumor-associated antigen is CD70. In certain embodiments, the CAR can be co-expressed with a cytokine to improve persistence in the presence of low amounts of tumor-associated antigens. For example, the CAR can be co-expressed with IL-15.

[0089] In some embodiments, a CAR having a CD70 antigen binding domain comprises, consists of, or consists essentially of a sequence that is 75%, 80%, 85%, 90%, 95%, or 100% (and any value in between) identical to SEQ ID NO: 49. In some embodiments, a CAR having a CD70 antigen binding domain is encoded by a nucleic acid that comprises, consists of, or consists essentially of a sequence that is 75%, 80%, 85%, 90%, 95%, or 100% (and any value in between) identical to SEQ ID NO: 51. In some embodiments, a CAR having a CD70 antigen binding domain comprises a truncated CD27 (tCD27) extracellular domain that comprises, consists of, or consists essentially of a sequence that is 75%, 80%, 85%, 90%, 95%, or 100% (and any value in between) identical to SEQ ID NO: 48. In some embodiments, a CAR with a CD70 antigen binding domain comprises a tCD27 extracellular domain encoded by a nucleic acid comprising, consisting of, or consisting essentially of a sequence that is 75%, 80%, 85%, 90%, 95%, or 100% identical (and any value in between) to SEQ ID NO:52.

[0090] The variable regions of the antigen-binding domain of the polypeptide of the present disclosure can be modified by mutating amino acid residues in the VH and / or VL CDR1, CDR2 and / or CDR3 regions to improve one or more binding properties (e.g., affinity) of the antibody. The term "CDR" refers to the complementarity determining region based on a portion of the variable chain in immunoglobulins (antibodies) and T cell receptors, which are produced by B cells and T cells, respectively, and these molecules bind to their specific antigens. Because many sequence variations associated with immunoglobulins and T cell receptors are found in the CDRs, these regions are sometimes referred to as hypervariable regions. Mutations can be introduced by site-directed mutagenesis or PCR-mediated mutagenesis, and the effect on antibody binding, or other functional properties of interest, can be evaluated in suitable in vitro or in vivo assays. Preferably, conservative modifications are introduced, typically changing no more than 1, 2, 3, 4 or 5 residues in the CDR regions. Mutations can be amino acid substitutions, additions or deletions.

[0091] For example, framework modifications can be made to the antibody to reduce immunogenicity by "backmutating" one or more framework residues to the corresponding germline sequence.

[0092] It is also contemplated that an antigen-binding domain may be multispecific or multivalent by multimerizing antigen-binding domains having VH and VL domain pairs that bind either the same antigen (multivalent) or different antigens (multispecific).

[0093] The binding affinity of an antigen-binding region, such as a variable region (heavy and / or light chain variable region), or of a CDR is at least 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, or 10 -13In some embodiments, the KD of an antigen-binding region, such as a variable region (heavy and / or light chain variable region), or of a CDR is at least 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, or 10 -13 M (or any derived range therein).

[0094] The binding affinity, K, or K can be determined by methods known in the art, such as surface plasmon resonance (SPR)-based biosensors, equilibrium exclusion spectroscopy (KinExA), polarization-modulated oblique incidence reflectance differential (OI-RD), or ELISA.

[0095] In some embodiments, a polypeptide comprising a humanized binding region has a binding affinity and / or expression level in a host cell that is equivalent to, better than, or is at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 104, 106, 106, 108, 109, 110, 115, or 120% of that of a polypeptide comprising a non-humanized binding region, such as a binding region derived from a mouse.

[0096] In some embodiments, the framework regions such as FR1, FR2, FR3, and / or FR4 of the human framework are at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 108, 109, 110, 111, 112 1, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 1 09, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 16 2, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 (or any range derivable therein) amino acid substitutions, consecutive amino acid additions, or consecutive amino acid deletions.

[0097] In some embodiments, the framework regions such as FR1, FR2, FR3, and / or FR4 of the mouse framework are at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 108, 109, 110, 111, 112 1, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 1 09, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 16 2, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 (or any range derivable therein) amino acid substitutions, consecutive amino acid additions, or consecutive amino acid deletions.

[0098] The substitutions may be made at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 108, 109, 110, 111, 112, 13, 14, 15, 16, 17, 18, 19, 20 The number may be 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

[0099] In some embodiments, the antigen binding domain of the CAR is an Fc region binding domain (e.g., binds to an immunoglobulin Fc domain). In certain embodiments, the antigen binding domain of the CAR is derived from an immunoglobulin Fc receptor (FcR). In certain embodiments, the antigen binding domain of the CAR that binds to an Fc region may be derived from a CD16 gene sequence. In certain embodiments, the FcR is related to IgG (e.g., FcγRI / CD64, FcγRII / CD32, and FcγRIII / CD16), IgE (e.g., FcεRI), IgA (e.g., FcαRI / CD89), IgM (e.g., FcμR), and / or IgA / IgM (e.g., Fcα / μR). In some embodiments, the FcR comprises, consists of, or consists essentially of a sequence that is 75%, 80%, 85%, 90%, 95%, or 100% identical (and any value therebetween) to SEQ ID NOs: 9-17. In some embodiments, a CAR having an FcR antigen binding domain is encoded by a nucleic acid comprising, consisting of, or consisting essentially of a sequence that is 75%, 80%, 85%, 90%, 95%, or 100% identical (and any value in between) to SEQ ID NO:40.

[0100] In some embodiments, the CAR with FcR antigen binding domain is used together with additional immunotherapy, for example, antibody-based therapy.In some embodiments, the CAR with FcR antigen binding domain provides a universal CAR that can be used with any antibody therapy, as long as it can target the antibody as Fc domain.In some embodiments, the FcR CAR can also act as a CAR and as a transduction marker and / or a safety switch.

[0101] 3. Peptide spacer A peptide spacer (e.g., a spacer), such as an extracellular spacer, can link the antigen-binding domain to the transmembrane domain. In some embodiments, the peptide spacer is flexible enough to orient the antigen-binding domain in different directions to facilitate antigen binding. In some embodiments, the peptide spacer is a "hinge", e.g., a flexible polypeptide connecting region that connects one or more domains of a CAR to one or more other domains of a CAR. As used herein, the term "hinge" refers to a flexible polypeptide connecting region (also referred to herein as a "hinge region") that provides structural flexibility and spaces between polypeptide regions, and may be composed of natural or synthetic polypeptides.

[0102] In some embodiments, the peptide spacer comprises, consists essentially of, or consists of a sequence derived from the CD30 gene. In some embodiments, the peptide spacer comprises, consists essentially of, or consists of a sequence derived from a mammalian CD30 gene. In some embodiments, the peptide spacer comprises, consists essentially of, or consists of a sequence derived from a mouse CD30 gene. In some embodiments, the peptide spacer comprises, consists essentially of, or consists of a sequence derived from the human CD30 gene. In some embodiments, the peptide spacer comprises, consists essentially of, or consists of a sequence derived from the human CD30 coding region. In some embodiments, the peptide spacer comprises, consists essentially of, or consists of a sequence derived from the human CD30 transcript isoform 1 coding region.

[0103] In some embodiments, the spacer comprises a hinge region derived from IgG. In some embodiments, the spacer comprises or further comprises a CH2CH3 region and a portion of CD3 of an immunoglobulin. In some embodiments, the CH2CH3 region may have a L235E / N297Q or L235D / N297Q modification, or at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity of the CH2CH3 region. In some embodiments, the spacer is derived from IgG4. The extracellular spacer may comprise a hinge region.

[0104] The "hinge" from an immunoglobulin (e.g., IgG1) is generally defined as the stretch from Glu216 to Pro230 of human IgG1 (Burton (1985) Molec. Immunol., 22:161-206). Hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues that form inter-heavy chain disulfide (SS) bonds in the same positions.

[0105] In some embodiments, the hinge region may be naturally occurring or non-naturally occurring, including, but not limited to, modified hinge regions as described in U.S. Patent No. 5,677,425, which is incorporated herein by reference. In some embodiments, the hinge region may comprise a complete hinge region derived from an antibody of a different class or subclass than that of the CH1 domain. In some embodiments, the terms "hinge" and / or "peptide spacer" may also include regions derived from CD8 and other receptors that provide similar functions of providing flexibility and spacing between flanking regions.

[0106] In certain embodiments, the hinge region is derived from CD30. In certain embodiments, the hinge does not contain cysteine. In certain embodiments, the hinge is enriched for G and / or S amino acids compared to other hinges known in the art. In certain embodiments, the hinge and the transmembrane domain are derived from the same gene. In some embodiments, the hinge and the transmembrane domain are derived from the same coding sequence. In some embodiments, when an immune cell is designed to express more than one CAR, a unique hinge region is used for each CAR, for example, one CD30 hinge, one CD8 hinge, etc.

[0107] In some embodiments, the extracellular peptide spacer, including the hinge, may have a length of at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids. In some embodiments, the peptide spacer is 42 amino acids in length.

[0108] In some embodiments, the extracellular peptide spacer comprising a hinge comprises at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 65, 70, 75, 100, 110, 119, 120, 130, 140, 150, 160, 170, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 260, 270, 280, 290, 300, 325, 350, or 400 amino acids in length (or any range derivable therein).

[0109] In some embodiments, the extracellular spacer comprises, consists essentially of, or consists of a hinge region derived from an immunoglobulin (e.g., IgG). Immunoglobulin hinge region amino acid sequences are known in the art; see, e.g., Tan et al. (1990) Proc. Natl. Acad. Sci. USA 87:162; and Huck et al. (1986) Nucl. Acids Res.

[0110] The length of the extracellular spacer may have an effect on the signaling activity of the CAR, the expression level (transcription and / or translation) of the CAR, the cytotoxicity and / or cancer cell killing efficacy, and / or the expansion properties of the CAR cells in response to antigen-stimulated CAR signaling. In some embodiments, the extracellular spacer sequence of the CAR depends on the location of the target antigen. In some embodiments, a longer extracellular spacer is used when the target antigen is proximal to the cell membrane. In some embodiments, a shorter extracellular spacer is used when the target antigen is distal to the cell membrane. In some embodiments, a longer extracellular spacer is used when a more flexible CAR is desired. In some embodiments, a shorter extracellular spacer is used when a more rigid CAR is desired.

[0111] In some embodiments, shorter spacers are used, such as less than 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acids. In some embodiments, shorter spacers may have advantages in CAR-mediated signaling activity, expression levels of CAR (transcriptional and / or translational), cytotoxicity and / or cancer cell killing efficacy, and / or expansion properties of CAR cells in response to antigen-stimulated CAR signaling.

[0112] In some embodiments, at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, Longer spacers are used, such as those that are 6, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 260, 270, 280, or 290 amino acids. In some embodiments, longer spacers may have advantages in CAR-mediated signaling activity, expression levels of the CAR (transcriptional and / or translational), cytotoxicity and / or cancer cell killing efficacy, and / or expansion properties of CAR cells in response to antigen-stimulated CAR signaling.

[0113] When the extracellular spacer includes multiple portions, there may be anywhere from 0 to 50 amino acids between the various portions. For example, there may be at least, at most, or exactly 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, or 50 amino acids (or any range derived therein) between the hinge and other region (e.g., between the CH2 or CH3 region or regions if both are present, and the CH2 and CH3 regions). In some embodiments, the extracellular spacer consists essentially of the hinge, CH2, and / or CH3 regions, meaning that the hinge, CH2, and / or CH3 regions are the only identifiable regions present, and all other domains or regions are not included, although additional amino acids that are not part of an identifiable region may be present.

[0114] In certain embodiments, the peptide spacer may come from any suitable source, but in certain embodiments, the peptide spacer is selected from the group consisting of CD30, CD8a, CD28, PD-1, CTLA4, the alpha, beta or zeta chain of the T cell receptor, CD2, CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta, CD45, CD4, CD5, CD8b, CD9, CD16, CD22, CD27, CD32, CD33 , CD37, CD64, CD80, CD86, OX-40 (CD134), CD137, CD154, CD160, BTLA, LAIR1, TIGIT, TIM4, ICOS / CD278, GITR / CD357, NKG2D, LAG-3, PD-L1, PD-1, TIM-3, HVEM, LIGHT, DR3, CD30, CD224, CD244, SLAM, CD226, DAP, or combinations or others thereof. In certain embodiments, the peptide spacer is derived from CD30. In certain embodiments, the peptide spacer is derived from an immunoglobulin light chain binding region.

[0115] 4. Transmembrane domain The polypeptides of the present disclosure may include a transmembrane domain. In some embodiments, the transmembrane domain is a hydrophobic alpha helix that spans the membrane. Different transmembrane domains may confer different receptor stabilities.

[0116] In some embodiments, the transmembrane domain is located between the extracellular spacer and the cytoplasmic region. In some embodiments, the transmembrane domain is located between the extracellular spacer and one or more costimulatory regions. In some embodiments, a linker is located between the transmembrane domain and one or more costimulatory regions.

[0117] In some embodiments, the transmembrane domain comprises, consists essentially of, or consists of a sequence derived from a CD30 gene. In some embodiments, the transmembrane domain comprises, consists essentially of, or consists of a sequence derived from a mammalian CD30 gene. In some embodiments, the transmembrane domain comprises, consists essentially of, or consists of a sequence derived from a mouse CD30 gene. In some embodiments, the transmembrane domain comprises, consists essentially of, or consists of a sequence derived from a human CD30 gene. In some embodiments, the transmembrane domain comprises, consists essentially of, or consists of a sequence derived from a human CD30 coding region. In some embodiments, the transmembrane domain comprises, consists essentially of, or consists of a sequence derived from a human CD30 transcript isoform 1 coding region.

[0118] In certain embodiments, the transmembrane domain does not contain cysteine. In certain embodiments, the hinge and transmembrane domain are derived from the same gene. In some embodiments, the hinge and transmembrane domain are derived from the same coding sequence. In certain embodiments, the hinge and transmembrane domain are consecutive amino acids derived from a wild-type gene. In some embodiments, when an immune cell is designed to express more than one CAR, a unique transmembrane region is used for each CAR, for example, one CD30 transmembrane region, one CD8 transmembrane region, etc.

[0119] In some embodiments, the transmembrane domain may have a length of at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids. In some embodiments, the transmembrane domain is 21 amino acids in length. In some embodiments, the transmembrane domain is not 28, 27, 26, 25, 24, 23, or 22 amino acids in length.

[0120] In some embodiments, the transmembrane domain comprises, consists essentially of, or consists of a sequence that is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:4.

[0121] In certain embodiments, the transmembrane domain lacks the amino acid sequence PVLDAG. In certain embodiments, the transmembrane domain lacks the amino acid sequence VLDAG. In certain embodiments, the transmembrane domain lacks the amino acid sequence LDAG. In certain embodiments, the transmembrane domain lacks the amino acid sequence DAG. In certain embodiments, the transmembrane domain lacks the amino acid sequence AG.

[0122] In some embodiments, the transmembrane domain and peptide spacer comprise, consist essentially of, or consist of a sequence that is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:1.

[0123] In some embodiments, the transmembrane domain and / or peptide spacer does not comprise 3 or more consecutive amino acids set forth in SEQ ID NO:5.

[0124] In some embodiments, any transmembrane domain that provides for insertion of the polypeptide into the cell membrane of a eukaryotic (e.g., mammalian) cell may be suitable for use. In some embodiments, the transmembrane domain is derived from CD30, CD28, CD8, CD4, CD3 zeta, OX-40 (CD134), or CD7. In some embodiments, the transmembrane domain is derived from the alpha, beta or zeta chain of the T cell receptor, CD28, CD2, CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta, CD45, CD4, CD5, CD8 (including CD8 alpha), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, OX-40 (CD134), 4-1BB (CD137, CD154, ICOS / CD278, GITR / CD357, NKG2D, PD-1, CTLA-4, and DAP molecules. In some embodiments, the transmembrane domain is synthetic. In some aspects, synthetic transmembrane domains comprise primarily hydrophobic residues such as leucine and valine. In some aspects, triplets of phenylalanine, tryptophan and valine can be found at each end of the synthetic transmembrane domain.

[0125] 5.Cytoplasmic region After antigen recognition, the receptor of the present disclosure may be clustered, and the signal is transmitted to the cell through the cytoplasmic region. In some embodiments, the costimulatory domain described herein is a part of the cytoplasmic region. In some embodiments, the cytoplasmic region comprises an intracellular signaling domain. The intracellular signaling domain may comprise a primary signaling domain and one or more costimulatory domains.

[0126] The cytoplasmic region and / or costimulatory region suitable for use in the polypeptides of the present disclosure include any desired signaling domain that provides a distinct detectable signal (e.g., increased production of one or more cytokines by the cell; altered transcription of a target gene; altered activity of a protein; altered cell behavior, e.g., cell death; cell proliferation; cell differentiation; cell survival; modulation of a cell signaling response) in response to activation by binding of an antigen to the antigen-binding domain. In some embodiments, the cytoplasmic region comprises at least one (e.g., one, two, three, four, five, six, etc.) ITAM motif described herein. In some embodiments, the cytoplasmic region comprises a DAP10 / CD28-type signaling chain.

[0127] Cytoplasmic regions suitable for use in the polypeptides of the present disclosure include intracellular signaling polypeptides containing an immunoreceptor tyrosine-based activation motif (ITAM). The ITAM motif is YX1X2(L / I), where X1 and X2 are independently any amino acid. In some cases, the cytoplasmic region contains 1, 2, 3, 4, or 5 ITAM motifs. In some cases, the ITAM motif is repeated twice in the intracellular domain, where the first and second instances of the ITAM motif are separated from each other by 6-8 amino acids, e.g., (YX1X2(L / I))(X3)n(YX1X2(L / I)), where n is an integer between 6 and 8, and each of the 6-8 X3 may be any amino acid.

[0128] In some embodiments, a suitable cytoplasmic region is an ITAM motif-containing portion derived from a polypeptide containing an ITAM motif. For example, a suitable cytoplasmic region may be an ITAM motif-containing domain derived from any ITAM motif-containing protein. Thus, a suitable intracellular domain does not need to contain the entire sequence of the entire protein from which it is derived. Examples of suitable ITAM motif-containing polypeptides include, but are not limited to, DAP12, DAP10, FCER1G (Fc epsilon receptor I gamma chain); CD3D (CD3 delta); CD3E (CD3 epsilon); CD3G (CD3 gamma); CD3 zeta; and CD79A (antigen receptor complex-associated protein alpha chain).

[0129] Exemplary cytoplasmic regions are known in the art. The cytoplasmic regions set forth below, in some embodiments, also provide examples of regions that are included in the CARs of the present disclosure.

[0130] In some embodiments, a suitable cytoplasmic region may comprise an ITAM motif-containing portion of the full-length DAP12 amino acid sequence. In some embodiments, the cytoplasmic region is derived from FCER1G (also known as FCRG; Fc epsilon receptor I gamma chain; Fc receptor gamma chain; fc epsilon R1 gamma; fcR gamma; fceRI gamma; high affinity immunoglobulin epsilon receptor subunit gamma; immunoglobulin E receptor, high affinity, gamma chain, etc.). In some embodiments, a suitable cytoplasmic region may comprise an ITAM motif-containing portion of the full-length FCER1G amino acid sequence.

