Chimeric antigen receptors based on CD30 hinge and / or transmembrane domain

Abstract

Aspects of the present disclosure include improvements in chimeric antigen receptor (CAR) constructs, immune cells comprising CARs, and methods of making and using such constructs and / or cells in the treatment of diseases (e.g., cancer). Disclosed are immune cells comprising CD30-derived hinges and / or transmembrane CARs and methods of using such cells to treat malignancies (e.g., B-cell malignancies). Also disclosed are polynucleotides encoding a CAR comprising a CD30-derived hinge and / or a transmembrane domain, as well as cells comprising such polynucleotides and pharmaceutical compositions comprising such cells.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 335,560, filed on April 27, 2022, which is incorporated herein by reference in its entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing that has been submitted in ST26 format and is hereby incorporated by reference in its entirety. The ST26 copy created on April 25, 2023 is named MDAC_1327WO_Sequence_Listing.xml and is 53,662 bytes in size.

[0005] background I. Technical field

[0006] The present disclosure relates generally to at least the fields of cancer biology, immunology, and medicine.

[0007] II. Background

[0008] Chimeric antigen receptor (CAR) cell therapy is generally considered an effective solution for relapsed or refractory tumors, especially for hematological malignancies. Although commercial anti-CD19 CAR-T therapies have produced impressive results, setbacks such as high relapse rates and drug resistance are also common. There is a need to discover more effective engineered CAR components to improve the use of effector cell therapy and further improve patient outcomes.

[0009] Overview

[0010] Described herein are novel functional components of a chimeric antigen receptor (CAR), CARs comprising one or more of the functional components, engineered cells comprising one or more of the functional components, compositions comprising one or more of the functional components, and methods of using the same.

[0011] Embodiments of the present disclosure include nucleic acids, polynucleotides, polypeptides, proteins, peptides, constructs, vectors, cells, therapeutic cells, immune cells, engineered cells, methods for producing engineered cells, methods for detecting engineered cells, methods for separating engineered cells, methods for eliminating engineered cells, and methods for purifying engineered cells. The nucleic acids of the present disclosure can encode one or more polypeptides of the present disclosure, including one or more functional components of chimeric polypeptides. In some embodiments, the nucleic acid molecules of the present disclosure encode chimeric polypeptides. In some embodiments, the nucleic acid molecules of the present disclosure encode two or more chimeric polypeptides. The chimeric polypeptides of the present disclosure can include at least 1, 2, 3 or more of the following regions or domains: signal peptide, extracellular domain, hinge region, transmembrane domain, and intracellular region. The engineered cells of the present disclosure can include 1, 2, 3, 4 or more polynucleotides and / or polypeptides of the present disclosure. The methods of the present disclosure may include at least 1, 2, 3, 4 or more of the following steps: introducing a polynucleotide into cells, introducing a vector into cells, introducing a polypeptide into cells, expressing the polypeptide in cells, expanding a cell population, contacting cells with an antigen binding protein, contacting cells with an antibody drug conjugate, and detecting cells with an imaging agent.

[0012] In certain embodiments, the present disclosure provides functional components of a chimeric antigen receptor (CAR), wherein the functional components include a CD30 (also known as TNFRSF8, D1S166E, and Ki-1) 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 co-stimulatory domain; and v) an intracellular stimulatory domain. In certain embodiments, the CD30 hinge domain comprises less than 51 consecutive amino acids and at least 7 consecutive amino acids of the CD30 extracellular domain. In certain embodiments, the CD30 transmembrane domain comprises no more than 27 consecutive amino acids. In certain embodiments, the CD30 hinge domain comprises less than 51 consecutive amino acids and at least 7 consecutive amino acids of the CD30 extracellular domain, and the CD30 transmembrane domain comprises no more than 27 consecutive amino acids.

[0013] In certain embodiments, the CD30 hinge and / or transmembrane domain does not contain cysteine. In certain embodiments, the CD30 hinge domain 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 3 or more consecutive amino acids according to 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.

[0014] In some embodiments, the present disclosure provides a CAR comprising a CD30 hinge domain and / or a transmembrane domain, and targeting 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 at least one antigen binding domain of BAFF-R. In certain embodiments, the disclosure provides CARs comprising a CD30 hinge and / or a transmembrane domain, and targeting CD19, CD20, CD22, CD70, CD79B, CD79A, ROR1, BCMA, BAFF receptor, GD2 and / or claudin18.2 at least one antigen binding domain. In certain embodiments, the antigen binding domain targets CD19, CD79B and / or CD70.

[0015] In certain embodiments, the disclosure provides CAR, which comprises a CD30 hinge and / or a transmembrane domain and at least one intracellular co-stimulatory domain, wherein the intracellular co-stimulatory domain includes 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 intracellular co-stimulatory domain. In certain embodiments, at least one intracellular co-stimulatory domain includes CD28.

[0016] In certain embodiments, the present disclosure provides a CAR comprising a CD30 hinge and / or transmembrane domain and at least one intracellular stimulatory domain, the intracellular stimulatory domain comprising DAP12, DAP10, FCER1G (Fcε receptor Iγ chain), CD3δ (CD3delta), CD3ε (CD3 epsilon), CD3γ (CD3 gamma), CD3ζ (CD3 zeta) or CD79A intracellular stimulatory domain. In certain embodiments, at least one intracellular stimulatory domain comprises a CD3ζ (CD3zeta) intracellular stimulatory domain.

[0017] In certain embodiments, the present disclosure provides a CAR comprising a CD30 hinge and / or transmembrane domain, at least one intracellular co-stimulatory domain, and at least one intracellular stimulatory domain, wherein the polypeptide sequence comprising the same is at least 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:2.

[0018] In certain embodiments, the disclosure provides cells comprising CAR, the CAR comprising a CD30 hinge and / or a 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, T cells include CD4+T cells, CD8+T cells, iNKT cells, NKT cells, γδT cells, regulatory T cells, innate lymphoid cells, or a combination thereof. In certain embodiments, T cells are γδT cells. In certain embodiments, immune cells are immune cells as described in International Publication No. WO2021034982A1, which is incorporated herein by reference in its entirety for this purpose.

[0019] In some embodiments, the disclosure provides cells comprising CAR and at least one additional transgenic. In certain embodiments, at least one additional transgenic encodes an immunomodulatory gene. In some embodiments, the immunomodulatory gene is a survival-promoting 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.

[0020] 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 play 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 that can exert 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.

[0021] In certain embodiments, the cell comprises at least one safety switch. In some embodiments, the safety switch is a truncated EGFR (e.g., an EGFR lacking domains 1 and 2). In certain embodiments, the cell is an immune cell (e.g., a T cell, an innate lymphocyte, and / or a NK cell) that expresses IL-2, IL-15, other growth or differentiation factors, or a combination thereof.

[0022] In certain embodiments, the cells maintain the 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.

[0023] 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 lymphocytes, NK cells, or mixtures thereof).

[0024] In certain embodiments, vectors are provided herein. In certain embodiments, the vector is a lentiviral vector. In certain embodiments, the vector is introduced into cells and is included in the presence of IL-2 and / or other growth factors and transduced with a lentiviral vector. In certain embodiments, the concentration of IL-2 is 10IU / mL to 1000IU / mL, such as 10-50IU / mL, 50-75IU / mL, 75-100IU / mL, 100-250IU / mL, 250-500IU / mL, 500-750IU / mL or 750-1000IU / mL. In certain embodiments, the concentration of IL-2 is 100, 200, 300, 400 or 500IU / mL.

[0025] In certain embodiments, provided herein are methods for treating immune-related disorders, infectious diseases, and / or cancers, comprising treating with at least one cell disclosed herein. In some embodiments, the disease or disorder is an infectious disease, cancer, and / or immune-related disorder. In certain embodiments, immune-related diseases are autoimmune diseases, graft-versus-host disease, allogeneic transplant rejection, or other inflammatory conditions. In some embodiments, immune cells are allogeneic. In some embodiments, immune-related diseases are cancers. In some embodiments, cancers are solid cancers or hematologic malignancies. In some embodiments, cancers are hematologic malignancies.

[0026] In some embodiments, the method of treatment comprises treatment with at least one cell disclosed herein and further comprises treatment with at least one additional therapeutic agent. In some embodiments, at least one additional therapeutic agent comprises chemotherapy, immunotherapy, surgery, radiotherapy, drug therapy, hormone therapy, biological therapy, or a combination thereof.

[0027] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the measurement or quantitative method.

[0028] When used in conjunction with the term "comprising", the use of the term "a" or "an" may mean "one", but it is also consistent with the meaning of "one or more", "at least one" and "one or more than one".

[0029] The phrase "and / or" means "and" or "or". For example, A, B, and / or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C. In other words, "and / or" serves as an inclusive or.

[0030] The words "comprise" (and any form of including, such as "comprises" and "comprises"), "have" (and any form of having, such as "have" and "has"), "include" (and any form of including, such as "includes" and "include"), or "contain" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended, and do not exclude additional, unrecited elements or method steps.

[0031] The compositions and methods of use thereof may "comprise," "consist essentially of," or "consist of" any ingredient or step disclosed in the specification. Compositions and methods consisting "essentially of" any disclosed ingredient or step limit the scope of the claims to specific materials or steps that do not materially affect the basic and novel characteristics of the claimed invention.

[0032] 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. In addition, the compositions of the invention can be used to implement the methods of the invention.

[0033] Other objects, features and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, although indicating specific embodiments of the present invention, are given by way of illustration only, because various changes and modifications within the spirit and scope of the present invention will be apparent to those skilled in the art based on the detailed description.

[0034] BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The following drawings constitute part of this specification and are included to further illustrate certain aspects of the present invention. The present 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.

[0036] Figure 1 Depicted are exemplary lentiviral vector diagrams for selecting different hinges, different transmembrane domains, and / or different costimulatory domain combinations. Different hinge, transmembrane, and / or costimulatory domain combinations were cloned between the FMC63 scFV and CD3ζ (CD3 zeta) domains to construct different CARs. Plasmids expressing different CARs were transfected into 293T cells using lipofectamine 3000 to screen for CARs with acceptable CD19 antigen binding ability. LTR = long terminal repeat; HTM = hinge and transmembrane domain; costim = costimulatory domain.

[0037] Figure 2 An exemplary lentiviral vector map of a CD19-targeted CAR for transducing T cells (e.g., infinite γδ T cells) generated from healthy donor T cells is depicted. sEF1a = short (weak) EF1α promoter; tPGK = TetO-PGK promoter; HTM = hinge and transmembrane domain; costim = co-stimulatory domain; tCD34 = CD34 with truncated cytoplasmic tail.

[0038] Figure 3A-3BThe expression of anti-CD19 CAR with different hinge and transmembrane domains in T cells (e.g., infinite γδT cells) is depicted. 3A) T cells (e.g., infinite γδT cells) produced by healthy donor T cells are transduced with lentiviral vectors expressing anti-CD19 CAR with CD28hTM-CD28 costim, PDL1hTM-CD28 costim, and CD30hTM-CD28 costim. The CAR expression cassette is driven by a weak (short EF1a promoter) or a strong promoter (PDL1 hTM CAR uses an MSCV promoter, and CD30 hTM CAR uses a composite PGK promoter). The expression of anti-CD19 CAR is determined by staining with FITC-coupled CD19 antigen. All cells are sorted using FITC-coupled CD19 antigen. Flow cytometry data are 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. 3B) Median fluorescence intensity (MFI) of the CAR-positive population of transduced cells (quantification of 3A). For the two weak promoter-driven CARs, the MFI of the CD30 hTM-CD28 costim CAR population was higher than that of the CD28hTM-CD28 costim CAR before and after sorting. For the two strong promoter-driven CARs, the MFI of the CD30 hTM-CD28 costim CAR population was higher than that of the PDL1hTM-CD28 costim CAR before and after sorting. Data represent one of three independent experiments. HTM = hinge and transmembrane domain; costim = co-stimulatory domain.

[0039] Figure 4 Depicted is the cytotoxic activity of T cells containing anti-CD19 CAR with CD30 hTM-CD28-CD3z against cancer cells. T cells (e.g., infinite γδT cells) generated by healthy donor T cells were transduced with a lentiviral vector expressing an anti-CD19 CAR (tPGK promoter) based on CD30hTM-CD28 costim. The CAR expression cassette is driven by a strong composite PGK promoter. After the transduced T cells were amplified, the CAR positive percentage before sorting was about 20%. These CART cells (e.g., CAR infinite γδT cells) were co-cultured with Nalm6 tumor cells in the presence of 200IU exogenous IL-2 in the culture medium, and the percentage changes of Nalm6 cells were monitored within 3 days. The results showed that the percentage of live Nalm6 tumor cells decreased rapidly within 3 days. HTM = hinge and transmembrane domain; costim = co-stimulatory domain. tPGK = TetO-PGK promoter.

[0040] Figures 5A-5C Comparison of cytotoxic activity of anti-CD19 CARs with different hinge and transmembrane domains is depicted. 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, CD30hTM-CD28 costim (vector diagram see Figure 2 ). After sorting, CAR-positive cells were co-cultured with Nalm6 tumor cells expressing RFP-luciferase in duplicate wells. TM Absolute counting beads were used to calculate the absolute number of live Nalm6 cells on days 0, 1, and 2. 5A) shows that under co-culture conditions without exogenous IL-2 in the culture medium, the anti-CD19 CAR based on CD28hTM-CD28 costim inhibited the proliferation of Nalm6 cells better than the anti-CD19 CAR based on CD8hTM-CD28 costim. For co-culture experiments containing 200 IU / mL IL-2 in the culture medium, 5B) shows that the anti-CD19 CAR based on CD30hTM-CD28 costim has stronger cytotoxicity than the anti-CD19 CAR based on CD28hTM-CD28 costim. 5C) shows that the anti-CD19 CAR based on CD30hTM-CD28 costim has stronger cytotoxicity than the anti-CD19 CAR based on PDL1hTM-CD28 costim. The data represent one of two independent experiments. Statistical significance between experimental conditions was determined by paired t-test (P<0.05). HTM = hinge and transmembrane domain; costim = co-stimulatory domain.

[0041] Figure 6 The expression of anti-CD79B CAR with CD30 hinge and transmembrane domain is depicted. 293T cells were transfected with lentiviral plasmids expressing CD30hTM-CD28 costim anti-CD79B CAR-one containing scFv from SN8 clone of CD79B antibody and the other containing scFv from 2F2 clone of CD79B antibody. Both CAR expression cassettes are driven by a composite human PGK promoter. 24 hours after transfection, the expression of anti-79B CAR was determined by staining with APC-coupled CD79B antigen. Flow cytometry data were analyzed using FlowJo software. The results show that CD30 hinge and transmembrane domains also work well with scFv from other antibodies (e.g., not just those targeting CD19 using FMC63 scFv antibodies). HTM = hinge and transmembrane domain; costim = co-stimulatory domain.

[0042] Figure 7 The expression of anti-CD19 CARs with different hinge and transmembrane domains in 293T cells is depicted. 293T cells were transfected with lentiviral plasmids expressing anti-CD19 CARs containing different hinge domains, transmembrane domains, and / or co-stimulatory domains. The CAR expression cassettes were all driven by the MSCV promoter (see vector diagram for details). Figure 1 ). Transfection efficiency was determined by AF647-coupled anti-EGFR antibody, and anti-CD19 CAR expression was determined by staining transduced 293T cells with FITC-coupled CD19 antigen. The data show that the hinge and transmembrane domains of some transmembrane receptors do not support optimal CAR expression (e.g., CD79A hTM-CD28costim, Long CTLA4 hTM-CD28 costim, TIM3 hTM-CD28 costim). In addition, the data show that the entire construct with hinge and transmembrane domains and costimulatory domains is essential for optimal CAR expression. This is based on the observation that combining the CD28 costimulatory domain with PD1 or PDL1 hTM produces good CAR expression on the cell surface, but does not produce good CAR expression in combination with CTLA4-, TIM3-, and CD79A-derived hTM domains. HTM = hinge and transmembrane domain; costim = costimulatory domain.

[0043] Figure 8 The signaling capabilities of anti-CD19 CARs with different hinge and transmembrane domains are depicted. CAR plasmids that showed acceptable expression in 293T cells were used to generate lentiviral vectors and transduced into Jurkat-Lucia TM In the NFAT reporter cell line (InvivoGen), the cell line was 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 although all CARs can be specifically activated by Raji cells, the CD30hTM-CD28 CAR induced the highest activity. In addition, based on the activity observed in the absence of Raji cells, the PD1hTM-CD28 CAR has higher tonic signaling than other CARs. The data represent one of two independent experiments. hTM = hinge and transmembrane domain; costi = co-stimulatory domain.

[0044] Figures 9A-9BDepicts the expression of anti-CD19 CARs with different hinges and transmembrane domains in infinite αβT cells. 9A) αβT cells generated by healthy donor T cells were transduced with lentiviral vectors expressing anti-CD19 CARs containing different hinges and transmembrane domains and co-stimulatory domains. All CAR expression cassettes are driven by the MSCV promoter. Anti-CD19 CAR expression was determined by staining transduced T cells with FITC-coupled CD19 antigen. CD30 hTM-OX40 costim CAR showed an excellent CAR-positive population, similar to CD28 HTM-CD28 costim and CD8 HTM-BAFF-R costim CAR. CAR expression of other constructs was low or absent. 9B) Anti-CD19 CAR expression on transduced cells in 9A) was determined by staining with FITC-coupled CD19 antigen. The MFI of the CD30 HTM-OX40costim CAR+ population was the highest of all constructs tested in this experiment. Taken together, these results indicate that the entire construct with the hinge and transmembrane domains as well as the co-stimulatory domain is important for optimal CAR folding and surface expression. HTM = hinge and transmembrane domain; costim = co-stimulatory domain.

[0045] Figures 10A-10E The signaling capabilities of different CARs with CD30 hinge and transmembrane domains (HTM) and CD28-CD3z signaling domains are depicted. Lentiviral vectors expressing different CAR constructs were transduced into Jurkat-Lucia TM NFAT reporter cell line, CAR+ cells were sorted, and CAR-induced signaling was quantified by measuring luciferase activity after 24 hours of co-culture with Daudi lymphoma cells (effector:target ratio of 1:1). 10A, 10B, and 10D) show that both FMC63scFv-CD30HTM-CD28costim (CD19-CD30HTM-CD28 CAR) and SN8 scFv-CD30HTM-CD28 costim (CD79b-CD30HTM-CD28 CAR) only signal in the presence of cells expressing CD19 and / or CD79b (e.g., Daudi tumor cells). 10C) Jurkat-Lucia TMNFAT reporter cells were also transduced with an Fc receptor CAR (FcR CAR-CD16V-CD30HTM-CD28). The data showed that the FcR CAR only signaled in the presence of rituximab (an anti-CD20 antibody) and Daudi tumor cells, but not when co-cultured with Daudi tumor cells alone. 10E, scFv-CD30HTM-41BB CAR (CD19-CD30HTM-41BB CAR) was transduced into Jurkat-Lucia TM In the NFAT reporter cell line, CAR+ cells were sorted and CAR-induced signaling was quantified by measuring luciferase activity in the presence or absence of co-culture with PDX203 lymphoma cells, a high-grade B-cell lymphoma cell line developed by the inventor's laboratory from patient-derived xenografts. 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. The data represent one of two or three independent experiments. Overall, these results indicate that the CD30 HTM domain can be used as an effective HTM component in the context of a variety of CAR designs and / or different costimulatory domains targeting different antigens on tumor cells.

[0046] Figures 11A-11C A comparison of the cytotoxic activity of anti-CD19 CARs with CD28 or CD30 hinge and transmembrane (HTM) domains is depicted. T cells generated from healthy donors M4 and M5 (e.g., infinite γδ T cells) were transduced with lentiviral vectors expressing anti-CD19 CARs with CD28 HTM-CD28 co-stimulatory domains or CD30 HTM-CD28 co-stimulatory domains, respectively, and then the cells were sorted as CAR+ cells and co-cultured with Nalm6 acute lymphoblastic leukemia tumor cells expressing luciferase-RFP at an effector:target ratio of 5:1 in duplicate wells. CountBright TM The absolute counting beads were used to calculate the absolute number of live Nalm6 tumor cells on day 0 and day 1, 11A) depicts the change in the absolute number of live Nalm6 cells, and 11B) and 11C) depict the change in the percentage of live tumor cells. The results showed that the CD19 CAR with the CD30 HTM domain had significantly stronger cytotoxicity than the CD19 CAR with the CD28 HTM domain. The P value was calculated by unpaired t-test.

