Anti-CD19 and anti-CD79b chimeric antigen receptors and methods of use thereof

Abstract

Aspects of the present disclosure describe dual chimeric antigen receptor (CAR) immune cells and methods for making such cells and for using in the treatment of cancer.Disclosed are immune cells that contain both anti-CD19 CAR and anti-CD79b CAR and methods for using in the treatment of B-cell malignancies.Also disclosed are polynucleotides that code for anti-CD19 CAR and anti-CD79 CAR, as well as cells that contain such polynucleotides, and pharmaceutical compositions that contain such cells. TIFF2025508961000021.tif31170

Description

[Technical field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 316,311, filed March 3, 2022. U.S. Provisional Patent Application No. 63 / 316,311 is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application has been filed electronically in accordance with ST.26 format and contains a Sequence Listing, which is incorporated herein by reference in its entirety. The Sequence Listing was created on Mar. 3, 2023, is named MDAC.P1328WO.xml, and is 87,011 bytes in size.

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

[0004] II. Background Recent studies have demonstrated that CD19-targeted chimeric antigen receptor (CAR)-modified T cell therapy induces high response rates in the majority of patients with refractory B cell malignancies, including follicular lymphoma (FL), mantle cell lymphoma (MCL), diffuse large B cell lymphoma (DLBCL), and chronic lymphocytic leukemia (CLL). 1 Some of these patients experience long-term remission, suggesting that adoptive T-cell therapy may be effective in patients resistant to existing therapies. However, >50% of patients relapse or progress after CD19 CAR T-cell therapy. The main cause of failure appears to be due to loss of CD19 expression on tumor cells. 2,3 Thus, there is a need to develop CAR immune cell (e.g., CAR T cell) therapies that minimize immune escape and improve efficacy compared to existing CAR T cell therapies. Summary of the Invention

[0005] overview In some aspects, dual CAR immune cells that target at least CD19 and CD79b and methods of use thereof are described herein, including methods of use for treating cancer. Also disclosed are polynucleotides encoding anti-CD19 CAR and anti-CD79b CAR, and methods for making engineered immune cells that contain such polynucleotides. In some aspects, the polynucleotides of the present disclosure are or are part of a bicistronic vector that expresses both anti-CD19 CAR and anti-CD79b CAR. In some cases, the anti-CD19 CAR and anti-CD79b CAR are separated by one or more elements so that the anti-CD19 CAR and anti-CD79b CAR are expressed as separate molecules. In certain embodiments, the element is a self-cleaving peptide, such as a 2A element. Further aspects of the present disclosure are described herein.

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

[0007] In some aspects, disclosed herein is a polynucleotide comprising: (a) a first sequence encoding an anti-CD19 chimeric antigen receptor (CAR), wherein the anti-CD19 CAR comprises: (i) GM-CSF receptor alpha signal peptide; (ii) CD19 binding domain; (iii) CD8α hinge region; (iv) CD8α transmembrane domain; (v) 4-1BB signaling domain; and (vi) CD3ζ signaling domain; and (b) a second sequence encoding an anti-CD79b CAR, wherein the anti-CD79b CAR comprises: (i) CD8α signal peptide; (ii) CD79b binding domain; (iii) CD8α hinge region; (iv) CD8α transmembrane domain; (v) OX40 signaling domain; and (vi) CD3ζ signaling domain.In some aspects, the first sequence is 5' to the second sequence. In some aspects, the polynucleotide further comprises a third sequence encoding a T2A peptide. In some aspects, the third sequence is 3' to the first sequence and 5' to the second sequence. In some aspects, the T2A peptide comprises SEQ ID NO:9.

[0008] In some aspects, the GM-CSF receptor signal peptide comprises SEQ ID NO:1. In some aspects, the CD19 binding domain is an scFv of an anti-CD19 antibody. In some aspects, the CD19 binding domain comprises a VL having SEQ ID NO:2 and a VH having SEQ ID NO:4. In some aspects, the CD19 binding domain further comprises a linker connecting the VL and VH. In some aspects, the linker comprises SEQ ID NO:3. In some aspects, the CD8α hinge region of (a) comprises SEQ ID NO:5. In some aspects, the CD8α transmembrane domain of (a) comprises SEQ ID NO:6. In some aspects, the 4-1BB signaling domain comprises SEQ ID NO:7. In some aspects, the CD3ζ signaling domain of (a) comprises SEQ ID NO:8.

[0009] In some aspects, the CD8α signal peptide comprises SEQ ID NO:10. In some aspects, the CD79b binding domain is an scFv of an anti-CD79 antibody. In some aspects, the CD79b binding domain comprises a VL having SEQ ID NO:11 and a VH having SEQ ID NO:13. In some aspects, the CD79b binding domain further comprises a linker connecting the VL and the VH. In some aspects, the CD79b binding domain comprises a linker having SEQ ID NO:12. In some aspects, the CD8α hinge region of (b) comprises SEQ ID NO:5. In some aspects, the CD8α transmembrane domain of (b) comprises SEQ ID NO:6. In some aspects, the OX40 signaling domain comprises SEQ ID NO:14. In some aspects, the CD3ζ signaling domain of (b) comprises SEQ ID NO:8.

[0010] In some aspects, the polynucleotide comprises a sequence encoding a polypeptide having SEQ ID NO:15. In some aspects, the polynucleotide comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity to SEQ ID NO:30, or any range or value derivable therein. In some aspects, the polynucleotide further comprises a promoter. In some aspects, the promoter is an EF1α promoter.

[0011] Further disclosed is a vector comprising the polynucleotide disclosed herein.Also provided is a polypeptide encoded by the polynucleotide or vector disclosed herein.Further described is a method for producing an engineered cell, comprising introducing the polynucleotide, vector or polypeptide disclosed herein into a cell.

[0012] In some aspects, engineered cells are also disclosed, comprising the polynucleotide, vector, or polypeptide disclosed herein. In some aspects, the engineered cells are T cells. In some aspects, the engineered cells are natural killer (NK) cells. Also contemplated herein is a population of engineered cells, comprising the engineered cells disclosed herein. In some aspects, at least 35% of the engineered cells in the population express both anti-CD19 CAR and anti-CD79b CAR. In some aspects, the population of engineered cells includes a subset that expresses either anti-CD19 CAR or anti-CD79b CAR, and at least 80% of the subset expresses both anti-CD19 CAR and anti-CD79b CAR. Also contemplated is a pharmaceutical composition, comprising a population of engineered cells disclosed herein.