[0131] In some embodiments, the cytoplasmic region is derived from the T cell surface glycoprotein CD3 delta chain (also known as CD3D; CD3-DELTA; T3D; CD3 antigen delta subunit; CD3 delta; CD3 delta; CD3d antigen delta polypeptide (TiT3 complex); OKT3 delta chain; T cell receptor T3 delta chain; T cell surface glycoprotein CD3 delta chain, etc.). In some embodiments, a suitable cytoplasmic region may comprise an ITAM motif-containing portion of the full-length CD3 delta amino acid sequence. In some embodiments, the cytoplasmic region is derived from the T cell surface glycoprotein CD3 epsilon chain (also known as CD3e, CD3ε; T cell surface antigen T3 / Leu-4 epsilon chain, T cell surface glycoprotein CD3 epsilon chain, AI504783, CD3, CD3 epsilon, T3e, etc.). In some embodiments, a suitable cytoplasmic region may comprise an ITAM motif-containing portion of the full-length CD3 epsilon amino acid sequence. In some embodiments, the cytoplasmic region is derived from the T cell surface glycoprotein CD3 gamma chain (also known as CD3G, CD3γ, T cell receptor T3 gamma chain, CD3-GAMMA, T3G, gamma polypeptide (TiT3 complex), etc.). In some embodiments, a suitable cytoplasmic region may comprise an ITAM motif-containing portion of the full-length CD3 gamma amino acid sequence. In some embodiments, the cytoplasmic region is derived from the T cell surface glycoprotein CD3 zeta chain (also known as CD3Z, CD3ζ, T cell surface receptor T3 zeta chain, CD247, CD3-ZETA, CD3H, CD3Q, T3Z, TCRZ, etc.). In some embodiments, a suitable cytoplasmic region may comprise an ITAM motif-containing portion of the full-length CD3 zeta amino acid sequence.

[0132] In some embodiments, the cytoplasmic region is derived from CD79A (also known as B-cell antigen receptor complex-associated protein alpha chain; CD79a antigen (immunoglobulin-associated alpha); MB-1 membrane glycoprotein; ig-alpha; membrane-associated immunoglobulin-associated protein; surface IgM-associated protein, etc.). In some embodiments, a suitable cytoplasmic region may comprise an ITAM motif-containing portion of the full-length CD79A amino acid sequence.

[0133] 6. Co-stimulation area Non-limiting examples of suitable costimulatory regions, such as those contained in the cytoplasmic region, include, but are not limited to, polypeptides derived from 4-1BB (CD137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, and HVEM. In some embodiments, the costimulatory region is derived from CD8, 4-1BB (CD137), CD27, CD28, CD30, OX-40 (CD134), CD3ε, CD3ζ, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, or CD154. In some embodiments, the costimulatory domain includes, but is not limited to, one or more of CD28, CD27, OX-40 (CD134), ICOS, HVEM, GITR, LIGHT, CD40L, DR3, CD30, SLAM, CD2, CD226 (DNAM-1), MyD88, CD244, TMIGD2, BTNL3, NKG2D, DAP10, DAP12, 4-1BB (CD137), or synthetic molecules. In some embodiments, in addition to the primary signal initiated by CD3zeta, additional signals provided by the costimulatory receptor inserted into the CAR are important for the complete activation of immune cells and may help improve the in vivo persistence and therapeutic success of cell therapy.

[0134] The costimulatory region may have a length of at least, at most, or exactly 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, or 300 amino acids or any range derived therein. In some embodiments, the costimulatory region is derived from the intracellular portion of the transmembrane protein 4-1BB (also known as TNFRSF9; CD137; CDwl37; ILA, etc.). In some embodiments, the costimulatory region is derived from the intracellular portion of the transmembrane protein CD28 (also known as Tp44). In some embodiments, the costimulatory region is derived from the intracellular portion of the transmembrane protein ICOS (also known as AILIM, CD278, and CVID1). In some embodiments, the costimulatory region is derived from the intracellular portion of the transmembrane protein OX-40 (also known as TNFRSF4, RP5-902P8.3, ACT35, CD134, OX40, TXGP1L). In some embodiments, the costimulatory region is derived from the intracellular portion of the transmembrane protein BTLA (also known as BTLA1 and CD272). In some embodiments, the costimulatory region is derived from the intracellular portion of the transmembrane protein CD27 (also known as S152, T14, TNFRSF7, and Tp55). In some embodiments, the costimulatory region is derived from the intracellular portion of the transmembrane protein CD30 (also known as TNFRSF8, D1S166E, and Ki-1). In some embodiments, the costimulatory region is derived from the intracellular portion of the transmembrane protein GITR (also known as TNFRSF18, RP-5-902P8.2, AITR, CD357, and GITR-D). In some embodiments, the costimulatory region is derived from the intracellular portion of the transmembrane protein HVEM (also known as TNFRSF14, RP3-395M20.6, ATAR, CD270, HVEA, HVEM, LIGHTR, and TR2).

[0135] In some embodiments, the polypeptides described herein may further comprise a detection peptide. Suitable detection peptides include hemagglutinin (HA; e.g., YPYDVPDYA (SEQ ID NO: 28)), FLAG (e.g., DYKDDDDK (SEQ ID NO: 29)), c-myc (e.g., EQKLISEEDL; SEQ ID NO: 30)), and the like. Other suitable detection peptides are known in the art.

[0136] In some embodiments, the CAR of the present disclosure comprises a CD28 costimulatory domain. In some embodiments, the CD28 costimulatory domain comprises SEQ ID NO: 15. In some embodiments, the CAR of the present disclosure comprises a 4-1BB costimulatory domain. In some embodiments, the 4-1BB costimulatory domain comprises SEQ ID NO: 25. In some embodiments, the CAR of the present disclosure comprises an OX40 costimulatory domain. In some embodiments, the OX40 costimulatory domain comprises SEQ ID NO: 26.

[0137] 7. Peptide Linker In some embodiments, the polypeptides of the present disclosure include a peptide linker (sometimes referred to as a linker). In some embodiments, a peptide linker is used to separate any of the peptide domains / regions described herein. By way of example, in some embodiments, the linker is between the signal peptide and the antigen binding domain, between the VH and VL of the antigen binding domain, between the antigen binding domain and the peptide spacer, between the peptide spacer and the transmembrane domain, sandwiching the costimulatory region or on the N or C region of the costimulatory region, and / or between the transmembrane domain and the intracellular domain. The peptide linker may have any of a variety of amino acid sequences. Generally, the domains and regions can be joined by peptide linkers, which are flexible, although other chemical bonds are not excluded. The linker may be a peptide about 6 to about 40 amino acids in length, or about 6 to about 25 amino acids in length. These linkers can be produced by joining proteins using synthetic, linker-encoding oligonucleotides.

[0138] Peptide linkers with some degree of flexibility can be used. Peptide linkers can have virtually any amino acid sequence, with the understanding that suitable peptide linkers will generally have sequences that result in flexible peptides. The use of small amino acids such as glycine and alanine is useful in creating flexible peptides. Creating such sequences is routine for those skilled in the art. In certain embodiments, it may be appropriate for those skilled in the art to adjust the length of the peptide linker to optimize the performance of one or more CAR molecules. Routine optimization of the length of the peptide linker is within the skill set of those skilled in the art.

[0139] Suitable linkers can be readily selected and can be of any suitable length, such as 1 amino acid (e.g., Gly) to 20 amino acids, 2 to 15 amino acids, 3 to 12 amino acids, including 4 to 10 amino acids, 5 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0140] Suitable linkers can be readily selected and can be of any suitable different length, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including from 4 amino acids to 10 amino acids, from 5 amino acids to 9 amino acids, from 6 amino acids to 8 amino acids, or from 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0141] Examples of flexible linkers include glycine polymers (G), glycine-serine polymers (including, for example, (GS), (GSGGS) (SEQ ID NO: 31), (G4S) and (GGGS) (SEQ ID NO: 32), where n is an integer of at least 1). In some embodiments, n is at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (or any range derived therein). Glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers can be used; both Gly and Ser are relatively unstructured and therefore can serve as neutral tethers between components. Glycine polymers can be used; glycine has access to significantly more phi-psi space than the equivalent alanine and is much less restrictive than residues with longer side chains. Exemplary spacers may comprise amino acid sequences including, but not limited to, GGSG (SEQ ID NO:33), GGSGG (SEQ ID NO:34), GSGSG (SEQ ID NO:35), GSGGG (SEQ ID NO:36), GGGSG (SEQ ID NO:37), or GSSSG (SEQ ID NO:38).

[0142] II. Nucleic acids In certain embodiments, the nucleic acid sequence may be present in various instances, such as an isolated segment of an integrated sequence or recombinant polynucleotide and recombinant vector encoding one or both chains of an antibody, or a fragment, derivative, mutein, or variant thereof, a polynucleotide sufficient for use as a hybridization probe, PCR primer or sequencing primer for identifying, analyzing, mutating, or amplifying a polynucleotide encoding a polypeptide, an antisense nucleic acid for inhibiting expression of a polynucleotide, and a complementary sequence of the above described herein. In some embodiments, nucleic acids encoding epitopes for certain polypeptides provided herein are also provided. In some embodiments, nucleic acids encoding fusion proteins comprising these peptides are also provided. The nucleic acid may be single-stranded or double-stranded, and may be an RNA and / or DNA molecule, as well as artificial variants thereof (e.g., peptide nucleic acid).

[0143] The term "polynucleotide" refers to a nucleic acid molecule that is recombinant or isolated from total genomic nucleic acid. The term "polynucleotide" includes oligonucleotides (nucleic acids of 100 residues or less in length), recombinant vectors including, for example, plasmids, cosmids, phages, viruses, and the like. A polynucleotide, in certain embodiments, includes a regulatory sequence or a protein-coding sequence that is substantially isolated from a naturally occurring gene. A polynucleotide may be single-stranded (coding or antisense) or double-stranded, and may be RNA, DNA (genomic, cDNA, or synthetic), analogs thereof, or combinations thereof. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide.

[0144] In this regard, the terms "gene", "polynucleotide" or "nucleic acid" are used to refer to a nucleic acid (including any sequences required for proper transcription, post-translational modification, or localization) that encodes a protein, polypeptide, or peptide. As will be understood by those skilled in the art, this term encompasses genomic sequences, expression cassettes, cDNA sequences, and smaller engineered nucleic acid segments that express, or can be adapted to express, proteins, polypeptides, domains, peptides, fusion proteins, and mutants. A nucleic acid that encodes all or a portion of a polypeptide may contain a contiguous nucleic acid sequence that encodes all or a portion of such a polypeptide. It is also contemplated that a particular polypeptide may be encoded by a nucleic acid that contains variations having slightly different nucleic acid sequences, but that nevertheless encodes the same or substantially similar protein.

[0145] In certain embodiments, there are polynucleotide variants that have substantial identity to the sequences disclosed herein; those that contain at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity (including all values ​​and ranges therebetween) compared to the polynucleotide sequences provided herein using the methods described herein (e.g., BLAST analysis using standard parameters). In certain aspects, the isolated polynucleotide will comprise a nucleotide sequence that encodes a polypeptide that has at least 90%, preferably 95% or more identity to the amino acid sequence described herein over the entire length of the sequence; or a nucleotide sequence that is complementary to the isolated polynucleotide.

[0146] Regardless of the length of the coding sequence itself, the nucleic acid segment may be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the overall length may vary considerably. Nucleic acids may be of any length. They may be, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 3000, 5000 nucleotides or more in length, and / or may include one or more additional sequences, e.g., regulatory sequences, and / or may be part of a larger nucleic acid, e.g., a vector. Thus, it is contemplated that nucleic acid fragments of almost any length may be used, with the total length preferably being limited by the ease of preparation and use in the intended recombinant nucleic acid protocol. In some cases, the nucleic acid sequence may encode a polypeptide sequence that includes additional heterologous coding sequences, for example to allow purification, transport, secretion, post-translational modification of the polypeptide, or for therapeutic benefits such as targeting or efficacy. As discussed above, tags or other heterologous polypeptides may be added to the modified polypeptide coding sequence, where "heterologous" refers to a polypeptide that is not the same as the modified polypeptide.

[0147] A. Vector The polypeptide of the present disclosure, including the CAR of the present disclosure, can be delivered to recipient immune cells by any suitable vector, including viral vector or non-viral vector.Examples of viral vector include at least retrovirus, lentivirus, adenovirus, or adeno-associated virus vector.Examples of non-viral vector include at least plasmid, transposon, lipid, nanoparticle, etc.

[0148] In some embodiments, suitable methods for nucleic acid delivery to effect expression of the compositions are expected to include virtually any method by which a nucleic acid (e.g., DNA, including viral and non-viral vectors) can be introduced into a cell, tissue, or organism, as described herein or known to one of skill in the art.Such methods include, but are not limited to, microinjection (Harland and Weintraub, 1985; U.S. Pat. No. 5,789,215, each of which is incorporated herein by reference), injection (U.S. Pat. Nos. 5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466, and 5,580,859, each of which is incorporated herein by reference); electroporation (U.S. Pat. No. 5,384,253, each of which is incorporated herein by reference); calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990); the use of DEAE dextran followed by polyethylene glycol (Gopal, 1985); direct ultrasound loading (Fechheimer et al., 1987); liposome-mediated transfection (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al., 1991); microprojectile bombardment (PCT Application Nos. WO 94 / 09699 and 95 / 06128, each of which is incorporated herein by reference; U.S. Patent Nos. 5,610,042, 5,322,783, 5,563,055, 5,572,102, and 5,610,042, respectively). Nos. 5,0318, 5,538,877 and 5,538,880); agitation with silicon carbide fibers (Kaeppler et al., 1990; U.S. Pat. Nos. 5,302,523 and 5,464,765, each of which is incorporated herein by reference); Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,591,616 and 5,563,055, each of which is incorporated herein by reference); or direct delivery of DNA by PEG-mediated transformation of protoplasts (Omirulleh et al., 1993; U.S. Pat. Nos. 4,684,611 and 4,952,500, each of which is incorporated herein by reference); desiccation / inhibition-mediated DNA uptake (Potrykus et al., 1985), and the like.Other methods include viral transduction, such as gene transfer by lentiviral or retroviral transduction.

[0149] In some embodiments, immune cells are transduced with a vector encoding one or more antigen-targeting CARs. In some embodiments, the CARs may or may not be included on or with the same vector. In some embodiments, one or more CARs are expressed from the same vector molecule, such as the same viral vector molecule. In some embodiments, the expression of one or more CARs may or may not be regulated by the same regulatory element. In some embodiments, when more than one CAR is included on the same vector, the more than one CAR may or may not be expressed as separate polypeptides. In some embodiments, when more than one CAR is included on the same vector and more than one CAR is expressed as separate polypeptides, they are separated on the vector by, for example, 2A elements and / or IRES elements (or both types are used once or more than once on the same vector).

[0150] In some embodiments, the CAR-expressing vector is a polycistronic (e.g., bicistronic) vector. In some embodiments, the CAR-expressing vector is a polycistronic vector that expresses both an anti-CD19 CAR and an anti-CD79B CAR separated by a 2A element. In some embodiments, the CAR-expressing vector is a polycistronic vector that encodes both an anti-CD19 CAR and an anti-CD79A CAR separated by a 2A element. In some embodiments, the CAR-expressing vector is a polycistronic vector that encodes both an FcR CAR and an anti-CD79B CAR separated by a 2A element. In some embodiments, the CAR-expressing vector is a polycistronic vector that encodes both an FcR CAR and an anti-CD19 CAR separated by a 2A element. In some embodiments, the CAR-expressing vector is a polycistronic vector that encodes a CD70 CAR and one or more additional CAR molecules. In some embodiments, the CAR-expressing vector is a polycistronic vector encoding an anti-CD19 CAR, an anti-CD79B CAR, an anti-CD79A CAR, an FcR CAR, and / or an anti-CD70 CAR (e.g., a tCD27-CAR).

[0151] In some embodiments, the 2A element is a T2A element. In some embodiments, the 2A element is a P2A element. In some embodiments, the 2A element is an E2A element.

[0152] B. Sequence Certain nucleotide sequences of polynucleotides, including polynucleotides that express chimeric antigen receptors and portions and regions thereof, are provided in Table 2. Table 2 - Polynucleotide sequences [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0153] C. Mutation By introducing changes into nucleic acid by mutation, it can lead to changes in the amino acid sequence of the polypeptide it encodes. Any technique known in the art can be used to introduce mutations. In one embodiment, one or more specific amino acid residues are changed, for example, using a site-directed mutagenesis protocol. In another embodiment, one or more randomly selected residues are changed, for example, using a random mutagenesis protocol. However, mutant polypeptides can be expressed and screened for desired properties.

[0154] Certain mutations can be introduced into the nucleic acid without significantly altering the biological activity of the encoded polypeptide. For example, one skilled in the art can make nucleotide substitutions that result in amino acid substitutions at non-essential amino acid residues. Alternatively, one or more mutations can be introduced into the nucleic acid that selectively alter the biological activity of the encoded polypeptide. See, for example, Romain Studer et al., Biochem. J. 449:581-594 (2013). For example, mutations can be made to quantitatively or qualitatively alter biological activity. Examples of quantitative changes include increasing, decreasing, or eliminating activity. Examples of qualitative changes include altering the antigen specificity of an antibody.

[0155] III. Therapy In some embodiments, disclosed herein are treatment methods that include the disclosed compositions, such as compositions that include CAR with CD30-derived hinge and / or transmembrane domains. In some embodiments, the treatment methods are directed against cancer, while in other embodiments. In some embodiments, the treatment methods are directed against disease pathogenicity and / or exogenous promoters.

[0156] In some embodiments, the method further comprises administering a cancer therapy to the patient. The cancer therapy can be selected based on the expression level measurements alone or in combination with the clinical risk score calculated for the patient. In some embodiments, the cancer therapy comprises a local cancer therapy. In some embodiments, the cancer therapy does not comprise a systemic cancer therapy. In some embodiments, the cancer therapy does not comprise a local therapy. In some embodiments, the cancer therapy comprises a local cancer therapy without administering a systemic cancer therapy. In some embodiments, the cancer therapy comprises an immune therapy, which may be an immune checkpoint therapy. These cancer therapies may also exclude any of them. A combination of these therapies may be administered.

[0157] As used herein, the term "cancer" can be used to describe solid tumors, metastatic cancer, or non-metastatic cancer. In certain embodiments, the cancer may occur in the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, colon, rectum, anus, gums, head, kidney, liver, lung, nasopharynx, cervix, ovary, pancreas, prostate, skin, stomach, testes, tongue, or uterus. In some embodiments, the cancer is a recurrent cancer. In some embodiments, the cancer is a stage I cancer. In some embodiments, the cancer is a stage II cancer. In some embodiments, the cancer is a stage III cancer. In some embodiments, the cancer is a stage IV cancer.