[0047] Figures 12A-12BThe in vivo antitumor effect of scFv CD30HTM CAR-transduced infinite γδCAR T cells was shown. Luciferase-labeled Daudi Burkitt lymphoma tumor cells (2×10 4 8×10 tumor cells / mouse) were injected intravenously into 3 groups of human IL-15 transgenic NSG mice (secreting physiological levels of human IL-15). 6 Infinite γδT or infinite anti-CD19 CD30HTM-CD28Cos CAR-γδT were injected into mice three times at a dose of 1 T cell / mouse / injection. The results showed that CD30HTM-CD28Cos CAR-γδT cells slowed the progression of lymphoma. Tumor burden was assessed by bioluminescence imaging12A), and the average total flux of each group was quantified12B). The data showed that the anti-tumor effect of infinite γδCAR-T cells was higher than that of infinite γδT cells without CD30HTM-CAR.

[0048] Figures 13A-13B The transduction, signaling, and CD70 binding abilities of a truncated CD27 (tCD27) CAR with a CD30 hinge and transmembrane domain are shown. 13A), CD27-based anti-CD70 CAR was made by fusing a truncated CD27 extracellular domain (SEQ ID NO: 48) with the CD30 hinge and TM domains, the CD28 co-stimulatory domain, and the CD3z signaling domain. Jurkat-Lucia TM The NFAT reporter cell line (Invivogen) was used to determine the signal transduction capacity. The lentiviral vector expressing this CAR construct was transduced into Jurkat-Lucia TMIn the NFAT reporter cell line, CAR+ cells were sorted and CAR-induced signaling was quantified by measuring luciferase activity in the presence or absence of co-culture with a CD70-positive T cell line at a 1:1 effector: target ratio. After 24 hours, the luciferase activity in the supernatant was measured. As shown in the figure, tCD27-CD30HTM-CD28cos-CD3z CAR only emits signals in the presence of cells expressing CD70. The results show that the CD30 HTM domain can be used as an effective HTM component in the case of a variety of CAR designs and / or different costimulatory domains targeting different antigens on tumor cells. RLU = relative light unit. 13B) shows the expression of the above-mentioned lentiviral vector (tCD27-CD30HTM-CD28cos-CD3z CAR) transduced into primary human T cells. Ten days after transduction, CD27 and CD70 cell surface expression on CAR-T cells was measured. The ability of CAR to bind to CD70 was also tested by staining with recombinant CD70 protein coupled with a fluorescent dye. Untransduced T cells and 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 (upper row). CD70 protein staining results showed that tCD27 CAR bound to recombinant CD70 protein (middle row). Anti-CD70 antibody staining showed that CD70+ cells were almost absent in tCD27 CAR-transduced T cells, indicating that cells expressing CD70 were effectively eliminated or that CD70 on the cell surface was masked by cis binding (lower row).

[0049] Detailed Description

[0050] Herein, the inventors describe a number of studies involving testing the scoring of hinges and / or transmembrane domains to determine whether they are suitable for CAR construction. Surprisingly, the inventors show that relatively few hinges and / or transmembrane domains can be used to construct functional and / or effective CAR molecules. In the component examples tested, hinges and / or transmembrane domains derived from CD30 are effective. Although not limited by theory, one reason for observing this effectiveness may be the potential primary amino acid sequence of the CD30 hinge and / or transmembrane domain. In some embodiments, CD30-derived hinges and / or transmembrane domains have more amino acids (e.g., G or S) that provide flexibility than amino acids derived from CD28 or CD8a (the most commonly used hinge and / or transmembrane domains in CAR). In addition, in some embodiments, the hinge and / or transmembrane domain derived from CD30 does not contain cysteine ​​(C) in the hinge region or the transmembrane region. In contrast, the CD28 hinge and transmembrane domain have 2 cysteines, while the CD8a hinge and transmembrane domain have 3 cysteines. It is known that proteins or peptides containing cysteine ​​tend to form homodimers between them or heterodimers with other transmembrane proteins, which may lead to unpredictable consequences for the folding of the antigen binding domain (e.g., scFv) of CAR expression and CAR molecules. Therefore, the unique sequence of the hinge and / or transmembrane domain derived from CD30 may allow for better folding of the antigen binding domain (e.g., scFv) of the CAR molecule, which explains the observed increase in cell surface expression of the CAR comprising the CD30 hinge and / or transmembrane domain described herein. Consistent with this, the results described herein show that the anti-CD19 CAR comprising CD30h / TM-CD28-CD3z has strong surface expression in T cells, has strong signal transduction ability (e.g., the highest signal transduction ability of the tested vector) in the Jurkat reporter cell line, and exhibits strong cytotoxicity to B cell leukemia.

[0051] The discovery of new effective hinge and / or transmembrane domains provides the field with an additional tool that can be utilized, in certain embodiments, to construct dual, triple, quadruple, etc. CARs against two or more different antigens, since it is well known that identical DNA sequences in lentiviral vectors can adversely affect the stability of the underlying constructs. Therefore, the significantly different DNA sequences encoding the CD30 hinge and / or transmembrane domains compared to the established CD8a or CD28 hinge and transmembrane domains facilitate the generation of stable lentiviral vectors to generate bispecific, trispecific, etc. CARs to simultaneously target multiple antigens.

[0052] 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 hinges and / or transmembrane (TM) domains that can be used to construct chimeric antigen receptors (CARs). In certain embodiments, hinges and / or TM domains are partially derived from the extracellular domain and complete transmembrane domain of human CD30 molecules. In certain embodiments, when connected to a costimulatory domain (e.g., but not limited to OX40 or CD28), CD30 hinges and / or TM domains can support strong CAR expression and effector cell cytotoxicity against tumor cells.

[0053] In some embodiments, a CAR construct comprising CD30 h / TM-CD28-CD3z can be used in Jurkat-Lucia cells compared to a similar CAR construct comprising CD28 h / TM-CD28-CD3z. TM Inducing stronger CD3z downstream signaling in reporter cell lines (as an example). In some embodiments, immune cells containing a CAR construct containing CD30 h / TM-CD28-CD3z have better cytotoxicity to cancer cells than immune cells containing a CAR construct containing CD28 h / TM-CD28-CD3z.

[0054] I. Peptides

[0055] As used herein, "protein" or "polypeptide" refers to a molecule comprising at least five amino acid residues. As used herein, the term "wild type" refers to the endogenous version of a molecule that occurs naturally in an organism. In some embodiments, a wild type version of a protein or polypeptide is employed, however, in many embodiments of the present disclosure, a modified protein or polypeptide is employed 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, in particular, its amino acid sequence, is altered relative 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 particularly contemplated that a modified / variant protein or polypeptide may be altered in one activity or function but retain a wild type activity or function in other aspects (e.g., immunogenicity).

[0056] When a protein is specifically mentioned herein, it generally refers to a natural (wild-type) or recombinant (modified) protein, or optionally, to a protein in which any signal sequence has been removed. The protein can be isolated directly from its native organism, produced by recombinant DNA / exogenous expression methods, or produced by solid phase peptide synthesis (SPPS) or other in vitro methods. In specific embodiments, there are isolated nucleic acid fragments and recombinant vectors that combine nucleic acid sequences encoding polypeptides (e.g., antibodies or fragments thereof). The term "recombinant" can be used together with the name of a polypeptide or a specific polypeptide, and generally refers to a polypeptide produced by a nucleic acid molecule manipulated in vitro or a replication product of such a molecule.

[0057] In certain embodiments, the size of a protein or polypeptide (wild type or modified) can include, but is not limited to, 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 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、65 0, 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 therein, or derivatives of the corresponding amino acid sequences described or referenced herein. It is contemplated that a polypeptide may be mutated by truncation to make it shorter than its corresponding wild-type form, or may be altered by fusion or conjugation to a heterologous protein or polypeptide sequence having a specific function (e.g., for targeting or localization, for enhancing immunogenicity, for purification purposes, etc.). As used herein, the term "domain" refers to any distinct functional or structural unit of a protein or polypeptide, and generally refers to an amino acid sequence having a structure or function recognizable by those skilled in the art.

[0058] 45, 46, 47, 48, 49, or 50 (or any range derivable therein) or more variant amino acid or nucleic acid substitutions,or at least 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, 142, 143, 144 4, 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, 400, 500, 550, 1000 or more consecutive amino acids or nucleic acids or any range derivable therein of at least 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 any range derivable therein) are similar, identical or homologous. ,

[0059] In some embodiments, the protein or polypeptide may comprise SEQ ID NO: 1-38, 43, 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 7, 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、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 5. 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 85、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).

[0060] In some embodiments, the protein, polypeptide or nucleic acid may comprise SEQ ID NO: 1-38, 43, 45 and 47-50 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 8, 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 4, 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, 2 05, 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.

[0061] In some embodiments, the polypeptide, protein or nucleic acid may comprise 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, or 100% (or any range derivable therein) similar, identical or homologous to one of SEQ ID NOs: 1-52. 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, 1 59, 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、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.

[0062] In some aspects, there is a NO: 1-52 any one of position 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, 1 16、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 8, 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, 2 39, 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, 28, 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, 88 6. 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 and includes SEQ At least, at most, or exactly 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、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、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 3. 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.

[0063] Nucleotide and protein, polypeptide and peptide sequences of various genes have been previously disclosed and can be found in recognized computer databases. Two commonly used databases are the Genbank and GenPept databases of the National Center for Biotechnology Information (ncbi.nlm.nih.gov / on the World Wide Web) and the Universal Protein Resource (UniProt; uniprot.org on the World Wide Web). The coding regions of these genes can be amplified and / or expressed using the techniques disclosed herein or techniques known to those of ordinary skill in the art.

[0064] It is contemplated that in certain embodiments of the compositions of the present disclosure, about 0.001 mg to about 10 mg of total polypeptide, peptide and / or protein are present per milliliter. The protein concentration in the composition can 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).

[0065] A.Sequence

[0066] The amino acid sequences of certain polypeptides (including chimeric antigen receptors and portions, regions and / or domains thereof) are listed in Table 1.

[0067] Table 1 - Amino Acid Sequence

[0068]

[0069]

[0070]

[0071]

[0072] B. Variant Polypeptides

[0073] The following is a discussion about changing the amino acid subunits of a protein to produce an equivalent or even improved second generation variant polypeptide or peptide. For example, certain amino acids can replace other amino acids in a protein or peptide sequence with or without a significant loss of interaction binding ability with a structure (e.g., an antigen binding region of an antibody or a binding site on a substrate molecule). Since the interaction ability and properties of a protein determine the functional activity of the protein, certain amino acid substitutions can be made in a protein sequence and its corresponding DNA coding sequence, and still produce a protein with similar or desired properties. Therefore, the inventors have considered that various changes can be made in the DNA sequence of a gene encoding a protein without significantly losing its biological utility or activity.

[0074] The term "functionally equivalent codons" is used herein to refer to codons encoding the same amino acid, such as six different codons for arginine. Also contemplated are "neutral substitutions" or "neutral mutations," which refer to changes in one or more codons encoding biologically equivalent amino acids.

[0075] The amino acid sequence variants of the present disclosure can be substitution, insertion or deletion variants. Compared with the wild type, the variation of the polypeptide of the present disclosure can 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 discontinuous or continuous amino acids of the protein or polypeptide. The variant can include at least 50%, 60%, 70%, 80% or 90% (including all values ​​and ranges therebetween) of any sequence provided or cited herein. The variant may include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more substituted amino acids.

[0076] It is also understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids, or 5' or 3' sequences, but still be substantially identical to one of the sequences disclosed herein, as long as the sequence meets the above criteria, including maintaining biological protein activity when it comes to protein expression. Adding terminal sequences is particularly applicable to nucleic acid sequences, for example, the nucleic acid sequence may include various non-coding sequences flanking the 5' or 3' portion of the coding region.

[0077] Deletion variants generally lack one or more residues of a native or wild-type protein. Single residues may be deleted, or multiple consecutive amino acids may be deleted. Stop codons may be introduced (by substitution or insertion) into the encoding nucleic acid sequence to generate truncated proteins.

[0078] Insertion mutants generally involve the addition of amino acid residues at non-terminal points of a polypeptide. This may include the insertion of one or more amino acid residues. Terminal additions may also be generated, and may include fusion proteins that are multimers or concatemers of one or more peptides or polypeptides described or referenced herein.

[0079] Substitution variants typically involve replacing one amino acid with another at one or more sites within a protein or polypeptide, and can be designed to modulate one or more properties of a polypeptide, with or without loss of other functions or properties. Substitutions can be conservative, i.e., replacing one amino acid with an amino acid having similar chemical properties. "Conservative amino acid substitutions" can involve exchanging a member of one amino acid class with another member of the same class. Conservative substitutions are well known in the art, and include, for example, the following changes: 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; valine to isoleucine or leucine. Conservative amino acid substitutions may encompass non-naturally occurring amino acid residues that are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptide mimetics or other reversed or inverted forms of amino acid moieties.

[0080] Alternatively, the substitution may be "non-conservative", such that the function or activity of the polypeptide is affected. Non-conservative changes generally involve replacing an amino acid residue with a chemically different amino acid residue, such as replacing a non-polar or uncharged amino acid with a polar or charged amino acid, or vice versa. Non-conservative substitutions may involve exchanging a member of one amino acid class for a member of another class.

[0081] 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 in the molecule that can be changed without destroying the activity by targeting regions that are considered to be unimportant to the activity. The technician can also identify amino acid residues and molecular parts that are conserved in similar proteins or polypeptides. In further embodiments, regions that may be important for biological activity or structure can undergo conservative amino acid substitutions without significantly changing the biological activity or adversely affecting the protein or polypeptide structure.

[0082] In making such changes, the hydropathic index of amino acids can be considered. The hydropathic characteristics of a protein are 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); glutamate (-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 recognized that the relative hydrophilicity of amino acids contributes to the secondary structure of the resulting protein or polypeptide, which in turn determines the interaction of the protein or polypeptide with other molecules (e.g., enzymes, substrates, receptors, DNA, antibodies, antigens, etc.). It is also known that certain amino acids can replace other amino acids with similar hydropathic indexes or scores and still retain similar biological activity. When making changes based on the hydropathic index, in certain embodiments, substitutions of amino acids with hydropathic indices within ±2 are included. In certain aspects of the present disclosure, substitutions of amino acids within ±1 are included, while in other aspects of the present disclosure, substitutions of amino acids within ±0.5 are included.

[0083] It is also understood in the art that substitution of similar amino acids can be effectively made based on hydrophilicity. U.S. Pat. No. 4,554,101 (incorporated herein by reference) indicates that the greatest local average hydrophilicity of a protein (determined by the hydrophilicity of its adjacent amino acids) is correlated with the biological properties of the protein. In certain embodiments, the greatest local average hydrophilicity of a protein (determined by the hydrophilicity of its adjacent amino acids) is correlated with its immunogenicity and antigen binding, i.e., as a biological property 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); glutamate (+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). When making changes based on similar hydrophilicity values, in certain embodiments, amino acid substitutions within ±2 of hydrophilicity values ​​are included, in other embodiments, amino acid substitutions within ±1 of hydrophilicity values ​​are included, and in yet other embodiments, amino acid substitutions within ±0.5 of hydrophilicity values ​​are included. In some cases, epitopes can also be identified from the primary amino acid sequence based on hydrophilicity. These regions are also referred to as "epitope core regions." It is understood that one amino acid can be substituted for another amino acid with a similar hydrophilicity value and still produce a biologically equivalent and immunologically equivalent protein.

[0084] In addition, one skilled in the art can review structure-function studies to identify residues in similar polypeptides or proteins that are important for activity or structure. Given this comparison, one can predict the importance of amino acid residues in a protein that correspond to amino acid residues in similar proteins that are important for activity or structure. One skilled in the art can select chemically similar amino acids to replace these predicted important amino acid residues.

[0085] One skilled in the art can also analyze the three-dimensional structure and amino acid sequence associated with the structure in similar proteins or polypeptides. Given such information, one skilled in the art can predict the arrangement of the amino acid residues of an antibody relative to its three-dimensional structure. One skilled in the art can choose not to make changes to amino acid residues predicted to be located on the surface of the protein because such residues may be involved in important interactions with other molecules. In addition, one skilled in the art can generate test variants containing a single amino acid substitution at each desired amino acid residue. These variants can then be screened for binding and / or activity using standard assays, thereby generating information collected from such routine experiments that allows one skilled in the art to determine amino acid positions that should be avoided from further substitutions, either alone or in combination with other mutations. Various tools for determining secondary structure can be found on the World Wide Web at expasy.org / proteomics / protein_structure.

[0086] In some embodiments of the present disclosure, amino acid substitutions are made to: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter ligand or antigen binding affinity, and / or (5) confer or alter other physicochemical or functional properties of 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 the antibody that are located outside of the domains that form intermolecular contacts. In such embodiments, conservative amino acid substitutions can be used that do not substantially change the structural characteristics of the protein or polypeptide (e.g., one or more replacement amino acids that do not disrupt the secondary structure that characterizes the native antibody).

[0087] C. Chimeric Antigen Receptor

[0088] In some aspects, chimeric antigen receptor (CAR) is disclosed. CAR generally includes an extracellular antigen (or ligand) binding domain connected to one or more intracellular signaling components, which is realized by a joint and / or a transmembrane domain in some aspects. Such molecules generally simulate or approximate the signal of a natural antigen receptor, the signal of such receptors combined with a costimulatory receptor, and / or the signal of a costimulatory receptor alone.

[0089] It is contemplated that the chimeric construct can be introduced into immune cells as naked DNA or in a suitable vector. Methods for stably transfecting cells using naked DNA by electroporation are known in the art. See, for example, U.S. Patent No. 6,410,319. Naked DNA generally refers to DNA encoding a chimeric receptor, which is contained in a plasmid expression vector and has an orientation suitable for expression.

[0090] Alternatively, a viral vector (e.g., a retroviral vector, an adenoviral vector, an adeno-associated viral vector, or a lentiviral vector) can be used to introduce the CAR construct into immune cells. Suitable vectors used according to the method of the present disclosure do not replicate in immune cells. A large number of viral-based vectors are known, in which the viral copy number maintained in the cell is low enough to maintain the viability of the cell, such as vectors based on HIV, SV40, EBV, HSV, or BPV.

[0091] Certain embodiments of the present disclosure relate to the use of nucleic acids, including nucleic acids encoding cancer antigen-specific CAR polypeptides, including CAR (hCAR) that has been humanized to reduce immunogenicity in some cases, which comprises at least one intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising one or more signaling motifs. In certain embodiments, the binding region may include a complementary 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) bound to a receptor.

[0092] It is considered that CAR nucleic acid can be a human gene for enhancing cellular immunotherapy of human patients. In a specific embodiment, the present disclosure includes full-length CAR cDNA or coding region. Antigen binding region or domain may include VH and VL chain fragments of single-chain variable fragments (scFv) derived from specific human monoclonal antibodies (e.g., anti-CD19 antibodies, such as FMC63.3 and / or anti-CD79b antibodies, such as antibodies described in PCT patent application publication WO2021 / 222944). In some embodiments, the fragment can also be any number of different antigen binding domains of human antigen-specific antibodies. In a more specific embodiment, the fragment is a cancer antigen-specific scFv encoded by a sequence optimized for human codon usage to be expressed in human cells. In some embodiments, the antigen binding region includes a protein or polypeptide as a ligand and / or receptor for another protein and / or polypeptide.

[0093] In some embodiments, the arrangement can be multimeric, such as a diabody or a multimer. A multimer is most likely formed by cross-pairing the variable portions of light and heavy chains into a diabody. The hinge portion of the construct can have a variety of substitution schemes, from completely deleting, to retaining the first cysteine, to replacing with proline instead of serine, to truncating to the first cysteine. In some embodiments, the Fc portion can be deleted. In some embodiments, any stable and / or dimerized protein can be used for this purpose. In some embodiments, only one of the Fc domains is used, such as the CH2 or CH3 domains from human immunoglobulin. In some embodiments, the hinge, CH2 and CH3 regions of human immunoglobulin modified to improve dimerization can be used. In some embodiments, only the hinge portion of an immunoglobulin can be used.

[0094] The sequence of the open reading frame encoding the chimeric receptor can be obtained from a genomic DNA source, a cDNA source, or can be 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, as introns have been found to stabilize mRNA. In addition, it may be advantageous to use endogenous or exogenous non-coding regions to stabilize mRNA.

[0095] In some aspects, antigen-specific binding (e.g., anti-CD19, anti-CD79b, anti-CD70, etc.) or recognition components are connected to one or more transmembrane and intracellular signaling domains. In some embodiments, CAR includes a transmembrane domain fused to the extracellular domain of CAR. In some embodiments, a transmembrane domain naturally associated with one of the domains in CAR is used. In some embodiments, a transmembrane domain not naturally associated with one of the domains of CAR is used. In some cases, a transmembrane domain is selected or modified by amino acid substitution to avoid such domains from being combined with the 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 from a natural or synthetic source. In some embodiments, when the source is natural, the transmembrane domain is from any membrane-bound or transmembrane protein. In some embodiments, the transmembrane region includes a transmembrane region derived from (i.e., at least including a transmembrane region; also referred to as "from") a T cell receptor, CD28, DAP12, DAP10, NKG2D, CD3ζ, CD3ε, CD3γ, CD3δ, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD30, CD33, CD37, CD64, CD80, CD86, OX-40 (CD134), 4-1BB (CD137), CD154, ICOS / CD278, etc. In some embodiments, the transmembrane domain of the present disclosure is a transmembrane domain from CD8α. In some embodiments, the transmembrane domain of the present disclosure is a transmembrane domain from CD30.