[0013] In some aspects, a method for treating a subject for cancer is also described, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition disclosed herein (e.g., a pharmaceutical composition comprising a population of engineered cells, such as dual CAR immune cells). In some aspects, the subject has a B-cell malignancy. In some aspects, the subject has 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, or chronic lymphocytic leukemia. In some aspects, the subject has CD19 + CD79b + Having cancer.

[0014] In some aspects, polynucleotides encoding a polypeptide having SEQ ID NO: 15 are also disclosed. In some aspects, the polynucleotides comprise a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity to SEQ ID NO: 30, or any range or value derivable therein. In some aspects, the polynucleotide comprises SEQ ID NO: 30.

[0015] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for any measuring or quantitating method.

[0016] The use of the words "a" and "an," when used in conjunction with the term "comprising," can mean "one," but is also consistent with the meanings "one or more," "at least one," and "one or more than one."

[0017] The phrase "and / or" means "and" or "or." By way of example, A, B, and / or C includes A only, B only, C only, the combination of A and B, the combination of A and C, the combination of B and C, or the combination of A, B and C. In other words, "and / or" operates as an inclusive or.

[0018] The words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (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.

[0019] The compositions and methods for using the compositions may "comprise," "consist essentially of," or "consist of" any of the components or steps disclosed throughout this specification. Compositions and methods "consisting essentially of" any of the components or steps disclosed limit the claims to the specified materials or steps that do not materially affect the basic and novel characteristics of the invention.

[0020] It is contemplated that any embodiment discussed herein can be practiced with any method or composition of the invention, and vice versa. Further, a composition of the invention can be used to practice a method of the invention.

[0021] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating particular embodiments of the present invention, are given by way of illustration only, since various modifications and changes within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief description of the drawings]