[0158] Cancer may be of the following histological types, specifically but not limited to: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilonidal carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; cavernous adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma of adenomatous polyps; adenocarcinoma, familial polyposis coli; solid tumors; malignant carcinoid tumor; lobular-alveolar adenocarcinoma; papillary adenocarcinoma; chromatophore carcinoma; Eosinophilic carcinoma;Eosinophilic adenocarcinoma;Basophilic carcinoma;Clear cell adenocarcinoma;Granular cell carcinoma;Follicle adenocarcinoma;Papillary and follicular adenocarcinoma;Nonencapsulated sclerosing carcinoma;Adrenal cortical carcinoma;Endometrial carcinoma;Cutaneous adnexal carcinoma;Apocrine adenocarcinoma;Sebaceous gland carcinoma;Keratin adenocarcinoma;Mucoepidermoid carcinoma;Cystadenocarcinoma;Papillary cystadenocarcinoma;Papillary serous cystadenocarcinoma;Mucinous cystadenocarcinoma;Mucinous adenocarcinoma;Signet ring cell carcinoma;Invasive ductal carcinoma;Medullary carcinoma;Lobular carcinoma;Inflammatory carcinoma;Paget's disease, breast;Acinic cell carcinoma;Adenosquamous carcinoma;Adenocarcinoma with squamous metaplasia;Thymoma, malignant;Ovarian stromal tumor, malignant;Sarcoma, malignant;Granulosa cell Tumor, malignant;Androblastoma, malignant;Sertoli cell carcinoma;Leydig cell tumor, malignant;Lipocytoma, malignant;Paraganoneuroma, malignant;Extramammary paraganglioma, malignant;Pheochromocytoma;Angiosarcoma;Malignant melanoma;Amelanotic melanoma;Superficial spreading melanoma;Malignant melanoma of giant pigmented nevus;Epithelioid cell melanoma;Blue nevus, malignant;Sarcoma;Fibrosarcoma;Malignant fibrous histiocytoma;Myxosarcoma;Liposarcoma;Leiomyosarcoma;Rhabdomyosarcoma;Embryonic rhabdomyosarcoma;Alveolar rhabdomyosarcoma;Stromatous sarcoma;Mixed tumor, malignant;Müllerian mixed tumor;Nephroblastoma;Hepatoblastoma;Carcinosarcoma;Mesenchymoma, malignant;Brenner tumor, malignant;F Alodes tumor, malignant;synovial sarcoma;mesothelioma, malignant;dysembryopathies;embryonic carcinoma;teratoma, malignant;ovarian goiter, malignant;choriocarcinoma;mesostosis, malignant;angiosarcoma;hemangioendothelioma, malignant;Kaposi's sarcoma;hemangiopericytoma, malignant;lymphangiosarcoma;osteosarcoma;cortical osteosarcoma;chondrosarcoma;chondroblastoma, malignant;mesenchymal chondrosarcoma;giant cell tumor of bone;Ewing's sarcoma;odontogenic tumor, malignant;ameloblastoma, malignant;ameloblastoma, malignant;ameloblastic fibrosarcoma;pinealoma, malignant;chordoma;glioma, malignant;ependymoma;astrocytoma;protoplasmic astrocytoma;fibrous astrocytoma;astroblastoma;glioblastoma;oligodendroglioma;Oligodendroblastoma;Primitive neuroectodermal tumor;Cerebellar sarcoma;Ganglioneuroblastoma;Neuroblastoma;Retinoblastoma;Olfactory neurogenic tumor;Meningioma, malignant;Neurofibrosarcoma;Schwannoma, malignant;Granular cell tumor, malignant;Malignant lymphoma;Hodgkin's disease;Hodgkin's;Paragranuloma;Malignant lymphoma, small lymphocytic;Malignant lymphoma, large cell, diffuse;Malignant lymphoma, follicular;Mycosis sarcomas;other specified non-Hodgkin's lymphoma;malignant histiocytosis;multiple myeloma;mast cell sarcoma;immunoproliferative small intestinal disease;leukemia;lymphocytic leukemia;plasma cell leukemia;erythroid leukemia;lymphoblastic cell leukemia;myeloid leukemia;basophilic leukemia;eosinophilic leukemia;monocytic leukemia;mast cell leukemia;megakaryocytic leukemia;myeloid sarcoma;and hairy cell leukemia.;

[0159] In some embodiments, the present disclosure provides a method for immunotherapy comprising administering an effective amount of a composition comprising a CAR of the present disclosure. In one embodiment, a medical disease or disorder is treated by administering a cell population expressing a CAR that elicits an immune response. In certain embodiments of the present disclosure, cancer is treated by administering a CAR immune cell population that elicits an immune response. Provided herein is a method for treating or delaying the progression of cancer in an individual, comprising administering to the individual an effective amount of an antigen-specific cell therapy. The method can be applied to the treatment of immune disorders, solid cancers, and hematological cancers, as examples. In particular, the cancer may be a B-cell malignancy, such as diffuse large B-cell lymphoma, high-grade B-cell lymphoma, follicular lymphoma, marginal zone lymphoma, mantle cell lymphoma, Burkitt's lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia, and chronic lymphocytic leukemia.

[0160] Certain embodiments relate to methods of treating leukemia. Leukemia is a cancer of the blood or bone marrow, characterized by abnormal proliferation (production by multiplication) of blood cells, usually white blood cells (leukocytes). It is part of a broad group of diseases called hematological neoplasms. Leukemia is a broad term that covers a spectrum of diseases. Leukemia is divided clinically and pathologically into acute and chronic forms.

[0161] In some embodiments of the method of the present disclosure, the activated CD4 and / or CD8 T cells in the individual are characterized by enhanced cytolytic activity compared to before administration of the CD4 and / or CD8 T cells and / or combination that produce γ-IFN. γ-IFN can be measured by any means known in the art, including intracellular cytokine staining (ICS), including, for example, cell fixation, permeabilization, and staining with an antibody against γ-IFN. Cytolytic activity can be measured by any means known in the art, for example, using a cell killing assay with a mixture of effector and target cells.

[0162] In some embodiments, the subject may be administered a non-myeloablative lymphodepleting chemotherapy prior to T cell therapy. The non-myeloablative lymphodepleting chemotherapy may be any suitable such therapy that may be administered by any suitable route. The non-myeloablative lymphodepleting chemotherapy may include, for example, administration of cyclophosphamide and fludarabine, particularly when the cancer is melanoma, which may be metastatic. An exemplary route for administering cyclophosphamide and fludarabine is intravenously. Similarly, any suitable dose of cyclophosphamide and fludarabine may be administered. In certain aspects, the dose is about 25 mg / m 2 of fludarabine for 5 days, followed by approximately 60 mg / kg of cyclophosphamide for 2 days.

[0163] In certain embodiments, a T cell growth factor that promotes the proliferation and activation of autologous T cells is administered to the subject at the same time as the autologous T cells or after the autologous T cells. The T cell growth factor may be any suitable growth factor that promotes the proliferation and activation of autologous T cells. Examples of suitable T cell growth factors include interleukin (IL)-2, IL-7, IL-15, and / or IL-12, which can be used alone or in various combinations, such as IL-2 and IL-7, IL-2 and IL-15, IL-7 and IL-15, IL-2, IL-7 and IL-15, IL-12 and IL-7, IL-12 and IL-15, or IL-12 and IL2.

[0164] Therapeutically effective amounts of immune cells can be administered by several routes, including parenteral administration, for example, intravenous, intraperitoneal, intramuscular, intrasternal, or intraarticular injection, or infusion.

[0165] Intratumoral injection, or injection into the tumor vasculature, is specifically contemplated for individual, solid, accessible tumors. Local, regional, or systemic administration may also be appropriate. In some embodiments, for tumors larger than 4 cm, the volume administered is about 4-10 ml (particularly 10 ml), while for tumors smaller than 4 cm, a volume of about 1-3 ml (particularly 3 ml) is used. In some embodiments, multiple injections delivered as a single dose may include a volume of about 0.1 to about 0.5 ml.

[0166] In some embodiments, the cell population can be administered in a treatment regimen consistent with the disease, for example, one or several doses over a period of one to several days to improve the disease state, or regular doses over a long period to inhibit disease progression and prevent disease recurrence. The exact dose used in the formulation also depends on the route of administration and the severity of the disease or disorder, and should be determined according to the judgment of the physician and each patient's circumstances. The therapeutically effective amount of cells will depend on the subject being treated, the severity and type of the affliction, and the mode of administration. In some embodiments, the dose that can be used in the treatment of a human subject is at least 3.8×10 4 , at least 3.8 × 10 5 , at least 3.8 × 10 6 , at least 3.8 × 10 7 , at least 3.8 × 10 8 , at least 3.8 × 10 9 , or at least 3.8 × 10 10 cells / m 2 In certain embodiments, the dose used in treating a human subject is in the range of about 3.8×10 9 ~Approx. 3.8×10 10 cells / m 2In a further embodiment, the therapeutically effective amount of cells is in the range of about 5×10 6 cells / kg body weight~approx. 7.5×10 8 Cells / kg body weight, e.g., about 2×10 7 Cells ~ approx. 5 x 10 8 cells / kg body weight, or approximately 5 x 10 7 cells ~ approx. 2 x 10 8 Cells / kg body weight may vary. Those skilled in the art can easily determine the exact amount of cells based on the age, weight, sex and physiological condition of the subject. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0167] In certain embodiments of the present disclosure, an effective amount of CAR-expressing immune cells is delivered to an individual in need thereof, such as an individual suffering from cancer. In some embodiments, the cells can boost the individual's immune system to attack cancer cells. In some cases, the individual is provided with one or more doses of immune cells (e.g., those described herein). In some embodiments, when more than one dose of immune cells is administered to an individual, the period between doses should be sufficient to allow time for proliferation in the individual, and in specific embodiments, the period between doses is 1, 2, 3, 4, 5, 6, 7, or more days.

[0168] In certain embodiments, the cells engineered to express a CAR are administered in a therapeutically effective amount (eg, 10-20 mg / mL) that ameliorates at least one symptom associated with cancer cells in an individual. 3 ~10 10 A therapeutically effective amount is provided to an individual in the range of 10 3 ~10 10 , 10 3 ~10 9 , 10 3 ~10 8 , 10 3 ~10 7 , 10 3 ~10 6 , 10 3 ~10 5 , 10 3 ~10 4 , 104 ~10 10 , 10 4 ~10 9 , 10 4 ~10 8 , 10 4 ~10 7 , 10 4 ~10 6 , 10 4 ~10 5 , 10 5 ~10 10 , 10 5 ~10 9 , 10 5 ~10 8 , 10 5 ~10 7 , 10 5 ~10 6 , 10 6 ~10 10 , 10 6 ~10 9 , 10 6 ~10 8 , 10 6 ~10 7 , 10 7 ~10 10 , 10 7 ~10 9 , 10 7 ~10 8 , 10 8 ~10 10 , 10 8 ~10 9 , or 10 9 ~10 10 Thus, in certain embodiments, an individual with a particular cancer is provided one or more times with a therapeutically effective amount of cells expressing one or more CARs.

[0169] Some of the cancer antigens targeted by the CAR of the present disclosure are expressed in the context of the disease, condition, or cell type targeted via adoptive cell therapy. Among the diseases and conditions are proliferative, neoplastic, and malignant diseases and disorders, including hematological cancers, lymphomas, leukemias, and / or cancers of the immune system, such as B, T, and myeloid leukemias, lymphomas, and myelomas, such as multiple myeloma. In some embodiments, the antigen is selectively expressed or overexpressed on disease or condition cells, such as tumor or pathogenic cells, compared to normal or non-target cells or tissues. In other embodiments, the antigen is expressed on normal cells and / or expressed on engineered cells.

[0170] In some embodiments, any suitable antigen may be used in the context of the present disclosure. Exemplary antigens include, but are not limited to, antigenic molecules derived from infectious agents, self / autoantigens, tumor / cancer-associated antigens, tumor neoantigens (Linnemann et al., 2015). In certain aspects, antigens include CD19, CD20, CD22, CD30, CD70, CD79a, CD79b, SLAM-F7NY-ESO, EGFRvIII, Muc-1, Her2, CA-125, WT-1, Mage-A3, Mage-A4, Mage-A10, TRAIL / DR4, CEA. In certain embodiments, the antigens for one or more antigen receptors include, but are not limited to, CD19, EBNA, WT1, CD123, NY-ESO, EGFRvIII, MUC1, HER2, CA-125, WT1, Mage-A3, Mage-A4, Mage-A10, TRAIL / DR4, and / or CEA. The sequences of these antigens include, for example, CD19 (accession number: NG_007275.1), EBNA (accession number: NG_002392.2), WT1 (accession number: NG_009272.1), CD123 (accession number: NC_000023.11), NY-ESO (accession number: NC_000023.11), EGFRvIII (accession number: NG_007726.3), MUC1 (accession number: NG_029383.1), HER2 (accession number: NG_007 503.1), CA-125 (Accession No. NG_055257.1), WT1 (Accession No. NG_009272.1), Mage-A3 (Accession No. NG_013244.1), Mage-A4 (Accession No. NG_013245.1), Mage-A10 (Accession No. NC_000023.11), TRAIL / DR4 (Accession No. NC_000003.12), and / or CEA (Accession No. NC_000019.10), are known in the art.

[0171] In some embodiments, the tumor-associated antigen may be derived from prostate cancer, breast cancer, colorectal cancer, lung cancer, pancreatic cancer, renal cancer, mesothelioma cancer, ovarian cancer, or melanoma cancer. Exemplary tumor-associated antigens or tumor cell-derived antigens include MAGE1, 3, and MAGE4 (or other MAGE antigens as disclosed in International Patent Publication No. WO99 / 40188); PRAME; BAGE; RAGE, Lage (also known as NY ESO 1); SAGE; and HAGE or GAGE. These non-limiting examples of tumor antigens are expressed in a wide range of tumor types, such as melanoma, lung cancer, sarcoma, and bladder cancer. See, for example, U.S. Patent No. 6,544,518. Prostate cancer tumor-associated antigens include, for example, prostate-specific membrane antigen (PSMA), prostate-specific antigen (PSA), prostatic acid phosphate, NKX3.1, and six-stage membrane epithelial antigen of the prostate (STEAP).

[0172] In some embodiments, tumor-associated antigens include Plu-1, HASH-1, HasH-2, Cripto, Criptin, etc. Additionally, tumor antigens may be self-peptide hormones, such as gonadotrophin-releasing hormone (GnRH), a short peptide with a full length of 10 amino acids, which is useful in the treatment of many cancers.

[0173] In some embodiments, the tumor antigen comprises a tumor antigen derived from a cancer characterized by a tumor-associated antigen, such as HER-2 / neu expression. Tumor-associated antigens of interest include lineage-specific tumor antigens such as the melanocyte-melanoma lineage antigen MART-1 / Melan-A, gp100, gp75, mda-7, tyrosinase and tyrosinase-related proteins. Exemplary tumor-associated antigens include, but are not limited to, p53, Ras, c-Myc, cytoplasmic serine / threonine kinases (e.g., A-Raf, B-Raf, and C-Raf, cyclin-dependent kinases), MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, MART-1, BAGE, DAM-6, -10, GAGE-1, -2, -8, GAGE-3, -4, -5, -6, -7B, NA88-A, MART-1, MC1R, Gp100, PSA, PSM, tyrosinase, TRP-1, TRP-2, ART-4, CAMEL, CEA, Cyp-B, hTERT, hTRT, i CE, MUC1, MUC2, phosphoinositide-3-kinase (PI3K), TRK receptor, PRAME, P15, RU1, RU2, SART-1, SART-3, Wilms tumor antigen (WT1), AFP, -catenin / m, caspase-8 / m, CEA, CDK-4 / m, ELF2M, GnT-V, G250, HSP70-2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, myosin / m, RAGE, SART-2, TRP-2 / INT2, 707-AP, annexin II, CDC27 / m, TPI / mbcr-abl, BCR-ABL, interferon regulatory factor 4 (IRF4), ETV6 / AML, LDLR / FUT, Pm,l / RAR, tumor-associated calcium signaling transduction 1 (TACSTD1), TACSTD2, receptor tyrosine kinases (e.g., epidermal growth factor receptor (EGFR) (especially EGFRvIII), platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR)), cytoplasmic tyrosine kinases (e.g., src family, syk-ZAP70 family), integrin-linked kinase (ILK), signal transducers and activators of transcription STAT3, STATS, and STATE, hypoxia-inducible factors (e.g., HIF-1 and HIF-2), nuclear factors -kappa B (NF-B), Notch receptors (e.g., Notch1-4), c-Met, mammalian target of rapamycin (mTOR), WNT, extracellular signal-regulated kinase (ERK) and their regulatory subunits, PMSA, PR-3, MDM2, mesothelin, renal cell carcinoma-5T4, SM22-alpha, carbonic anhydrase I (CAI) and IX (CAIX) (also known as G250), STEAD, TEL / AML1, GD2, proteinase 3, hTERT, sarcoma translocation breakpoints, EphA2, ML-IAP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, GD3, fucosyl GM1, mesothelial, PSCA, sLe, PLAC1, GM3, bORIS, Tn, GLoboH, NY-BR-1, RGsS, SART3, STn, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE1, B7H3, legumain, TIE2, Page4, MAD-CT-1, FAP, MAD-CT-2, fos-related antigen 1, CBX2, CLDN6, SPANX, TPTE, ACTL8, ANKRD30A, CDKN2A, MAD2L1, CTAG1B, SUNC1, LRRN1, and idiotype. ,

[0174] In some embodiments, antigens may include epitope regions or epitope peptides derived from genes mutated in tumor cells or transcribed at different levels in tumor cells compared to normal cells, such as telomerase enzyme, survivin, mesothelin, mutant ras, bcr / abl rearrangements, Her2 / neu, mutant or wild-type p53, cytochrome P450 1B1, and aberrantly expressed intronic sequences, e.g., N-acetylglucosaminyltransferase-V; clonal rearrangements of immunoglobulin genes that generate unique idiotypes in myelomas and B-cell lymphomas; tumor antigens that include epitope regions or epitope peptides derived from oncoviral processes, such as human papillomavirus proteins E6 and E7; Epstein-Barr virus protein LMP2; and non-mutated oncofetal proteins that exhibit tumor-selective expression, such as carcinoembryonic antigen and alpha-fetoprotein.

[0175] B. Non-cancer antigens In certain embodiments, the antigen may be of a microorganism. In some embodiments, the antigen is obtained or derived from a pathogenic or opportunistic pathogenic microorganism (also referred to herein as infectious disease microorganism), such as a virus, fungus, parasite, and bacteria. In certain embodiments, the antigen derived from such a microorganism comprises a full-length protein.

[0176] Exemplary pathogenic organisms for which antigens are contemplated for use in the methods described herein include human immunodeficiency virus (HIV), herpes simplex virus (HSV), respiratory syncytial virus (RSV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), influenza A, B, and C, vesicular stomatitis virus (VSV), polyomaviruses (e.g., BK virus and JC virus), adenovirus, coronaviruses such as SARS-CoV, SARS-CoV-2, or MERS, Staphylococcus species, including methicillin-resistant Staphylococcus aureus (MRSA), and Streptococcus species, including Streptococcus pneumoniae. As one of skill in the art will appreciate, proteins from these and other pathogenic microorganisms for use as antigens as described herein, as well as the nucleotide sequences encoding the proteins, can be identified in publications and public databases such as GENBANK®, SWISS-PROT®, and TREMBL®.

[0177] Antigens derived from Human Immunodeficiency Virus (HIV) include any of the HIV virion structural proteins (e.g., gp120, gp41, p17, p24), protease, reverse transcriptase, or the HIV proteins encoded by tat, rev, nef, vif, vpr, and vpu.