[0096] In some embodiments, CAR nucleic acid includes sequences encoding other co-stimulatory receptors, such as transmembrane domains and one or more intracellular signaling domains. In addition to the main T cell activation signal (e.g., it may be initiated by CD3ζ and / or FcεRIγ), chimeric receptors can also be used to bind to target antigens for immune effector cell proliferation and additional stimulation signals for effector functions. For example, part or all of the human co-stimulatory receptors for enhancing cell activation can be used, which can help improve in vivo persistence and improve the treatment success rate of adoptive immunotherapy. Examples include co-stimulatory domains 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 specific substitution embodiments, any of these domains listed may be excluded from CAR.

[0097] In certain embodiments, specific CAR molecules are contemplated herein. In a certain case, the antigen binding domain of CAR is scFv, and any scFv that binds to a cancer antigen can be used herein. In the case of using scFv in the extracellular domain of CAR, the variable heavy chain and variable light chain of scFv can be arranged in any order from the N-terminal to the C-terminal direction. For example, the variable heavy chain can be located on the N-terminal side of the variable light chain, and vice versa. The scFv and / or ligand binding to the antigen in CAR may or may not be codon optimized. In some embodiments, the antigen binding domain of CAR is a ligand of another protein (e.g., a "bait" protein), such as a CD27 molecule that acts on targeting CD70. In some embodiments, the CD27 molecule is a truncated CD27 extracellular binding domain (e.g., tCD27).

[0098] In certain embodiments, the carrier encodes cancer antigen-specific CAR, and also encodes one or more other molecules. For example, the carrier can encode the first CAR (e.g., anti-CD19 CAR) and the second CAR (e.g., anti-CD79B CAR, anti-CD70 CAR, etc.).

[0099] In some embodiments, on the same molecule, cancer antigen-specific CAR may include one or more antigen-specific extracellular domains, specific hinges, specific transmembrane domains, one or more specific costimulatory domains, and one or more specific activation signals. When multiple antigen-specific extracellular domains are used, for example, for targeting two different antigens, there may be a joint between two antigen-specific extracellular domains. The examples of CAR considered herein include but are not limited to CD19 specificity (also referred to as "anti-CD19") CAR, anti-CD70 CAR (also referred to as CD70 CAR or tCD27-CAR) and CD79b specificity (also referred to as "anti-CD79b") CAR.

[0100] In the specific embodiment of a specific CAR molecule, CAR can utilize CD28, DAP10, DAP12, 4-1BB, NKG2D, etc. or other costimulatory domains (which may be referred to herein as intracytoplasmic domains). In some cases, CD3ζ is used without using any costimulatory domains. In the specific embodiment of a specific CAR molecule, CAR can utilize any suitable transmembrane domain, for example, from CD30, DAP12, DAP10, 4-1BB, 2B4, OX40, CD27, NKG2D, CD8, CD28, IL12Rβ1 or IL12Rβ2. In the specific embodiment of a specific CAR molecule, CAR can utilize a transmembrane domain from CD30.

[0101] 1. Signal peptide

[0102] The polypeptides of the present disclosure may include signal peptides. "Signal peptide" refers to a peptide sequence that directs protein transport and localization in a cell, for example, directs protein transport and localization in an organelle (e.g., endoplasmic reticulum) and / or cell surface. In some embodiments, the signal peptide directs nascent protein to enter the endoplasmic reticulum. This is essential if the receptor is to be glycosylated and anchored in the cell membrane. Typically, a signal peptide that is naturally attached to the amino-terminal component is used (e.g., in a scFv with a light chain-linker-heavy chain orientation, the natural signal of the light chain is used).

[0103] In some embodiments, the signal peptide is cleaved after passing through the endoplasmic reticulum (ER), ie, is a cleavable signal peptide. In some embodiments, a restriction site is located at the carboxyl terminus of the signal peptide to facilitate cleavage.

[0104] 2. Antigen binding domain

[0105] The polypeptides of the present disclosure may comprise one or more antigen binding domains. An "antigen binding domain" describes a polypeptide region that is capable of binding 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., a CD20 antibody). In some embodiments, the antigen binding domain comprises a variable heavy chain (VH) region and a variable light chain (VL) region, wherein the VH and VL regions are located on the same polypeptide. In some embodiments, the antigen binding domain comprises a linker between the VH and VL regions. The linker enables the antigen binding domain to form a desired structure for antigen binding.

[0106] In certain embodiments of chimeric antigen receptors, the antigen-specific portion of the receptor (which may be referred to as an extracellular domain comprising an antigen binding region) comprises a tumor-associated antigen or a pathogen-specific antigen binding domain. Antigens include carbohydrate antigens recognized by pattern recognition receptors, such as Dectin-1. Tumor-associated antigens can be of any kind, as long as it is expressed on the cell surface of tumor cells. Exemplary embodiments of tumor-associated antigens include CD19, CD70, CD20, carcinoembryonic antigen, alpha-fetoprotein, CA-125, MUC-1, CD56, EGFR, c-Met, AKT, Her2, Her3, epithelial tumor antigens, melanoma-associated antigens, mutated p53, mutated ras, CD79a, CD79b, etc. 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, CAR can be co-expressed with cytokines to improve persistence when the amount of tumor-associated antigen is low. For example, CAR can be co-expressed with IL-15.

[0107] In some embodiments, the CAR with a CD70 antigen binding domain comprises, consists of, or is substantially composed of a sequence identical to SEQ ID NO: 49 75%, 80%, 85%, 90%, 95% or 100% (and any values ​​therebetween). In some embodiments, the CAR with a CD70 antigen binding domain is encoded by a nucleic acid comprising, consisting of, or is substantially composed of a sequence identical to SEQ ID NO: 51 75%, 80%, 85%, 90%, 95% or 100% (and any values ​​therebetween). In some embodiments, the CAR with a CD70 antigen binding domain comprises a truncated CD27 (tCD27) extracellular domain comprising, consisting of, or is substantially composed of a sequence identical to SEQ ID NO: 48 75%, 80%, 85%, 90%, 95% or 100% (and any values ​​therebetween). In some embodiments, the 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 to SEQ ID NO:52 (and any values ​​therebetween).

[0108] The variable region of the antigen-binding domain of the polypeptide of the present disclosure can be modified by mutating the 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 complementary determining region of a part of the variable chain in the immunoglobulin (antibody) and T cell receptor produced by B cells and T cells, respectively, wherein these molecules bind to their specific antigens. Since most of the sequence variations associated with immunoglobulins and T cell receptors are present in the CDR, these regions are sometimes referred to as hypervariable regions. Mutations can be introduced by site-directed mutagenesis or PCR-mediated mutagenesis, and the effects on antibody binding or other functional properties of interest can be evaluated in appropriate in vitro or in vivo assays. It is preferred to introduce conservative modifications, and generally no more than one, two, three, four or five residues in the CDR region are changed. Mutations can be amino acid substitutions, additions or deletions.

[0109] Framework modifications may be made to an antibody to reduce immunogenicity, for example by "backmutating" one or more framework residues to the corresponding germline sequence.

[0110] It is also contemplated that the antigen binding domains may be multispecific or multivalent by multimerizing the antigen binding domains with pairs of VH and VL regions that bind the same antigen (multivalent) or different antigens (multispecific).

[0111] The binding affinity of the antigen binding region (e.g., variable region (heavy chain and / or light chain variable region)) or CDR may be at least 10-5M, 10-6M, 10-7M, 10-8M, 10-9M, 10-10M, 10-11M, 10-12M, or 10-13M. In some embodiments, the KD of the antigen binding region (e.g., variable region (heavy chain and / or light chain variable region)) or CDR may be at least 10-5M, 10-6M, 10-7M, 10-8M, 10-9M, 10-10M, 10-11M, 10-12M, or 10-13M (or any range derivable therein).

[0112] Binding affinity, KA or KD can be determined by methods known in the art, such as by a biosensor based on surface plasmon resonance (SRP), by a kinetic exclusion assay (KinExA), by an optical scanner based on microarray detection of polarization-modulated oblique incidence reflectance difference (OI-RD), or by ELISA.

[0113] In some embodiments, a polypeptide comprising a humanized binding region has equal, better, or 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% binding affinity and / or expression level in a host cell as compared to a polypeptide comprising a non-humanized binding region (e.g., a binding region from a mouse).

[0114] In some embodiments, the framework regions (e.g., FR1, FR2, FR3, and / or FR4) of the human framework can each or collectively have 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, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 1 , 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.

[0115] In some embodiments, the framework regions (e.g., FR1, FR2, FR3, and / or FR4 of a mouse framework) can each or collectively have 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, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 1 , 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.

[0116] Substitutions may be in position 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 7, 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.

[0117] In some embodiments, the antigen binding domain of CAR is an Fc region binding domain (e.g., binding to an immunoglobulin Fc domain). In certain embodiments, the antigen binding domain of CAR is derived from an immunoglobulin Fc receptor (FcR). In certain embodiments, the antigen binding domain of CAR binding to the Fc region may be derived from a CD16 gene sequence. In certain embodiments, FcR is directed to IgG (e.g., FcγRI / CD64, FcγRII / CD32, and FcγRIII / CD16), IgE (e.g., Fc∈RI), IgA (e.g., FcαRI / CD89), IgM (e.g., FcμR) and / or IgA / IgM (e.g., Fcα / μR). In certain embodiments, FcR comprises a sequence identical to SEQ ID NO: 9-17 75%, 80%, 85%, 90%, 95% or 100% (and any value therebetween), consisting of, or consisting essentially of. In some embodiments, the 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 to SEQ ID NO:40 (and any values ​​therebetween).

[0118] In some embodiments, CAR with FcR antigen binding domains is used in combination with another immunotherapy (e.g., antibody-based therapy). In some embodiments, CAR with FcR antigen binding domains provides a universal CAR that can be used with any antibody therapy, as long as the antibody is a targetable Fc domain. In some embodiments, FcR CAR can be used as a CAR, and can also be used as a transduction marker and / or safety switch.

[0119] 3. Peptide Spacer

[0120] Peptide spacers (e.g., spacers), such as extracellular spacers, can connect antigen binding domains to transmembrane domains. In some embodiments, peptide spacers are flexible enough to allow antigen binding domains to be oriented in different directions to promote antigen binding. In some embodiments herein, peptide spacers are "hinge", for example, it is a flexible polypeptide connection region that connects one or more domains of CAR to one or more other domains of CAR. As used herein, the term "hinge" refers to a flexible polypeptide connection region (also referred to herein as "hinge region"), which provides structural flexibility and spacing for flanking polypeptide regions, and can be composed of natural or synthetic polypeptides.

[0121] In some embodiments, the peptide spacer comprises, consists essentially of, or consists of a sequence derived from a 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 a human CD30 gene. In some embodiments, the peptide spacer comprises, consists essentially of, or consists of a sequence derived from a human CD30 coding region. In some embodiments, the peptide spacer comprises, consists essentially of, or consists of a sequence derived from a human CD30 transcript isoform 1 coding region.

[0122] In some embodiments, the spacer includes a hinge region from IgG. In some embodiments, the spacer includes or further includes a CH2CH3 region of an immunoglobulin and a portion of CD3. In some embodiments, the CH2CH3 region may have L235E / N297Q or L235D / N297Q modifications, or have at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% amino acid sequence identity in the CH2CH3 region. In some embodiments, the spacer is from IgG4. The extracellular spacer may include a hinge region.

[0123] The "hinge" derived from an immunoglobulin (e.g., IgG1) is generally defined as extending from Glu216 to Pro230 of human IgG1 (Burton (1985) Molec. Immunol., 22: 161-206). The 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 bonds (SS) in the same position.

[0124] In some embodiments, the hinge region may be naturally occurring or non-naturally occurring, including but not limited to altered hinge regions as described in U.S. Pat. No. 5,677,425, which is incorporated herein by reference. In some embodiments, the hinge region may include a complete hinge region from an antibody of a different class or subclass from the CH1 domain. In some embodiments, the term "hinge" and / or "peptide spacer" may also include regions from CD8 and other receptors that provide similar functions in providing flexibility and spacing for the flanking regions.

[0125] In certain embodiments, the hinge region is from CD30. In certain embodiments, the hinge does not contain cysteine. In certain embodiments, the hinge is rich in G and / or S amino acids relative to other hinges known in the art. In certain embodiments, the hinge and the transmembrane domain are from the same gene. In some embodiments, the hinge and the transmembrane domain are from the same coding sequence. In some embodiments, when the immune cell is designed to express more than one CAR, each CAR uses a unique hinge region, such as a CD30 hinge, a CD8 hinge, etc.

[0126] In some embodiments, the length of the extracellular peptide spacer comprising the hinge can be 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 length of the peptide spacer is 42 amino acids.

[0127] In some embodiments, the length of the extracellular peptide spacer including the hinge can be 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, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 250, 260, 270, 280, 290, 300, 325, 350 or 400 amino acids (or any range derivable therein).

[0128] In some embodiments, the extracellular spacer comprises, consists essentially of, the hinge region of an immunoglobulin (e.g., IgG). The amino acid sequences of the hinge regions of immunoglobulins 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..

[0129] The length of the extracellular spacer can affect the signal transduction activity of CAR, the expression level (transcription and / or translation) of CAR, cytotoxicity and / or cancer cell killing efficacy, and / or CAR cell response antigen stimulation CAR signal transduction amplification characteristics. In some embodiments, the extracellular spacer sequence of CAR depends on the position of the target antigen. In some embodiments, when the target antigen is close to the cell membrane, a longer extracellular spacer is used. In some embodiments, when the target antigen is away from the cell membrane, a shorter extracellular spacer is used. In some embodiments, when a more flexible CAR is needed, a longer extracellular spacer is used. In some embodiments, when a more rigid CAR is needed, a shorter extracellular spacer is used.

[0130] In some embodiments, a shorter spacer is used, for example, 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, a shorter spacer may have advantages in the amplification properties of CAR signaling activity mediated by CAR, CAR expression level (transcription and / or translation), cytotoxicity and / or cancer cell killing efficacy, and / or CAR cell response antigen stimulation CAR signaling.

[0131] In some embodiments, longer spacers are used, for example, 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, 220, 2219, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246 18, 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, 260, 270, 280 or 290 amino acids. In some embodiments, longer spacers may have advantages in CAR-mediated signal transduction activity, CAR expression levels (transcription and / or translation), cytotoxicity and / or cancer cell killing efficacy, and / or CAR cell response antigen stimulation CAR signal transduction amplification characteristics.

[0132] When the extracellular spacer comprises multiple parts, there may be 0-50 amino acids between each part. For example, there may be at least, at most or just 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 derivable range thereof) between the hinge region and other regions (for example, CH2 or CH3 regions, or when CH2 and CH3 regions are present, between CH2 and CH3 regions). In some embodiments, the extracellular spacer is mainly composed of hinge, CH2 and / or CH3 regions, which means that hinge, CH2 and / or CH3 regions are the only identifiable regions that exist, and exclude all other domains or regions, but there may be other amino acids that are not a part of the identifiable region.

[0133] In certain embodiments, the peptide spacer can be from any suitable source, but in specific embodiments, the peptide spacer is from CD30, CD8a, CD28, PD-1, CTLA4, the alpha, beta or zeta chain of the T cell receptor, CD2, CD3ζ, CD3ε, CD3γ, CD3δ, 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 a combination thereof or others. In a specific embodiment, the peptide spacer is from CD30. In a specific embodiment, the peptide spacer is from an immunoglobulin light chain joining region.

[0134] 4. Transmembrane domain

[0135] 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 lead to different receptor stabilities.

[0136] In some embodiments, the transmembrane domain is between the extracellular spacer and the cytoplasmic region. In some embodiments, the transmembrane domain is between the extracellular spacer and one or more costimulatory regions. In some embodiments, the linker is located between the transmembrane domain and one or more costimulatory regions.

[0137] 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 set derived from the human CD30 transcript isoform 1 coding region.

[0138] In certain embodiments, the transmembrane domain does not contain cysteine. 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 certain embodiments, the hinge and the transmembrane domain are continuous amino acids derived from a wild-type gene. In some embodiments, when immune cells are designed to express more than one CAR, each CAR uses a unique transmembrane region, such as a CD30 transmembrane region, a CD8 transmembrane region, etc.

[0139] In some embodiments, the transmembrane domain can be 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 length. 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.

[0140] 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.

[0141] 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.

[0142] In some embodiments, the transmembrane domain and the 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.

[0143] In some embodiments, the transmembrane domain and / or the peptide spacer does not comprise 3 or more consecutive amino acids according to SEQ ID NO:5.

[0144] In some embodiments, any transmembrane domain for inserting a 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-ζ, OX-40 (CD134) or CD7. In some embodiments, the transmembrane domain is derived from the α, β or ζ chain of the T cell receptor, CD28, CD2, CD3ζ, CD3ε, CD3γ, CD3δ, CD45, CD4, CD5, CD8 (including CD8α), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, OX-40 (CD134), 4-1BB (CD137, CD154, ICOS / CD278, GITR / CD357, NKG2D, PD-1, CTLA4 and DAP molecules. In some embodiments, the transmembrane domain is synthetic. In some aspects, the synthetic transmembrane domain mainly comprises hydrophobic residues, such as leucine and valine. In some aspects, a tripartite of phenylalanine, tryptophan and valine will be located at both ends of the synthetic transmembrane domain.

[0145] 5. Cytoplasmic region

[0146] After antigen recognition, the receptors of the present disclosure can aggregate and transmit signals to cells through the cytoplasmic region. In some embodiments, the costimulatory domain described herein is part of the cytoplasmic region. In some embodiments, the cytoplasmic region includes an intracellular signaling domain. The intracellular signaling domain can include a primary signaling domain and one or more costimulatory domains.

[0147] The cytoplasmic region and / or co-stimulatory region of the polypeptides suitable for use in the present disclosure include any desired signaling domains that are activated in response to the binding of an antigen to an antigen binding domain, providing a unique and detectable signal (e.g., increased production of one or more cytokines by the cell; changes in target gene transcription; changes in protein activity; changes in cell behavior, such as cell death; cell proliferation; cell differentiation; cell survival; regulation of cell signaling responses; etc.). In some embodiments, the cytoplasmic region includes at least one (e.g., one, two, three, four, five, six, etc.) ITAM motif as described herein. In some embodiments, the cytoplasmic region includes a DAP10 / CD28 type signaling chain.

[0148] The cytoplasmic region of the polypeptide suitable for the present disclosure includes an intracellular signaling polypeptide containing an immunoreceptor tyrosine-based activation motif (ITAM). The ITAM motif is YX1X2(L / I), wherein X1 and X2 are independently any amino acid. In some cases, the cytoplasmic region comprises 1, 2, 3, 4 or 5 ITAM motifs. In some cases, the ITAM motif is repeated twice in the endodomain, wherein the first and second instances of the ITAM motif are separated by 6 to 8 amino acids from each other, for example, (YX1X2(L / I))(X3)n(YX1X2(L / I)), wherein n is an integer from 6 to 8, and each of 6-8 X3 can be any amino acid.

[0149] In some embodiments, a suitable cytoplasmic region is a portion containing an ITAM motif derived from a polypeptide containing an ITAM motif. For example, a suitable cytoplasmic region can be a domain containing an ITAM motif derived from any protein containing an ITAM motif. Therefore, a suitable internal domain does not need to contain the entire sequence of the entire protein from which it is derived. Examples of suitable polypeptides containing ITAM motifs include, but are not limited to: DAP12, DAP10, FCER1G (Fcε receptor Iγ chain); CD3D (CD3δ); CD3E (CD3ε); CD3G (CD3γ); CD3-ζ; and CD79A (antigen receptor complex-associated protein α chain).

[0150] Exemplary cytoplasmic regions are known in the art. The cytoplasmic regions shown below also provide examples of regions that are incorporated into the CAR of the present disclosure in some embodiments:

[0151] In some embodiments, a suitable cytoplasmic region may include a portion of the full-length DAP12 amino acid sequence containing an ITAM motif. In some embodiments, the cytoplasmic region is derived from FCER1G (also known as FCRG; Fcε receptor I γ chain; Fc receptor γ chain; fc-εR1-γ; fcRγ; fceRIγ; high affinity immunoglobulin epsilon receptor subunit γ; immunoglobulin E receptor, high affinity, γ chain; etc.). In some embodiments, a suitable cytoplasmic region may include a portion of the full-length FCER1G amino acid sequence containing an ITAM motif.