[0022] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0023] [Figure 1] Figures 1A-1B show an exemplary dual CD19-CD79b CAR construct and expression analysis. Figure 1A shows a schematic of a dual CD19-CD79b CAR construct consisting of an anti-CD19 CAR and an anti-CD79b CAR cloned into a third generation lentiviral vector under the control of the EF1α promoter. A T2A self-cleaving peptide links the two CARs, facilitating their simultaneous expression. Figure 1B shows the expression of CD19 and CD79b CARs on T cells transduced with the dual CAR construct shown in Figure 1A. This expression was assessed by staining with FITC-conjugated CD19 protein, which binds to the CD19 scFv, and PE-conjugated CD79b protein, which binds to the CD79b scFv. [Figure 2A] Figures 2A-2G show results demonstrating that dual CD19-CD79b CAR T cells degranulate in a CD19- or CD79b-dependent manner. Figure 2A shows that dual CD19-CD79b CD4+ CAR T cells degranulate in response to CD19+CD79b+ tumor cells. Untransduced T, CD19 CAR T, and dual CD19-CD79b CAR T were co-cultured with B-cell lymphoma cell lines (CD19+CD79b+: Daudi, SUDHL6, PDX203, and PDX300) at a 1:2 effector:target (E:T) ratio for 6 hours. Flow cytometry plots show high CD107a expression in the CD4+ subset of CD19 CAR and dual CD19-CD79b CAR T cells. [Figure 2B]Figures 2A-2G show results demonstrating that dual CD19-CD79b CAR T cells degranulate in a CD19- or CD79b-dependent manner. Figure 2B shows that dual CD19-CD79b CD8+ CAR T cells degranulate in response to CD19+CD79b+ tumor cells. The experiment was set up as described for Figure 2A to show CD107a expression in the CD8+ subset. [Figure 2C] Figures 2A-2G show results demonstrating that dual CD19-CD79b CAR T cells degranulate in a CD19- or CD79b-dependent manner. Figure 2C shows that dual CD19-CD79b CAR T cells degranulate in a CD19-dependent manner. Untransduced T, CD19 CAR T, and dual CD19-CD79b CAR T cells were co-cultured with a B-cell leukemia cell line, NALM6, which is CD19+CD79b-, at a 1:1 effector:target (E:T) ratio for 6 hours. CD107a expression in CD4+ and CD8+ T cells was shown. [Figure 2D] Figures 2A-2G show results demonstrating that dual CD19-CD79b CAR T cells degranulate in a CD19- or CD79b-dependent manner. Figure 2D shows that dual CD19-CD79b CAR T cells degranulate in a CD79b-dependent manner. Untransduced T, CD19 CAR T, and dual CD19-CD79b CAR T cells were co-cultured with SUDHL6-CD19KO cell line at a 1:1 effector:target (E:T) ratio for 6 hours. CD107a expression in CD4+ and CD8+ T cells was shown. [Figure 2E] Figures 2A-2G show results demonstrating that dual CD19-CD79b CAR T cells degranulate in a CD19- or CD79b-dependent manner. Figure 2E shows that dual CD19-CD79b CAR T cells do not respond to CD19-CD79b- tumor cells. Untransduced T, CD19 CAR T, and dual CD19-CD79b CAR T cells were co-cultured with CD19-CD79b-K562 tumor cells at a 1:1 effector:target (E:T) ratio for 6 hours, and CD107a expression was assessed by flow cytometry. [Figure 2F]Figures 2A-2G show results demonstrating that dual CD19-CD79b CAR T cells degranulate in a CD19- or CD79b-dependent manner. Figure 2F shows that cryopreserved and thawed dual CD19-CD79b CAR T cells degranulate in a CD79b-dependent manner. Thawed untransduced T cells, CD19 CAR T cells, CD79b CAR T cells, and CD19-CD79b dual CAR T cells were co-cultured with CD19KO B-cell lymphoma tumor cells (Daudi-CD19KO and SUDHL6-CD19KO) at a 2:1 effector:target (E:T) ratio for 6 hours, and CD107a expression was assessed by flow cytometry. [Figure 2G] Figures 2A-2G show results demonstrating that dual CD19-CD79b CAR T cells degranulate in a CD19- or CD79b-dependent manner. Figure 2G shows that cryopreserved and thawed dual CD19-CD79b CAR T cells degranulate in response to CD19+CD79b+ Daudi tumor cells. Thawed untransduced T cells, CD19 CAR T cells, CD79b CAR T cells, and CD19-CD79b dual CAR T cells were co-cultured with Daudi tumor cells at a 1:1 effector:target (E:T) ratio for 6 hours, and CD107a expression was assessed by flow cytometry. [Figure 3A] Figures 3A-3I show results demonstrating that dual CAR T cells mediate specific lysis of tumor cells in a CD19- or CD79b-dependent manner. Figure 3A shows that dual CAR T cells induced lysis of CD19+CD79b+ tumors. Untransduced T, CD19 CAR T, and dual CD19-CD79b CAR T cells were co-cultured with CD19+CD79b+ B-cell lymphoma cell lines (Daudi, SUDHL6, PDX203, and PDX300) at a 1:1 effector:target (E:T) ratio for 96 hours. The percentage of tumor viable cells at days 0 and 4 is shown for each co-culture condition. [Figure 3B]Figures 3A-3I show results demonstrating that dual CAR T cells mediate specific lysis of tumor cells in a CD19- or CD79b-dependent manner. Figure 3B shows that dual CAR T cells induced lysis of CD19+CD79b- tumor cells. Untransduced T and dual CD19-CD79b CAR T cells were co-cultured with a CD19+CD79b- B-cell leukemia cell line (NALM6) at a 1:1 effector:target (E:T) ratio for 96 hours. CD19 CAR T cells were co-cultured with NALM6 at a 2:1 (E:T) ratio. The percentage of tumor viable cells at days 0 and 4 is shown for each co-culture condition. [Figure 3C] Figures 3A-3I show results demonstrating that dual CAR T cells mediate specific lysis of tumor cells in a CD19- or CD79b-dependent manner. Figure 3C shows that dual CAR T cells induce specific cytotoxicity against CD19-CD79b+ tumors. Untransduced T, CD19 CAR T, and dual CD19-CD79b CAR T were co-cultured with CD19KO B-cell lymphoma cell lines (CD19-CD79b+: Daudi-CD19KO and SUDHL6-CD19KO) at a 1:1 effector:target (E:T) ratio, respectively, for 96 hours. The percentage of tumor viable cells at days 0 and 4 is shown for each co-culture condition. [Figure 3D] Figures 3A-3I show results demonstrating that dual CAR T cells mediate specific lysis of tumor cells in a CD19- or CD79b-dependent manner. Figure 3D shows that dual CAR T cells did not exhibit cytotoxicity against CD19-CD79b- tumor cells. Untransduced T, CD19 CAR T, and dual CD19-CD79b CAR T cells were co-cultured with CD19-CD79b-K562 tumor cell line at a 1:1 effector:target (E:T) ratio for 96 hours. The percentage of tumor viable cells at days 0 and 4 is shown for each co-culture condition. [Figure 3E]Figures 3A-3I show results demonstrating that dual CAR T cells mediate specific lysis of tumor cells in a CD19- or CD79b-dependent manner. Figure 3E shows results from an experiment in which non-transduced T, CD19 CAR T, and dual CD19-CD79b CAR T cells were co-cultured with the CD19+CD79b+ Daudi tumor cell line at a 1:1 effector:target (E:T) ratio for 24, 48, and 96 hours. Percent specific lysis was calculated based on the absolute number of tumor viable cells and normalization to non-transduced T cells (% specific lysis = 1-[target viable cells with CAR T cells / target viable cells with non-transduced T cells). [Figure 3F] Figures 3A-3I show results demonstrating that dual CAR T cells mediate specific lysis of tumor cells in a CD19- or CD79b-dependent manner. Figure 3F shows results from an experiment in which untransduced T, CD19 CAR T, and dual CD19-CD79b CAR T cells were co-cultured with the CD19+CD79b+SUDHL6 tumor cell line at a 1:1 effector:target (E:T) ratio for 24 h, 48 h, and 96 h. Percent specific lysis was calculated as described for Figure 3E. [Figure 3G] Figures 3A-3I show results demonstrating that dual CAR T cells mediate specific lysis of tumor cells in a CD19- or CD79b-dependent manner. Figure 3G shows results from an experiment in which untransduced T, CD19 CAR T, and dual CD19-CD79b CAR T cells were co-cultured with the CD19+CD79b-NALM6 tumor cell line at a 1:1 effector:target (E:T) ratio for 24 h, 48 h, and 96 h. Percent specific lysis was calculated as described for Figure 3E. [Figure 3H]Figures 3A-3I show results demonstrating that dual CAR T cells mediate specific lysis of tumor cells in a CD19- or CD79b-dependent manner. Figure 3H shows results from an experiment in which untransduced T, CD19 CAR T, and dual CD19-CD79b CAR T cells were co-cultured with the CD19-CD79b+Daudi-CD19KO tumor cell line at a 1:1 effector:target (E:T) ratio for 24 h, 48 h, and 96 h. Percent specific lysis was calculated as described for Figure 3E. [Figure 3I] Figures 3A-3I show results demonstrating that dual CAR T cells mediate specific lysis of tumor cells in a CD19- or CD79b-dependent manner. Figure 3I shows results from an experiment in which untransduced T, CD19 CAR T, and dual CD19-CD79b CAR T cells were co-cultured with CD19-CD79b+SUDHL6-CD19KO tumor cell lines at a 1:1 effector:target (E:T) ratio for 24 h, 48 h, and 96 h. Percent specific lysis was calculated as described for Figure 3E. [Figure 4A] Figures 4A-4H show results demonstrating that dual CD19-CD79b CAR T cells proliferate in response to B cell leukemia or lymphoma tumor cells in a CD19- or CD79b-dependent manner. Untransduced T, CD19 CAR T, and dual CD19-CD79b T cells labeled with Cell Trace™ Far Red were co-cultured with B cell lymphoma or leukemia tumor cells, Daudi (Figure 4A), SUDHL6 (Figure 4B), PDX203 (Figure 4C), PDX300 (Figure 4D), NALM6 (Figure 4E), Daudi-CD19KO (Figure 4F), SUDHL6-CD19KO (Figure 4G), and K562 (Figure 4H), at a 1:1 effector:target (E:T) ratio for 96 hours. On day 4, the percentage of proliferating T cells was determined based on the dilution of the dye and is shown in each plot. [Figure 4B] See legend to Figure 4A. [Figure 4C] See legend to Figure 4A. [Figure 4D] See legend to Figure 4A. [Figure 4E]See legend to Figure 4A. [Figure 4F] See legend to Figure 4A. [Figure 4G] See legend to Figure 4A. [Figure 4H] See legend to Figure 4A. [Figure 5A] Figures 5A-5B show the results from the design of an exemplary dual CD19 / CD79b CAR construct and the transfection efficiency in 293T cells. The percentage of 293T cells expressing CD19 CAR or CD79b CAR or both is shown. Figure 5B shows the results from the design of an exemplary dual CD19 / CD79b CAR construct and the transduction efficiency in primary T cells. The percentage of primary T cells expressing CD19 CAR or CD79b CAR or both is shown. [Figure 5B] See legend to Figure 5A. [Figure 6A] Figures 6A-6C show results demonstrating that CD19-CD79b dual CAR T cells produced effector cytokines in response to B cell lymphoma and leukemia cell lines in a CD19- or CD79b-dependent manner. Dual CD19-CD79b CAR T cells specifically recognized and released IFN-γ (Figure 6A), IL-2 (Figure 6B), and TNF-α (Figure 6C) in response to the CD19+CD79b+ B cell lymphoma cell lines Daudi, SUDHL6, PDX203-5D4, and PDX300-5E6, the CD19+CD79b- B cell leukemia cell line NALM6, and the CD19-CD79b+ Daudi-CD19KO and SUDHL6-CD19KO lymphoma cell lines, but not in response to CD19-CD79b-K562 tumor cells. CD19 CAR T cells were used as a control. In Figures 6A and 6B, there is no detectable untransduced (UTD), and the left bar in the set of two bars is the CD19 CAR. In Figure 6C, the UTD (if present) is the leftmost bar in the set of three bars. The CD19-CD79b CAR is the rightmost bar in the set of three bars. [Figure 6B]See legend to Figure 6A. [Figure 6C] See legend to Figure 6A. [Figure 7A] Figures 7A-7C show results demonstrating that CD19-CD79b dual CAR T cells have potent antitumor activity in vivo in a NALM6 CD19+CD79b- cell line xenograft model. NOD.Cg-PrkdcscidIl2rgtm1Wjl / SzJ (NSG) mice were intravenously injected with 0.2x106 firefly luciferase-expressing NALM6 tumor cells that are CD19+CD79b-. Four days later, 5x106 CAR+ cells per mouse of CD19 CAR T or CD19-CD79b dual CAR T cells and corresponding non-transduced T cells were injected into tumor-bearing mice via tail vein injection. Tumor burden was measured by bioluminescence imaging on the indicated days (Figure 7A) and quantified by total flux (n=5 mice / group, significance between treatment groups was determined by Mann-Whitney test, p=ns, **, p<0.01) (Figure 7B); CD19 CAR and CD19-CD79b CAR lines are along the x-axis). Kaplan-Meier survival curves for mice treated with non-transduced T, CD19 CAR T, and CD19-CD79b dual CAR T (n=5 mice / group). Significance between treatment groups was determined by log-rank (Mantel-Cox) test (p=ns, **, p<0.01) (Figure 7C); CD19 CAR and CD19-CD79b CAR lines are along the top of the image). [Figure 7B] See legend to Figure 7A. [Figure 7C] See legend to Figure 7A. [Figure 8]Figure 8 shows results demonstrating that CD19-CD79b dual CAR T cells have potent antitumor activity in vivo in a patient-derived xenograft model with PDX203-5D4 CD19KO, which is CD19-CD79b+. NOD.Cg-PrkdcscidIl2rgtm1Wjl / SzJ (NSG) mice were intravenously injected with 0.2x106 firefly luciferase-expressing PDX203-5D4 CD19KO cells. 15 days later, tumor-bearing mice were injected via tail vein with 3x106 CAR+ cells per mouse of CD19 CAR T, CD79b CAR T, or CD19-CD79b dual CAR T cells and corresponding non-transduced T cells. Tumor burden was assessed by bioluminescence imaging on days 0, 7, 14, and 21. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] Detailed Description Together with CD19, CD79b is a pan-B cell antigen that is expressed at high levels in 95-100% of most B cell lymphomas, including DLBCL, FL, MCL, Burkitt lymphoma, marginal zone lymphoma, and lymphoplasmacytic lymphoma. 11-15 CD79b expression is also high in hairy cell leukemia, but is reduced in intensity in chronic lymphocytic leukemia (CLL) compared with normal B cells. 11 Thus, in certain aspects, the disclosure provides dual chimeric antigen receptor (CAR) immune cells (e.g., dual CAR T cells) that target CD19 and CD79b, and methods of their use in treating B cell malignancies, such as malignancies that express CD19 and CD79b, including diffuse large B cell lymphoma, high-grade B cell lymphoma, follicular lymphoma, marginal zone lymphoma, mantle cell lymphoma, Burkitt's lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia, and chronic lymphocytic leukemia. Aspects of the disclosure include polynucleotides encoding anti-CD19 CARs and anti-CD79b CARs, as well as methods for using such polynucleotides in the generation of dual CAR T cells.