[0178] Antigens derived from herpes simplex viruses (e.g., HSV1 and HSV2) include, but are not limited to, proteins expressed from HSV late genes. The late group of genes mainly encode proteins that form virion particles. Such proteins include five proteins that form the viral capsid (UL): UL6, UL18, UL35, UL38 and major capsid proteins UL19, UL45, and UL27, each of which can be used as an antigen as described herein. Other exemplary HSV proteins contemplated for use as antigens herein include ICP27 (H1, H2), glycoprotein B (gB) and glycoprotein D (gD) proteins. The HSV genome contains at least 74 genes, each of which encodes a protein that can potentially be used as an antigen.

[0179] Antigens derived from cytomegalovirus (CMV) include CMV structural proteins, viral antigens expressed in the immediate early and early stages of viral replication, glycoproteins I and III, capsid proteins, tegument proteins, small matrix proteins pp65 (ppUL83), p52 (ppUL44), IE1 and 1E2 (UL123 and UL122), protein products from the gene cluster derived from UL128-UL150 (Rykman et al., 2006), envelope glycoproteins B (gB), gH, gN, and pp150. As one of skill in the art will appreciate, CMV proteins for use as antigens as described herein can be identified in public databases such as GENBANK®, SWISS-PROT®, and TREMBL® (see, e.g., Bennekov et al., 2004; Loewendorf et al., 2010; Marschall et al., 2009).

[0180] Antigens derived from Epstein-Barr virus (EBV) contemplated for use in certain embodiments include EBV proteins produced during latent cycle infection, including EBV lysis proteins gp350 and gp110, Epstein-Barr nuclear antigen (EBNA)-1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C, EBNA leader protein (EBNA-LP), and latent membrane protein (LMP)-1, LMP-2, and LMP-2B (see, e.g., Lockey et al., 2008).

[0181] Antigens derived from respiratory syncytial virus (RSV) contemplated for use herein include any of 11 proteins, or antigenic fragments thereof, encoded by the RSV genome: NS1, NS2, N (nucleocapsid protein), M (matrix protein) SH, G and F (viral coat proteins), M2 (second matrix protein), M2-1 (elongation factor), M2-2 (transcriptional regulation), RNA polymerase, and phosphoprotein P.

[0182] Antigens derived from vesicular stomatitis virus (VSV) contemplated for use include any one of the five major proteins encoded by the VSV genome and antigenic fragments thereof: large protein (L), glycoprotein (G), nucleoprotein (N), phosphoprotein (P), and matrix protein (M) (see, e.g., Rieder et al., 1999).

[0183] Antigens derived from influenza virus contemplated for use in certain embodiments include hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix proteins M1 and M2, NS1, NS2 (NEP), PA, PB1, PB1-F2, and PB2.

[0184] Exemplary viral antigens include, but are not limited to, adenovirus polypeptides, alphavirus polypeptides, calicivirus polypeptides (e.g., calicivirus capsid antigen), coronavirus polypeptides, distemper virus polypeptides, ebolavirus polypeptides, enterovirus polypeptides, flavivirus polypeptides, hepatitis virus (AE) polypeptides (hepatitis B core or surface antigen, hepatitis C virus E1 or E2 glycoproteins, core, or nonstructural proteins), herpesvirus polypeptides (herpes simplex virus or varicella zoster virus glycoproteins), infectious peritonitis virus polypeptides, leukemia virus polypeptides, Peptides include Marburg virus polypeptides, orthomyxovirus polypeptides, papillomavirus polypeptides, parainfluenza virus polypeptides (e.g., hemagglutinin and neuraminidase polypeptides), paramyxovirus polypeptides, parvovirus polypeptides, pestivirus polypeptides, picornavirus polypeptides (e.g., poliovirus capsid polypeptides), poxvirus polypeptides (e.g., vaccinia virus polypeptides), rabies virus polypeptides (e.g., rabies virus glycoprotein G), reovirus polypeptides, retrovirus polypeptides, and rotavirus polypeptides.

[0185] In certain embodiments, the antigen may be a bacterial antigen. In certain embodiments, the bacterial antigen of interest may be a secreted polypeptide. In other certain embodiments, the bacterial antigen includes an antigen that has a portion or portions of a polypeptide exposed on the outer cell surface of the bacteria.

[0186] Antigens from Staphylococcus species, including Methicillin-resistant Staphylococcus aureus (MRSA), contemplated for use include virulence regulators such as the Agr system, Sar and Sae, the Arl system, Sar homologs (Rot, MgrA, SarS, SarR, SarT, SarU, SarV, SarX, SarZ and TcaR), the Srr system and TRAP.Other Staphylococcus proteins that can serve as antigens include Clp proteins, HtrA, MsrR, aconitase, CcpA, SvrA, Msa, CfvA and CfvB (see, for example, Staphylococcus: Molecular Genetics, 2008 Caister Academic Press (ed.), Jodi Lindsay). Two S. aureus genomes (N315 and Mu50) have been sequenced and are publicly available, for example, at PATRIC (PATRIC: The VBI PathoSystems Resource Integration Center, Snyder et al., 2007). As one of skill in the art will appreciate, Staphylococcal proteins for use as antigens can also be identified in other public databases, such as GenBank®, Swiss-Prot®, and TrEMBL®.

[0187] Antigens from Streptococcus pneumoniae contemplated for use in certain embodiments described herein include pneumolysin, PspA, choline-binding protein (CbpA), NanA, NanB, SpnHL, PavA, LytA, Pht, and pilin proteins (RrgA; RrgB; RrgC). Antigenic proteins of Streptococcus pneumoniae are also known in the art and can be used as antigens in some embodiments (see, e.g., Zysk et al., 2000). The complete genome sequence of virulent strains of Streptococcus pneumoniae has been sequenced, and as one of skill in the art can appreciate, S. pneumoniae proteins for use herein can also be identified in other public databases such as GENBANK®, SWISS-PROT®, and TREMBL®. Proteins of particular interest for antigens according to the present disclosure include virulence factors and proteins predicted to be exposed on the surface of pneumococci (see, e.g., Frolet et al., 2010).

[0188] Examples of bacterial antigens that can be used as antigens include, but are not limited to, Actinomyces polypeptides, Bacillus polypeptides, Bacteroides polypeptides, Bordetella polypeptides, Bartonella polypeptides, Borrelia polypeptides (e.g., B. burgdorferi OspA), Brucella polypeptides, Campylobacter polypeptides, Capnocytophaga polypeptides, Chlamydia (C hlamydia polypeptides, Corynebacterium polypeptides, Coxiella polypeptides, Dermatophilus polypeptides, Enterococcus polypeptides, Ehrlichia polypeptides, Escherichia polypeptides, Francisella polypeptides, Fusobacterium polypeptides, Haemobartonella polypeptides, Haemophilus polypeptides (e.g., H. influenzae (H.influenzae type b outer membrane protein), Helicobacter polypeptide, Klebsiella polypeptide, L-type bacterial polypeptide, Leptospira polypeptide, Listeria polypeptide, Mycobacteria polypeptide, Mycoplasma polypeptide, Neisseria polypeptide, Neorickettsia polypeptide, Nocardia polypeptide, Pasteurella polypeptide, Peptococcus polypeptide, Peptostreptococcus polypeptide, Pneumococcus polypeptide (i.e. These include S. pneumoniae polypeptides (see description herein), Proteus polypeptides, Pseudomonas polypeptides, Rickettsia polypeptides, Rochalimaea polypeptides, Salmonella polypeptides, Shigella polypeptides, Staphylococcus polypeptides, Group A Streptococcus polypeptides (e.g., S. pyogenes M protein), Group B Streptococcus polypeptides (e.g., S. agalactiae polypeptides, Treponema polypeptides, and Yersinia polypeptides (e.g., Y. pestis F1 and V antigens).

[0189] Examples of fungal antigens include, but are not limited to, Absidia polypeptides, Acremonium polypeptides, Alternaria polypeptides, Aspergillus polypeptides, Basidiobolus polypeptides, Bipolaris polypeptides, Blastomyces polypeptides, Candida polypeptides, Coccidioides polypeptides, Conidiobolus polypeptides, Cryptococcus polypeptides, Curvalaria polypeptides, Epidermophyton polypeptides, Exophiala polypeptides, Geotrichum polypeptides, Histoplasma polypeptides, Madurella polypeptides, Malassezia polypeptides, Microsporum polypeptides, Moniliella polypeptides, Mortierella polypeptides, Mucor polypeptides, Paecilomyces polypeptides, Penicillium polypeptides, Phialemonium polypeptides, Phialophora polypeptides, Prototheca polypeptides, Pseudallescheria polypeptides, Pseudomicrodochium polypeptides, Pythium polypeptides, Rhinosporidium polypeptides, Rhizopus polypeptides, Scolecobasidium polypeptides, Sporothrix polypeptides, Stemphylium polypeptides, Trichophyton polypeptides,Trichosporon polypeptides, and Xylohypha polypeptides.

[0190] Examples of protozoan parasite antigens include, but are not limited to, Babesia polypeptides, Balantidium polypeptides, Besnoitia polypeptides, Cryptosporidium polypeptides, Eimeria polypeptides, Encephalitozoon polypeptides, Entamoeba polypeptides, Giardia polypeptides, Hammondia polypeptides, Hepatozoon polypeptides, Isospora polypeptides, Leishmania polypeptides, Microsporidia polypeptides, Neospora polypeptides, Nosema polypeptides, Pentatrichomonas polypeptides, Plasmodium polypeptides. Examples of parasitic helminth parasite antigens include, but are not limited to, Acanthocheilonema polypeptides, Aelurostrongylus polypeptides, Ancylostoma polypeptides, Angiostrongylus polypeptides, Ascaris polypeptides, Brugia polypeptides, Bunostomum polypeptides, Capillaria polypeptides, Shoveltia polypeptides, and the like. Chabertia polypeptides, Cooperia polypeptides, Crenosoma polypeptides, Dictyocaulus polypeptides, Dioctophyme polypeptides, Dipetalonema polypeptides, Diphyllobothrium polypeptides, Diplydium polypeptides, Dirofilaria polypeptides, Dracunculus polypeptides,Enterobius Polypeptides, Filaroides Polypeptides, Haemonchus Polypeptides, Lagochilascaris Polypeptides, Loa Polypeptides, Mansonella Polypeptides, Muellerius Polypeptides, Nanophyetus Polypeptides, Necator Polypeptides, Nematodirus Polypeptides, Oesophagostomum Polypeptides, Onchocerca Polypeptides, Opisthorchis Polypeptides, Ostertagia Polypeptides, Parafilaria Polypeptides, Paragonimus Polypeptides, Parascaris Polypeptides, Physaloptera Polypeptides, Protostrongylus Polypeptides, ostrongylus polypeptides, Setaria polypeptides, Spirocerca polypeptides, Spirometra polypeptides, Stephanofilaria polypeptides, Strongyloides polypeptides, Strongylus polypeptides, Thelazia polypeptides, Toxascaris polypeptides, Toxocara polypeptides, Trichinella polypeptides, Trichostrongylus polypeptides, Trichuris polypeptides, Uncinaria polypeptides, and Wuchereria polypeptides (e.g., P. falciparum circumsporozoite protein (PfCSP)), sporozoite surface protein 2 (PfSSP2), carboxyl terminus of liver status antigen 1 (PfLSA1 c-term), and transport protein 1 (PfExp-1),Pneumocystis polypeptides, Sarcocystis polypeptides, Schistosoma polypeptides, Theileria polypeptides, Toxoplasma polypeptides, and Trypanosoma polypeptides.

[0191] Examples of ectoparasite antigens include, but are not limited to, polypeptides (including antigens and allergens) derived from fleas; ticks, including hard and soft mites; midges, mosquitoes, sandflies, black flies, horseflies, horn flies, deer flies, tsetse flies, stable flies, flies causing myiasis and biting nuts; ants; spiders, lice; mites; and hemipteran insects such as bedbugs and assassin bugs.

[0192] IV. Cell therapy Certain embodiments relate to cells comprising the polypeptide or nucleic acid of the present disclosure. In some embodiments, the cell is an immune cell. In certain embodiments, as used herein, "T cell" includes any type of immune cell that expresses CD3, including helper T cells, invariant natural killer T (iNKT) cells, cytotoxic T cells, regulatory T cells (Treg), gamma delta T cells, natural killer (NK) cells, and neutrophils. T cell may refer to CD4+ or CD8+ T cells.

[0193] Suitable mammalian cells include primary cells and immortalized cell lines. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, and the like. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), human embryonic kidney (HEK) 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RATI cells, mouse L cells (ATCC No. CCLI.3), HLHepG2 cells, Hut-78, Jurkat, HL-60, NK cell lines (e.g., NKL, NK92, and YTS), and the like.

[0194] In some cases, the cell is not an immortalized cell line, but instead is a cell (e.g., a primary cell) obtained from an individual. For example, in some cases, the cell is an immune cell obtained from an individual. For example, the cell is a T lymphocyte obtained from an individual. In another example, the cell is a cytotoxic cell obtained from an individual. In another example, the cell is a stem cell (e.g., a peripheral blood stem cell) or progenitor cell obtained from an individual.

[0195] A. Infinite immune cells Certain embodiments of the present disclosure relate to immune cells that are engineered to express one or more genes. The expression of one or more genes directly or indirectly results in an increased life span of the cell compared to a cell lacking expression of the one or more genes. In certain embodiments, the cell is engineered to express one or more genes, including one or more heterologous genes. In other cases, the cell is engineered to have upregulation of expression of one or more genes that are endogenous to the cell, such as by manipulation of one or more regulatory elements of one or more endogenous genes to the cell. In certain embodiments, methods and compositions related to infinite immune cells are described in PCT Patent Application Publication No. WO2021 / 034982, the entirety of which is incorporated herein by reference.

[0196] In certain embodiments, immune cells are engineered to express BCL6 and one or more pro-survival or anti-apoptotic or cell survival genes (and there may or may not be overlap in genes classified as pro-survival or anti-apoptotic or cell survival). As used herein, pro-survival genes refer to nucleic acid polymers that can exert anti-apoptotic functions or promote survival by any mechanism. Nucleic acid polymers that can exert anti-apoptotic functions may be one or more Bcl2 family genes, such as BCL-xL, BCL-2, MCL-1, Bcl-w, Bfl-1, BCL-B. Nucleic acid polymers that can exert anti-apoptotic functions may be one or more inhibitors of apoptosis (IAP) family genes, such as XIAP, c-IAP1, C-IAP2, NAIP, and survivin. The nucleic acid polymer capable of exerting anti-apoptotic function may be capable of inhibiting or knocking out the expression of one or more caspases that play a role in apoptosis, such as caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14. The nucleic acid polymer for knockdown or knockout may be an shRNA expression cassette, or these caspase genes may be knocked out by gene editing methods (CRISPR, TALEN, zinc finger method, etc.). The nucleic acid polymer capable of exerting anti-apoptotic function may be capable of inhibiting or knocking out the expression of one or more pro-apoptotic genes, such as BIM, Puma, Noxa, Bik, Bmf, Bad, Hrk, Bid, BAX, BAK, BOK, etc.Nucleic acid polymers capable of exerting anti-apoptotic functions may have anti-apoptotic effects, such as insulin-like growth factor (IGG-1), Hsp70, Hsp27, cFLIP, BNIP3, FADD, Akt, and NF-κB, Raf-1 and MEK1, p90Rsk, C-Jun, BNIP2, BAG1, HSPA9, HSP90B1, miRNA21, miR-106b-25, miR-206, miR-221 / 222, miR-17-92, miR-133, miR-143, miR-145, miR-155, miR-330, etc.

[0197] Infinite T cells can be generated using either wild-type or mutant BCL6. The inventors have determined that infinite T cells can be generated using either wild-type BCL6 or mutant BCL6 with a single specific nucleotide difference (the codon for amino acid 395 in wild-type BCL6 is CCT (encoding proline / P), and the codon for amino acid 395 in mutant BCL6 is CTT (encoding leucine / L). The nucleotide and amino acid sequences for the two BCL6 genes are shown below (the position of the mutation in the wild-type sequence is underlined): SEQ ID NO:43 - Amino acid sequence of wild type BCL6: MASPADSCIQFTRHASDVLLNLNRLRSRDILTDVVIVVSREQFRAHKTVLMACSGLFYSIFTDQLKCNLSVINLDPEINPEGFCILLDFMYTSRLNLREGNIMAVMATAMYLQMEHVVDTCRKFIKASEAEMVSAIKPPREEFLNSRMLMPQDIMAYRGREVVENNLPLRSAPGCESRAFAPSLYSGLSTPPASYSM YSHLPVSSLLFSDEEFRDVRMPVANPFPKERALPCDSARPVPGEYSRPTLEVSPNVCHSNIYSPKETIPEEARSDMHYSVAEGLKPAAPSARNAPYFPCDKASKEEERPSSEDEIALHFEPNAPLNRKGLVSPQSPQKSDCQPNSPTESCSSKNACILQASGSPPAKSPTDPKACNWKKYKFIVLNSLNQNAKPEG P EQAELGRLSPRAYTAPPACQPPMEPENLDLQSPTKLSASGEDSTIPQASRLNNIVNRSMTGSPRSSSESHSPLYMHPPKCTSCGSQSPQHAEMCLHTAGPTFPEEMGETQSEYSDSSCENGAFFCNECDCRFSEEASLKRHTLQTHSDKPYKCDRCQASFRYKGNLASHKTVHTGEKPYRCNICGAQFNRPANLKTHTRIHSGEKPYKCETCGARFVQVAHLRAHVLIHTGEKPYPCEICGTRFRHLQTLKSHLRIHTGEKPYHCEKCNLHFRHKSQLRLHLRQKHGAITNTKVQYRVSATDLPPELPKAC (SEQ ID NO: 43) SEQ ID NO:44 - Nucleotide sequence of wild type BCL6 (mutation point codons in the wild type sequence are underlined): cCt gagcaggctgagctgggccgcctttccccacgagcctacacggccccacctgcctgccagccacccatggagcctgagaaccttgacctccagtccccaaccaagctgagtgccagcggggaggactccaccatcccacaagccagccggctcaataacatcgttaacaggtccatgacgggctctccccgcagcagcagcgagagccactcaccactctacatgcaccccccgaagtgcacgtcctgcggctctcagtccccacagcatgcagagatgtgcctccacaccgctggccccacgttccctgaggagatgggagagacccagtctgagtactcagattctagctgtgagaacggggccttcttctgcaatgagtgtgactgccgcttctctgaggaggcctcactcaagaggcacacgctgcagacccacagtgacaaaccctacaagtgtgaccgctgccaggcctccttccgctacaagggcaacctcgccagccacaagaccgtccataccggtgagaaaccctatcgttgcaacatctgtggggcccagttcaaccggccagccaacctgaaaacccacactcgaattcactctggagagaagccctacaaatgcgaaacctgcggagccagatttgtacaggtggcccacctccgtgcccatgtgcttatccacactggtgagaagccctatccctgtgaaatctgtggcacccgtttccggcaccttcagactctgaagagccacctgcgaatccacacaggagagaaaccttaccattgtgagaagtgtaacctgcatttccgtcacaaaagccagctgcgacttcacttgcgccagaagcatggcgccatcaccaacaccaaggtgcaataccgcgtgtcagccactgacctgcctccggagctccccaaagcctgc(SEQ ID NO: 44) SEQ ID NO:45 - Amino acid sequence of mutated BCL6 (leucine mutations are underlined): MASPADSCIQFTRHASDVLLNLNRLRSRDILTDVVIVVSREQFRAHKTVLMACSGLFYSIFTDQLKCNLSVINLDPEINPEGFCILLDFMYTSRLNLREGNIMAVMATAMYLQMEHVVDTCRKFIKASEAEMVSAIKPPREEFLNSRMLMPQDIMAYRGREVVENNLPLRSAPGCESRAFAPSLYSGLSTPPASYSM YSHLPVSSLLFSDEEFRDVRMPVANPFPKERALPCDSARPVPGEYSRPTLEVSPNVCHSNIYSPKETIPEEARSDMHYSVAEGLKPAAPSARNAPYFPCDKASKEEERPSSEDEIALHFEPNAPLNRKGLVSPQSPQKSDCQPNSPTESCSSKNACILQASGSPPAKSPTDPKACNWKKYKFIVLNSLNQNAKPEG L EQAELGRLSPRAYTAPPACQPPMEPENLDLQSPTKLSASGEDSTIPQASRLNNIVNRSMTGSPRSSSESHSPLYMHPPKCTSCGSQSPQHAEMCLHTAGPTFPEEMGETQSEYSDSSCENGAFFCNECDCRFSEEASLKRHTLQTHSDKPYKCDRCQASFRYKGNLASHKTVHTGEKPYRCNICGAQFNRPANLKTHTRIHSGEKPYKCETCGARFVQVAHLRAHVLIHTGEKPYPCEICGTRFRHLQTLKSHLRIHTGEKPYHCEKCNLHFRHKSQLRLHLRQKHGAITNTKVQYRVSATDLPPELPKAC (SEQ ID NO: 45) SEQ ID NO:46 - Nucleotide sequence of mutant BCL6 (leucine codons are underlined): cTt gagcaggctgagctgggccgcctttccccacgagcctacacggccccacctgcctgccagccacccatggagcctgagaaccttgacctccagtccccaaccaagctgagtgccagcggggaggactccaccatcccacaagccagccggctcaataacatcgttaacaggtccatgacgggctctccccgcagcagcagcgagagccactcaccactctacatgcaccccccgaagtgcacgtcctgcggctctcagtccccacagcatgcagagatgtgcctccacaccgctggccccacgttccctgaggagatgggagagacccagtctgagtactcagattctagctgtgagaacggggccttcttctgcaatgagtgtgactgccgcttctctgaggaggcctcactcaagaggcacacgctgcagacccacagtgacaaaccctacaagtgtgaccgctgccaggcctccttccgctacaagggcaacctcgccagccacaagaccgtccataccggtgagaaaccctatcgttgcaacatctgtggggcccagttcaaccggccagccaacctgaaaacccacactcgaattcactctggagagaagccctacaaatgcgaaacctgcggagccagatttgtacaggtggcccacctccgtgcccatgtgcttatccacactggtgagaagccctatccctgtgaaatctgtggcacccgtttccggcaccttcagactctgaagagccacctgcgaatccacacaggagagaaaccttaccattgtgagaagtgtaacctgcatttccgtcacaaaagccagctgcgacttcacttgcgccagaagcatggcgccatcaccaacaccaaggtgcaataccgcgtgtcagccactgacctgcctccggagctccccaaagcctgc(SEQ ID NO: 46)