[0152] In some embodiments, the cytoplasmic region is derived from the T cell surface glycoprotein CD3 δ chain (also known as CD3D; CD3-DELTA; T3D; CD3 antigen, δ subunit; CD3δ; CD3d antigen, δ polypeptide (TiT3 complex); OKT3, δ chain; T cell receptor T3 δ chain; T cell surface glycoprotein CD3 δ chain; etc.). In some embodiments, a suitable cytoplasmic region may include an ITAM motif-containing portion of a full-length CD3 δ amino acid sequence. In some embodiments, the cytoplasmic region is derived from the T cell surface glycoprotein CD3 ε chain (also known as CD3e, CD3 ε; T cell surface antigen T3 / Leu-4 ε chain, T cell surface glycoprotein CD3 ε chain, AI504783, CD3, CD3-ε, T3e, etc.). In some embodiments, a suitable cytoplasmic region may include an ITAM motif-containing portion of a full-length CD3 ε amino acid sequence. In some embodiments, the cytoplasmic region is derived from the T cell surface glycoprotein CD3γ chain (also known as CD3G, CD3γ, T cell receptor T3γ chain, CD3-GAMMA, T3G, γ polypeptide (TiT3 complex), etc.). In some embodiments, the suitable cytoplasmic region may include the ITAM motif-containing portion of the full-length CD3γ amino acid sequence. In some embodiments, the cytoplasmic region is derived from the T cell surface glycoprotein CD3ζ chain (also known as CD3Z, CD3ζ, T cell receptor T3ζ chain, CD247, CD3-ZETA, CD3H, CD3Q, T3Z, TCRZ, etc.). In some embodiments, the suitable cytoplasmic region may include the ITAM motif-containing portion of the full-length CD3ζ amino acid sequence.

[0153] 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-α; membrane-bound 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.

[0154] 6. Co-stimulatory Zone

[0155] The non-limiting examples of suitable costimulatory regions (such as costimulatory regions included in cytoplasmic regions) include, but are not limited to, polypeptides 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 co-stimulatory 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 main signal initiated by CD3ζ, the additional signal provided by the co-stimulatory receptor inserted into the CAR is also important for the complete activation of immune cells and can help improve the in vivo persistence and treatment success rate of cell therapy.

[0156] The length of the costimulatory region can be at least, at most or just 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200 or 300 amino acids or any range that can be derived therefrom. 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 referred to as BTLA1 and CD272). In some embodiments, the costimulatory region is derived from the intracellular portion of the transmembrane protein CD27 (also referred to as S152, T14, TNFRSF7 and Tp55). In some embodiments, the costimulatory region is derived from the intracellular portion of the transmembrane protein CD30 (also referred to as TNFRSF8, D1S166E and Ki-1). In some embodiments, the costimulatory region is derived from the intracellular portion of the transmembrane protein GITR (also referred to as TNFRSF18, RP5-902P8.2, AITR, CD357 and GITR-D). In some embodiments, the costimulatory region is derived from the intracellular portion of the transmembrane protein HVEM (also referred to as TNFRSF14, RP3-395M20.6, ATAR, CD270, HVEA, HVEM, LIGHTR and TR2).

[0157] 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.

[0158] In some aspects, the CAR of the present disclosure comprises a CD28 co-stimulatory domain. In some aspects, the CD28 co-stimulatory domain comprises SEQ ID NO: 15. In some aspects, the CAR of the present disclosure comprises a 4-1BB co-stimulatory domain. In some aspects, the 4-1BB co-stimulatory domain comprises SEQ ID NO: 25. In some aspects, the CAR of the present disclosure comprises an OX40 co-stimulatory domain. In some aspects, the OX40 co-stimulatory domain comprises SEQ ID NO: 26.

[0159] 7. Peptide linkers

[0160] In some embodiments, the polypeptide of the present disclosure includes a peptide linker (sometimes referred to as a linker). In some embodiments, a peptide linker is used to separate any peptide domain / region described herein. For example, in some embodiments, a linker is located between a signal peptide and an antigen binding domain, between the VH and VL of an antigen binding domain, between an antigen binding domain and a peptide spacer, between a peptide spacer and a transmembrane domain, on the N- or C-region of a costimulatory region flank or a costimulatory region and / or between a transmembrane domain and an internal domain. A peptide linker can have any of a variety of amino acid sequences. Domains and regions can be connected by peptide linkers that are generally flexible, but other chemical connections are not excluded. A linker can be a peptide of about 6 to about 40 amino acids in length, or a peptide of about 6 to about 25 amino acids in length. These linkers can be produced by coupling proteins using synthetic linker encoding oligonucleotides.

[0161] A peptide linker with a certain degree of flexibility can be used. The peptide linker can have almost any amino acid sequence, but it should be remembered that a suitable peptide linker will have a sequence that results in a generally flexible peptide. Small amino acids (such as glycine and alanine) can be used to produce flexible peptides. The generation of such sequences is conventional for those skilled in the art. In certain embodiments, it may be suitable for those skilled in the art to adjust the length of the peptide linker to optimize the performance of one or more CAR components. The conventional optimization of the length of the peptide linker belongs to the skill range of those skilled in the art.

[0162] Suitable linkers can be readily selected and can be of any suitable length, for example 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 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6 or 7 amino acids.

[0163] Suitable linkers can be readily selected and can be of any suitable different lengths, for example 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 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6 or 7 amino acids.

[0164] Exemplary flexible linkers include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, (GSGGS)n (SEQ ID NO: 31), (G4S)n, and (GGGS)n (SEQ ID NO: 32), wherein 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 derivable 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 thus can serve as neutral tethers between components. Glycine polymers can be used; glycine can access significantly more phi-psi space than alanine and is less restricted than residues with longer side chains. Exemplary spacers can 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).

[0165] II. Nucleic Acids

[0166] In certain embodiments, the nucleic acid sequence can be present in a variety of situations, such as: an insertion sequence encoding an antibody or its fragment, derivative, mutant protein or variant of one or two chains or a fragment and recombinant vector of a recombinant polynucleotide, a polynucleotide sufficient to be used as a hybridization probe, a PCR primer or sequencing primer for identifying, analyzing, mutating or amplifying a polynucleotide encoding a polypeptide, an antisense nucleic acid for suppressing polynucleotide expression, and the aforementioned complementary sequence herein. In some embodiments, nucleic acids encoding epitopes for certain polypeptides provided herein are also provided. In some embodiments, nucleic acids encoding fusion proteins including these peptides are also provided. Nucleic acids can be single-stranded or double-stranded, and can include RNA and / or DNA nucleotides and artificial variants thereof (e.g., peptide nucleic acids).

[0167] The term "polynucleotide" refers to a nucleic acid molecule that is recombinant or has been separated from a 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, etc. In certain aspects, a polynucleotide includes a regulatory sequence that is substantially separated from its naturally occurring gene or protein coding sequence. A polynucleotide can be single-stranded (coding or antisense) or double-stranded, and can be RNA, DNA (genomic, cDNA or synthetic), its analogs or combinations thereof. Other coding or non-coding sequences may be present in a polynucleotide, but are not required.

[0168] In this regard, the term "gene", "polynucleotide" or "nucleic acid" is used to refer to a nucleic acid encoding a protein, polypeptide or peptide (including any sequence required for correct transcription, post-translational modification or localization). As will be appreciated by those skilled in the art, the term encompasses genomic sequences, expression cassettes, cDNA sequences and smaller engineered nucleic acid fragments that express or can be adapted to express proteins, polypeptides, domains, peptides, fusion proteins and mutants. A nucleic acid encoding all or part of a polypeptide may comprise a continuous nucleic acid sequence encoding all or part of such a polypeptide. It is also contemplated that a particular polypeptide may be encoded by a nucleic acid containing a variant having a slightly different nucleic acid sequence, but nonetheless encoding the same or substantially similar protein.

[0169] In certain embodiments, there are polynucleotide variants with substantial identity to the sequences disclosed herein; using methods described herein (e.g., BLAST analysis using standard parameters), compared to the polynucleotide sequences provided herein, comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or higher sequence identity (including all values ​​and ranges therebetween) those. In certain aspects, the isolated polynucleotide will comprise a nucleotide sequence encoding a polypeptide having at least 90%, preferably 95% and above identity to the amino acid sequence described herein over the entire sequence length; or a nucleotide sequence complementary to the isolated polynucleotide.

[0170] Regardless of the length of the coding sequence itself, the nucleic acid fragment can be combined with other nucleic acid sequences (e.g., promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding fragments, etc.) so that their total length can vary greatly. The nucleic acids can be of any length. Their length can 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 or more nucleotides, and / or can contain one or more additional sequences, such as regulatory sequences, and / or be part of a larger nucleic acid (e.g., vector). Therefore, it is contemplated that nucleic acid fragments of almost any length can be used, wherein the total length is preferably limited by the ease of preparation and use in the expected recombinant nucleic acid protocol. In some cases, the nucleic acid sequence may encode a polypeptide sequence with additional heterologous coding sequences, for example to allow for purification of the polypeptide, transport, secretion, post-translational modification, or for therapeutic benefit, such as targeting or efficacy. As described above, a tag or other heterologous polypeptide may be added to the modified polypeptide coding sequence, where "heterologous" refers to a polypeptide that is different from the modified polypeptide.

[0171] A. Carrier

[0172] The polypeptides of the present disclosure, including the CARs of the present disclosure, can be delivered to the recipient immune cells via any suitable vector, including viral vectors or non-viral vectors. Examples of viral vectors include at least retrovirus, lentivirus, adenovirus or adeno-associated virus vectors. Examples of non-viral vectors include at least plasmids, transposons, lipids, nanoparticles, etc.

[0173] In some embodiments, suitable methods for delivering nucleic acids to achieve expression of the composition are contemplated to include virtually any method by which nucleic acids (e.g., DNA, including viral and non-viral vectors) can be introduced into cells, tissues, or organisms, as described herein or as known to those of ordinary skill in the art. Such methods include, but are not limited to, direct delivery of DNA, such as by 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), including microinjection (Harland and Weintraub, 1985; U.S. Pat. No. 5,789,215, incorporated herein by reference); by electroporation (U.S. Pat. No. 5,384,253, incorporated herein by reference); by calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990); by using DEAE dextran followed by polyethylene glycol (Gopal, 1985); by direct ultrasonic loading (Fechheimer et al., 1987); by liposome-mediated transfection (Nicolau and Sene, 1982; Fraley et al., 1990); 1979; Nicolau et al., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al., 1991); by particle bombardment (PCT Application Nos. WO94 / 09699 and 95 / 06128; U.S. Patents 5,610,042; 5,322,783; 5,563,055; 5,550,318; 5,538,877; and 5,538,880, each of which is incorporated herein by reference); by stirring 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); by Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,591,616 and 5,563,055, each of which is incorporated herein by reference); or by PEG-mediated protoplast transformation (Omirulleh et al., 1993; U.S. Pat. Nos. 4,684,611 and 4,952,500, each of which is incorporated herein by reference); by desiccation / inhibition-mediated DNA uptake (Potrykus et al., 1985). Other methods include viral transduction, such as gene transfer by lentiviral or retroviral transduction.

[0174] In some embodiments, immune cells are transduced with a vector encoding one or more antigen targeting CARs. In some embodiments, CAR may or may not be included on the same vector or included with the same vector. In some embodiments, one or more CARs are expressed by the same vector molecule (eg, 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, more than one CAR may or may not be expressed as a separate polypeptide. In some embodiments, when more than one CAR is included on the same vector and more than one CAR is expressed as a separate polypeptide, they are separated by, for example, 2A elements and / or IRES elements on the vector (or two types are used once or more than once on the same vector).

[0175] In some embodiments, the CAR expression vector is a polycistronic (e.g., bicistronic) vector. In some embodiments, the CAR expression vector is a polycistronic vector expressing anti-CD19 CAR and anti-CD79B CAR separated by 2A elements. In some embodiments, the CAR expression vector is a polycistronic vector encoding anti-CD19 CAR and anti-CD79A CAR separated by 2A elements. In some embodiments, the CAR expression vector is a polycistronic vector encoding FcR CAR and anti-CD79B CAR separated by 2A elements. In some embodiments, the CAR expression vector is a polycistronic vector encoding FcR CAR and anti-CD19 CAR separated by 2A elements. In some embodiments, the CAR expression vector is a polycistronic vector encoding FcR CAR and anti-CD19 CAR separated by 2A elements. In some embodiments, the CAR expression vector is a polycistronic vector encoding CD70 CAR and one or more additional CAR molecules. In some embodiments, the CAR expression 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., tCD27-CAR).

[0176] 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.

[0177] B. Sequence

[0178] Table 2 provides certain nucleotide sequences of polynucleotides, including polynucleotides expressing chimeric antigen receptors, and portions and regions thereof.

[0179] Table 2 - Polynucleotide sequences

[0180]

[0181]

[0182]

[0183]

[0184] C. Mutation

[0185] Can change be introduced into nucleic acid by sudden change, thereby cause the amino acid sequence of its encoded polypeptide to change.Can use any technology known in the art to introduce sudden change.In one embodiment, use for example site-directed mutagenesis scheme to change one or more specific amino acid residues.In another embodiment, use for example random mutagenesis scheme to change one or more randomly selected residues.No matter how to make, can express mutant polypeptide and screen desired properties.

[0186] Certain mutations can be introduced into a nucleic acid without significantly changing the biological activity of the polypeptide encoded therein. For example, nucleotide substitutions can be performed, resulting in amino acid substitutions at non-essential amino acid residues. Alternatively, one or more mutations can be introduced into a nucleic acid to selectively change the biological activity of the polypeptide encoded therein. See, for example, Romain Studer et al., Biochem. J. 449: 581-594 (2013). For example, mutations can quantitatively or qualitatively change biological activity. Examples of quantitative changes include increasing, decreasing or eliminating activity. Examples of qualitative changes include changing the antigenic specificity of an antibody.

[0187] III. Therapy

[0188] In some embodiments, disclosed herein are methods of treatment, including disclosed compositions, e.g., compositions comprising CARs having CD30-derived hinge and / or transmembrane domains. In some embodiments, the methods of treatment are directed to cancer, while in other embodiments. In some embodiments, the methods of treatment are directed to pathogenic and / or exogenous drivers of the disease.

[0189] In some embodiments, the method further comprises administering a cancer therapy to the patient. Cancer therapy can be selected based on expression level measurements alone or in combination with a clinical risk score calculated for the patient. In some embodiments, cancer therapy comprises local cancer therapy. In some embodiments, cancer therapy does not include systemic cancer therapy. In some embodiments, cancer therapy does not include local therapy. In some embodiments, cancer therapy comprises local cancer therapy without administering systemic cancer therapy. In some embodiments, cancer therapy comprises immunotherapy, which may be immune checkpoint therapy. Any of these cancer therapies may also be excluded. Combinations of these therapies may also be administered.

[0190] The term "cancer" as used herein can be used to describe solid tumors, metastatic cancers, or non-metastatic cancers. In certain embodiments, cancer may originate from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, colon, rectum, anus, gums, head, kidney, liver, lung, nasopharynx, neck, ovary, pancreas, prostate, skin, stomach, testicles, tongue, or uterus. In some embodiments, cancer is a recurrent cancer. In some embodiments, cancer is a phase I cancer. In some embodiments, the cancer is a phase II cancer. In some embodiments, the cancer is a phase III cancer. In some embodiments, the cancer is a phase IV cancer.

[0191] The cancer may specifically be of the following histological types, although not limited thereto: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatricoma carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; bronchoalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe cell carcinoma; oncocytic carcinoma; aerobic adenocarcinoma; basophilic cell carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinomas; non-encapsulated sclerosing carcinoma; adrenal cortex carcinoma; endometrioid carcinoma; carcinoma of skin appendages; apocrine carcinoma; sebaceous carcinoma; cervical adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease, breast; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; theca cell tumor, malignant; granulosa cell tumor, malignant; male cell tumor, malignant; sertoli cell carcinoma; Leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extramammary paraganglioma, malignant; pheochromocytoma; glomus sarcoma; malignant melanoma; amelanotic melanoma melanoma; superficial spreading melanoma; malignant melanoma in giant nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrohistiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mixed Müllerian tumor; Wilms tumor; hepatoblastoma carcinoma; carcinosarcoma; stromal tumor, malignant; Brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; ovarian goiter, malignant; choriocarcinoma; mesonephroblastoma, malignant; angiosarcoma; hemangioendothelioma, malignant; Kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; Osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic odontosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrous astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroglioma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmary tumor, 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 fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative intestinal disease; leukemias; lymphoid leukemias; plasma cell leukemias; erythroleukemias; lymphosarcoma cell leukemias; myeloid leukemias; basophilic leukemias; eosinophilic leukemias; monocytic leukemias; mast cell leukemias; megakaryoblastic leukemias; myeloid sarcomas; and hairy cell leukemias. ;

[0192] In some embodiments, the present disclosure provides an immunotherapy method, including administering an effective amount of a composition comprising a CAR of the present disclosure. In one embodiment, a medical disease or condition is treated by administering a CAR-expressing cell group that elicits an immune response. In certain embodiments of the present disclosure, cancer is treated by administering a CAR immune cell group that elicits an immune response. Provided herein is a method for treating or delaying the progression of cancer in an individual, comprising administering an effective amount of antigen-specific cell therapy to the individual. As an example, the present method can be applied to treat immune disorders, solid cancers, and hematological cancers. Specifically, cancer can 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 lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia, and chronic lymphocytic leukemia.

[0193] 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. It is part of a larger class of diseases called hematological neoplasms. Leukemia is a broad term that encompasses a range of diseases. Leukemia is clinically and pathologically classified into two forms: acute and chronic.

[0194] In some embodiments of the methods of the present disclosure, activated CD4 and / or CD8 T cells in an individual are characterized by CD4 and / or CD8 T cells producing γ-IFN and / or enhanced cytolytic activity relative to before administration of the combination. γ-IFN can be measured by any method known in the art, including, for example, intracellular cytokine staining (ICS) involving cell fixation, permeabilization, and staining with anti-γ-IFN antibodies. Cytolytic activity can be measured by any method known in the art, such as using a cell killing assay utilizing mixed effector cells and target cells.

[0195] In some embodiments, non-myeloablative lymphodepleting chemotherapy may be administered to the subject prior to T cell therapy. The non-myeloablative lymphodepleting chemotherapy may be any suitable such therapy, which may be administered by any suitable route. Non-myeloablative lymphodepleting chemotherapy may include, for example, administration of cyclophosphamide and fludarabine, particularly if the cancer is melanoma, which may be metastatic. An exemplary route of administration of cyclophosphamide and fludarabine is intravenous. Likewise, any suitable dose of cyclophosphamide and fludarabine may be administered. In a specific aspect, about 60 mg / kg of cyclophosphamide is administered for two days, followed by about 25 mg / m 2 Fludarabine for five days.

[0196] In certain embodiments, the T cell growth factor that promotes autologous T cell growth and activation is administered to the subject simultaneously with the autologous T cells or is subsequently administered to the subject. The T cell growth factor can be any suitable growth factor that promotes autologous T cell growth and activation. The example of a suitable T cell growth factor includes 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.

[0197] Therapeutically effective amounts of immune cells can be administered by a variety of routes, including parenteral administration, such as intravenous, intraperitoneal, intramuscular, intrasternal, or intraarticular injection or infusion.

[0198] For discrete, solid, accessible tumors, intratumoral injection or injection into tumor blood vessels is particularly contemplated. Local, regional or systemic administration may also be suitable. In some embodiments, for tumors >4 cm, the volume to be administered will be about 4-10 ml (particularly 10 ml), while for tumors <4 cm, a volume of about 1-3 ml (particularly 3 ml) will be used. In some embodiments, multiple injections delivered as a single dose may include a volume of about 0.1 to about 0.5 ml.

[0199] In some embodiments, the cell population can be administered according to a treatment regimen consistent with the disease, such as a single dose or a small number of doses administered over one to several days to improve the disease state or regularly administered over an extended period of time to inhibit disease progression and prevent disease recurrence. The precise dose used in the formulation also depends on the route of administration and the severity of the disease or condition, and should be determined based on the judgment of the practitioner and the circumstances of each patient. The therapeutically effective amount of cells depends on the subject being treated, the severity and type of the disease, and the mode of administration. In some embodiments, the dosage range that can be used to treat human subjects is at least 3.8×104 , 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 one embodiment, the dosage range for treating human subjects is about 3.8×10 9 About 3.8×10 10 Cells / m 2 In other embodiments, the therapeutically effective amount of cells may be from about 5×10 6 cells / kg body weight to about 7.5×10 8 cells / kg body weight change, for example, about 2×10 7 cells / kg body weight to about 5×10 8 cells / kg body weight, or about 5×10 7 cells / kg body weight is about 2×10 8 Cells / kg body weight. The exact number of cells can be easily determined by a person skilled in the art based on the age, weight, sex and physiological condition of the subject. The effective dose can be inferred from a dose-response curve derived from an in vitro or animal model test system.

[0200] In certain embodiments of the present disclosure, an effective amount of CAR-expressing immune cells are delivered to individuals in need, such as individuals with cancer. In some embodiments, cells can then enhance the immune system of an individual to attack cancer cells. In some cases, one or more doses of immune cells (such as immune cells described herein) are provided to an individual. In some embodiments, when two or more doses of immune cells are provided to an individual, the interval between two administrations should be sufficient to allow time for breeding in an individual, and in a specific embodiment, the interval between two doses is 1, 2, 3, 4, 5, 6, 7 or more days.