[0025] I. Polypeptides As used herein, a "protein" or "polypeptide" refers to a molecule that comprises at least five amino acid residues. As used herein, the term "wild type" refers to the endogenous version of a molecule that occurs naturally in an organism. In some embodiments, a wild type version of a protein or polypeptide is used. However, in many embodiments of the present disclosure, modified proteins or polypeptides are used to generate an immune response. The above terms may be used interchangeably. "Modified protein" or "modified polypeptide" or "variant" refers to a protein or polypeptide whose chemical structure, particularly its amino acid sequence, has been altered relative to the wild type protein or polypeptide. In some embodiments, a modified / variant protein or polypeptide has at least one modified activity or function (recognizing that a protein or polypeptide may have multiple activities or functions). It is specifically contemplated that a modified / variant protein or polypeptide may be altered in some activities or functions but retain wild type activities or functions in other respects.

[0026] When a protein is specifically mentioned herein, this generally refers to a natural (wild type) or recombinant (modified) protein, or a protein that, optionally, has had any signal sequence removed. A protein may be directly isolated from an organism, the protein may be natural and produced by recombinant DNA / exogenous expression methods, or may be produced by solid phase peptide synthesis (SPPS) or other in vitro methods. In certain embodiments, there are isolated nucleic acid segments and recombinant vectors that incorporate a nucleic acid sequence that encodes a polypeptide (e.g., an antibody or fragment thereof). The term "recombinant" may be used in conjunction with a polypeptide or the name of a particular polypeptide, generally referring to a polypeptide produced from a nucleic acid molecule that has been manipulated in vitro or is the product of replication of such a molecule.