[0198] Immune cells include T cells (e.g. regulatory T cells, CD4 + T cells, CD8 + The immune cells may be any type of immune cell, including T cells, alpha beta T cells, gamma delta T cells, or a mixture thereof), NK cells, invariant NKT cells, NKT cells, innate lymphocytes, or a mixture thereof. The immune cells may be virus-specific, express a CAR, and / or express a TCR. In some embodiments, the cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells (DCs), mast cells, eosinophils, and / or basophils. Also provided herein are methods of producing and manipulating immune cells and using and administering cells for adoptive immunotherapy, where the cells may be autologous or allogeneic. Thus, the immune cells can be used as immunotherapy, such as to target cancer cells. These immune cells can be used as a single cell type or as a combination of multiple immune cell types for therapy. In certain embodiments, the immune cells are CD3+, CD4+, CD8+, CD16+, or a mixture thereof.

[0199] Immune cells can be isolated from subjects, particularly human subjects. Immune cells can be obtained from subjects of interest, such as subjects suspected of having a particular disease or condition, subjects suspected of having a predisposition to a particular disease or condition, or subjects undergoing therapy for a particular disease or condition. Immune cells can be collected from any location where they are present in subjects, including but not limited to blood, umbilical cord blood, spleen, thymus, lymph nodes, and bone marrow. Immune cells isolated can be used directly, or they can be stored for a period of time, such as by freezing. Immune cells can be enriched / purified from any tissue in which they are present, including, but not limited to, blood (including blood collected by blood banks or umbilical cord blood banks), spleen, bone marrow, tissues removed and / or exposed during surgical procedures, and tissues obtained by biopsy procedures. The tissues / organs from which immune cells are enriched, isolated and / or purified may be isolated from both living and non-living subjects, where the non-living subject is an organ donor. In certain embodiments, immune cells are isolated from blood, such as peripheral blood or umbilical cord blood. In some aspects, immune cells isolated from umbilical cord blood have enhanced immune modulation capabilities, as measured by CD4 or CD8 positive T cell suppression. In certain aspects, immune cells are isolated from pooled blood, particularly pooled umbilical cord blood, for enhanced immune modulation capabilities. Pooled blood may be derived from two or more sources, such as 3, 4, 5, 6, 7, 8, 9, 10 or more sources (e.g., donor subjects).

[0200] The population of immune cells can be obtained from a subject who needs therapy or a subject who suffers from a disease associated with reduced immune cell activity. Thus, the cells will be autologous to the subject who needs therapy. Alternatively, the population of immune cells can be obtained from a donor, such as a partially or fully histocompatible donor or a fully histocompatible mismatched donor. The immune cell population can be harvested from peripheral blood, umbilical cord blood, bone marrow, spleen, or any other organ / tissue in which immune cells are present in the subject or donor. The immune cells can be isolated from a pool of subjects and / or donors, such as pooled umbilical cord blood.

[0201] When the population of immune cells is obtained from a donor different from the subject, the donor may be allogeneic, provided that the cells obtained are subject-compatible in that they can be introduced into the subject. Allogeneic donor cells may or may not be human leukocyte antigen (HLA) compatible.

[0202] Further methods and compositions related to Infinite Immune Cells are described in PCT Patent Application Publication No. WO2021 / 034982, which is incorporated by reference in its entirety.

[0203] 1.T cells In some embodiments, the immune cells are T cells. Several basic approaches for the induction, activation and expansion of functional anti-tumor effector cells have been described in the last two decades. These include autologous cells such as tumor infiltrating lymphocytes (TILs); T cells activated ex vivo using autologous DCs or PBMCs, lymphocytes, artificial antigen presenting cells (APCs) or beads coated with T cell ligands and activating antibodies, or cells isolated by capturing target cell membranes; allogeneic cells that naturally express anti-host tumor T cell receptors (TCRs); and non-tumor specific autologous or allogeneic cells that have been genetically reprogrammed or "targeted" to express tumor-reactive TCRs or chimeric TCR molecules that exhibit tumor recognition capabilities such as antibodies known as "T-bodies". These approaches have given rise to several protocols for T cell preparation and immunization that can be used in the methods described herein.

[0204] In some embodiments, the T cells are derived from blood, bone marrow, lymph, umbilical cord, or lymphoid organs. In some aspects, the cells are human cells. The cells are typically primary cells, such as those isolated directly from a subject and / or isolated and frozen from a subject. In some embodiments, the cells are total T cell populations, CD4 + cells, CD8 +The present invention includes one or more subsets of T cells or other cell types, such as cells and subpopulations thereof, such as those defined by function, activation state, maturity, differentiation potential, expansion, recirculation, localization, and / or persistence ability, antigen specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and / or degree of differentiation. With reference to the subject to be treated, the cells may be allogeneic and / or autologous. In some aspects, such as for off-shelf technologies, the cells are pluripotent and / or multipotent, such as stem cells, such as induced pluripotent stem cells (iPSCs). In some embodiments, the method includes isolating the cells from the subject, preparing, processing, culturing, and / or manipulating them as described herein, and reintroducing them into the same patient before or after cryopreservation.

[0205] In particular, T cells (e.g., CD4 + and / or CD8 + T cell) subtypes and subpopulations include naive T (T N ) cells, effector T cells (T EFF ), memory T cells and their subtypes, such as stem cell memory T (TSC M ), Central Memory T (TC M ), Effector Memory T (T EM ), or terminally differentiated effector memory T cells, tumor infiltrating lymphocytes (TILs), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha / beta T cells, and gamma / delta T cells. In certain embodiments, the T cells are gamma / delta T cells.

[0206] In some embodiments, one or more T cell populations are enriched for or depleted of cells that are positive for or negative for a particular marker, such as a surface marker. In some cases, such markers are absent or expressed at relatively low levels on certain populations of T cells (e.g., non-memory cells) but present or expressed at relatively high levels on certain other populations of T cells (e.g., memory cells).

[0207] In some embodiments, T cells are separated from the PBMC sample by negative selection of markers expressed on B cells, non-T cells such as monocytes, or other white blood cells such as CD14. + or CD8 + Using a selection step, CD4 + Helper T cells and CD8 + Cytotoxic T cells are isolated from CD4 + and CD8 + The population can be further sorted into subpopulations by positive or negative selection for markers expressed, or expressed to a relatively high extent, on one or more naive, memory, and / or effector T cell subpopulations.

[0208] In some embodiments, CD8 + The T cells are further enriched for or depleted of naive, central memory, effector memory, and / or central memory stem cells, such as by positive or negative selection based on surface antigens associated with each subpopulation. In some embodiments, central memory T (T CM Enrichment for primary or stem cell memory cells is performed to increase efficacy, such as to improve long-term survival, expansion, and / or engraftment following administration, and in some embodiments is particularly robust in such subpopulations.

[0209] In some embodiments, the T cells are autologous T cells. In this method, a tumor sample is obtained from a patient and a single cell suspension is obtained. The single cell suspension can be obtained in any suitable manner, for example, mechanically (e.g., using a gentleMACS™ Dissociator, Miltenyi Biotec, Auburn, Calif. to disintegrate the tumor) or enzymatically (e.g., collagenase or DNase). The single cell suspension of the tumor enzymatic digest is cultured in interleukin-2 (IL-2) or other growth factors.

[0210] The cultured T cells can be pooled and rapidly expanded. Rapid expansion results in an increase in the number of antigen-specific T cells of at least about 50-fold (e.g., 50, 60, 70, 80, 90, or 100-fold or more) over a period of about 10 to about 14 days. More preferably, rapid expansion results in an increase in the number of antigen-specific T cells of at least about 200-fold (e.g., 200, 300, 400, 500, 600, 700, 800, 900-fold or more) over a period of about 10 to about 14 days.

[0211] Expansion can be achieved by any of several methods as known in the art. For example, T cells can be rapidly expanded using non-specific T cell receptor stimulation in the presence of feeder lymphocytes and interleukin-2 (IL-2) or interleukin-15 (IL-15), with IL-2 being preferred. Non-specific T cell receptor stimulation may include about 30 ng / ml OKT3 (available from Ortho-McNeil®, Raritan, NJ), a mouse monoclonal anti-CD3 antibody. Alternatively, T cells can be rapidly expanded by stimulation of peripheral blood mononuclear cells (PBMCs) in vitro with one or more antigens of the cancer (including antigenic portions thereof, such as epitopes, or cells), which can be optionally expressed from a vector, such as a human leukocyte antigen A2 (HLA-A2) binding peptide or peptides that bind to other MHC class I or class II molecules, in the presence of a T cell growth factor, such as 300 IU / ml IL-2 or IL-15, with IL-2 being preferred. In vitro induced T cells are rapidly expanded by restimulation with the same cancer antigen pulsed on antigen presenting cells expressing HLA-A2 or other HLA molecules. In vitro induced T cells can also be expanded in the absence of antigen presenting cells.

[0212] Autologous T cells can be modified to express T cell growth or differentiation factors that promote the proliferation, differentiation, and activation of autologous T cells. Suitable T cell growth factors include, for example, interleukin (IL)-2, IL-7, IL-15, IL-18, IL-21, and IL-12. Suitable modification methods are known in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. In certain embodiments, modified autologous T cells express high levels of T cell growth factors. T cell growth factor coding sequences, such as those of IL-12, are readily available in the art, as are promoters whose operably linked to T cell growth factor coding sequences promote high level expression.

[0213] 2.NK cells In some embodiments, the immune cell is a natural killer (NK) cell. NK cells are a subpopulation of lymphocytes that have spontaneous cytotoxicity against various tumor cells, virus-infected cells, and some normal cells in bone marrow and thymus. NK cells differentiate and mature in bone marrow, lymph nodes, spleen, tonsils, and thymus. NK cells can be detected by specific surface markers such as CD16, CD56, and CD8 in humans. NK cells do not express T cell antigen receptor, pan-T cell marker CD3, or surface immunoglobulin B cell receptor.

[0214] In certain embodiments, NK cells are derived from human peripheral blood mononuclear cells (PBMCs), unstimulated leukoreduction products (PBSCs), human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs), bone marrow, tissue, or umbilical cord blood by methods well known in the art.

[0215] 3.NKT cells Natural killer T (NKT) cells are a heterogeneous group of T cells that share characteristics of both T cells and natural killer cells. Many of these cells recognize the non-polymorphic CD1d molecule, an antigen-presenting molecule that binds self and foreign lipids and glycolipids. They account for only approximately 0.1% of all peripheral blood T cells. NKT cells are a subset of T cells that not only co-express the αβ T cell receptor, but also express various molecular markers typically associated with NK cells, such as NK1.1. Invariant natural killer T (iNKT) cells express and depend on high levels of the transcriptional regulator promyelocytic leukemia zinc finger for their development. Currently, there are five major distinct iNKT cell subsets. These subset cells produce distinct sets of cytokines once activated. The subtypes iNKT1, iNKT2, and iNKT17 closely resemble Th cell subsets in cytokine production. In addition, there are subtypes that are specialized for follicular helper T cell-like functions and IL-10-dependent regulatory functions.

[0216] 4. Innate lymphocytes Innate lymphoid cells (ILCs) are a population of innate immune cells that originate from common lymphoid progenitors (CLPs) and belong to the lymphoid lineage. These cells are defined by the absence of antigen-specific B or T cell receptors due to the lack of recombination activating genes (RAGs). ILCs do not express myeloid or dendritic cell markers. They play a role in the regulation of protective immunity as well as homeostasis and inflammation, and therefore, their dysregulation can result in immunopathologies such as allergy, bronchial asthma and autoimmune diseases. ILCs can be divided based on the cytokines they produce and the transcription factors that regulate their development and function.

[0217] B. Cell culture In some embodiments, the cells may be cultured for at least about 10 to about 40 days, at least about 15 to about 35 days, at least about 15 to 21 days, for example, at least about 15, 16, 17, 18, 19, or 21 days. In some embodiments, the cells of the present disclosure may be cultured for 60 days or less, or 50 days or less, or 45 days or less. The cells may be cultured for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days. The cells may be cultured in the presence of a liquid culture medium. Typically, the medium may include a basal medium formulation as known in the art. Many basal medium formulations can be used to culture the cells of the present invention, including, but not limited to, Eagle's Minimum Essential Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), alpha modified minimum essential medium (alpha-MEM), essential basal medium (BME), Iscove's Modified Dulbecco's Medium (IMDM), BGJb's medium, F-12 nutrient mixture (Ham), Liebovitz L-15, DMEM / F-12, essential modified Eagle's medium (EMEM), RPMI-1640, and modifications and / or combinations thereof. The compositions of the above basal media are generally known in the art, and it is within the skill of the artisan to modify or modulate the concentrations of the media and / or media additives as needed for the cells to be cultured. In some embodiments, the culture medium formulation may be explant medium (CEM) composed of IMDM supplemented with 10% fetal bovine serum (FBS), 100 U / ml penicillin G, 100 μg / ml streptomycin, and 2 mmol / L L-glutamine. Other embodiments may use additional basal medium formulations, such as those selected from one of the above.

[0218] Any medium capable of supporting cells in vitro may be used to culture the cells. Media formulations capable of supporting cell growth include, but are not limited to, Dulbecco's Modified Eagle Medium (DMEM), alpha modified minimum essential medium (αMEM), and Roswell Park Memorial Institute Medium 1640 (RPMI Medium 1640). Typically, up to 20% fetal bovine serum (FBS) or 1-20% horse serum is added to the above media to support cell growth. However, defined media may be used if the growth factors, cytokines, and hormones required to culture the cells are provided in the media at appropriate concentrations. Media useful in the methods of the present disclosure may include one or more compounds of interest, including, but not limited to, antibiotics, mitogenic compounds, or differentiation compounds useful for culturing the cells. Cells may be grown at temperatures between 27°C and 40°C, such as between 31°C and 37°C, and placed in a humidified incubator. Carbon dioxide content may be maintained at 2% to 10% and oxygen content may be maintained at 1% to 22%. However, this disclosure should not be construed in any way as being limited to any one method of isolating and culturing cells, but rather, any method of isolating and culturing cells should be construed as being included in this disclosure.

[0219] For use in cell culture, the medium can be provided with one or more additional components. For example, additional supplements can be used to provide cells with the necessary trace elements and materials for optimal growth and expansion. Such supplements include insulin, transferrin, selenium salts, and combinations thereof. These components can be contained in salt solutions, such as, but not limited to, Hanks' Balanced Salt Solution (HBSS), Eagle's Salt Solution, etc. Additional antioxidant supplements, such as β-mercaptoethanol, can be added. Many media already contain amino acids, but some amino acids, such as L-glutamine, which are known to be less stable when in solution, can be supplemented later. The medium may further be supplemented with antibiotic and / or antifungal compounds, such as, typically, a mixture of penicillin and streptomycin, and / or other compounds, such as, but not limited to, amphotericin, ampicillin, gentamicin, bleomycin, hygromycin, kanamycin, mitomycin, mycophenolic acid, nalidixic acid, neomycin, nystatin, paromomycin, polymyxin, puromycin, rifampicin, spectinomycin, tetracycline, tylosin, and zeocin. Also contemplated is the supplementation of cell culture medium with mammalian plasma or serum. Plasma or serum often contains cellular factors and components necessary for survival and expansion. The use of suitable serum substitutes is also contemplated.

[0220] References to specific buffers, media, reagents, cells, culture conditions, etc., or some subclasses thereof, are not intended to be limiting and should be read to include all such related materials that a person skilled in the art would recognize as being of interest or useful in the particular context in which the discussion is presented. For example, it is often possible to substitute one buffer system or culture medium for another, so that a different but known method is used to achieve the same goal that the proposed method, material or composition is intended to achieve. In certain embodiments, cells are preferably cultured in a cell culture system that includes a cell culture medium in the culture solution, in particular a cell culture medium supplemented with a substance suitable and determined for protecting cells from in vitro senescence and / or for inducing non-specific or specific reprogramming.