[0201] In a specific embodiment, cells engineered to express CAR are injected into the cell culture medium in a therapeutically effective amount (at 10 3 Up to 10 10 The therapeutically effective amount may be 10 3 Up to 10 10 , 10 3 Up to 10 9 , 10 3 Up to 10 8 , 10 3 Up to 10 7, 10 3 Up to 10 6 , 10 3 Up to 10 5 , 10 3 Up to 10 4 , 10 4 Up to 10 10 , 10 4 Up to 10 9 , 10 4 Up to 10 8 , 10 4 Up to 10 7 , 10 4 Up to 10 6 , 10 4 Up to 10 5 , 10 5 Up to 10 10 , 10 5 Up to 10 9 , 10 5 Up to 10 8 , 10 5 Up to 10 7 , 10 5 Up to 10 6 , 10 6 Up to 10 10 , 10 6 Up to 10 9 , 10 6 Up to 10 8 , 10 6 Up to 10 7 , 10 7 Up to 10 10 , 10 7 Up to 10 9 , 10 7 Up to 10 8 , 10 8 Up to 10 10 , 10 8 Up to 10 9 or 10 9 Up to 10 10 Thus, in certain embodiments, a therapeutically effective amount of cells expressing one or more CARs is provided to an individual with a certain cancer one or more times.

[0202] A. Cancer Antigens

[0203] The cancer antigens targeted by the CAR of the present disclosure are those expressed in the context of diseases, disorders or cell types to be targeted by adoptive cell therapy. Diseases and disorders include hyperplastic, neoplastic and malignant diseases and disorders, including cancers and tumors, including blood cancers, immune system cancers, such as lymphomas, leukemias and / or myeloma, such as B, T and myeloid leukemias, lymphomas and multiple myeloma. In some embodiments, compared with normal or non-targeted cells or tissues, antigens are selectively expressed or overexpressed on cells (such as tumors or pathogenic cells) of diseases or disorders. In other embodiments, antigens are expressed on normal cells and / or expressed on engineered cells.

[0204] In some embodiments, any suitable antigen can be used in the context of the present disclosure. Exemplary antigens include, but are not limited to, antigen molecules, self / autoantigens, tumor / cancer-associated antigens and tumor neoantigens (Linnemann et al., 2015) from infectious agents. In particular 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 particular aspects, antigens for one or two 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 are known in the art, for example, CD19 (Accession No. NG_007275.1), EBNA (Accession No. NG_002392.2), WT1 (Accession No. NG_009272.1), CD123 (Accession No. NC_000023.11), NY-ESO (Accession No. NC_000023.11), EGFRvIII (Accession No. NG_007726.3), MUC1 (Accession No. NG_029383.1), HER2 (Accession No. NG_009272.1), NG_007503.1), 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).

[0205] In some embodiments, the tumor-associated antigen may be derived from prostate cancer, breast cancer, colorectal cancer, lung cancer, pancreatic cancer, renal cancer, mesothelioma, ovarian cancer or melanoma. Exemplary tumor-associated antigens or tumor cell-derived antigens include MAGE1, 3 and MAGE 4 (or other MAGE antigens, such as those disclosed in International Patent Publication No. WO99 / 40188); PRAME; BAGE; RAGE, Lage (also known as NY ESO 1); SAGE; and HAGE or GAGE. Non-limiting examples of these tumor antigens are expressed in a variety 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 prostate six transmembrane epithelial antigen (STEAP).

[0206] In some embodiments, tumor-associated antigens include Plu-1, HASH-1, HasH-2, Cripto, and Criptin. In addition, tumor antigens can be self-peptide hormones, such as full-length gonadotropin-releasing hormone (GnRH), a short peptide of 10 amino acids long that can be used to treat many cancers.

[0207] In some embodiments, tumor antigens include tumor antigens derived from cancer characterized by tumor-associated antigen expression (e.g., HER-2 / neu expression). Interested tumor-associated antigens include lineage-specific tumor antigens, such as melanocyte-melanoma lineage antigens MART-1 / Melan-A, gp100, gp75, mda-7, tyrosinase, and tyrosinase-associated proteins. Exemplary tumor-associated antigens include, but are not limited to, being derived from or comprising one or more of the following tumor antigens: 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, iCE, MUC1, MUC2, phosphatidylinositol 3-kinases (PI3Ks), TRK receptors, 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, HSP7 0-2M, 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, Pml / RAR, tumor-associated calcium signal transducer 1 (TACSTD1) TACSTD2, receptor tyrosine kinases (e.g., epidermal growth factor receptor (EGFR) (particularly 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 transducer and transcription activator STAT3, STATS and STATE, hypoxia-inducible factors (e.g., HIF-1 and HIF-2), nuclear factor-κB (NF-B), Notch receptors (e.g., Notch1-4), c-Met, mammalian target of rapamycin (mTOR), WNT, extracellular signal-regulated kinase (ERK) and its regulatory subunits,PMSA, PR-3, MDM2, mesothelin, renal cell carcinoma-5T4, SM22-α, 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 cells, 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.

[0208] In some embodiments, antigens may include epitope regions or epitope peptides derived from genes mutated in tumor cells or from genes transcribed at different levels in tumor cells compared to normal cells, such as telomerase, survivin, mesothelin, mutated ras, bcr / abl rearrangement, Her2 / neu, mutated or wild-type p53, cytochrome P450 1B1, and abnormally expressed intronic sequences such as N-acetylglucosamine transferase-V; clonal rearrangements of immunoglobulin genes that produce unique idiotypes in myeloma and B-cell lymphoma; tumor antigens including epitope regions or epitope peptides derived from oncoviral processes, such as human papillomavirus proteins E6 and E7; Epstein bar virus protein LMP2; non-mutated oncofetal proteins with tumor-selective expression, such as carcinoembryonic antigen and alpha-fetoprotein.

[0209] B. Non-cancer antigens

[0210] In certain embodiments, antigen can be a microorganism. In some embodiments, antigen is obtained or derived from pathogenic microorganisms or opportunistic pathogenic microorganisms (also referred to herein as infectious microorganisms) (e.g., viruses, fungi, parasites, and bacteria). In certain embodiments, antigen derived from this microorganism includes full-length protein.

[0211] Exemplary pathogenic organisms whose 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 viruses, vesicular stomatitis virus (VSV), vesicular stomatitis virus (VSV), polyomaviruses (e.g., BK virus and JC virus), adenoviruses, coronaviruses (e.g., SARS-CoV, SARS-CoV-2, or MERS), Staphylococcus species (including methicillin-resistant Staphylococcus aureus (MRSA)), and Streptococcus species (including Streptococcus pneumoniae). It will be appreciated by those skilled in the art that antigens of pathogenic organisms may be found in publications and public databases (e.g., ) are used to identify proteins derived from these and other pathogenic microorganisms and nucleotide sequences encoding these proteins for use as antigens described herein.

[0212] Antigens derived from human immunodeficiency virus (HIV) include any HIV virion structural protein (e.g., gp120, gp41, p17, p24), protease, reverse transcriptase, or HIV proteins encoded by tat, rev, nef, vif, vpr, and vpu.

[0213] Antigens derived from herpes simplex viruses (e.g., HSV1 and HSV2) include, but are not limited to, proteins expressed by HSV late genes. The late genome primarily encodes proteins that form viral particles. Such proteins include five proteins from (UL), which form viral capsids: 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 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 may be used as an antigen.

[0214] Antigens derived from cytomegalovirus (CMV) include CMV structural proteins, viral antigens expressed in the early and early stages of viral replication, glycoproteins I and III, capsid proteins, coat proteins, lower matrix proteins pp65 (ppUL83), p52 (ppUL44), IE1 and 1E2 (UL123 and UL122), protein products from the UL128-UL150 gene cluster (Rykman et al., 2006), envelope glycoprotein B (gB), gH, gN and pp150. It will be appreciated by those skilled in the art that CMV proteins used as antigens described herein can be identified in public databases, such as (See, e.g., Bennekov et al., 2004; Loewendorf et al., 2010; Marschall et al., 2009).

[0215] Antigens derived from Epstein-Ban virus (EBV) contemplated for use in certain embodiments include EBV lytic proteins gp350 and gp110, EBV proteins produced during latent infection, including Epstein-Ban nuclear antigen (EBNA)-1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C, EBNA-leader protein (EBNA-LP), and latent membrane protein (LMP)-1, LMP-2A, and LMP-2B (see, e.g., Lockey et al., 2008).

[0216] Antigens derived from respiratory syncytial virus (RSV) contemplated for use herein include any of the 11 proteins encoded by the RSV genome, or antigenic fragments thereof: NS1, NS2, N (nucleocapsid protein), M (matrix protein) SH, G and F (viral capsid proteins), M2 (second matrix protein), M2-1 (elongation factor), M2-2 (transcription regulation), RNA polymerase, and phosphoprotein P.

[0217] Antigens derived from vesicular stomatitis virus (VSV) contemplated for use herein include any 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).

[0218] Antigens derived from influenza viruses 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.

[0219] Exemplary viral antigens also include, but are not limited to, adenovirus polypeptides, alphavirus polypeptides, calicivirus polypeptides (e.g., calicivirus capsid antigens), coronavirus polypeptides, canine distemper virus polypeptides, Ebola virus polypeptides, enterovirus polypeptides, flavivirus polypeptides, hepatitis virus (AE) polypeptides (hepatitis B core or surface antigens, hepatitis C virus E1 or E2 glycoproteins, core or nonstructural proteins), herpesvirus polypeptides (including herpes simplex virus or varicella zoster virus glycoproteins), infectious peritonitis virus polypeptides, leukemia virus polypeptides, Marburg virus polypeptides, orthomyxovirus polypeptides, papillomavirus polypeptides, parainfluenza virus polypeptides (e.g., hemagglutinin and neuraminidase polypeptides), paramyxovirus polypeptides, parvovirus polypeptides, swine fever virus 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.

[0220] In certain embodiments, the antigen may be a bacterial antigen. In certain embodiments, the bacterial antigen of interest may be a secreted polypeptide. In certain other embodiments, the bacterial antigen includes an antigen that is exposed on the outer cell surface of the bacteria by one or more portions of the polypeptide.

[0221] 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 Staphylococcal proteins that may be used as antigens include the Clp protein, HtrA, MsrR, aconitase, CcpA, SvrA, Msa, CfvA, and CfvB (see, e.g., Staphylococcus: Molecular Genetics, 2008 Caister Academic Press, Ed. Jodi Lindsay). The genomes of two Staphylococcus aureus (N315 and Mu50) have been sequenced and are publicly available, e.g., at PATRIC (PATRIC: The VBI PathoSystems Resource Integration Center, Snyder et al., 2007). As will be appreciated by those skilled in the art, Staphylococcal proteins used as antigens may also be found in other public databases (e.g. ) was identified.

[0222] Antigens derived from Streptococcus pneumoniae contemplated for use in certain embodiments described herein include pneumolysin, PspA, choline binding protein A (CbpA), NanA, NanB, SpnHL, PavA, LytA, Pht, and pili proteins (RrgA; RrgB; RrgC). Antigenic proteins of Streptococcus pneumoniae are also known in the art and can be used as antigens in certain embodiments (see, e.g., Zysk et al., 2000). The complete genome sequence of a virulent strain of Streptococcus pneumoniae has been sequenced, and as will be appreciated by those skilled in the art, the Streptococcus pneumoniae proteins used herein can also be identified in other public databases, such as According to the present disclosure, proteins of particular interest as antigens include virulence factors and proteins predicted to be exposed on the surface of pneumococci (see, e.g., Frolet et al., 2010).

[0223] 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 polypeptides, Corynebacterium polypeptides, The polypeptides include, but are not limited to, polypeptides of Corynebacterium, Coxiella, Dermatophilus, Enterococcus, Ehrlichia, Escherichia, Francisella, Fusobacterium, Haemobartonella, Haemophilus, Helicobacter, Klebsiella, and Helicobacter. Klebsiella polypeptides, L-type bacterial polypeptides, Leptospira polypeptides, Listeria polypeptides, Mycobacteria polypeptides, Mycoplasma polypeptides, Neisseria polypeptides, Neorickettsia polypeptides, Nocardia polypeptides, Pasteurella polypeptides, Peptococcus polypeptides, Peptostreptococcus polypeptides, Pneumococcus polypeptides (i.e., Pneumonia polypeptides) Streptococcus polypeptides) (see description herein), Proteus polypeptides, Pseudomonas polypeptides, Rickettsia polypeptides, Rochalimaea polypeptides, Salmonella polypeptides, Shigella polypeptides, Staphylococcus polypeptides, Group A Streptococcus polypeptides (e.g., Streptococcus pyogenes M protein), Group B Streptococcus (S. agalactiae) polypeptides, Treponema polypeptides, and Yersinia polypeptides (e.g., Yersinia pestis F1 and V antigens).

[0224] 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, peptides, Moniliella polypeptides, Mortierella polypeptides, Mucor polypeptides, Paecilomyces polypeptides, Penicillium polypeptides, Philalemonium polypeptides, Phialophora polypeptides, Prototheca polypeptides, Pseudallescheria polypeptides, Pseudo microdochium polypeptides, Pythium polypeptides, Rhinosporidium polypeptides, Rhizopus polypeptides, Scolecobasidium polypeptides, Sporothrix polypeptides, Stemphylium polypeptides, Trichophyton polypeptides, Trichosporon polypeptides, and Xylohypha polypeptides.

[0225] 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 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, Chabertia polypeptides, Cooperia polypeptides, Crenosoma polypeptides, Dictyocaulus polypeptides, 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, Nan ophyetus polypeptide, Necator polypeptide, Nematodirus polypeptide, Oesophagostomum polypeptide, Onchocerca polypeptide, Opisthorchis polypeptide, Ostertagia polypeptide, Parafilaria polypeptide, Paragonimus polypeptide, Parascaris polypeptide, Physaloptera polypeptide, Protostrongylus polypeptide, Setaria polypeptide , 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.falciparum circumsporozoite (PfCSP)), sporozoite surface protein 2 (PfSSP2), carboxyl terminus of liver state antigen 1 (PfLSA1 c-term) and exportin 1 (PfExp-1), Pneumocystis polypeptides, Sarcocystis polypeptides, Schistosoma polypeptides, Theileria polypeptides, Toxoplasma polypeptides and Trypanosoma polypeptides.

[0226] Examples of ectoparasite antigens include, but are not limited to, polypeptides (including antigens as well as allergens) from: fleas; ticks, including hard ticks and soft ticks; flies, such as midges, mosquitoes, sand flies, black flies, horse flies, horn flies, deer flies, tsetse flies, biting flies, myiasis-causing flies and biting mosquitoes; ants; spiders, lice; mites; and true bugs, such as bed bugs and kissing bugs.

[0227] IV. Cell Therapy

[0228] Certain embodiments relate to cells comprising polypeptides or nucleic acids of the present disclosure. In some embodiments, the cell is an immune cell. In certain embodiments, as used herein, "T cells" include all types of immune cells expressing CD3, including T helper cells, invariant natural killer T (iNKT) cells, cytotoxic T cells, T regulatory cells (Treg) γ-δ T cells, natural killer (NK) cells and neutrophils. T cells may refer to CD4+ or CD8+ T cells.

[0229] Suitable mammalian cells include primary cells and immortalized cell lines. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (eg, 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.

[0230] In some cases, the cell is not an immortalized cell line, but 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. As an example, the cell is a T lymphocyte obtained from an individual. As another example, the cell is a cytotoxic cell obtained from an individual. As another example, the cell is a stem cell (e.g., a peripheral blood stem cell) or a progenitor cell obtained from an individual.

[0231] A. Unlimited immune cells

[0232] Certain embodiments of the present disclosure relate to immune cells engineered to express one or more genes. Compared with cells lacking one or more gene expression, the expression of one or more genes directly or indirectly leads to an increase in cell lifespan. In specific embodiments, cells are manipulated to express one or more genes, including one or more heterologous genes. In other cases, cells are manipulated to upregulate the expression of one or more genes endogenous to the cell, for example, by manipulating one or more regulatory elements of one or more genes endogenous to the cell. In certain embodiments, methods and compositions related to unlimited immune cells are described in PCT Patent Application Publication No. WO / 2021 / 034982, which is incorporated herein by reference in its entirety.

[0233] In a specific embodiment, immune cells are manipulated to express BCL6 and one or more pro-survival genes or anti-apoptotic genes or genes that promote cell survival (and there may or may not be overlap in genes classified as pro-survival or anti-apoptotic or promoting cell survival). As used herein, a pro-survival gene refers to a nucleic acid polymer that can play an anti-apoptotic function or promote survival by any mechanism. The nucleic acid polymer that can play an anti-apoptotic function can be one or more of the Bcl2 family genes, such as BCL-xL, BCL-2, MCL-1, Bcl-w, Bfl-1, BCL-B, etc. The nucleic acid polymer that can play an anti-apoptotic function can be one or more of the inhibitor of apoptosis (IAP) family genes, such as XIAP, c-IAP1, C-IAP2, NAIP, survivin, etc. Nucleic acid polymers that can play an anti-apoptotic function can inhibit or knock 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. Nucleic acid polymers for knocking down or knocking out can be shRNA expression cassettes, or these caspase genes can be knocked out by gene editing methods (CRISPR, TALEN, zinc finger methods, etc.). Nucleic acid polymers that can play an anti-apoptotic function can inhibit or knock 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 that can exert anti-apoptotic functions can have anti-apoptotic effects, such as insulin-like growth factor (IGF-1), Hsp70, Hsp27, cFLIP, BNIP3, FADD, Akt, 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.

[0234] Infinite T cells can be generated from wild-type or mutant BCL6. The inventors determined that infinite T cells can be generated from 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 of the two BCL6 genes are shown below (the mutation points in the wild-type sequence are underlined).

[0235] SEQ ID NO:43—Amino acid sequence of wild-type BCL6:

[0236]

[0237] SEQ ID NO:44—Nucleotide sequence of wild-type BCL6 (the codons at the mutation point in the wild-type sequence are underlined):

[0238]

[0239]

[0240] SEQ ID NO:45—Amino acid sequence of mutant BCL6 (leucine mutation is underlined):

[0241]

[0242] SEQ ID NO:46—Nucleotide sequence of mutant BCL6 (the codon for leucine is underlined):

[0243]

[0244]

[0245] Immune cells can be any kind of immune cells, including T cells (e.g., regulatory T cells, CD4+ T cells, CD8+ T cells, αβ T cells, γ-δ T cells, or mixtures thereof), NK cells, invariant NKT cells, NKT cells, innate lymphocytes, or mixtures thereof. Immune cells can be virus-specific, express CAR and / or express TCR. In some embodiments, the cell is a monocyte or granulocyte, for example, a bone marrow cell, a macrophage, a neutrophil, a dendritic cell (DC), a mast cell, an eosinophil, and / or a basophil. Also provided herein are methods for producing and engineering immune cells and methods for using and administering cells for adoptive cell therapy, in which case the cells can be autologous or allogeneic. Therefore, immune cells can be used as immunotherapy, such as targeting cancer cells. These immune cells can be used for treatment as a single cell type or a combination of multiple immune cell types. In a specific embodiment, the immune cell is CD3+, CD4+, CD8+, CD16+, or a mixture thereof.

[0246] 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 tendency to a particular disease or condition, or subjects receiving treatment for a particular disease or condition. Immune cells can be collected from any location in the subject where they reside (including but not limited to blood, cord blood, spleen, thymus, lymph nodes, and bone marrow). Isolation of immune cells can be used directly, or they can be stored for a period of time, such as by freezing.

[0247] Immune cells can be enriched / purified from any tissue in which they reside (including but not limited to blood (including blood collected by a blood bank or cord blood bank), spleen, bone marrow, tissue removed and / or exposed during an operation procedure, and tissue obtained by a biopsy procedure). The tissue / organ from which the immune cells are enriched, separated and / or purified can be separated from living and non-living subjects, wherein the non-living subject is an organ donor. In a specific embodiment, immune cells are separated from blood such as peripheral blood or cord blood. In some aspects, the immune cells separated from cord blood have enhanced immunomodulatory ability, such as measured by CD4 or CD8 positive T cell suppression. In a specific aspect, immune cells are separated from pooled blood, particularly pooled cord blood, to enhance immunomodulatory ability. The blood collected can come from 2 or more sources, such as 3, 4, 5, 6, 7, 8, 9, 10 or more sources (e.g., donor subjects).

[0248] The colony of immunocytes can be obtained from a subject who needs to treat or suffers from a disease associated with the immunocyte activity of reduction. Therefore, these cells can be autologous for the subject who needs to treat. Alternatively, the colony of immunocytes can be obtained from a donor such as a donor of partial or complete tissue compatibility matching or a donor of complete tissue compatibility mismatch. The immunocyte colony can be harvested from any other organ / tissue in which peripheral blood, cord blood, bone marrow, spleen or immunocytes reside in the subject or donor. Immune cells can be separated from the pooled library of the subject and / or donor, for example, separated from the cord blood collected.