[0027] In certain embodiments, the size of a protein or polypeptide (wild type or modified) is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 108, 109, 109, 108, 110, 111, 112, 113, 114, 115, 116, 117, 1 5 pieces, 46 pieces, 47 pieces, 48 ​​pieces, 49 pieces, 50 pieces, 51 pieces, 52 pieces, 53 pieces, 54 pieces, 55 pieces, 56 pieces, 57 pieces, 58 pieces, 59 pieces, 60 pieces, 61 pieces, 62 pieces, 63 pieces, 64 pieces, 65 pieces, 66 pieces, 67 pieces, 68 pieces, 69 pieces , 70 pieces, 71 pieces, 72 pieces, 73 pieces, 74 pieces, 75 pieces, 76 pieces, 77 pieces, 78 pieces, 79 pieces, 80 pieces, 81 pieces, 82 pieces, 83 pieces, 84 pieces, 85 pieces, 86 pieces, 87 pieces, 88 pieces, 89 pieces, 90 pieces, 91 pieces, 92 pieces, 93 pieces, 9 4 pieces, 95 pieces, 96 pieces, 97 pieces, 98 pieces, 99 pieces, 100 pieces, 110 pieces, 120 pieces, 130 pieces, 140 pieces, 150 pieces, 160 pieces, 170 pieces, 180 pieces, 190 pieces, 200 pieces, 210 pieces, 220 pieces, 230 pieces, 240 pieces, 250 pieces, 275 pieces, 300 pieces, 325 pieces, 350 pieces, 375 pieces, 400 pieces, 425 pieces, 450 pieces, 475 pieces, 500 pieces, 525 pieces, 550 pieces, 575 pieces, 600 pieces, 625 pieces, 650 pieces, 675 pieces, 700 pieces, 725 The polypeptides may include, but are not limited to, 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 also contemplated that the polypeptides may be mutated by truncation that renders the polypeptide shorter than the corresponding wild type, and may also be altered by fusing or conjugating heterologous proteins or polypeptide sequences having a particular function (e.g., for targeting or localization, for enhanced immunogenicity, purification purposes, etc.).The term "domain" as used herein refers to any distinct functional or structural unit of a protein or polypeptide, and generally refers to a sequence of amino acids having a structure or function recognizable by one of skill in the art.

[0028] Polypeptides, proteins of the disclosure, or polynucleotides encoding such polypeptides or proteins, may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (or any range derivable therein) or more variant amino acid or nucleic acid substitutions, or may be a variant amino acid or nucleic acid substitution of any of SEQ ID NOs: 1-64. 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 pieces, 36 pieces, 37 pieces, 38 pieces, 39 pieces, 40 pieces, 41 pieces, 42 pieces, 43 pieces, 44 pieces, 45 pieces, 46 pieces, 47 pieces, 48 ​​pieces, 49 pieces, 5 0 pieces, 51 pieces, 52 pieces, 53 pieces, 54 pieces, 55 pieces, 56 pieces, 57 pieces, 58 pieces, 59 pieces, 60 pieces, 61 pieces, 62 pieces, 63 pieces, 64 pieces, 65 pieces, 66 pieces, 67 pieces, 68 pieces, 69 pieces, 70 pieces, 71 pieces, 72 pieces, 73 pieces, 74 pieces, 75 pieces, 76 pieces, 77 pieces, 78 pieces, 79 pieces, 80 pieces, 81 pieces, 82 pieces, 83 pieces, 84 pieces, 85 pieces, 86 pieces, 87 pieces, 88 pieces, 89 pieces, 90 pieces, 91 pieces, 92 pieces, 93 pieces, 94 pieces, 95 pieces, 96 pieces, 9 7 pieces, 98 pieces, 99 pieces, 100 pieces, 101 pieces, 102 pieces, 103 pieces, 104 pieces, 105 pieces, 106 pieces, 107 pieces, 108 pieces, 109 pieces, 1 10 pieces, 111 pieces, 112 pieces, 113 pieces, 114 pieces, 115 pieces, 116 pieces, 117 pieces, 118 pieces, 119 pieces, 120 pieces, 121 pieces, 122 pieces , 123 pieces, 124 pieces, 125 pieces, 126 pieces, 127 pieces, 128 pieces, 129 pieces, 130 pieces, 131 pieces, 132 pieces, 133 pieces, 134 pieces, 1 35 pieces, 136 pieces, 137 pieces, 138 pieces, 139 pieces, 140 pieces, 141 pieces, 142 pieces, 143 pieces, 144 pieces, 145 pieces, 146 pieces, 147 pieces , 148 pieces, 149 pieces, 150 pieces, 151 pieces, 152 pieces, 153 pieces, 154 pieces, 155 pieces, 156 pieces, 157 pieces, 158 pieces, 159 pieces, 1 60 pieces, 161 pieces, 162 pieces, 163 pieces, 164 pieces, 165 pieces, 166 pieces, 167 pieces, 168 pieces, 169 pieces, 170 pieces, 171 pieces, 172 pieces , 173 pieces, 174 pieces, 175 pieces, 176 pieces, 177 pieces, 178 pieces, 179 pieces, 180 pieces, 181 pieces, 182 pieces, 183 pieces, 184 pieces, 1 85 pieces, 186 pieces, 187 pieces, 188 pieces, 189 pieces, 190 pieces, 191 pieces, 192 pieces, 193 pieces, 194 pieces, 195 pieces, 196 pieces, 197 pieces , 198 pieces, 199 pieces, 200 pieces, 201 pieces, 202 pieces, 203 pieces, 204 pieces, 205 pieces, 206 pieces, 207 pieces, 208 pieces, 209 pieces, 21 0 pieces, 211 pieces, 212 pieces, 213 pieces, 214 pieces, 215 pieces, 216 pieces, 217 pieces, 218 pieces, 219 pieces, 220 pieces, 221 pieces, 222 pieces,223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 300, 400, 500, 550, 1000 or more; or At most 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 pieces, 36 pieces, 37 pieces, 38 pieces, 39 pieces, 40 pieces, 41 pieces, 42 pieces, 43 pieces, 44 pieces, 45 pieces, 46 pieces, 47 pieces, 48 ​​pieces, 49 pieces, 50 pieces pieces, 51 pieces, 52 pieces, 53 pieces, 54 pieces, 55 pieces, 56 pieces, 57 pieces, 58 pieces, 59 pieces, 60 pieces, 61 pieces, 62 pieces, 63 pieces, 64 pieces, 65 pieces, 6 6 pieces, 67 pieces, 68 pieces, 69 pieces, 70 pieces, 71 pieces, 72 pieces, 73 pieces, 74 pieces, 75 pieces, 76 pieces, 77 pieces, 78 pieces, 79 pieces, 80 pieces, 81 pieces , 82 pieces, 83 pieces, 84 pieces, 85 pieces, 86 pieces, 87 pieces, 88 pieces, 89 pieces, 90 pieces, 91 pieces, 92 pieces, 93 pieces, 94 pieces, 95 pieces, 96 pieces, 97 pieces, 98 pieces, 99 pieces, 100 pieces, 101 pieces, 102 pieces, 103 pieces, 104 pieces, 105 pieces, 106 pieces, 107 pieces, 108 pieces, 109 pieces, 11 0 pieces, 111 pieces, 112 pieces, 113 pieces, 114 pieces, 115 pieces, 116 pieces, 117 pieces, 118 pieces, 119 pieces, 120 pieces, 121 pieces, 122 pieces, 123 pieces, 124 pieces, 125 pieces, 126 pieces, 127 pieces, 128 pieces, 129 pieces, 130 pieces, 131 pieces, 132 pieces, 133 pieces, 134 pieces, 13 5 pieces, 136 pieces, 137 pieces, 138 pieces, 139 pieces, 140 pieces, 141 pieces, 142 pieces, 143 pieces, 144 pieces, 145 pieces, 146 pieces, 147 pieces, 148 pieces, 149 pieces, 150 pieces, 151 pieces, 152 pieces, 153 pieces, 154 pieces, 155 pieces, 156 pieces, 157 pieces, 158 pieces, 159 pieces, 16 0 pieces, 161 pieces, 162 pieces, 163 pieces, 164 pieces, 165 pieces, 166 pieces, 167 pieces, 168 pieces, 169 pieces, 170 pieces, 171 pieces, 172 pieces, 173 pieces, 174 pieces, 175 pieces, 176 pieces, 177 pieces, 178 pieces, 179 pieces, 180 pieces, 181 pieces, 182 pieces, 183 pieces, 184 pieces, 18 5 pieces, 186 pieces, 187 pieces, 188 pieces, 189 pieces, 190 pieces, 191 pieces, 192 pieces, 193 pieces, 194 pieces, 195 pieces, 196 pieces, 197 pieces, 198 pieces, 199 pieces, 200 pieces, 201 pieces, 202 pieces, 203 pieces, 204 pieces, 205 pieces, 206 pieces, 207 pieces, 208 pieces, 209 pieces, 21 0 pieces, 211 pieces, 212 pieces, 213 pieces, 214 pieces, 215 pieces, 216 pieces, 217 pieces, 218 pieces, 219 pieces, 220 pieces, 221 pieces, 222 pieces,223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 300, 400, 500, 550, 1000 or more, %, 97%, 98%, 99%, or 100% (or any range derivable therein) similar, identical, or homologous to the contiguous amino acids or nucleic acids of, or any range derivable therein.