[0221] C. Cell Generation Certain methods of the present disclosure relate to culturing cells obtained from a human tissue sample. In certain embodiments of the present disclosure, the cells are seeded on a substrate that allows for cell adhesion. This can be carried out, for example, by seeding the cells in a culture plate that displays one or more substrate surfaces compatible with cell adhesion. When one or more substrate surfaces come into contact with a suspension of cells (e.g., a suspension in a medium) introduced into the culture system, cell adhesion between the cells and the substrate surface can result. Thus, in certain embodiments, the cells are introduced into a culture system that generally features at least one substrate surface that is compatible with the adhesion of cells thereto, such that the seeded cells can come into contact with said substrate surface, and such embodiments include seeding on a substrate that allows for the adhesion of cells thereto.

[0222] The cells of the present disclosure can be identified and characterized by the expression of certain marker proteins, such as cell surface markers. Detection and isolation of these cells can be achieved, for example, by flow cytometry, ELISA, and / or magnetic beads. Reverse transcription polymerase chain reaction (RT-PCR) can be used to quantify cell-specific genes and / or monitor changes in gene expression in response to differentiation. In certain embodiments, the marker proteins used to identify and characterize cells are selected from the list consisting of c-Kit, Nanog, Sox2, Hey1, SMA, Vimentin, Cyclin D2, Snail, E-cadherin, Nkx2.5, GATA4, CD105, CD90, CD29, CD73, Wt1, CD34, CD45, and combinations thereof.

[0223] D. Pharmaceutical Compositions In certain embodiments, compositions or agents for use in methods such as cell-based therapy are preferably contained in a pharma-ceutically acceptable carrier. The carrier is selected to be non-toxic, biocompatible, and not to adversely affect the biological activity of the agent. The agents in some embodiments of the present disclosure can be formulated into preparations for local delivery (i.e., to a specific location in the body, such as a tumor site, or other tissue) or systemic delivery in solid, semi-solid, gel, liquid, or gaseous forms, such as tablets, capsules, powders, granules, ointments, liquids, suppositories, inhalants, and injections, that allow oral, parenteral, or surgical administration. Certain embodiments of the present disclosure also contemplate local administration of the composition by coating a pharmaceutical device, etc.

[0224] Suitable carriers for parenteral delivery by injection, infusion or irrigation and local delivery include distilled water, physiological phosphate buffered saline, normal or lactated Ringer's solution, dextrose solution, Hank's solution, or propanediol.In addition, sterile fixed oils can be used as a solvent or suspension medium.For this purpose, any biocompatible oil can be used, including synthetic mono- or diglycerides.In addition, fatty acids such as oleic acid are useful in the preparation of injections.The carrier and drug can be mixed as a liquid, suspension, polymeric or non-polymeric gel, paste, or ointment.

[0225] Carriers may also include delivery vehicles to sustain (i.e., extend, delay, or modulate) delivery of an agent or to enhance delivery, uptake, stability, or pharmacokinetics of a therapeutic agent. Such delivery vehicles may include, by way of non-limiting example, microparticles, microspheres, nanospheres, or nanoparticles composed of proteins, liposomes, carbohydrates, synthetic organic compounds, inorganic compounds, polymeric or copolymeric hydrogels, and polymeric micelles.

[0226] In certain embodiments, the actual dosage of the composition administered to a patient or subject can be determined by physical and physiological factors such as body weight, severity of the condition, type of disease being treated, previous or current therapeutic interventions, idiopathic disease of the patient, and route of administration. The physician responsible for administration will, in any event, determine the concentration of active ingredient in the composition and the appropriate dose for the individual subject.

[0227] Solutions of the pharmaceutical compositions can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose.Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, as well as in oils.Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0228] In certain embodiments, pharmaceutical compositions are advantageously administered in the form of injectable compositions as liquid solutions or suspensions; suitable solid forms or solutions or suspensions in liquids can also be prepared prior to injection. These preparations can also be emulsified. Typical compositions for such purposes include pharmaceutically acceptable carriers. For example, the composition can contain up to 10 mg, 25 mg, 50 mg or up to about 100 mg of human serum albumin per milliliter of phosphate buffered saline. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers, etc.

[0229] Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils and injectable organic esters such as ethyl oleate. Aqueous carriers include parenteral vehicles such as water, alcoholic / aqueous solutions, saline solutions, sodium chloride, Ringer's dextrose, etc. Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antimicrobial agents, antifungal agents, antioxidants, chelating agents and inert gases. The pH and exact concentration of the various components of the pharmaceutical composition are adjusted according to well-known parameters.

[0230] Further formulations are suitable for oral administration. Oral formulations include typical excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. The compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders.

[0231] In further embodiments, the pharmaceutical composition may include classical pharmaceutical preparations. Administration of the pharmaceutical composition according to certain embodiments may be by any common route, so long as the target tissue is available by that route. This may include oral, nasal, buccal, rectal, vaginal or topical. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions will usually be administered as a pharma- ceutically acceptable composition, including a physiologically acceptable carrier, buffer or other excipient. For the treatment of pulmonary conditions, aerosol delivery may be used. The volume of the aerosol may be, for example, about 0.01 ml to 0.5 ml.

[0232] The effective amount of pharmaceutical composition is determined based on the intended goal.The term "unit dose" or "dosage" refers to a physically separate unit suitable for use in a subject, each containing a predetermined amount of pharmaceutical composition calculated to bring about the desired response discussed above in connection with its administration, i.e., appropriate route and treatment regimen.The amount administered, both according to the number of treatments and unit dose, depends on protection or desired effect.

[0233] The precise amount of the pharmaceutical composition also depends on the judgment of the practitioner and is peculiar to each individual. Factors influencing the dosage include the physical and clinical condition of the patient, the route of administration, the intended goal of the treatment (e.g., relief of symptoms versus cure), and the efficacy, stability, and toxicity of the particular therapeutic agent.

[0234] V. Preparation and Culture of Cells In certain embodiments, the cells of the present disclosure can be specifically formulated and / or they can be cultured in a particular medium. The cells can be formulated in such a manner that they are suitable for delivery to a recipient without adverse effects.

[0235] In certain embodiments, the medium can be prepared using a medium used to culture animal cells as its base medium, such as AIM V, X-VIVO-15, NeuroBasal, EGM2, TeSR, BME, BGJb, CMRL 1066, Glasgow MEM, Improved MEM Zinc Option, IMDM, Medium 199, Eagle MEM, αMEM, DMEM, Ham, RPMI-1640, and Fischer medium, and any combination thereof, but the medium may not be particularly limited thereto as long as it can be used to culture animal cells. In particular, the medium may be xeno-free or chemically defined.

[0236] The medium may be serum-containing or serum-free, or xeno-free. In order to prevent contamination with heterologous animal-derived components, serum can be derived from the same animal as that of stem cells. Serum-free medium refers to a medium that does not contain unprocessed or unpurified serum, and therefore may include a medium that contains purified blood-derived components or animal tissue-derived components (such as growth factors).

[0237] The medium may or may not contain a serum replacement. The serum replacement may include materials that suitably contain albumin (such as lipid-rich albumin, bovine albumin, recombinant or humanized albumin, albumin substitutes such as vegetable starch, dextran and protein hydrolysates), transferrin (or other iron transport factors), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, or their equivalents. The serum replacement may be prepared, for example, by the method disclosed in International Publication No. 98 / 30679 (incorporated herein in its entirety). Alternatively, for further convenience, any commercially available material may be used. Commercially available materials include Knockout Serum Replacement (KSR), Chemically Defined Lipid Concentrate (Gibco), and Glutamax (Gibco).

[0238] In certain embodiments, the medium may include one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more of the following: vitamins, such as biotin; DL-alpha tocopherol acetate; DL-alpha tocopherol; vitamin A (acetate); proteins, such as BSA (bovine serum albumin) or human albumin, fatty acid-free fraction V; catalase; human recombinant insulin; human transferrin; superoxide dismutase; other components, such as corticosterone; D-galactose; ethanolamine HCl; glutathione (reduced); L-carnitine HCl; linoleic acid; linolenic acid; progesterone; putrescine 2HCl; sodium selenite; and / or T3 (triodo-I-thyronine). In certain embodiments, one or more of these may be explicitly excluded.

[0239] In some embodiments, the medium further comprises vitamins. In some embodiments, the medium comprises one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or thirteen of the following (and any range therefrom): biotin, DL-alpha tocopherol acetate, DL-alpha tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, nicotinamide folate, pyridoxine, riboflavin, thiamine, inositol, vitamin B12, or the medium comprises combinations thereof or salts thereof. In some embodiments, the medium comprises or consists essentially of biotin, DL-alpha tocopherol acetate, DL-alpha tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, nicotinamide folate, pyridoxine, riboflavin, thiamine, inositol, and vitamin B12. In some embodiments, the vitamin comprises or consists essentially of biotin, DL alpha tocopherol acetate, DL alpha tocopherol, vitamin A, or combinations thereof or salts thereof. In some embodiments, the medium further comprises a protein. In some embodiments, the protein comprises albumin or bovine serum albumin, a fraction of BSA, catalase, insulin, transferrin, superoxide dismutase, or combinations thereof. In some embodiments, the medium further comprises one or more of the following: corticosterone, D-galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triodo-I-thyronine, or combinations thereof. In some embodiments, the medium comprises one or more of the following: B-27® supplement, Xenofree B-27® supplement, GS21™ supplement, or combinations thereof. In some embodiments, the medium comprises or further comprises amino acids, monosaccharides, inorganic ions. In some embodiments, the amino acids include arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine, or a combination thereof.In some embodiments, the inorganic ions include sodium, potassium, calcium, magnesium, nitrogen, or phosphorus, or combinations or salts thereof. In some embodiments, the medium further includes one or more of the following: molybdenum, vanadium, iron, zinc, selenium, copper, or manganese, or combinations thereof. In certain embodiments, the medium comprises or consists essentially of one or more vitamins discussed herein and / or one or more proteins discussed herein, and / or one or more of the following: corticosterone, D-galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triiodo-I-thyronine, B-27® supplement, XenoFree B-27® supplement, GS21™ supplement, amino acids (arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine), monosaccharides, inorganic ions (such as sodium, potassium, calcium, magnesium, nitrogen, and / or phosphorus) or salts thereof, and / or molybdenum, vanadium, iron, zinc, selenium, copper, or manganese. In certain embodiments, one or more of these may be explicitly excluded.

[0240] The medium may also contain one or more exogenously added fatty acids or lipids, amino acids (such as non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, 2-mercaptoethanol, pyruvic acid, buffers, and / or inorganic salts, in certain embodiments one or more of which may be explicitly excluded.

[0241] One or more media components may be added at a concentration of at least, at most, or about 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 180, 200, 250 ng / L, ng / ml, μg / ml, mg / ml, or any range derivable therein.

[0242] In certain embodiments, the cells of the present disclosure are specifically formulated. They may or may not be formulated as a cell suspension. In certain cases, they are formulated in a single dose form. They may be formulated for systemic or local administration. In some cases, the cells are formulated for storage prior to use, and the cell formulation may include one or more cryopreservatives, such as DMSO (e.g., in 5% DMSO). The cell formulation may include albumin, including human albumin, and certain formulations include 2.5% human albumin. The cells can be specifically formulated for intravenous administration; for example, they are formulated for intravenous administration in less than 1 hour. In certain embodiments, the cells are in a formulated cell suspension that is stable at room temperature for 1, 2, 3, or 4 hours from the time of thawing.

[0243] In certain embodiments, the cells of the present disclosure include an exogenous TCR, which may be of a defined antigen specificity. In some embodiments, the TCR can be selected based on the absence or reduction of alloreactivity to the intended recipient. In the example where the exogenous TCR is non-alloreactive, during T cell differentiation, the exogenous TCR suppresses the rearrangement and / or expression of the endogenous TCR locus by a developmental process called allelic elimination, resulting in T cells that express only the non-alloreactive exogenous TCR, thus being non-alloreactive. In some embodiments, the selection of the exogenous TCR may not necessarily be defined based on the lack of alloreactivity. In some embodiments, the endogenous TCR genes are modified by gene editing such that they do not express proteins. Methods of gene editing, such as those using the CRISPR / Cas9 system, are known in the art and are described herein. In some embodiments, the cells of the present disclosure further comprise one or more chimeric antigen receptors (CARs). Examples of tumor cell antigens to which the CAR can be directed include, for example, at least 5T4, 8H9, αvβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CA9, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family, e.g., ErbB2 (HER2), EGFRvIII, EGP2, EGP40, ERBB3, ERBB4, ErbB3 / 4, EPCAM, EphA2, E pCAM, folate receptor-a, FAP, FBP, fetal AchR, FR, GD2, G250 / CAIX, GD3, glypican-3 (GPC3), Her2, IL-13Rα2, Lambda, Lewis-Y, Kappa, KDR, MAGE, MCSP, mesothelin, Muc1, Muc16, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSC1, PSCA, PSMA, ROR1, SP17, survivin, TAG72, TEM, carcinoembryonic antigen, HMW-MAA, AFP, CA-125, ETA, tyrosinase, MAGE, laminin receptor, HPV Examples of the CAR include E6, E7, BING-4, calcium-activated chloride channel 2, cyclin-B1, 9D7, EphA3, telomerase, SAP-1, BAGE family, CAGE family, GAGE ​​family, MAGE family, SAGE family, XAGE family, NY-ESO-1 / LAGE-1, PAME, SSX-2, Melan-A / MART-1, GP100 / pmel17, TRP-1 / -2, P. polypeptide, MC1R, prostate specific antigen, β-catenin, BRCA1 / 2, CML66, fibronectin, MART-2, TGF-βRII, or VEGF receptor (e.g., VEGFR2). The CAR may be a first, second, third, or higher generation CAR. The CAR may be bispecific for any two non-identical antigens, or it may be specific for more than two non-identical antigens.

[0244] VI. Administration of the Compositions The treatment methods described herein may include administration of a combination of therapeutic agents, such as a single agent therapy or a first cancer therapy (e.g., a cell therapy) and a second cancer therapy (e.g., a common pharmaceutical composition). Therapies can be administered in any suitable manner known in the art. For example, the first and second cancer treatments can be administered sequentially (at different times) or simultaneously (at the same time). In some embodiments, the first and second cancer treatments are administered in separate compositions. In some embodiments, the first and second cancer treatments are in the same composition.

[0245] In some embodiments, the pharmaceutical composition is administered to a subject. Different aspects may include administering an effective amount of the composition to a subject. In some embodiments, a cell therapy (e.g., immune cells comprising one or more CARs) is administered to a subject to protect against or treat a condition (e.g., cancer). Furthermore, such compositions can be administered in combination with additional therapeutic agents (e.g., chemotherapeutic agents, immunotherapeutic agents, biotherapeutic agents, etc.). Such compositions will generally be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

[0246] The phrase "pharmacologically acceptable" or "pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic or other undesirable reactions when administered to animals or humans. As used herein, "pharmacologically acceptable carriers" include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharma-ceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in immunogenic and therapeutic compositions is contemplated. Supplementary active ingredients, such as other anti-infective agents and vaccines, can also be incorporated into the composition.

[0247] Active compound can be formulated for parenteral administration, for example, by injection via intravenous, intramuscular, subcutaneous or intraperitoneal route.Typically, such composition can be prepared as liquid solution or suspension;Can also prepare solid form suitable for use in preparing solution or suspension when adding liquid before injection;Also, preparation can be emulsified.

[0248] Suitable pharmaceutical forms for injectable use include sterile aqueous solutions or dispersions; formulations, for example, containing aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.In all cases, the form must be sterile and must be fluid to the extent that it can be easily injected.It must also be stable under the conditions of manufacture and storage, and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0249] Proteinaceous compositions can be formulated in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of protein) formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxides, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc.

[0250] The pharmaceutical composition may contain a solvent or dispersion medium, for example, containing water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include an isotonic agent, for example, sugar or sodium chloride. Prolonged absorption of the injectable composition can be brought about by the use of an agent that delays absorption, for example, aluminum monostearate and gelatin, in the composition.

[0251] Sterile injectable solution is prepared by incorporating the active compound in the required amount in a suitable solvent together with various other components listed above as necessary, and then carrying out filtration sterilization or equivalent procedures.Generally, dispersion is prepared by incorporating various sterilized active components into a sterile vehicle that contains basic dispersion medium and other necessary components from those listed above.In the case of sterile powder for preparing sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying technology, which obtains powder of active component and any additional desired components from its previously sterile filtered solution.

[0252] Administration of the composition will typically be by any common route, including, but not limited to, oral or intravenous administration. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, or intranasal administration. Such compositions will usually be administered as pharma- ceutically acceptable compositions that include physiologically acceptable carriers, buffers, or other excipients.

[0253] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as will be therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the types of injectable solutions described above.

[0254] In some embodiments, the immune cells can be administered in conjunction with one or more other therapeutic agents for the treatment of an immune-mediated disorder. Combination therapy may include, but is not limited to, one or more antimicrobial agents (e.g., antibiotics, antiviral agents, and antifungal agents), anti-tumor agents (e.g., monoclonal antibodies such as rituximab, trastuzumab, fluorouracil, methotrexate, paclitaxel, fludarabine, etoposide, doxorubicin, or vincristine), immunodepleting agents (e.g., fludarabine, etoposide, doxorubicin, or vincristine), immunosuppressants (e.g., azathioprine, or glucocorticoids such as dexamethasone or prednisone), anti-inflammatory agents (e.g., glucocorticoids such as hydrocortisone, dexamethasone, or prednisone, or nonsteroidal anti-inflammatory agents such as acetylsalicylic acid, ibuprofen, or naproxen sodium), cytokines (e.g., interleukin-10 or transforming growth factor-beta), hormones (e.g., estrogen), or vaccines. In addition, immunosuppressants or tolerogenic agents can be administered, including, but not limited to, calcineurin inhibitors (e.g., cyclosporine and tacrolimus); mTOR inhibitors (e.g., rapamycin); mycophenolate mofetil, antibodies (e.g., recognizing CD3, CD4, CD40, CD154, CD45, IVIG, or B cells); chemotherapeutic agents (e.g., methotrexate, treosulfan, busulfan); radiation; or chemokines, interleukins or their inhibitors (e.g., BAFF, IL-2, anti-IL-2R, IL-4, JAK kinase inhibitors). Such additional pharmaceutical agents can be administered before, during, or after the administration of immune cells, depending on the desired effect. This administration of cells and agents can be by the same route or by different routes, and at the same site or at different sites.

[0255] In some embodiments, the first cancer therapy and the second cancer therapy are administered substantially simultaneously. In some embodiments, the first cancer therapy and the second cancer therapy are administered sequentially. In some embodiments, the first cancer therapy, the second cancer therapy, and the third therapy are administered sequentially. In some embodiments, the first cancer therapy is administered before the administration of the second cancer therapy. In some embodiments, the first cancer therapy is administered after the administration of the second cancer therapy.

[0256] The embodiments of the present disclosure relate to compositions and methods, including therapeutic compositions. Different therapies may be administered in one composition, or in more than one composition, such as two compositions, three compositions, or four compositions. Combinations of various drugs may be used.

[0257] The therapeutic agents of the present disclosure may be administered by the same or different routes of administration. In some embodiments, the cancer therapeutic agent may be administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. In some embodiments, the antibiotic is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. The appropriate dosage may be determined based on the type of disease being treated, the severity and course of the disease, the individual's clinical condition, the individual's clinical history and response to treatment, and the discretion of the attending physician.