[0249] When the population of immune cells is obtained from a donor different from the subject, the donor may be allogeneic, provided that the obtained cells are subject-compatible in the subject into which they may be introduced. The allogeneic donor cells may or may not be compatible with human leukocyte antigens (HLA).

[0250] Other methods and compositions related to unlimited immune cells are described in PCT Patent Application Publication No. WO / 2021 / 034982, which is incorporated herein by reference in its entirety.

[0251] 1. T cells

[0252] In some embodiments, immune cells are T cells. In the past two decades, several basic methods for deriving, activating and amplifying functional anti-tumor effector cells have been described. These include: autologous cells, such as tumor infiltrating lymphocytes (TILs); using autologous DC or PBMC, lymphocytes, artificial antigen presenting cells (APCs) or beads coated with T cell ligands and activating antibodies or cells separated by capturing target cell membranes to activate T cells in vitro; allogeneic cells that naturally express anti-host tumor T cell receptors (TCRs); and autologous or allogeneic cells that are genetically reprogrammed or "redirected" to express non-tumor-specific autologous or allogeneic cells of tumor-reactive TCRs or chimeric TCR molecules (referred to as "T bodies") that display antibody-like tumor recognition capabilities. These methods have produced many schemes for T cell preparation and immunity that can be used for methods described herein.

[0253] In some embodiments, T cells are derived from blood, bone marrow, lymph, umbilical cord or lymphoid organs. In some aspects, cells are human cells. Cells are typically primary cells, such as those directly separated from a subject and / or separated and frozen from a subject. In some embodiments, cells include one or more subsets of T cells or other cell types, such as complete T cell populations, CD4+ cells, CD8+ cells and subgroups thereof, such as those defined by function, activation state, maturity, differentiation potential, amplification, recycling, positioning and / or persistence, antigen specificity, antigen receptor type, presence in a specific organ or compartment, markers or cytokine secretion profiles and / or differentiation degree. With respect to a subject to be treated, cells can be allogeneic and / or autologous. In some aspects, for example, for existing technology, cells are pluripotent and / or multipotent, such as stem cells, such as induced pluripotent stem cells (iPSC). In some embodiments, the method includes separating cells from a subject as described herein, preparing, processing, culturing and / or engineering them, and reintroducing them into the same patient before or after cryopreservation.

[0254] Subtypes and subpopulations of T cells (e.g., CD4+ and / or CD8+ T cells) include naive T (TN) cells, effector T cells (TEFF), memory T cells and subtypes thereof, such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM) or terminally differentiated effector memory T cells, tumor infiltrating lymphocytes (TIL), 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, α / βT cells and γ / δT cells. In certain embodiments, T cells are γ / δT cells.

[0255] In some embodiments, one or more T cell populations are enriched or depleted for cells that are positive for a particular marker, such as a surface marker, or that are negative for a particular marker. In some cases, such markers are markers that are absent or expressed at relatively low levels on certain T cell populations (e.g., non-memory cells) but present or expressed at relatively high levels on certain other T cell populations (e.g., memory cells).

[0256] In some embodiments, T cells are isolated from PBMC samples by negative selection for markers expressed on non-T cells (e.g., B cells, monocytes, or other leukocytes), such as CD14. + or CD8 + Selection step for separation of CD4 +Helper T cells and CD8 + Cytotoxic T cells. This type of 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 degree on one or more naive, memory and / or effector T cell subpopulations.

[0257] In some embodiments, CD8+ T cells are further enriched or depleted for naive, central memory, effector memory, and / or central memory stem cells, for example, by positive or negative selection based on surface antigens associated with the corresponding subpopulations. CM ) cells or enrichment of stem cell memory cells to improve efficacy, for example to improve long-term survival, expansion and / or engraftment after administration, which in some aspects are particularly robust in such subpopulations.

[0258] 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, such as mechanically (using, for example, gentleMACS TM Dissociator, Miltenyi Biotec, Auburn, Calif.) or enzymatically (e.g., collagenase or DNase). Single-cell suspensions of enzymatic digests of tumors are incubated in the presence of interleukin 2 (IL-2) or other growth factors.

[0259] The T cells of culture can be merged and rapidly expanded. Rapid expansion increases the number of antigen-specific T cells by at least about 50 times (e.g., 50, 60, 70, 80, 90 or 100 times or more) within about 10 to about 14 days. More preferably, rapid expansion provides an increase of at least about 200 times (e.g., 200, 300, 400, 500, 600, 700, 800, 900 times or more) within about 10 to about 14 days.

[0260] Amplification can be achieved by any of a variety of methods known in the art. For example, in the presence of feeder lymphocytes and interleukin-2 (IL-2) or interleukin-15 (IL-15) (preferably IL-2), T cells can be rapidly amplified using non-specific T cell receptor stimulation. Non-specific T cell receptor stimulators may include about 30 ng / ml of OKT3, which is a mouse monoclonal anti-CD3 antibody (available from Ortho-Raritan, NJ). Alternatively, T cells can be rapidly amplified by stimulating peripheral blood mononuclear cells (PBMCs) in vitro with one or more cancer antigens (including antigenic portions thereof, such as epitopes or cells) in the presence of T cell growth factors (e.g., 300 IU / ml IL-2 or IL-15, preferably IL-2) (which can optionally be expressed from a vector, such as human leukocyte antigen A2 (HLA-A2) binding peptides or peptides that bind to other MHC class I or class II molecules). The in vitro induced T cells are rapidly expanded by re-stimulation with the same cancer antigen loaded onto antigen presenting cells expressing HLA-A2 or antigen presenting cells expressing other HLA molecules. The in vitro induced T cells can also be expanded in the absence of antigen presenting cells.

[0261] Autologous T cells can be modified to express T cell growth or differentiation factors that promote the growth, 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 a particular aspect, the modified autologous T cells express T cell growth factors at high levels. T cell growth factor coding sequences (such as IL-12) and promoters (operably connected to T cell growth factor coding sequences to promote high-level expression) are readily available in the art.

[0262] 2. NK cells

[0263] In some embodiments, immune cells are natural killer (NK) cells. NK cells are a subgroup of lymphocytes, which have spontaneous cytotoxicity against a variety of tumor cells, virus-infected cells, and some normal cells in the bone marrow and thymus. NK cells differentiate and mature in the 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 receptors, pan-T markers CD3, or surface immunoglobulin B cell receptors.

[0264] In certain embodiments, NK cells are derived from human peripheral blood mononuclear cells (PBMC), unstimulated leukocyte separation products (PBSC), human embryonic stem cells (hESC), induced pluripotent stem cells (iPSC), bone marrow, tissue or umbilical cord blood by methods well known in the art.

[0265] 3. NKT cells

[0266] Natural killer T (NKT) cells are a heterogeneous group of T cells that have properties 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 only account for about 0.1% of all peripheral blood T cells. NKT cells are a subset of T cells that co-express the αβ T cell receptor but also express various molecular markers that are usually associated with NK cells, such as NK1.1. Invariant natural killer T (iNKT) cells express high levels of the transcriptional regulator promyelocytic leukemia zinc finger and are dependent on this transcriptional regulator for development. Currently, there are five major different subsets of iNKT cells. These subsets of cells produce a different set of cytokines once activated. The subtypes iNKT1, iNKT2, and iNKT17 are similar to Th cell subsets in terms of cytokine production. In addition, there are subtypes that specialize in T follicular helper-like functions and IL-10-dependent regulatory functions.

[0267] 4. Innate lymphoid cells

[0268] Innate lymphoid cells (ILCs) are a group of innate immune cells that are derived from common lymphoid progenitors (CLPs) and belong to the lymphoid lineage. These cells are defined by the lack of antigen-specific B or T cell receptors due to the absence of recombination activation genes (RAGs). ILCs do not express myeloid or dendritic cell markers. They play a role in protective immunity and in the regulation of homeostasis and inflammation, so their dysregulation can lead to immunopathologies such as allergies, bronchial asthma and autoimmune diseases. ILCs can be divided according to the cytokines they can produce and the transcription factors that regulate their development and function.

[0269] B. Cell Culture

[0270] In some embodiments, cells can be cultured for at least about 10 days to about 40 days, at least about 15 days to about 35 days, at least about 15 days to 21 days, for example, at least about 15, 16, 17, 18, 19 or 21 days. In some embodiments, cells of the present disclosure can be cultured for no more than 60 days, or no more than 50 days, or no more than 45 days. Cells can 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. Cells can be cultured in liquid culture medium. Typically, culture medium may include basal culture medium formulations known in the art. A variety of basal culture medium formulations can be used to culture the cells herein, including but not limited to Eagle's Minimum Essential Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), α-modified Minimum Essential Medium (α-MEM), Basic Essential Medium (BME), Iscove's Modified Dulbecco's Medium (IMDM), BGJb medium, F-12 nutrient mixture (Ham), Liebovitz L-15, DMEM / F-12, Essential Modified Eagle Medium (EMEM), RPMI-1640 and modifications and / or combinations thereof. The composition of the above-mentioned basal culture medium is generally known in the art, and those skilled in the art can modify or adjust the concentration of the culture medium and / or culture medium supplement according to the needs of culturing cells. In some embodiments, the culture medium formulation can be an explant culture medium (CEM), which is composed of IMDM supplemented with 10% fetal bovine serum (FBS), 100U / ml penicillin G, 100μg / ml streptomycin and 2mmol / L L-glutamine. Other embodiments can use other basal culture medium formulations, such as those selected from the above-mentioned formulations.

[0271] Any culture medium capable of supporting cells in vitro can be used to culture cells. Culture medium formulas that can support cell growth include, but are not limited to, Dulbecco's modified Eagle medium (DMEM), α-modified minimum essential medium (αMEM), and Roswell Park Memorial Institute culture medium 1640 (RPMI culture medium 1640), etc. Typically, up to 20% of fetal bovine serum (FBS) or 1-20% of horse serum is added to the above culture medium to support cell growth. However, if the growth factors, cytokines, and hormones required for culturing cells are provided in the culture medium at appropriate concentrations, a culture medium with defined components can also be used. The culture medium that can be used for the method of the present disclosure may contain one or more compounds of interest, including but not limited to antibiotics, mitogen compounds, or differentiation compounds that can be used to culture cells. Cells can be grown at a temperature between 27°C and 40°C, such as 31°C to 37°C, and can be in a humidified incubator. The carbon dioxide content can be maintained between 2% and 10%, and the oxygen content can be maintained between 1% and 22%. However, the present disclosure should never be interpreted as being limited to any one method of separating and culturing cells. On the contrary, any method of isolating and culturing cells should be construed as being included in the present disclosure.

[0272] For use in cell culture, culture medium can be supplied with one or more other components. For example, additional supplements can be used to provide cells with trace elements and substances required for optimal growth and expansion. Such supplements include insulin, transferrin, selenium salts and combinations thereof. These components can be included in saline solutions, such as but not limited to Hanks balanced salt solution (HBSS), Earle salt solution. Other antioxidant supplements can be added, such as β-mercaptoethanol. Although many culture media already contain amino acids, some amino acids can be supplemented later, such as L-glutamine, which is known to be less stable in solution. Culture medium can further provide antibiotics and / or antifungal compounds, such as 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. It is also contemplated to supplement cell culture medium with mammalian plasma or serum. Plasma or serum generally contains cytokines and components that are essential for cell survival and expansion. The use of appropriate serum replacements is also contemplated.

[0273] Reference to specific buffers, media, reagents, cells, culture conditions, etc., or to some subclass of the same, is not intended to be limiting, but should be understood to include all relevant materials deemed by one of ordinary skill in the art to be of interest or value in the specific context under discussion. For example, one buffer system or culture medium can generally be substituted for another, so that a different but known method can be used to achieve the same goal as that for which the proposed method, material or composition is used. In a specific embodiment, cells are cultured in a cell culture system comprising a cell culture medium, preferably in a culture vessel, particularly in a cell culture medium supplemented with a substance suitable and determined for protecting cells from senescence in vitro and / or inducing reprogramming in a non-specific or specific manner.

[0274] C. Cell generation

[0275] Some methods of the present disclosure relate to the cells obtained from human tissue samples by cultivation. In the specific embodiments of the present disclosure, cells are seeded on the matrix allowing cell adhesion. This can be achieved by, for example, seeding cells into a culture plate showing one or more matrix surfaces compatible with cell adhesion. When one or more matrix surfaces contact the cell suspension (for example, the suspension in the culture medium) introduced into a culture system, cell adhesion may occur between the cell and the matrix surface. Therefore, in certain embodiments, cells are introduced into a culture system, the system has at least one matrix surface compatible with cell adhesion usually, so that the cells of inoculation can contact the matrix surface, and such an embodiment includes being seeded on the matrix allowing cell adhesion.

[0276] The cells of the present disclosure can be identified and characterized by the expression of their specific marker proteins (e.g., cell surface markers). The detection and separation of these cells can be achieved by, for example, 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 differentiation reactions. In certain embodiments, the marker proteins used to identify and characterize cells are selected from a 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.

[0277] D. Pharmaceutical Composition

[0278] In some aspects, the composition or medicament for method (e.g., cell-based therapy) is suitably contained in a pharmaceutically acceptable carrier. The carrier is nontoxic, biocompatible and is selected to not adversely affect the biological activity of the medicament. The medicament in some aspects of the present disclosure can be formulated into a preparation for local delivery (i.e., to a specific location of the body, such as at a tumor site or other tissues) or systemic delivery, in a solid, semisolid, gel, liquid or gaseous form, such as tablets, capsules, powders, granules, ointments, solutions, reservoirs, inhalants and injections, which allow oral, parenteral or surgical administration. Some aspects of the present disclosure also contemplate that the composition is locally applied by coating medical equipment, etc.

[0279] Carriers for parenteral delivery by injection, infusion or lavage and local delivery include distilled water, physiological phosphate buffered saline, normal or lactated Ringer's solution, dextrose solution, Hank's solution or propylene glycol. In addition, sterile fixed oils can be used as solvents or suspension media. For this reason, any biocompatible oil can be used, including synthetic monoglycerides or diglycerides. In addition, fatty acids such as oleic acid can be used to prepare injections. The carrier and the medicament can be mixed into a liquid, a suspension, a polymerizable or non-polymerizable gel, a paste or an ointment.

[0280] The carrier may also include a delivery vehicle to maintain (i.e., extend, delay or modulate) the delivery of the agent or enhance the delivery, uptake, stability or pharmacokinetics of the therapeutic agent. Such delivery vehicles may include, but are not limited to, microparticles, microspheres, nanospheres or nanoparticles composed of proteins, liposomes, carbohydrates, synthetic organic compounds, inorganic compounds, polymer or copolymer hydrogels and polymer micelles.

[0281] In certain aspects, the actual dosage of the composition administered to a patient or subject may be determined by physical and physiological factors, such as body weight, severity of the condition, type of disease being treated, previous or concurrent therapeutic interventions, idiopathic conditions of the patient, and route of administration. The practitioner responsible for administration will in any case determine the concentration of the active ingredient in the composition and the appropriate dosage for the individual subject.

[0282] Solutions of pharmaceutical compositions can be prepared in water suitably mixed with a surfactant (e.g., hydroxypropylcellulose). Dispersions can also be prepared in glycerol, liquid polyethylene glycol, mixtures thereof, and oils. Under normal conditions of storage and use, these preparations contain preservatives to prevent the growth of microorganisms.

[0283] In certain aspects, the pharmaceutical composition is advantageously administered in the form of an injectable composition, as a liquid solution or suspension; suitable solid forms or solutions or suspensions in liquids prior to injection may also be prepared. These formulations may also be emulsified. Typical compositions for this purpose include a pharmaceutically acceptable carrier. For example, the composition may contain 10 mg or less, 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, nontoxic excipients, including salts, preservatives, buffers, and the like.

[0284] Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters, such as ethyl oleate. Aqueous carriers include water, alcohol / water solutions, saline solutions, parenteral carriers, such as sodium chloride, Ringer's dextrose, and the like. Intravenous carriers include liquids and nutritional supplements. Preservatives include antibacterial agents, antifungal agents, antioxidants, chelating agents, and inert gases. The pH and exact concentrations of the various ingredients in the pharmaceutical composition are adjusted according to well-known parameters.

[0285] Other formulations are suitable for oral administration. Oral formulations contain typical excipients, such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. The composition is in the form of a solution, suspension, tablet, pill, capsule, sustained-release formulation or powder.

[0286] In further aspects, the pharmaceutical composition may include a classical pharmaceutical preparation. According to certain aspects, the administration of the pharmaceutical composition may be by any common route, as long as the target tissue can be obtained by the route. This may include oral, nasal, oral, rectal, vaginal or topical. Alternatively, administration may be by in situ, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions are typically administered as pharmaceutically acceptable compositions comprising physiologically acceptable carriers, buffers or other excipients. For the treatment of pulmonary diseases, aerosol delivery may be used. For example, the volume of the aerosol may be between approximately 0.01 ml and 0.5 ml.

[0287] The effective amount of the pharmaceutical composition is determined based on the intended goal. The term "unit dose" or "dose" refers to a physically discrete unit suitable for a subject, each unit containing a predetermined amount of a pharmaceutical composition calculated to produce the above-mentioned desired response associated with its administration (i.e., appropriate route and treatment regimen). Depending on the number of treatments and the unit dose, the amount to be administered depends on the desired protection or effect.

[0288] The exact amount of the pharmaceutical composition also depends on the judgment of the practitioner and varies from person to person. Factors affecting dosage include the patient's physical and clinical state, route of administration, the intended goal of treatment (e.g., symptom relief rather than cure), and the potency, stability, and toxicity of the specific therapeutic substance.

[0289] V. Cell Formulation and Culture

[0290] In certain embodiments, the cells of the present disclosure may be specially formulated and / or they may be cultured in a specific medium. The cells may be formulated in a manner suitable for delivery to a recipient without causing deleterious effects.

[0291] In certain aspects, the culture medium can be prepared using a culture medium for culturing animal cells as its basal medium, such as AIM V, X-VIVO-15, NeuroBasal, EGM2, TeSR, BME, BGJb, CMRL1066, Glasgow MEM, modified MEM Zinc Option, IMDM, Medium 199, Eagle MEM, αMEM, DMEM, Ham, RPMI-1640, and Fischer medium, and any combination thereof, but the culture medium may not be particularly limited thereto, as long as it can be used for culturing animal cells. Specifically, the culture medium may be xeno-free or chemically defined.

[0292] The culture medium can be a serum-containing or serum-free medium, or a xeno-free medium. From the perspective of preventing contamination by heterogeneous animal-derived components, the serum can come from the same animal as the stem cells. Serum-free medium refers to a culture medium without unprocessed or unpurified serum, and therefore can include a culture medium with purified blood-derived components or animal tissue-derived components (e.g., growth factors).

[0293] The culture medium may or may not contain any serum replacement. Serum replacements may include materials that appropriately contain albumin (e.g., lipid-rich albumin, bovine albumin, albumin replacements (e.g., recombinant albumin or humanized albumin), plant starch, dextran, and protein hydrolyzate), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thioglycerol, or their equivalents. Serum replacements may be prepared by methods disclosed in, for example, International Publication No. 98 / 30679 (incorporated herein in its entirety). Alternatively, for greater convenience, any commercially available materials may be used. Commercially available materials include knockout serum replacement (KSR), chemically-defined Lipid concentrated (Gibco), and Glutamax (Gibco).

[0294] In certain embodiments, the culture 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 substances: 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 (triiodo-1-thyronine). In specific embodiments, one or more of these may be explicitly excluded.

[0295] In some embodiments, the culture medium also includes vitamins. In some embodiments, the culture medium includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 of the following substances (and any range that can be derived therein): biotin, DLα-tocopherol acetate, DLα-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid nicotinamide, pyridoxine, riboflavin, thiamine, inositol, vitamin B12, or the culture medium includes a combination thereof or a salt thereof. In some embodiments, the culture medium includes biotin, DLα-tocopherol acetate, DLα-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid nicotinamide, pyridoxine, riboflavin, thiamine, inositol and vitamin B12 or is substantially composed thereof. In some embodiments, vitamins include biotin, DLα-tocopherol acetate, DLα-tocopherol, vitamin A or a combination thereof or salt thereof or is substantially composed thereof. In some embodiments, the culture medium also includes protein. In some embodiments, the protein comprises albumin or bovine serum albumin, BSA fractions, catalase, insulin, transferrin, superoxide dismutase, or a combination thereof. In some embodiments, the culture medium further comprises one or more of the following substances: corticosterone, D-galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triiodo-1-thyronine, or a combination thereof. In some embodiments, the culture medium comprises one or more of the following substances: Supplements, Xeno-Free Supplement, GS21TM supplement, or a combination thereof. In some embodiments, the culture medium comprises or further comprises amino acids, monosaccharides, inorganic ions. In some embodiments, amino acids include arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine or valine, or a combination thereof. In some embodiments, inorganic ions include sodium, potassium, calcium, magnesium, nitrogen or phosphorus, or a combination or salt thereof. In some embodiments, the culture medium further comprises one or more of the following: molybdenum, vanadium, iron, zinc, selenium, copper or manganese, or a combination thereof. In certain embodiments, the culture medium comprises or is substantially composed 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, Supplements, Xeno-Free Supplement, GS21TM supplement, amino acid (for example arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine or valine), monosaccharide, inorganic ion (for example sodium, potassium, calcium, magnesium, nitrogen and / or phosphorus) or its salt and / or molybdenum, vanadium, iron, zinc, selenium, copper or manganese. In a specific embodiment, one or more of them can be explicitly excluded.