[0029] In some embodiments, the protein or polypeptide comprises amino acids 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 110, 111, 112, 13, 14, 15, 16, 17, 18, 20, 21, 22, 2 , 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、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).

[0030] In some embodiments, the protein, polypeptide, or nucleic acid is selected from the group consisting of SEQ ID NOs: 1-64. 1 piece, 2 pieces, 3 pieces, 4 pieces, 5 pieces, 6 pieces, 7 pieces, 8 pieces, 9 pieces, 10 pieces, 11 pieces, 12 pieces, 13 pieces, 14 pieces, 15 pieces, 16 pieces, 17 pieces, 18 pieces, 19 pieces, 20 pieces, 21 pieces, 22 pieces, 23 pieces, 24 pieces, 25 pieces, 26 pieces, 27 pieces , 28 pieces, 29 pieces, 30 pieces, 31 pieces, 32 pieces, 33 pieces, 34 pieces, 35 pieces, 36 pieces, 37 pieces, 38 pieces, 39 pieces, 40 pieces, 41 pieces, 42 pieces, 43 pieces, 44 pieces, 45 pieces, 46 pieces, 47 pieces, 48 ​​pieces, 49 pieces, 50 pieces, 51 pieces, 5 2 pieces, 53 pieces, 54 pieces, 55 pieces, 56 pieces, 57 pieces, 58 pieces, 59 pieces, 60 pieces, 61 pieces, 62 pieces, 63 pieces, 64 pieces, 65 pieces, 66 pieces, 67 pieces, 68 pieces, 69 pieces, 70 pieces, 71 pieces, 72 pieces, 73 pieces, 74 pieces, 75 pieces, 76 pieces , 77 pieces, 78 pieces, 79 pieces, 80 pieces, 81 pieces, 82 pieces, 83 pieces, 84 pieces, 85 pieces, 86 pieces, 87 pieces, 88 pieces, 89 pieces, 90 pieces, 91 pieces, 92 pieces, 93 pieces, 94 pieces, 95 pieces, 96 pieces, 97 pieces, 98 pieces, 99 pieces, 100 pieces, 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, 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 pieces, 902 pieces, 903 pieces, 904 pieces, 905 pieces, 906 pieces, 907 pieces, 908 pieces, 909 pieces, 910 pieces, 911 pieces, 912 pieces, 913 pieces, 914 pieces, 915 pieces, 916 pieces, 917 pieces, 918 pieces, 919 pieces, 920 pieces, 921 pieces, 922 pieces, 923 pieces, 924 pieces, 925 pieces, 926 pieces, 927 pieces, 92 pieces 8 pieces, 929 pieces, 930 pieces, 931 pieces, 932 pieces, 933 pieces, 934 pieces, 935 pieces, 936 pieces, 937 pieces, 938 pieces, 939 pieces, 940 pieces, 941 pieces, 942 pieces, 943 pieces, 944 pieces, 945 pieces, 946 pieces, 947 pieces, 948 pieces, 949 pieces, 950 pieces, 951 pieces, 952 pieces, 953 pieces, 954 pieces, 955 pieces , 956 pieces, 957 pieces, 958 pieces, 959 pieces, 960 pieces, 961 pieces, 962 pieces, 963 pieces, 964 pieces, 965 pieces, 966 pieces, 967 pieces, 968 pieces, 96 9 pieces, 970 pieces, 971 pieces, 972 pieces, 973 pieces, 974 pieces, 975 pieces, 976 pieces, 977 pieces, 978 pieces, 979 pieces, 980 pieces, 981 pieces, 982 pieces, 9 It may comprise 83, 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.