[0258] The therapeutic agent may include various "unit doses". A unit dose is defined as containing a predetermined amount of the therapeutic composition. The amount to be administered, as well as the specific route and formulation, are within the judgement of those skilled in the art of clinical medicine. A unit dose need not be administered as a single injection, but may include continuous infusion over a period of time. In some embodiments, a unit dose includes a single administrable dose.

[0259] The dosage depends on the desired therapeutic effect, both in number of treatments and unit dose. Effective amount is understood to refer to the amount required to achieve a specific effect. In practice, it is contemplated that in certain embodiments, a dosage ranging from 10mg / kg to 200mg / kg can affect the protective capacity of these agents. Thus, dosages include about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200, 300, 400, 500, 1000 μg / kg, mg / kg, μg / day, or mg / day, or any range derivable therein. Furthermore, such dosages can be administered multiple times during the day and / or on multiple days, weeks, or months.

[0260] In certain embodiments, an effective amount of the pharmaceutical composition is one that can provide a blood level of about 1 μM to 150 μM. In other embodiments, an effective amount provides a blood level of about 4 μM to 100 μM; or about 1 μM to 100 μM; or about 1 μM to 50 μM; or about 1 μM to 40 μM; or about 1 μM to 30 μM; or about 1 μM to 20 μM; or about 1 μM to 10 μM; or about 10 μM to 150 μM; or about 10 μM to 100 μM; or about 10 μM to about 50 μM; or about 25 μM to about 150 μM; or about 25 μM to about 100 μM; or about 25 μM to about 50 μM; or about 50 μM to about 150 μM; or about 50 μM to about 100 μM (or any range derivable therein). In other embodiments, the dose is about, at least, or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, The blood concentration may be 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 μM, or any range derivable therein. In certain embodiments, the therapeutic agent administered to the subject is metabolized in the body to become a metabolized therapeutic agent, in which case the blood concentration may refer to the amount of the therapeutic agent. Alternatively, to the extent that the therapeutic agent is not metabolized by the subject, the blood concentration discussed herein may refer to the therapeutic agent that is not metabolized.

[0261] The exact amount of the therapeutic composition also depends on the judgment of the practitioner and is specific to each individual. Factors influencing the dosage include the physical and clinical condition of the patient, the route of administration, the intended goal of the treatment (relief of symptoms versus cure), and the efficacy, stability, and toxicity of the particular therapeutic agent or other therapy that the subject may be subjected to.

[0262] Those skilled in the art will understand and appreciate that dosage units of μg / kg body weight or mg / kg body weight can be converted and expressed in comparable concentration units of μg / ml or mM (blood levels), such as 4 μM to 100 μM. It is also understood that uptake is species and organ / tissue dependent. The applicable conversion factors and physiological assumptions made regarding uptake and concentration measurements are well known, and those skilled in the art can convert one concentration measurement to another to make reasonable comparisons and conclusions regarding doses, efficacy and results described herein.

[0263] In certain instances, it may be desirable to administer multiple doses of the composition, e.g., 2, 3, 4, 5, 6 or more doses, which may be 1, 2, 3, 4, 5, 6, 7, 8 to 5, 6, 7, 8, 9, 10, 11, or 12 week intervals (including all ranges therebetween).

[0264] The phrase "pharmacologically acceptable" or "pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic or other undesirable reactions when administered to animals or humans. As used herein, "pharmacologically acceptable carriers" include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharma-ceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in immunogenic and therapeutic compositions is contemplated. Supplementary active ingredients, such as other anti-infective agents and vaccines, can also be incorporated into the composition.

[0265] Active compound can be formulated for parenteral administration, for example, by injection via intravenous, intramuscular, subcutaneous or intraperitoneal route.Typically, such composition can be prepared as liquid solution or suspension;Can also prepare solid form suitable for use in preparing solution or suspension when adding liquid before injection;Also, preparation can be emulsified.

[0266] Suitable pharmaceutical forms for injectable use include sterile aqueous solutions or dispersions; formulations, for example, containing aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.In all cases, the form must be sterile and must be fluid to the extent that it can be easily injected.It must also be stable under the conditions of manufacture and storage, and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0267] Proteinaceous compositions can be formulated in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of protein) formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxides, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc.

[0268] The pharmaceutical compositions may contain solvents or dispersion media, including, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars and sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents that delay absorption, for example, aluminum monostearate and gelatin.

[0269] Sterile injectable solution is prepared by incorporating the required amount of active compound into a suitable solvent, with various other ingredients as listed above as necessary, and then sterilizing by filtration.Generally, dispersion is prepared by incorporating various sterilized active ingredients into a sterile vehicle that contains a basic dispersion medium and other ingredients as listed above.For the preparation of sterile powder for sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying technology, which allows the powder of active ingredient to be added to the solution that is previously sterile filtered and then desired additional ingredient can be obtained.

[0270] Administration of the composition will typically be by any common route, including, but not limited to, oral or intravenous administration. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, or intranasal administration. Such compositions will usually be administered as pharma- ceutically acceptable compositions that include physiologically acceptable carriers, buffers, or other excipients.

[0271] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as will be therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the types of injectable solutions described above.

[0272] A. Chemotherapy A wide variety of chemotherapeutic agents can be used in accordance with this embodiment. The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapeutic agent" is used to refer to a compound or composition administered in the treatment of cancer. These agents or drugs are classified according to their mode of activity within the cell, for example, whether and at what stage they affect the cell cycle. Alternatively, agents can be characterized based on their ability to directly crosslink DNA, to intercalate into DNA, or to induce chromosomal or mitotic abnormalities by affecting nucleic acid synthesis.

[0273] Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa and uredopa; ethylenimines and methylamines such as altretamine, triethylenemelamine, triethylenephosphoramide, triethyleneethylethiophosphoramide and trimethylolmelamine; acetogenins (particularly bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatin; kallistatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin and bizelesin); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycins (synthetic analogs , KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictine; spongiostatins; nitrogen mustards such as chlorambucil, chlornaphazine, colofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembitine, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimnustine; antibiotics such as the enediyne antibiotics (e.g. calicheamicin, especially calicheamicin gamma II and calicheamicin omega II); dynemicins such as dynemicin A; bisphosphonates such as clodronate; esperamicin;and neocarzinostatin chromophore and related chromoprotein enediine antibiotic chromophores, aclacinomycin, actinomycin, ausularisin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, deoxydoxorubicin). ), mitomycins such as epirubicin, esorubicin, idarubicin, marcelomycin, and mitomycin C, mycophenolic acid, nogalarnisin, olivomycin, peplomycin, potfilomycin, puromycin, keramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; metabolic inhibitors such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, pteropterin, and trimetrexate; fludarabine, 6-mercapto Purine analogues such as toprine, thiamiprine, and thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; antiadrenergic drugs such as mitotane and trilostane; folic acid supplements such as floric acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; enyluracil; amsacrine ;Bestravcil;Bisantrene;Edatraxate;Defofamine;Demecolcine;Diazicon;Elformitin;Elliptinium acetate;Epothilone;Etoglucide;Gallium nitrate;Hydroxyurea;Lentinan;Lonidynin;Maytansinoids such as maytansine and ansamitocin;Mitoguazone;Mitoxantrone;Mopidammol;Nitraerin;Pentostatin;Fenamet;Pirarubicin;Rosoxantrone;Podophyllic acid;2-Ethylhydrazide;Procarbazine;PSK polysaccharide complex;Razoxane;Rhizoxin;Schizofiran;Spirogermanium;Tenuazonic acid;Triazicon;2,2',2''-Trichlorotriethylamine;Tricothecenes (especially T-2 toxin, veraculin A, roridin A and anguidine);Urethane;Vindesine;Dacarbazine;Mannomustine;Mitobronitol;Mitolactol;Pipobroman;Gasitosine;Arabinoside ("Ara-C");Cyclophosphamide;Taxoids such as paclitaxel and docetaxel;Gemcitabine;6-Thioguanine;Mercaptopurine;Platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin;Vinblastine;Platinum;Etoposide (VP -16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitors RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabine, navelbine, farnesyl-protein transferase inhibitors, transplatinum, and pharmaceutically acceptable salts, acids, or derivatives of any of the above.;

[0274] B. Radiation therapy Other agents that cause DNA damage and have been used extensively include what are commonly known as gamma radiation, X-rays, and / or the directed delivery of radioisotopes to tumor cells. Other DNA damaging agents are also possible, such as microwaves, proton beam radiation, and ultraviolet radiation. All of these agents likely cause widespread damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. X-ray doses range from daily doses of 50-200 roentgens over prolonged periods (3-4 weeks) to single doses of 2000-6000 roentgens. Dose ranges for radioisotopes vary and depend on the half-life of the isotope, the strength and type of radiation, and uptake by tumor cells.

[0275] C. Immunotherapy Those skilled in the art will appreciate that additional immunotherapies may be used in conjunction or in combination with the methods and compositions of the present disclosure. In the context of cancer therapy, immunotherapeutics generally rely on the use of immune effector cells or molecules to target and destroy cancer cells. Rituximab (RITUXAN®) is an example. Immune effectors are, for example, antibodies specific for tumor cell surface markers. Antibodies may function alone as therapeutic effectors or may recruit other cells to actually affect cell killing. Antibodies may also be conjugated to drugs or toxins (chemotherapeutic agents, radionuclides, ricin A chain, cholera toxin, pertussis toxin, etc.) and function as targeting agents. Alternatively, effectors may be lymphocytes bearing surface molecules that interact directly or indirectly with tumor cell targets. Various effector cells include cytotoxic T cells, NKT cells, innate lymphocytes, and NK cells.

[0276] Antibody-drug conjugates (ADCs) contain monoclonal antibodies (MAbs) covalently linked to cytocidal drugs and can be used in combination therapy. This approach combines the high specificity of MAbs for antigen targets with potent cytotoxic agents, resulting in "armed" MAbs that deliver the payload (drug) to tumor cells where the antigen concentration is concentrated. Targeted delivery of the drug also minimizes drug exposure in normal tissues, reducing toxicity and improving the therapeutic index. Examples of ADC drugs are ADCETRIS® (brentuximab vedotin) and KADCYLA® (trastuzumab emtansine or T-DM1).

[0277] In one aspect of immunotherapy, the tumor cells must have some marker suitable for targeting, i.e., not present on the majority of other cells. Many tumor markers exist, any of which are suitable for targeting in the context of this embodiment. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, laminin receptor, erb B, p155, and the like. Another aspect of immunotherapy is to combine anti-cancer and immune stimulatory effects. There are also immune stimulatory molecules, including cytokines such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, chemokines such as MIP-1, MCP-1, IL-8, growth factors such as FLT3 ligand, and the like.

[0278] Examples of immunotherapies include immune adjuvants, such as Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds; cytokine therapy, such as interferon alpha, beta, and gamma, IL-1, GM-CSF, and TNF; gene therapy, such as TNF, IL-1, IL-2, and p53; and monoclonal antibodies, such as anti-CD20, anti-ganglioside GM2, and anti-p185. It is contemplated that one or more anti-cancer drug therapies may be employed in conjunction with the antibody therapies described herein.

[0279] In some embodiments, the immunotherapy is an immune checkpoint inhibitor. Immune checkpoints either raise or lower signals (e.g., costimulatory molecules). Inhibitory immune checkpoints that can be targeted by immune checkpoint inhibitors include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuating factor (BTLA), cytotoxic T lymphocyte-associated protein 4 (CTLA-4, also known as CD152), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin (KIR), lymphocyte activation gene-3 (LAG3), programmed death 1 (PD-1), T cell immunoglobulin domain and mucin domain 3 (TIM-3), and V domain Ig suppressor of T cell activation (VISTA). In particular, immune checkpoint inhibitors target the PD-1 axis and / or CTLA-4.

[0280] The immune checkpoint inhibitor may be a drug, such as a small molecule, a recombinant form of a ligand or receptor, or an antibody, such as a human antibody. A known inhibitor of immune checkpoint protein or its analog may be used, and in particular, an antibody in chimeric, humanized or human form may be used. As known by those skilled in the art, alternative names and / or equivalent names may be used for certain antibodies described in this disclosure. Such alternative names and / or equivalent names are interchangeable in the context of this disclosure. For example, it is known that lambrolizumab is also known under the alternative and equivalent names MK-3475 and pembrolizumab.

[0281] In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In certain aspects, the PD-1 ligand binding partner is PDL1 and / or PDL2. In another embodiment, the PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partner. In certain aspects, the PDL1 binding partner is PD-1 and / or B7-1. In another embodiment, the PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partner. In certain aspects, the PDL2 binding partner is PD-1. The antagonist may be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide.

[0282] In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011. In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some embodiments, the PD-1 binding antagonist is AMP-224. Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, are anti-PD-1 antibodies that may be used. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an exemplary anti-PD-1 antibody. CT-011, also known as hBAT or hBAT-1, is also an anti-PD-1 antibody. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor.

[0283] Another immune checkpoint that can be targeted in the methods provided herein is cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has Genbank accession number L15006. CTLA-4 is found on the surface of T cells and acts as an "off" switch when bound to CD80 or CD86 on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of helper T cells and transmits inhibitory signals to T cells. CTLA4 is similar to CD28, a T cell costimulatory protein, and both molecules bind to CD80 and CD86, also called B7-1 and B7-2, respectively, on antigen-presenting cells. CTLA4 transmits inhibitory signals to T cells, while CD28 transmits stimulatory signals. Intracellular CTLA4 is also found in regulatory T cells and may be important for their function. T cell activation via the T cell receptor and CD28 leads to increased expression of the B7 molecule inhibitory receptor, CTLA-4.

[0284] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide.

[0285] Anti-human CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present method can be generated using methods well known in the art. Alternatively, art-recognized anti-CTLA-4 antibodies can be used. Exemplary anti-CTLA-4 antibodies are ipilimumab (10D1, MDX-010, MDX-101, and Yervoy®) or antigen-binding fragments and variants thereof. In other embodiments, the antibody comprises the heavy and light chain CDRs or VRs of ipilimumab. Thus, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of ipilimumab, and the CDR1, CDR2, and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody competes for binding to and / or binds to the same epitope on CTLA-4 as the above antibodies. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with an antibody described above (eg, at least about 90%, 95%, or 99% variable region identity with ipilimumab).

[0286] D.Surgery Approximately 60% of cancer patients undergo some type of surgery, including preventive surgery, diagnostic or staging surgery, curative surgery, and palliative surgery. Curative surgery includes resection surgery, which physically removes, excises, and / or destroys all or part of the cancerous tissue, and may be combined with other therapies, such as the present treatment, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies. Tumor resection refers to the physical removal of at least a portion of the tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs' surgery).

[0287] When cancer cells, tissue, or tumors are partially or completely removed, a cavity may form in the body. Treatment can be by perfusion, direct injection, or local application of anticancer therapy to the site. Such treatments can be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, or 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments are also available in a variety of dosages.

[0288] E. Other drugs It is contemplated that other agents may be used in combination with certain aspects of the present embodiment to improve the efficacy of the treatment. These additional agents include agents that affect the upregulation of cell surface receptors and GAP junctions, cell activators and differentiation promoters, cell adhesion inhibitors, agents that increase the sensitivity of hyperproliferative cells to apoptosis inducers, or other biological agents. Increasing intercellular signaling by increasing the number of GAP junctions will increase the anti-hyperproliferative effect on adjacent hyperproliferative cell populations. In other embodiments, cell activators or differentiation promoters may be used in combination with certain aspects of the present embodiment to improve the anti-hyperproliferative effect of the treatment. Cell adhesion inhibitors are contemplated to improve the efficacy of the present embodiment. Examples of cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and lovastatin. It is further contemplated that other agents that increase the sensitivity of hyperproliferative cells to apoptosis, such as the antibody c225, may be used in combination with certain aspects of the present embodiment to improve the efficacy of the treatment.

[0289] VII. Products or Kits An article of manufacture or kit is provided that includes a composition or a method of using the composition provided herein. For example, a kit is also provided herein that includes one or more immune cells that include a CAR that includes a CD30-derived hinge and / or transmembrane domain. The article of manufacture or kit may further include a package insert that includes instructions for using the immune cells to treat or delay the progression of cancer in an individual, or to enhance the immune function of an individual with cancer. Any of the antigen-specific immune cells described herein can be included in the article of manufacture or kit. Suitable containers include, for example, bottles, vials, bags, and syringes. The containers can be formed from a variety of materials, such as glass, plastic (such as polyvinyl chloride or polyolefin), or metal alloys (stainless steel or Hastelloy). In some embodiments, the container holds the formulation and a label on or associated with the container that may indicate instructions for use. The article of manufacture or kit may further include other materials that are desirable from a commercial and user standpoint, such as other buffers, diluents, fillers, needles, syringes, and package inserts, along with instructions for use. In some embodiments, the article of manufacture further comprises one or more additional agents (e.g., chemotherapeutic agents, and anti-neoplastic agents). Suitable containers for the agent or agents include, for example, bottles, vials, bags, and syringes. EXAMPLES

[0290] The following examples are included to demonstrate preferred embodiments of the invention. Those skilled in the art should understand that the techniques disclosed in the examples that follow represent techniques that the inventors have discovered to work well in the practice of the invention, and therefore can be considered to constitute preferred modes for its practice. However, those skilled in the art should understand in light of this disclosure that many changes can be made in the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the invention.

[0291] Example 1 - Generation of vectors containing different combinations of hinge, transmembrane, and / or costimulatory domains DNA fragments containing different combinations of hinge, transmembrane, and / or costimulatory domains were synthesized as Gblocks and cloned between FMC63 scFv and CD3ζ (CD3 zeta) domains using the Gibson assembly method to construct different CARs (see FIG. 1). Plasmids expressing different CARs were transfected into 293T cells using Lipofectamine 3000 for screening of CARs with acceptable CD19 antigen binding capacity. FIG. 2 depicts a subset of exemplary lentiviral vectors containing CD19-targeted CARs that were used to transduce T cells (e.g., infinite γδ T cells) generated from healthy donor T cells. The vectors were designed according to the general scheme as shown in FIG. 2 and driven by various promoters such as MSCV, sEF1a = short (weak) EF1 alpha promoter, or tPGK = TetO-PGK promoter. We tested over 50 different combinations of different promoters, hinges, transmembrane domains, and costimulatory domains (subset of data shown, additional data available upon request).

[0292] Example 2 – Determination of vector expression in transduced cells The expression of anti-CD19 CARs with different hinge and transmembrane domains was determined using T cells. Briefly, T cells (e.g., indefinite γδ T cells) were generated from T cells of healthy donors by transducing lentiviral vectors expressing BCL6 and BCL2L1. After expanding the BCL6- and BCL2L1-transduced cells for 4–6 weeks, they were transduced with lentiviral vectors expressing anti-CD19 CARs with hinge (h), transmembrane (TM), and costimulatory (costim) domains constructed as the following: CD28 hTM-CD28 costim, PDL1hTM-CD28 costim, and CD30hTM-CD28 costim. The CAR expression cassettes were driven by weak (short EF1a promoter) or strong promoters (PDL1hTM CAR used the MSCV promoter, and CD30hTM CAR used the composite PGK promoter). Anti-CD19 CAR expression was determined by staining with FITC-conjugated CD19 antigen. All cells were sorted using FITC-conjugated CD19 antigen. Flow cytometry data was analyzed using FlowJo software. All constructs showed clear CAR-positive populations before and after sorting, but the CD30hTMCD28 costim CAR with the TRE-PGK promoter showed the highest CD19 CAR expression (see Figures 3A-3B). Expression was quantified and the median fluorescence intensity of the CAR-positive population derived from the transduced cells was determined (see Figures 3A-3B). For the two weak promoter-driven CARs, the MFI of the CD30hTM-CD28 costim CAR population was higher than the CD28hTM-CD28 costim CAR before and after sorting. For CARs driven by two strong promoters, the MFI of the CD30hTM-CD28 costim CAR population was higher than that of the PDL1hTM-CD28 costim CAR before and after sorting.