[0296] The culture medium may also contain one or more externally added fatty acids or lipids, amino acids (e.g., non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, 2-mercaptoethanol, pyruvic acid, buffers and / or inorganic salts. In a specific embodiment, one or more of these may be explicitly excluded.

[0297] One or more media components can 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.

[0298] In certain embodiments, the cells of the present disclosure are specially 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 before use, and the cell preparation may contain one or more cryopreservatives, such as DMSO (e.g., in 5% DMSO). The cell preparation may contain albumin, including human albumin, wherein a specific preparation contains 2.5% human albumin. The cells may be specially formulated for intravenous administration; for example, they are formulated for intravenous administration in less than one 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 or more from the time of thawing.

[0299] In certain embodiments, the cell of the present disclosure includes exogenous TCR, which may have a determined antigen specificity. In some embodiments, TCR may be selected based on the alloreactivity of the missing or reduced expected receptor. In the example where exogenous TCR is non-alloreactivity, during T cell differentiation, exogenous TCR suppresses the rearrangement and / or expression of endogenous TCR locus by a developmental process known as allele exclusion, causing T cells to express only non-alloreactivity exogenous TCR, therefore non-alloreactivity. In some embodiments, the selection of exogenous TCR may not necessarily be defined based on lack of alloreactivity. In some embodiments, endogenous TCR genes have been modified by genome editing so that they do not express protein. Gene editing methods (such as methods using CRISPR / Cas9 systems) are known in the art and described herein.

[0300] In some embodiments, the cells of the present disclosure further comprise one or more chimeric antigen receptors (CARs). Examples of tumor cell antigens that CARs can target 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 including ErbB2 (HER2), EGF RvIII, EGP2, EGP40, ERBB3, ERBB4, ErbB3 / 4, EPCAM, EphA2, EpCAM, folate receptor-a, FAP, FBP, fetal AchR, FR, GD2, G250 / CAIX, GD3, phosphatidylinositol glycan-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, HPVE6, 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 (eg, VEGFR2). CAR can be a first generation, second generation, third generation or more generation CAR. CAR can have bispecificity to any two different antigens, or can have specificity to more than two different antigens.

[0301] VI. Administration of the Composition

[0302] The treatment methods described herein can include monotherapy, or a combination of therapeutic agents, such as a first cancer therapy (e.g., a cell therapy) and a second cancer therapy (e.g., a general pharmaceutical composition). Therapy 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.

[0303] In some embodiments, the pharmaceutical composition is administered to a subject. Different aspects may involve administering an effective amount of a composition to a subject. In some embodiments, cell therapy (e.g., immune cells comprising one or more CARs) is administered to a subject to prevent or treat a condition (e.g., cancer). In addition, such compositions can be administered in combination with other therapeutic agents (e.g., chemotherapeutic agents, immunotherapeutic agents, biotherapeutic agents, etc.). Such compositions are typically dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

[0304] The phrases "pharmaceutically acceptable" or "pharmacologically acceptable" refer to molecular entities and compositions that do not produce adverse, allergic or other untoward reactions when administered to animals or humans. As used herein, "pharmaceutically 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 pharmaceutically active substances is well known in the art. Unless any conventional media or agents are incompatible with the active ingredient, their use in immunogenic and therapeutic compositions is contemplated. Supplementary active ingredients, such as other anti-infective agents and vaccines, may also be incorporated into the compositions.

[0305] The active compound can be formulated for parenteral administration, for example, for injection by intravenous, intramuscular, subcutaneous or intraperitoneal routes. Generally, such compositions can be prepared as liquid solutions or suspensions; solid forms suitable for adding liquids to prepare solutions or suspensions before injection can also be prepared; and the preparations can also be emulsified.

[0306] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations, including, for example, aqueous propylene glycol solutions; 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 facilitate injection. It should 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.

[0307] The protein composition can be formulated in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of proteins) and are formed with inorganic acids (e.g., hydrochloric acid or phosphoric acid) or organic acids (e.g., 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 hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and organic bases, such as isopropylamine, trimethylamine, histidine, procaine, etc.

[0308] The pharmaceutical composition may include a solvent or dispersion medium containing, for example, water, ethanol, a polyol (such as glycerol, propylene glycol and liquid polyethylene glycol, etc.), a suitable mixture thereof and vegetable oil. Suitable fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of a dispersion and by using a surfactant. The effects of microorganisms can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, isotonic agents are preferably included, such as sugar or sodium chloride. The absorption of the injectable composition can be extended by using delayed absorption agents such as aluminum monostearate and gelatin in the composition.

[0309] Sterile injection solutions are prepared by incorporating the desired amount of the active compound with the various other ingredients listed above (as required) in an appropriate solvent, followed by filtration sterilization or an equivalent procedure. Generally speaking, dispersions are prepared by incorporating various sterile active ingredients into a sterile carrier containing a basic dispersion medium and other ingredients required from those listed above. In the case of sterile powders for the preparation of sterile injection solutions, preferred preparation methods are vacuum drying and freeze drying techniques, which produce a powder of the active ingredient, plus any other required ingredients from its previously sterile filtered solution.

[0310] Administration of the composition is generally carried out by any common route. This includes, but is not limited to, oral or intravenous administration. Alternatively, administration may be carried out by in situ, intradermal, subcutaneous, intramuscular, intraperitoneal or intranasal administration. Such compositions are generally administered as pharmaceutically acceptable compositions comprising a physiologically acceptable carrier, buffer or other excipient.

[0311] After formulation, the solution will be administered in a manner compatible with the dosage formulation and in an amount effective for treatment or prevention. The preparation can be conveniently administered in a variety of dosage forms, such as the above-mentioned injection solution type.

[0312] In some embodiments, immune cells can be co-administered with one or more other therapeutic agents to treat immune-mediated diseases. Combination therapy can include, but is not limited to, one or more antimicrobial agents (e.g., antibiotics, antivirals, and antifungals), antitumor agents (e.g., monoclonal antibodies, such as rituximab, trastuzumab, etc., 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-β), hormones (e.g., for estrogen), or vaccines. In addition, immunosuppressants or tolerogenic agents may 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, trosoflavan, busulfan); radiation; or chemokines, interleukins, or inhibitors thereof (e.g., BAFF, IL-2, anti-IL-2R, IL-4, JAK kinase inhibitors). Such additional agents may be administered before, during, or after the administration of immune cells, depending on the desired effect. Administration of cells and agents may be performed by the same route or by different routes, and may be administered at the same site or at different sites.

[0313] 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 second cancer therapy is administered. In some embodiments, the first cancer therapy is administered after the second cancer therapy is administered.

[0314] Embodiments of the present disclosure relate to compositions and methods comprising therapeutic compositions. Different therapies can be administered in one composition or more than one composition, such as 2 compositions, 3 compositions, or 4 compositions. Various combinations of agents can be used.

[0315] The therapeutic agents of the present disclosure may be administered by the same route of administration or by different routes of administration. In some embodiments, cancer treatment is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. In some embodiments, antibiotics are administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. Appropriate dosage may be determined based on the type of disease to be treated, the severity and course of the disease, the clinical condition of the individual, the clinical history of the individual, and the response to treatment, as well as the judgment of the attending physician.

[0316] Treatment may include various "unit doses". A unit dose is defined as a therapeutic composition containing a predetermined amount. The amount to be administered, as well as the specific route and formulation, is determined within the skill of a person skilled in the clinical art. A unit dose need not be administered as a single injection, but may include continuous infusion over a set period of time. In some aspects, a unit dose includes a single administrable dose.

[0317] The amount to be administered according to the number of treatments and the unit dose depends on the desired therapeutic effect. An effective dose is understood to mean the amount required to achieve a specific effect. In the practice of certain embodiments, it is expected that a dose within the range of 10 mg / kg to 200 mg / kg may affect the protective ability of these agents. Thus, contemplated dosages include dosages of 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. In addition, such dosages may be administered multiple times a day, and / or may be administered over multiple days, weeks, or months.

[0318] In certain embodiments, the effective dose of the pharmaceutical composition is a dose that can provide a blood level of about 1 μM to 150 μM. In another embodiment, the effective dose 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 50 μM; or about 25 μM to 150 μM; or about 25 μM to 100 μM; or about 25 μM to 50 μM; or about 50 μM to 150 μM; or about 50 μM to 100 μM (or any range derivable therein). In other embodiments, the dose can provide a blood level of the therapeutic agent administered to a subject of about, at least about, 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, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65 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 or 100 μM or any range derivable therein. In certain embodiments, the therapeutic agent administered to the subject is metabolized in vivo to a metabolized therapeutic agent, in which case the blood level may refer to the amount of the agent. Alternatively, to the extent that the therapeutic agent is not metabolized by the subject, the blood levels discussed herein may refer to the unmetabolized therapeutic agent.

[0319] The precise amount of the therapeutic composition also depends on the judgment of the practitioner and is peculiar to each individual. Factors affecting dosage include the patient's physical and clinical state, the route of administration, the desired goal of treatment (relief of symptoms versus cure), and the potency, stability, and toxicity of the specific therapeutic substance or other therapies the subject may be receiving.

[0320] Those skilled in the art will understand and appreciate that dosage units of μg / kg or mg / kg body weight can be converted and expressed as comparable concentration units of μg / ml or mM (blood levels), e.g., 4 μM to 100 μM. It should also be understood that absorption is species and organ / tissue dependent. Applicable conversion factors and physiological assumptions regarding absorption and concentration measurements are well known and will allow those skilled in the art to convert one concentration measurement to another and make reasonable comparisons and conclusions about the dosages, efficacies, and results described herein.

[0321] In some cases, multiple administrations of the composition are required, such as 2, 3, 4, 5, 6 or more administrations. Administration can be performed at intervals of 1, 2, 3, 4, 5, 6, 7, 8 to 5, 6, 7, 8, 9, 10, 11 or 12 weeks, including all ranges therebetween.

[0322] The phrases "pharmaceutically acceptable" or "pharmacologically acceptable" refer to molecular entities and compositions that do not produce adverse, allergic or other untoward reactions when administered to animals or humans. As used herein, "pharmaceutically 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 pharmaceutically active substances is well known in the art. Unless any conventional media or agents are incompatible with the active ingredient, their use in immunogenic and therapeutic compositions is contemplated. Supplementary active ingredients, such as other anti-infective agents and vaccines, may also be incorporated into the composition.

[0323] The active compound can be formulated for parenteral administration, for example, for injection via intravenous, intrapleural, intramuscular, subcutaneous or intraperitoneal routes. Generally, such compositions can be prepared as liquid solutions or suspensions; solid forms suitable for adding liquids to prepare solutions or suspensions before injection can also be prepared; and the preparations can also be emulsified.

[0324] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations, including, for example, aqueous propylene glycol solutions; 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 facilitate injection. It should 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.

[0325] The protein composition can be formulated in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of proteins) and are formed with inorganic acids (e.g., hydrochloric acid or phosphoric acid) or organic acids (e.g., 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 hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and organic bases, such as isopropylamine, trimethylamine, histidine, procaine, etc.

[0326] The pharmaceutical composition may include a solvent or dispersion medium containing, for example, water, ethanol, a polyol (such as glycerol, propylene glycol and liquid polyethylene glycol, etc.), a suitable mixture thereof and vegetable oil. Suitable fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of a dispersion and by using a surfactant. The effects of microorganisms can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, isotonic agents are preferably included, such as sugar or sodium chloride. The absorption of the injectable composition can be extended by using delayed absorption agents such as aluminum monostearate and gelatin in the composition.

[0327] Sterile injection solutions are prepared by incorporating the desired amount of the active compound with the various other ingredients listed above (as required) in an appropriate solvent, followed by filtration sterilization or an equivalent procedure. Generally speaking, dispersions are prepared by incorporating various sterile active ingredients into a sterile carrier containing a basic dispersion medium and other ingredients required from those listed above. In the case of sterile powders for the preparation of sterile injection solutions, preferred preparation methods are vacuum drying and freeze drying techniques, which produce a powder of the active ingredient, plus any other required ingredients from its previously sterile filtered solution.

[0328] The composition is generally administered by any common route. This includes, but is not limited to, oral or intravenous administration. Alternatively, it may be administered by in situ, intradermal, subcutaneous, intramuscular, intraperitoneal or intranasal administration. Such compositions are generally administered as pharmaceutically acceptable compositions comprising a physiologically acceptable carrier, buffer or other excipient.

[0329] After formulation, the solution will be administered in a manner compatible with the dosage formulation and in an amount effective for treatment or prevention. The preparation can be conveniently administered in a variety of dosage forms, such as the above-mentioned injection solution type.

[0330] A. Chemotherapy

[0331] According to an embodiment of the present invention, a variety of chemotherapeutic agents can be used. The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapeutic agent" is used to represent a compound or composition used in the treatment of cancer. These agents or drugs are classified according to their intracellular activity mode (e.g., whether they affect the cell cycle and at what stage the cell cycle is affected). Alternatively, agents can be characterized based on their ability to directly cross-link DNA, insert DNA, or induce chromosome and mitotic aberrations by affecting nucleic acid synthesis.

[0332] Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclophosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylamelamines, including altretamine, triethylenemelamine, and melamine. , trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (particularly bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatin; callystatin; CC-1065 (including its adolesin derivatives); zelesin, carzelesin and bizelesin synthetic analogs); cryptophycin (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, naphthalene mustard, and chloramphenicol. aphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, and uracil mustard;Nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimnustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicins, particularly calicheamicin gamma II and calicheamicin omega II); dynemicins, including dynemicin A; bisphosphonates, such as clodronate; esperamicin; and neocarcinogen chromophores and related chromoprotein enediyne antibiotics. Chromophores, aclacinomysin, actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, lamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folate analogs such as leucovorin, pteropterin, and trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiopurine, and thioguanine;Pyrimidine analogs such as cyclocytidine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; adrenal antistagnation drugs such as mitotane and trilostane; folic acid supplements such as folinic acid; aceglatone; aldophosphamide glycosides; androgens such as oxadiazine, dapoxetine, dapoxetine, and dapoxetine. glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid acid; 2-ethylhydrazines; methylbenzylhydrazine; PSK polysaccharide complex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triazinon; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, mucin A, baculosporin A, and anguidine); urethan; vindesine; dacarbazine; mannomustine; dibromomannitol; dibromodulanol; pipobroman;gacytosine; cytarabine ("Ara-C"); cyclophosphamide; taxanes 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; daunorubicin; aminopterin; xeloda; ibandronic acid salts; irinotecan (e.g. CPT-11); topoisomerase inhibitor 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. ;

[0333] B. Radiotherapy

[0334] Other factors that cause DNA damage and have been widely used include the directional delivery of commonly known gamma rays, X-rays and / or radioisotopes to tumor cells. Other forms of DNA damaging factors, such as microwaves, proton beam irradiation and UV irradiation, may also be considered. Most likely, all of these factors affect the extensive damage to DNA, DNA precursors, replication and repair of DNA, and assembly and maintenance of chromosomes. The dosage range of X-rays ranges from daily doses of 50 to 200 roentgens for a long time (3-4 weeks) to single doses of 2000 to 6000 roentgens. The dosage range of radioisotopes varies widely and depends on the half-life of the isotope, the intensity and type of the irradiation emitted, and the absorption of tumor cells.

[0335] C. Immunotherapy

[0336] Those skilled in the art will appreciate that other immunotherapies may be combined or used in conjunction with the methods and compositions of the present disclosure. In the context of cancer treatment, immunotherapy generally relies on the use of immune effector cells and molecules to target and destroy cancer cells. Rituximab Is such an example.Immune effectors can be antibodies that are specific, for example, to certain markers on the surface of tumor cells. A single antibody can serve as an effector of therapy, or it can recruit other cells to actually affect cell killing. Antibodies can also be conjugated with drugs or toxins (chemotherapeutic agents, radionuclides, ricin A chains, cholera toxin, pertussis toxin, etc.), and used as targeting agents. Alternatively, effectors can be lymphocytes that carry 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.

[0337] Antibody-drug conjugates (ADCs) contain a monoclonal antibody (MAb) covalently linked to a cytocidal drug and can be used in combination therapy. This approach combines the high specificity of a MAb for its antigenic target with a highly potent cytotoxic drug, resulting in an "armed" MAb that can deliver the payload (drug) to tumor cells with abundant antigen levels. Targeted delivery of the drug also minimizes its exposure to normal tissues, thereby reducing toxicity and improving the therapeutic index. Exemplary ADC drugs include (brentuximab vedotin) and (Trastuzumab Emtansine or T-DM1).

[0338] In one aspect of immunotherapy, tumor cells must carry some markers that are easy to target, i.e., they are not present on most other cells. There are many tumor markers, and in the context of embodiments of the present invention, any of these tumor markers may be suitable for targeting. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialylated Lewis antigens, MucA, MucB, PLAP, laminin receptor, erbB and p155. An optional aspect of immunotherapy is to combine anticancer effects with immunostimulatory effects. There are also immunostimulatory molecules, including: cytokines, such as IL-2, IL-4, IL-12, GM-CSF, γ-IFN, chemokines, such as MIP-1, MCP-1, IL-8 and growth factors, such as FLT3 ligand.

[0339] Examples of immunotherapies include immune adjuvants, such as Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds); cytokine therapy, such as interferon α, β, and γ, 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 therapies may be used together with the antibody therapies described herein.

[0340] In some embodiments, the immunotherapy may be an immune checkpoint inhibitor. Immune checkpoints turn up signals (e.g., co-stimulatory molecules) or turn down signals. Inhibitory immune checkpoints that may be targeted by immune checkpoint blockade include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (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 inhibitor of T cell activation (VISTA). In particular, immune checkpoint inhibitors target the PD-1 axis and / or CTLA-4.

[0341] Immune checkpoint inhibitors can be drugs, such as small molecules, recombinant forms of ligands or receptors, or in particular antibodies, such as human antibodies. Known inhibitors of immune checkpoint proteins or their analogs can be used, in particular chimeric, humanized or human forms of antibodies can be used. As the technician will know, alternative and / or equivalent names can be used for certain antibodies mentioned in the present disclosure. In the context of the present disclosure, such alternative and / or equivalent names are interchangeable. For example, lambrolizumab is also known as alternative and equivalent names MK-3475 and pembrolizumab.

[0342] In some embodiments, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In a specific aspect, the PD-1 ligand binding partner is PDL1 and / or PDL2. In another embodiment, a PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partner. In a specific aspect, the PDL1 binding partner is PD-1 and / or B7-1. In another embodiment, a PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partner. In a specific aspect, the PDL2 binding partner is PD-1. The antagonist can be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein or an oligopeptide.

[0343] 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 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 ) is an anti-PD-1 antibody that can be used. Pembrolizumab (also known as MK-3475, Merck3475, lambrolizumab, and SCH-900475) are exemplary anti-PD-1 antibodies. 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.

[0344] Another immune checkpoint that can be targeted in the methods provided herein is cytotoxic T lymphocyte-associated protein 4 (CTLA-4), also referred to as CD152. The Genbank accession number of the complete cDNA sequence of human CTLA-4 is L15006. CTLA-4 is found on the surface of T cells and acts as a "close" switch when combined with CD80 or CD86 on the surface of antigen presenting cells. CTLA4 is a member of the immunoglobulin superfamily, which is expressed on the surface of helper T cells and transmits inhibitory signals to T cells. CTLA4 is similar to T cell co-stimulatory protein CD28, and both molecules are combined with CD80 and CD86 (also referred to as 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 present in regulatory T cells and may be important for their function. T cell activation by T cell receptors and CD28 leads to increased expression of CTLA-4 (inhibitory receptor for B7 molecules).

[0345] 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.

[0346] Anti-human-CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the methods of the invention can be produced using methods well known in the art. Alternatively, art-recognized anti-CTLA-4 antibodies can be used. Exemplary anti-CTLA-4 antibodies are ipilimumab (also known as 10D1, MDX-010, MDX-101 and ) or its antigen-binding fragments and variants. 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 the same epitope on CTLA-4 as the above-mentioned antibodies and / or binds to the same epitope on CTLA-4 as the above-mentioned antibodies. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with the above-mentioned antibodies (e.g., at least about 90%, 95% or 99% variable region identity with ipilimumab).

[0347] D. Surgery

[0348] Approximately 60% of cancer patients will undergo some type of surgery, including preventive, diagnostic or staging, curative, and palliative surgery. Curative surgery includes resection (in which all or part of the cancerous tissue is physically removed, excised, and / or destroyed) and may be used in combination with other therapies (e.g., treatment of the present embodiment, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapy). Tumor resection refers to the removal of at least a portion of a tumor. In addition to tumor resection, surgical treatments also include laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs surgery).

[0349] After removing part or all of the cancer cells, tissues or tumors, a cavity may be formed in the body. Treatment may be accomplished by perfusing, directly injecting or topically applying other anti-cancer therapies to the area. For example, this treatment may be repeated every 1, 2, 3, 4, 5, 6 or 7 days, or every 1, 2, 3, 4 and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months. These treatments may also have different dosages.

[0350] E. Other medicines

[0351] It is expected that other agents can be used in combination with certain aspects of this embodiment to improve the efficacy of treatment. These additional agents include agents that affect the upregulation of cell surface receptors and GAP connections, cell growth inhibitors and differentiation agents, cell adhesion inhibitors, agents that increase the sensitivity of overproliferation cells to apoptosis inducing agents or other biological agents. Increasing intercellular signal transduction by increasing the number of GAP connections will increase the anti-overproliferation effect on neighboring overproliferation cell groups. In other embodiments, cell growth inhibitors or differentiation agents can be used in combination with certain aspects of this embodiment to improve the anti-overproliferation efficacy of treatment. It is expected that inhibitors of cell adhesion can improve the efficacy of embodiments of the present invention. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. It is further considered that other agents (such as antibody c225) that increase the sensitivity of overproliferation cells to apoptosis can be used in combination with certain aspects of the present embodiment of the invention to improve the therapeutic efficacy.

[0352] VII. Preparation or Kit

[0353] A product or kit is provided, which comprises a composition provided herein or a method of using a composition. For example, a kit comprising one or more immune cells is also provided herein, the immune cells comprising a CAR containing a hinge and / or a transmembrane domain derived from CD30. The product or kit may also include a package insert containing instructions for using immune cells to treat or delay individual cancer progression or enhance the immune function of individuals with cancer. Any antigen-specific immune cells described herein may be included in a product or kit. Suitable containers include, for example, bottles, vials, bags, and syringes. The container may be formed of a variety of materials, such as glass, plastic (e.g., polyvinyl chloride or polyolefin) or metal alloys (e.g., stainless steel or Hastelloy). In some embodiments, the container retains the preparation and the label on or associated with the container may indicate instructions for use. The product or kit may also include other materials required from a commercial and user perspective, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. In some embodiments, the product also includes one or more other agents (e.g., chemotherapeutic agents and anti-tumor agents). Suitable containers for one or more agents include, for example, bottles, vials, bags, and syringes. Example

[0354] The following examples are included to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques that the inventors have found to work well in the practice of the present invention and therefore can be considered to constitute preferred modes of its practice. However, based on this disclosure, it will be appreciated by those skilled in the art that many changes may be made in the specific embodiments disclosed without departing from the spirit and scope of the present invention and that similar or similar results may still be obtained.

[0355] Example 1 - Creation of vectors containing various combinations of hinge domains, transmembrane domains and / or co-stimulatory domains.

[0356] DNA fragments containing different combinations of hinge, transmembrane and / or co-stimulatory domains were synthesized as Gblocks and cloned between the FMC63 scFv and CD3ζ (CD3 zeta) domains using the Gibson assembly method to construct different CARs (see Figure 1 Plasmids expressing different CARs were transfected into 293T cells using lipofectamine 3000 to screen CARs with acceptable CD19 antigen binding ability. Figure 2 Depicted is an exemplary subset of lentiviral vectors containing a CD19-targeted CAR for transduction of T cells (e.g., infinite γδ T cells) generated from healthy donor T cells. Figure 2 The general scheme shown is designed and driven by various promoters, such as MSCV, sEF1a = short (weak) EF1alpha promoter or tPGK = TetO-PGK promoter. The inventors tested more than 50 different promoter, hinge, transmembrane domain and co-stimulatory domain combinations (a subset of data is shown, additional data available on request).

[0357] Example 2 - Determination of vector expression in transduced cells

[0358] T cells were used to determine the expression of anti-CD19 CARs with different hinges and transmembrane domains. In short, T cells (e.g., infinite γδ T cells) were generated from healthy donor T cells by transduction with lentiviral vectors expressing BCL6 and BCL2L1. After the cells transduced with BCL6 and BCL2L1 were amplified for 4-6 weeks, they were transduced with lentiviral vectors expressing anti-CD19 CARs, in which the hinge (h), transmembrane (TM), and co-stimulatory (costim) domains were as follows: CD28hTM-CD28 costim, PDL1hTM-CD28costim, and CD30hTM-CD28costim. The CAR expression cassette was driven by a weak (short EF1a promoter) or a strong promoter (PDL1 hTM CAR used the MSCV promoter, and CD30 hTM CAR used the composite PGK promoter). Anti-CD19 CAR expression was determined by staining with FITC-coupled CD19 antigen. All cells were sorted using FITC-coupled CD19 antigens. Flow cytometry data were analyzed using FlowJo software. All constructs showed a clear CAR-positive population before and after sorting, but the CD30 hTMCD28 costim CAR with the TRE-PGK promoter showed the highest CD19 CAR expression (see Figure 3A-3B ). Expression was quantified and the median fluorescence intensity of the CAR-positive population of transduced cells was determined (see Figure 3A-3B ). For the two weak promoter-driven CARs, the MFI of the CD30 hTM-CD28 costim CAR population was higher than that of the CD28 hTM-CD28costim CAR before and after sorting. For the two strong promoter-driven CARs, the MFI of the CD30 hTM-CD28 costim CAR population was higher than that of the PDL1 hTM-CD28 costim CAR before and after sorting.

[0359] like Figure 6 As shown, 293T cells were used to determine the expression of anti-CD79B CARs with CD30 hinge and transmembrane domains. 293T cells were transfected with lentiviral plasmids expressing CD30hTM-CD28 costim anti-CD79BCARs - one containing scFv from the SN8 clone of CD79B antibody and the other containing scFv from the 2F2 clone of CD79B antibody. The CAR expression cassette is driven by a composite human PGK promoter. 24 hours after transfection, the expression of anti-79B CAR was determined by staining with APC-coupled CD79B antigen. Flow cytometry data were analyzed using FlowJo software. The results show that the CD30 hinge and transmembrane regions also work well with scFvs derived from non-CD19 targeting antibodies.

[0360] like Figure 7 As shown, 293T cells were used to determine the expression level of anti-CD19 CARs with different hinge and transmembrane regions. 293T cells were transfected with lentiviral plasmids expressing anti-CD19 CARs containing different hinge domains, transmembrane domains and / or co-stimulatory domains. The CAR expression cassettes were all driven by the MSCV promoter (see vector map for details). Figure 1 ). Transfection efficiency was determined using an 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 the hinge and transmembrane domains of some transmembrane receptors did not support optimal CAR expression. For example, CD79A hTM-CD28 co-stimulation, Long CTLA4 hTM-CD28 co-stimulation, and TIM3 hTM-CD28 co-stimulation all failed to show optimal CAR expression. In addition, the data showed that the entire construct with hinge and transmembrane domains and co-stimulatory domains is critical for optimal CAR expression. This is based on the observation that combining the CD28 co-stimulatory domain with PD1 or PDL1 hTM produced good CAR expression on the cell surface, but the CD28 co-stimulatory domains combined with CTLA4-, TIM3-, and CD79A-derived hTM domains, respectively, failed to show optimal CAR expression on the cell surface.

[0361] like Figures 9A-9B As shown, the expression of anti-CD19 CARs with different hinge and transmembrane domains in αβ T cells (e.g., infinite αβ T cells) was determined. Fig. 9A The results shown show that αβT cells (e.g., infinite αβT cells) generated from healthy donor T cells were successfully transduced with lentiviral vectors expressing anti-CD19 CARs with different hinge and transmembrane domains and co-stimulatory domains. All CAR expression cassettes are driven by the MSCV promoter. Anti-CD19 CAR expression was determined by staining the transduced T cells with FITC-coupled CD19 antigen. The results showed that CD30 hTM-OX40 costim CAR showed an excellent CAR-positive population, similar to CD28 HTM-CD28 costim and CD8HTM-BAFF-R costim CAR. In addition, the results showed that CAR expression of other constructs was low or absent. Fig. 9A Anti-CD19 CAR expression on transduced cells is shown in Fig. 9BQuantification was performed as shown. Quantification was determined by staining with FITC-coupled CD19 antigen. The MFI of the CD30 HTM-OX40costim CAR population was the highest of all constructs tested in this experiment. Together, these results suggest that the entire construct with the hinge and transmembrane domains and the co-stimulatory domain is important for optimal CAR folding and cell surface expression.

[0362] Example 3 - Determining the Cytotoxicity and / or Signaling Levels of Transduced Cells

[0363] The cytotoxicity of T cells containing anti-CD19 CAR and CD30hTM-CD28-CD3z against cancer cells was determined. T cells generated from healthy donor T cells (e.g., infinite γδ T cells) were transduced with a lentiviral vector expressing an anti-CD19 CAR (tPGK promoter) based on CD30hTM-CD28 costim. The CAR expression cassette is driven by a strong composite PGK promoter. After the transduced T cells were expanded, the CAR positive percentage was approximately 20% before sorting. These CAR T cells (e.g., CAR infinite γδ T cells) were co-cultured with Nalm6 cells in the presence of 200 IU exogenous IL-2 in the culture medium, and the changes in the percentage of Nalm6 cells were monitored over 3 days. The results showed that the percentage of live Nalm6 cells decreased rapidly within 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 generated from healthy donor T cells (e.g., unlimited γδ T cells) were transduced with a lentiviral vector expressing an anti-CD19 CAR with a hinge (h), transmembrane (TM), and co-stimulatory (costim) domain with the following structures: CD8hTM-CD28costim, CD28hTM-CD28 costim, PDL1hTM-CD28 costim, or CD30hTM-CD28 costim (see vector diagram Figure 2 ). After sorting, CAR-positive cells were co-cultured with Nalm6 cells expressing RFP-luciferase. TM Absolute counting beads were used to count the viable cell number of Nalm6 cells on day 0, day 1, and day 2. Figure 5A As shown, in the absence of exogenous IL-2 in the culture medium, the anti-CD19 CAR based on CD28hTM-CD28costim inhibited the proliferation of Nalm6 cells better than the anti-CD19 CAR based on CD8hTM-CD28 costim. The experiment was also performed in the culture medium with 200 IU / mL IL-2. Figure 5BAs shown, cells containing CD30hTM-CD28costim anti-CD19 CAR based on CD30hTM-CD28costim have stronger cytotoxicity than cells containing CD28hTM-CD28costim anti-CD19 CAR based on CD28hTM-CD28costim. Figure 5C As shown, cells containing anti-CD19 CAR based on CD30hTM-CD28costim were more cytotoxic than cells containing anti-CD19 CAR with PDL1hTM-CD28costim.

[0364] The cytotoxic activity of anti-CD19 CARs with CD28 or CD30 hinge and transmembrane (HTM) domains was directly compared. 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 co-stimulatory domains or CD30HTM-CD28 co-stimulatory domains, respectively, and then the cells were sorted to obtain CAR+ cells and co-cultured with Nalm6 acute lymphoblastic leukemia tumor cells expressing luciferase-RFP in duplicate wells with an effector:target ratio of 5:1. CountBright TM Absolute counting beads were used to count the viable cell number of Nalm6 cells on day 0 and day 1, as Fig.11A As shown in Figure 2, the absolute number of viable cells of Nalm6 was significantly changed. Figures 11B-11C As shown, the percentage of viable tumor cells varies depending on the hinge and transmembrane domain used. The data represent one of two independent experiments. The results show that cells containing CD19 CAR with CD30 HTM domain have significantly stronger cytotoxicity than cells containing CD19 CAR with CD28 HTM domain (P value calculated by unpaired T test).

[0365] 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 acceptable expression in 293T cells were used (see Figure 7 ) to produce a lentiviral vector, which is used to transfect Jurkat-Lucia TM NFAT reporter cell line (InvivoGen). Transfected cells 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 a 1:1 effector:target ratio. After 24 hours, luciferase activity was measured in the supernatant (see Figure 8). The results showed that while all CARs could be specifically activated by Raji cells, cells containing the CD30hTM-CD28 CAR had the highest activity. In addition, based on the activity observed in the absence of Raji cells, the PD1hTM-CD28 CAR had a more potent signaling than the other CARs.

[0366] 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 TM CAR+ cells were sorted in the NFAT reporter cell line and CAR-induced signaling was quantified by measuring luciferase activity in the absence or presence of co-culture with Daudi lymphoma cells at an effector:target ratio of 1:1. Luciferase activity was measured in the supernatant after 24 h. Fig. 10A , 10B As shown in Figures 10 and 10D, FMC63 scFv-CD30HTM-CD28costim (CD19-CD30HTM-CD28 CAR) and SN8 scFv-CD30HTM-CD28costim (CD79b-CD30HTM-CD28 CAR) only signal in the presence of cells expressing CD19 and / or CD79b (e.g., Daudi tumor cells). TM NFAT reporter cells were also transduced with an Fc receptor CAR (FcR CAR-CD16V-CD30HTM-CD28) (see Fig. 10C ), data showed that FcR CAR only signaled in the presence of rituximab (an anti-CD20 antibody) and Daudi tumor cells, but not when co-cultured with Daudi tumor cells alone. Fig.10E As shown, Jurkat-Lucia TM NFAT reporter cells were also transduced with FMC63 scFv-CD30HTM-CD28 costim (CD19-CD30HTM-CD28CAR) and exposed to a high-grade B-cell lymphoma cell line (PDX 203 lymphoma cells), and the data showed that the CD19-CD30HTM-41BB CAR only signaled in the presence of tumor cells. Overall, these results suggest that the CD30 HTM domain functions as an effective HTM component in the context of multiple CAR designs targeting different antigens on tumor cells and / or different costimulatory domains.

[0367] like Figures 12A-12BAs shown, the in vivo tumor cell control capacity of anti-CD19 CD30 HTM CAR-transduced unlimited γδ T cells was determined. 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. On days 0, 3, and 8, 8×10 6 Unlimited γδT or unlimited anti-CD19 CD30HTM-CD28CosCAR-γδT were injected into mice at a dose of 1 T cell / mouse / injection. The data showed that CD30HTM-CD28Cos CAR-γδT could slow the progression of lymphoma. Tumor burden was assessed by bioluminescence imaging ( Fig. 12A ), and the average total flux for each group is calculated and displayed ( Fig. 12B ). The data showed that the anti-tumor effect of infinite γδCAR T cells containing CD30 HTM was higher than that of infinite γδT cells without CAR.

[0368] like Figures 13A-13B As shown, a CD70-binding CAR comprising a truncated CD27 (tCD27) CAR having the CD30 hinge and transmembrane domains was successfully transduced, expressed, induced signals, and bound to CD70. Fig.13A Shown is how to make a CD27-based anti-CD70 CAR by fusing a truncated CD27 extracellular domain (SEQ ID NO: 52 encoding SEQ ID NO: 48) to the CD30 hinge and TM domains, the CD28 co-stimulatory domain, and the CD3z signaling domain. TM The signal transduction capacity was determined by using a NFAT reporter cell line (Invivogen). The lentiviral vector expressing the CAR construct (SEQ ID NO: 49) was transduced into Jurkat-Lucia TM CAR+ cells were sorted in the NFAT reporter cell line and CAR-induced signaling was quantified by measuring luciferase activity in cells co-cultured or not with a CD70-positive T cell line (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 signals in the presence of cells expressing CD70. The results suggest that the CD30 HTM domain can function as an effective HTM component in the context of multiple CAR designs targeting different antigens on tumor cells and / or different co-stimulatory domains. RLU = relative light unit. Fig. 13BThe above-mentioned lentiviral vector (tCD27-CD30HTM-CD28cos-CD3z CAR) is shown to be transduced into primary human T cells. Ten days after transduction, the 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 recombinant CD70 protein coupled with a fluorescent dye. Untransduced T cells and low-affinity anti-CD70scFv antibodies (clone 1F6) were used as controls. The anti-CD27 antibody staining results showed that tCD27 was properly folded and expressed on the surface of primary T cells (upper row). The CD70 protein staining results showed that tCD27 CAR bound to recombinant CD70 protein (middle row). Anti-CD70 antibody staining showed that CD70+ cells were almost absent in T cells transduced with tCD27 CAR, indicating that cells expressing CD70 were effectively eliminated, or that CD70 on the cell surface was masked by cis binding (bottom row).

[0369] Together, these results suggest that the CD30 HTM domain functions as a potent HTM component with diverse CAR designs targeting different antigens on tumor cells in vitro and in vivo.

[0370] ***

[0371] All of the methods disclosed and claimed herein may 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 changes may be made to the methods and the steps or order 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 reagents of both chemical and physiological relevance may be substituted for the reagents described herein and the same or similar results will be obtained. 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.

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Claims

1. A chimeric antigen receptor (CAR), comprising: i) antigen binding domain; ii) CD30 hinge domain; iii) CD30 transmembrane domain; iv) at least one intracellular co-stimulatory domain; and v) intracellular stimulatory domain; wherein the CD30 hinge domain comprises less than 51 consecutive amino acids and at least 7 consecutive amino acids of the extracellular domain of CD30; and The CD30 transmembrane domain comprises no more than 27 consecutive amino acids of CD30.

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

3. The CAR of claim 1 or 2, wherein the CD30 hinge is at least 80%, 85%, 90%, 95% or 98% identical to SEQ ID NO:

3.

4. The CAR of any one of claims 1-3, wherein the CD30 transmembrane domain is at least 80%, 85%, 90% or 95% identical to SEQ ID NO:

4.

5. The CAR of any one of claims 1-4, wherein the CD30 hinge domain and / or transmembrane domain lacks 3 or more consecutive amino acids according to SEQ ID NO:

6.

6. The CAR of any one of claims 1-5, wherein the CD30 hinge domain comprises SEQ ID NO:

3.

7. The CAR of any one of claims 1-6, wherein the CD30 transmembrane domain comprises SEQ ID NO:

4.

8. The CAR of any one of claims 1-7, wherein the CD30 domain and the 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.

9. The CAR of any one of claims 1-8, wherein the antigen binding domain targets CD19, CD20, CD22, CD70, CD79B, CD79A, ROR1, BCMA, BAFF receptor, GD2 or claudin18.

2.

10. The CAR of claim 9, wherein the antigen binding domain targets CD19, CD79B and / or CD70.

11. The CAR of any one of claims 1-9, wherein at least one intracellular co-stimulatory domain comprises a CD8, CD27, CD28, CD30, CD3ε, CD3ζ, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 (OX40), CD137 (4-1BB) or CD154 intracellular co-stimulatory domain.

12. The CAR of claim 11, wherein at least one intracellular co-stimulatory domain comprises CD28.

13. The CAR of any one of claims 1-12, wherein the intracellular stimulatory domain comprises DAP12, DAP10, FCER1G (Fcε receptor Iγ chain), CD3δ (CD3 delta), CD3ε (CD3 epsilon), CD3γ (CD3 gamma), CD3ζ (CD3zeta) or CD79A intracellular stimulatory domain.

14. The CAR of claim 13, wherein the intracellular stimulatory domain comprises CD3ζ (CD3zeta).

15. The CAR of any one of claims 1-14, 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%, 90%, 95%, 99% or 100% identical to SEQ ID NO:

2.

16. A method for producing cells, comprising transducing and / or transfecting cells with a CAR according to any one of claims 1-15.

17. A cell produced according to claim 16.

18. The cell of claim 17, wherein the cell is a T cell or a NK cell.

19. The cell of claim 18, wherein the cell is a γδ T cell.

20. The cell of any one of claims 17-19, wherein the cell comprises a BCL6 gene and at least one additional transgene encoding an immunomodulatory gene.

21. The cell of claim 20, wherein the at least one additional transgene encoding an immunomodulatory gene is a Bcl2 family gene.

22. The cell of any one of claims 17 to 21, wherein the cell comprises at least one man-made mutation in an endogenous gene, and / or at least one heterologous nucleic acid capable of altering the expression of at least one endogenous gene.

23. The cell of claim 22, wherein the endogenous gene is an immunomodulatory gene.

24. A composition comprising the cell of any one of claims 17-23.

25. A method of treating a blood cancer in a patient comprising administering to the patient the cell of any one of claims 17-23 or the composition of claim 24.

26. The method of claim 25, further comprising administering to the subject at least one second therapeutic agent.

27. The method of claim 26, wherein the at least one second therapeutic agent comprises chemotherapy, immunotherapy, surgery, radiation therapy, drug therapy, targeted therapy, hormone therapy, biological therapy, or a combination thereof.

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