[0293] As shown in Figure 6, 293T cells were used to determine the expression of anti-CD79B CAR with CD30 hinge and transmembrane domain. 293T cells were transfected with lentiviral plasmids expressing CD30hTM-CD28 costim anti-CD79B CAR (one containing scFv derived from SN8 clone of CD79B antibody, the other containing scFv derived from 2F2 clone of CD79B antibody). CAR expression cassettes were driven by a composite human PGK promoter. 24 hours after transfection, anti-79B CAR expression was determined by staining with APC-conjugated CD79B antigen. Flow cytometry data was analyzed using FlowJo software. The results showed that CD30 hinge and transmembrane domain also worked well with scFv derived from non-CD19 targeting antibody.

[0294] As shown in Figure 7, 293T cells were used to determine the expression levels of anti-CD19 CARs with different hinge and transmembrane domains. 293T cells were transfected with lentiviral plasmids expressing anti-CD19 CARs with different hinge, transmembrane, and / or costimulatory domains. The CAR expression cassettes were all driven by MSCV promoter (see Figure 1 for vector map). Transfection efficiency was determined using AF647-conjugated anti-EGFR antibody, and anti-CD19 CAR expression was determined by staining transduced 293T cells with FITC-conjugated CD19 antigen. The data showed that hinge and transmembrane domains derived from some transmembrane receptors did not support optimal CAR expression. For example, CD79A hTM-CD28 costim, Long CTLA4 hTM-CD28 costim, and TIM3 hTM-CD28 costim all failed to show optimal CAR expression. Furthermore, the data suggested that the entire construct with the hinge and transmembrane domains and the costimulatory domain together was important for optimal CAR expression. This was based on the observation that combining the CD28 costimulatory domain with PD1 or PDL1 hTM resulted in good CAR expression on the cell surface, whereas the CD28 costimulatory domain combined with the hTM domains from CTLA-4, TIM3, or CD79A, respectively, failed to show optimal CAR expression on the cell surface.

[0295] As shown in Figures 9A-9B, the expression of anti-CD19 CARs with different hinge and transmembrane domains in αβ T cells (e.g., infinite αβ T cells) was determined. The results depicted in Figure 9A showed that αβ T cells (e.g., infinite αβ T cells) generated from healthy donor T cells were successfully transduced with lentiviral vectors expressing anti-CD19 CARs with different hinge and transmembrane domains and costimulatory domains. All CAR expression cassettes were driven by the MSCV promoter. Anti-CD19 CAR expression was determined by staining transduced T cells with FITC-conjugated CD19 antigen. The results showed that the CD30hTM-OX40 costim CAR showed a superior CAR-positive population, as did the CD28HTM-CD28 costim and CD8HTM-BAFF-R costim CARs. Furthermore, the results showed that CAR expression was low or absent for the other constructs. Anti-CD19 CAR expression on the transduced cells shown in Figure 9A was quantified as shown in Figure 9B. Quantification was determined by staining with FITC-conjugated CD19 antigen. The MFI of the CD30HTM-OX40 costim CAR population was the highest among all constructs tested in this experiment. These results together suggested that all constructs with hinge and transmembrane domains and costimulatory domains are important for optimal CAR folding and cell surface expression.

[0296] Example 3 – Determination of cytotoxicity and / or signaling levels of transduced cells The cytotoxicity of T cells containing anti-CD19 CAR with CD30hTM-CD28-CD3z against cancer cells was determined. T cells (e.g., infinite γδ T cells) generated from healthy donor T cells were transduced with a lentiviral vector expressing anti-CD19 CAR (tPGK promoter) based on CD30hTM-CD28 costim. The CAR expression cassette was driven by a strong composite PGK promoter. After expansion of the transduced T cells, the percentage of CAR positive was about 20% before sorting. These CAR T cells (e.g., CAR infinite γδ T cells) were co-cultured with Nalm6 cells with 200 IU / mL of exogenous IL-2 in the medium, and the percentage change of Nalm6 cells was monitored over 3 days. The results showed that the percentage of live Nalm6 cells rapidly decreased over 3 days (see Figure 4). The cytotoxicity of T cells containing anti-CD19 CARs with different hinge and transmembrane domains was also determined. T cells (e.g., infinite γδ T cells) generated from healthy donor T cells were transduced with lentiviral vectors expressing anti-CD19 CARs with hinge (h), transmembrane (TM), and costimulatory (costim) domains constructed as follows: CD8hTM-CD28 costim, CD28hTM-CD28 costim, PDL1hTM-CD28 costim, or CD30hTM-CD28 costim, respectively (see Figure 2 for vector map). After sorting, CAR-positive cells were co-cultured with Nalm6 cells expressing RFP-luciferase. The viable cell count of Nalm6 cells was calculated using CountBright™ absolute counting beads on days 0, 1, and 2. As shown in Figure 5A, CD28hTM-CD28 costim-based anti-CD19 CAR inhibited the proliferation of Nalm6 cells better than CD8hTM-CD28 costim-based anti-CD19 CAR when co-cultured without exogenous IL-2 in the medium, and the experiment was performed with 200 IU / mL IL-2 in the medium.As shown in Figure 5B, cells containing CD30hTM-CD28 costim-based anti-CD19 CARs had stronger cytotoxicity than those containing CD28hTM-CD28 costim-based anti-CD19 CARs, and as shown in Figure 5C, cells containing CD30hTM-CD28 costim-based anti-CD19 CARs had stronger cytotoxicity than those containing PDL1hTM-CD28 costim-based anti-CD19 CARs.

[0297] A direct comparison between the cytotoxic activity of anti-CD19 CARs with CD28 or CD30 hinge and transmembrane (HTM) domains was depicted. T cells (e.g., infinite γδ T cells) generated from healthy donor M4 and donor M5 were transduced with lentiviral vectors expressing anti-CD19 CARs with CD28HTM-CD28 costimulatory domains or CD30HTM-CD28 costimulatory domains, respectively, and then the cells were sorted for CAR+ cells and co-cultured with luciferase-RFP-expressing Nalm6 acute lymphoblastic leukemia tumor cells in duplicate wells at an effector:target ratio of 5:1. The viable cell count of Nalm6 cells was calculated using CountBright™ absolute counting beads on days 0 and 1, and a change in the absolute number of live Nalm6 cells was evident, as shown in Figure 11A. As shown in Figures 11B-11C, the percentage of live tumor cells differed as a function of the hinge and transmembrane domains used. Data were representative of one of two independent experiments. Results showed that cells containing a CD19 CAR with a CD30HTM domain had significantly more potent (P value calculated by unpaired T-test) cytotoxicity than cells containing a CD19 CAR with a CD28HTM domain.

[0298] In addition to expression and cytotoxicity, the signaling capacity of cells containing various anti-CD19 CARs with different hinge and transmembrane domains was determined. CAR plasmids that showed permissive expression in 293T cells (see FIG. 7) were used to produce lentiviral vectors, which were then used to transfect Jurkat-Lucia™ NFAT receptor cell lines (InvivoGen). The transfected cells were used to quantify CAR-induced signaling by measuring luciferase activity. After sorting the CAR-positive populations, each population was co-cultured with Raji lymphoma cells at an effector:target ratio of 1:1. After 24 hours, luciferase activity was measured in the supernatant (see FIG. 8). The results showed that all CARs could be specifically activated by Raji cells, but cells containing CD30hTM-CD28 CAR had the highest activity. Furthermore, the PD1hTM-CD28 CAR had greater tonic signaling than the other CARs based on the observed activity in the absence of Raji cells.

[0299] The signaling capacity of different CARs with CD30 hinge and transmembrane domain (HTM) and CD28-CD3z signaling domain was determined. Lentiviral vectors expressing different CAR constructs were transduced into Jurkat-Lucia™ NFAT reporter cell line, CAR+ cells were selected, and CAR-induced signaling was quantified by measuring luciferase activity with or without co-culture with Daudi lymphoma cells at an effector:target ratio of 1:1. Luciferase activity was measured in the supernatant after 24 hours. As depicted in Figures 10A, 10B, and 10D, both FMC63 scFv-CD30HTM-CD28costim (CD19-CD30HTM-CD28 CAR) and SN8 scFv-CD30HTM-CD28costim (CD79b-CD30HTM-CD28 CAR) signaled only in the presence of cells expressing CD19 and / or CD79b (e.g., Daudi tumor cells). Jurkat-Lucia™ NFAT reporter cells were also transduced with Fc receptor CAR (FcR CAR - CD16V-CD30HTM-CD28) (see Figure 10C). This data indicated that FcR CAR signaled only in the presence of both rituximab (anti-CD20 antibody) and Daudi tumor cells, but not when co-cultured with Daudi tumor cells alone. As shown in Figure 10E, Jurkat-Lucia™ NFAT reporter cells were also transduced with FMC63 scFv-CD30HTM-CD28costim (CD19-CD30HTM-CD28 CAR) and exposed to a high-grade B cell lymphoma cell line (PDX203 lymphoma cells), and the data showed that the CD19-CD30HTM-41BB CAR only signaled in the presence of tumor cells. Overall, these results indicated that the CD30HTM domain functions as an efficient HTM component with multiple CAR designs and / or different co-stimulatory domains targeting different antigens on tumor cells.

[0300] As shown in Figures 12A-12B, the in vivo tumor cell control capacity of anti-CD19 CD30HTM CAR-transduced infinite γδ T cells was determined. Luciferase-labeled Daudi Burkitt lymphoma tumor cells (2 × 10 4 8 × 10 tumor cells / mouse) were injected intravenously into three groups of human IL-15 transgenic NSG mice (secreting physiological levels of human IL-15) on day -2. Three fusions of infinity γδT or infinity anti-CD19CD30HTM-CD28Cos CAR-γδT were administered at 8 × 10 6 Mice were injected with CD30HTM-CD28Cos CAR-γδ T at a dose of 10 T cells / mouse / injection on days 0, 3, and 8. The data showed that CD30HTM-CD28Cos CAR-γδ T could slow lymphoma progression. The tumor burden was assessed by bioluminescence imaging (Figure 12A), and the mean total flux in each group was calculated and shown (Figure 12B). The data show that the antitumor effect of infinite γδ CAR T cells was higher than that of infinite γδ T cells without CAR.

[0301] As shown in Figures 13A-13B, CD70-binding CARs, including a truncated CD27 (tCD27) CAR with CD30 hinge and transmembrane domains, were successfully transduced, expressed, and induced to signal and bind to CD70. Figure 13A shows how a CD27-based anti-CD70 CAR was created by fusing a truncated CD27 extracellular domain (SEQ ID NO:52, which encodes SEQ ID NO:48) with the CD30 hinge and TM domains, the CD28 costimulatory domain, and the CD3z signaling domain. Signaling capacity was determined using the Jurkat-Lucia™ NFAT reporter cell line (Invivogen). A lentiviral vector expressing this CAR construct (SEQ ID NO: 49) was transduced into a Jurkat-Lucia™ NFAT reporter cell line, CAR+ cells were sorted, and CAR-induced signaling was quantified by measuring luciferase activity with or without co-culture with a CD70-positive T cell line at an effector:target ratio of 1:1. After 24 hours, luciferase activity was measured in the supernatant. As shown, the tCD27-CD30HTM-CD28cos-CD3z CAR only signaled in the presence of cells expressing CD70. The results indicated that the CD30HTM domain functions as an efficient HTM component with multiple CAR designs and / or different co-stimulatory domains targeting different antigens on tumor cells. RLU = relative light units. Figure 13B shows the expression of the above lentiviral vector (tCD27-CD30HTM-CD28cos-CD3z CAR) transduced into primary human T cells. Ten days after transduction, cell surface expression of CD27 and CD70 on CAR-T cells was measured. The ability of CAR to bind to CD70 was also tested by staining with fluorescent dye-conjugated recombinant CD70 protein. Non-transduced T cells and low affinity anti-CD70 scFv antibody (clone 1F6) were used as controls. The results of anti-CD27 antibody staining showed that tCD27 was properly folded and expressed on the surface of primary T cells (top row).CD70 protein staining showed that tCD27 CAR bound to recombinant CD70 protein (middle row). Anti-CD70 antibody staining showed that CD70+ cells were nearly absent in tCD27 CAR-transduced T cells, indicating that CD70-expressing cells were efficiently eliminated or CD70 on the cell surface was hidden by cis binding (bottom row).

[0302] Overall, these results demonstrated that the CD30 HTM domain functions as an efficient HTM component with multiple CAR designs targeting different antigens on tumor cells in vitro and in vivo.

[0303] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. Although the compositions and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that modifications can be made to the methods described herein and in the steps or sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are chemically and physiologically related can be substituted for the agents described herein while achieving the same or similar results. All such similar substitutions and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

[0304] The references cited herein, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. U.S. Patent No. 4,554,101 U.S. Patent No. 4,684,611 U.S. Patent No. 4,952,500 U.S. Patent No. 5,302,523 U.S. Patent No. 5,322,783 U.S. Patent No. 5,384,253 U.S. Patent No. 5,464,765 U.S. Patent No. 5,538,877 U.S. Patent No. 5,538,880 U.S. Patent No. 5,550,318 U.S. Patent No. 5,563,055 U.S. Patent No. 5,563,055 U.S. Patent No. 5,580,859 U.S. Patent No. 5,589,466 U.S. Patent No. 5,591,616 U.S. Patent No. 5,610,042 U.S. Patent No. 5,656,610 U.S. Patent No. 5,677,425 U.S. Patent No. 5,702,932 U.S. Patent No. 5,736,524 U.S. Patent No. 5,780,448 U.S. Patent No. 5,789,215 U.S. Patent No. 5,945,100 U.S. Patent No. 5,981,274 U.S. Patent No. 5,994,624 U.S. Patent No. 6,410,319 U.S. Patent No. 6,544,518 WO / 2021 / 034982 WO2021 / 034982 WO2021 / 222944 WO94 / 09699 WO95 / 06128 WO99 / 40188 Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY (1994) Burton, Molec. Immunol., 22: 161- 206 (1985) Chen and Okayama (1987) Fechheimer et al. (1987) Fraley et al. (1979) Frolet et al. (2010) Graham and Van Der Eb (1973) Harland and Weintraub (1985) Huck et al., Nucl. Acids Res (1986) Kaeppler et al. (1990) Kaneda et al. (1989) Kato et al. (1991) Kyte et al., J. Mol. Biol. 157:105-131 (1982) Nicolau and Sene (1982) Nicolau et al. (1987) Omirulleh et al. (1993) Potrykus et al. (1985) Rieder et al. (1999) Rippe et al. (1990) Romain Studer et al., Biochem. J. 449:581-594 (2013) Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, (2001) Snyder et al., PATRIC: The VBI PathoSystems (2007) Tan et al., Proc. Natl. Acad. Sci. USA87: 162 (1990) Wong et al. (1980) Zysk et al. (2000)

Claims

1. i) Antigen-binding domain; ii) CD30 hinged domain; iii) CD30 transmembrane domain; iv) at least one intracellular co-stimulatory domain; and v) Intracellular stimulatory domains A chimeric antigen receptor (CAR) containing, The CD30 hinge domain comprises fewer than 51 consecutive amino acids and at least 7 consecutive amino acids of the extracellular domain of CD30; The CD30 transmembrane domain is a chimeric antigen receptor (CAR) containing 27 or fewer consecutive amino acids of CD30.

2. The CAR according to claim 1, wherein the CD30 hinge and / or CD30 transmembrane domain does not contain cysteine.

3. The CAR according to claim 1, wherein the CD30 hinge domain is at least 80% identical to that of sequence number 3.

4. The CAR according to claim 1, wherein the CD30 transmembrane domain is at least 80% identical to that of SEQ ID NO:

4.

5. The CAR according to claim 1, wherein the CD30 hinge domain and / or transmembrane domain lacks three or more consecutive amino acids as described in SEQ ID NO:

6.

6. The CAR according to claim 1, wherein the CAR comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:

1.

7. A polynucleotide encoding a CAR as described in Claim 1, The CD30 hinge domain and transmembrane domain are encoded by a nucleic acid sequence that is at least 75% identical to sequence number 39. Polynucleotide.

8. The CAR according to claim 1, wherein the antigen-binding domain is targeted to CD19, CD79B, CD70, CD20, CD22, CD79A, ROR1, BCMA, BAFF receptor, GD2, and / or claudin 18.

2.

9. The CAR according to claim 1, wherein the at least one intracellular co-stimulatory domain includes the intracellular co-stimulatory domains of CD28, CD8, CD27, CD30, CD3ε, CD3ζ, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 (OX40), CD137 (4-1BB), or CD154.

10. The CAR according to claim 1, wherein the intracellular stimulating domain includes the intracellular stimulating domains of CD3ζ (CD3 zeta), DAP12, DAP10, FCER1G (Fc epsilon receptor I gamma chain), CD3δ (CD3 delta), CD3ε (CD3 epsilon), CD3γ (CD3 gamma), or CD79A.

11. The CAR according to claim 1, wherein the CD30 hinge domain, the CD30 transmembrane domain, at least one intracellular co-stimulatory domain, and the intracellular stimulatory domain are at least 85% identical to those of SEQ ID NO:

2.

12. A method for producing cells in vitro, comprising transfecting and / or transfecting cells with the CAR and / or nucleic acid encoding the CAR as described in Claim 1.

13. A cell comprising the CAR according to claim 1 and / or the polynucleotide according to claim 7.

14. The cell according to claim 13, which is a T cell, a γδT cell, or an NK cell.

15. The cell according to claim 13, comprising the BCL6 gene and at least one further transgene encoding an immunomodulatory gene.

16. The cell according to claim 15, wherein the at least one further transgene encoding an immunomodulatory gene is a Bcl2 family gene.

17. The cell according to claim 13, comprising at least one artificial mutation in an endogenous gene and / or at least one heterogeneous nucleic acid encoding an shRNA expression cassette, CRISPR, TALEN, and / or a zinc finger.

18. A composition for treating blood cancer in a patient, comprising the cells described in claim 17.

19. The composition according to claim 18, wherein the composition is used in combination with at least a second treatment, the at least second treatment comprising chemotherapy, immunotherapy, surgery, radiotherapy, drug therapy, targeted therapy, hormone therapy, biotherapy, or a combination thereof.

20. A composition comprising a population of cells, The cell population comprises a chimeric antigen receptor (CAR) and / or a nucleic acid encoding the CAR. The aforementioned CAR is, i) Antigen-binding domain; ii) CD30 hinged domain; iii) CD30 transmembrane domain; iv) at least one intracellular co-stimulatory domain; and v) Intracellular stimulatory domains Includes, The CD30 hinge domain comprises fewer than 51 consecutive amino acids and at least 7 consecutive amino acids of the extracellular domain of CD30. The aforementioned CD30 transmembrane domain contains 27 or fewer consecutive amino acids of CD30. composition.

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