[0031] In some embodiments, the polypeptide or protein has a sequence similar to one of SEQ ID NOs: 1-15. 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), at most 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); or Exactly 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range derivable therein) Similar, identical, or homologous to SEQ ID NO:1-15 At least 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 pieces, 52 pieces, 53 pieces, 54 pieces, 55 pieces, 56 pieces, 57 pieces, 58 pieces, 59 pieces, 60 pieces, 61 pieces, 62 pieces, 63 pieces, 64 pieces, 65 pieces, 66 pieces, 67 pieces, 68 pieces, 69 pieces, 70 pieces, 71 pieces, 72 pieces, 73 pieces, 74 pieces, 75 pieces, 7 6 pieces, 77 pieces, 78 pieces, 79 pieces, 80 pieces, 81 pieces, 82 pieces, 83 pieces, 84 pieces, 85 pieces, 86 pieces, 87 pieces, 88 pieces, 89 pieces, 90 pieces, 91 pieces, 92 pieces, 93 pieces, 94 pieces, 95 pieces, 96 pieces, 97 pieces, 98 pieces, 99 pieces, 100 pieces, 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, 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 pieces, 902 pieces, 903 pieces, 904 pieces, 905 pieces, 906 pieces, 907 pieces, 908 pieces, 909 pieces, 910 pieces, 911 pieces, 912 pieces, 913 pieces, 9 14 pieces, 915 pieces, 916 pieces, 917 pieces, 918 pieces, 919 pieces, 920 pieces, 921 pieces, 922 pieces, 923 pieces, 924 pieces, 925 pieces, 926 pieces, 927 pieces, 928 pieces, 929 pieces, 930 pieces, 931 pieces, 932 pieces, 933 pieces, 934 pieces, 935 pieces, 936 pieces, 937 pieces, 938 pieces, 939 pieces, 940 pieces, 941 pieces, 942 pieces, 943 pieces, 944 pieces, 945 pieces, 946 pieces, 947 pieces, 948 pieces, 949 pieces, 950 pieces, 951 pieces, 952 pieces, 953 pieces, 95 4 pieces, 955 pieces, 956 pieces, 957 pieces, 958 pieces, 959 pieces, 960 pieces, 961 pieces, 962 pieces, 963 pieces, 964 pieces, 965 pieces, 966 pieces, 967 pieces, 968 pieces, 969 pieces, 970 pieces, 971 pieces, 972 pieces, 973 pieces, 974 pieces, 975 pieces, 976 pieces, 977 pieces, 978 pieces, 979 pieces, 980 pieces, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, or 1000 consecutive amino acids (or any range derivable therein), At most 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 pieces, 53 pieces, 54 pieces, 55 pieces, 56 pieces, 57 pieces, 58 pieces, 59 pieces, 60 pieces, 61 pieces, 62 pieces, 63 pieces, 64 pieces, 65 pieces, 66 pieces, 67 pieces, 68 pieces, 69 pieces, 70 pieces, 71 pieces, 72 pieces, 73 pieces, 74 pieces, 75 pieces, 76 pieces, 77 pieces, 78 pieces, 79 pieces, 80 pieces, 81 pieces, 82 pieces, 83 pieces, 84 pieces, 85 pieces, 86 pieces, 87 pieces, 88 pieces, 89 pieces, 90 pieces, 91 pieces, 92 pieces, 93 pieces, 94 pieces, 95 pieces, 96 pieces, 97 pieces, 98 pieces, 99 pieces, 100 pieces, 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, 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 pieces, 902 pieces, 903 pieces, 904 pieces, 905 pieces, 906 pieces, 907 pieces, 908 pieces, 909 pieces, 910 pieces, 911 pieces, 912 pieces, 913 pieces, 91 4 pieces, 915 pieces, 916 pieces, 917 pieces, 918 pieces, 919 pieces, 920 pieces, 921 pieces, 922 pieces, 923 pieces, 924 pieces, 925 pieces, 926 pieces, 927 pieces , 928 pieces, 929 pieces, 930 pieces, 931 pieces, 932 pieces, 933 pieces, 934 pieces, 935 pieces, 936 pieces, 937 pieces, 938 pieces, 939 pieces, 940 pieces, 9 41 pieces, 942 pieces, 943 pieces, 944 pieces, 945 pieces, 946 pieces, 947 pieces, 948 pieces, 949 pieces, 950 pieces, 951 pieces, 952 pieces, 953 pieces, 954 pieces, 955 pieces, 956 pieces, 957 pieces, 958 pieces, 959 pieces, 960 pieces, 961 pieces, 962 pieces, 963 pieces, 964 pieces, 965 pieces, 966 pieces, 967 pieces, 968 pieces, 969 pieces, 970 pieces, 971 pieces, 972 pieces, 973 pieces, 974 pieces, 975 pieces, 976 pieces, 977 pieces, 978 pieces, 979 pieces, 980 pieces, 98 1, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, or 1000 consecutive amino acids (or any range derivable therein); 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 pieces, 53 pieces, 54 pieces, 55 pieces, 56 pieces, 57 pieces, 58 pieces, 59 pieces, 60 pieces, 61 pieces, 62 pieces, 63 pieces, 64 pieces, 65 pieces, 66 pieces, 67 pieces, 68 pieces, 69 pieces, 70 pieces, 71 pieces, 72 pieces, 73 pieces, 74 pieces, 75 pieces, 76 pieces, 77 pieces, 78 pieces, 79 pieces, 80 pieces, 81 pieces, 82 pieces, 83 pieces, 84 pieces, 85 pieces, 86 pieces, 87 pieces, 88 pieces, 89 pieces, 90 pieces, 91 pieces, 92 pieces, 93 pieces, 94 pieces, 95 pieces, 96 pieces, 97 pieces, 98 pieces, 99 pieces, 100 pieces, 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, 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 pieces, 902 pieces, 903 pieces, 904 pieces, 905 pieces, 906 pieces, 907 pieces, 908 pieces, 909 pieces, 910 pieces, 911 pieces, 912 pieces, 913 pieces, 9 14 pieces, 915 pieces, 916 pieces, 917 pieces, 918 pieces, 919 pieces, 920 pieces, 921 pieces, 922 pieces, 923 pieces, 924 pieces, 925 pieces, 926 pieces, 927 pieces, 928 pieces, 929 pieces, 930 pieces, 931 pieces, 932 pieces, 933 pieces, 934 pieces, 935 pieces, 936 pieces, 937 pieces, 938 pieces, 939 pieces, 940 pieces , 941 pieces, 942 pieces, 943 pieces, 944 pieces, 945 pieces, 946 pieces, 947 pieces, 948 pieces, 949 pieces, 950 pieces, 951 pieces, 952 pieces, 953 pieces, 9 54 pieces, 955 pieces, 956 pieces, 957 pieces, 958 pieces, 959 pieces, 960 pieces, 961 pieces, 962 pieces, 963 pieces, 964 pieces, 965 pieces, 966 pieces, 96 7 pieces, 968 pieces, 969 pieces, 970 pieces, 971 pieces, 972 pieces, 973 pieces, 974 pieces, 975 pieces, 976 pieces, 977 pieces, 978 pieces, 979 pieces, 980 pieces , 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, or 1000 consecutive amino acids (or any range derivable therein). may include:

[0032] In some aspects, the amino acid sequence of any one of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 20, 21, 22 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、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, 95 2, 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 any of SEQ ID NOs: 1 to 64 At least 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 pieces, 53 pieces, 54 pieces, 55 pieces, 56 pieces, 57 pieces, 58 pieces, 59 pieces, 60 pieces, 61 pieces, 62 pieces, 63 pieces, 64 pieces, 65 pieces, 66 pieces, 67 pieces, 68 pieces, 69 pieces, 70 pieces, 71 pieces, 72 pieces, 73 pieces, 74 pieces, 75 pieces, 76 pieces, 77 pieces, 78 pieces, 79 pieces, 80 pieces, 81 pieces, 82 pieces, 83 pieces, 84 pieces, 85 pieces, 86 pieces, 87 pieces, 88 pieces, 89 pieces, 90 pieces, 91 pieces, 92 pieces, 93 pieces, 94 pieces, 95 pieces, 96 pieces, 97 pieces, 98 pieces, 99 pieces, 100 pieces, 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, 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 pieces, 902 pieces, 903 pieces, 904 pieces, 905 pieces, 906 pieces, 907 pieces, 908 pieces, 909 pieces, 910 pieces, 911 pieces, 912 pieces, 913 pieces, 914 pieces, 915 pieces, 916 pieces, 917 pieces, 918 pieces, 919 pieces, 920 pieces, 921 pieces, 922 pieces, 923 pieces, 924 pieces, 925 pieces, 926 pieces, 927 pieces, 9 28 pieces, 929 pieces, 930 pieces, 931 pieces, 932 pieces, 933 pieces, 934 pieces, 935 pieces, 936 pieces, 937 pieces, 938 pieces, 939 pieces, 940 pieces, 941 pieces , 942 pieces, 943 pieces, 944 pieces, 945 pieces, 946 pieces, 947 pieces, 948 pieces, 949 pieces, 950 pieces, 951 pieces, 952 pieces, 953 pieces, 954 pieces, 95 5 pieces, 956 pieces, 957 pieces, 958 pieces, 959 pieces, 960 pieces, 961 pieces, 962 pieces, 963 pieces, 964 pieces, 965 pieces, 966 pieces, 967 pieces, 968 pieces, 969 pieces, 970 pieces, 971 pieces, 972 pieces, 973 pieces, 974 pieces, 975 pieces, 976 pieces, 977 pieces, 978 pieces, 979 pieces, 980 pieces, 981 pieces, 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; At most 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 pieces, 53 pieces, 54 pieces, 55 pieces, 56 pieces, 57 pieces, 58 pieces, 59 pieces, 60 pieces, 61 pieces, 62 pieces, 63 pieces, 64 pieces, 65 pieces, 66 pieces, 67 pieces, 68 pieces, 69 pieces, 70 pieces, 71 pieces, 72 pieces, 73 pieces, 74 pieces, 75 pieces, 76 pieces, 77 pieces, 78 pieces, 79 pieces, 80 pieces, 81 pieces, 82 pieces, 83 pieces, 84 pieces, 85 pieces, 86 pieces, 87 pieces, 88 pieces, 89 pieces, 90 pieces, 91 pieces, 92 pieces, 93 pieces, 94 pieces, 95 pieces, 96 pieces, 97 pieces, 98 pieces, 99 pieces, 100 pieces, 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, 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 pieces, 902 pieces, 903 pieces, 904 pieces, 905 pieces, 906 pieces, 907 pieces, 908 pieces, 909 pieces, 910 pieces, 911 pieces, 912 pieces, 913 pieces, 914 pieces, 915 pieces, 916 pieces, 917 pieces, 918 pieces, 919 pieces, 920 pieces, 921 pieces, 922 pieces, 923 pieces, 924 pieces, 925 pieces, 926 pieces, 927 pieces, 9 28 pieces, 929 pieces, 930 pieces, 931 pieces, 932 pieces, 933 pieces, 934 pieces, 935 pieces, 936 pieces, 937 pieces, 938 pieces, 939 pieces, 940 pieces, 941 pieces , 942 pieces, 943 pieces, 944 pieces, 945 pieces, 946 pieces, 947 pieces, 948 pieces, 949 pieces, 950 pieces, 951 pieces, 952 pieces, 953 pieces, 954 pieces, 955 pieces, 956 pieces, 957 pieces, 958 pieces, 959 pieces, 960 pieces, 961 pieces, 962 pieces, 963 pieces, 964 pieces, 965 pieces, 966 pieces, 967 pieces, 968 pieces, 9 69 pieces, 970 pieces, 971 pieces, 972 pieces, 973 pieces, 974 pieces, 975 pieces, 976 pieces, 977 pieces, 978 pieces, 979 pieces, 980 pieces, 981 pieces, 982 pieces, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, or 1000 consecutive amino acids or nucleotides (or any range derivable therein); 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, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 108, 109, 109, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 2 pieces, 53 pieces, 54 pieces, 55 pieces, 56 pieces, 57 pieces, 58 pieces, 59 pieces, 60 pieces, 61 pieces, 62 pieces, 63 pieces, 64 pieces, 65 pieces, 66 pieces, 67 pieces, 68 pieces, 69 pieces, 70 pieces, 71 pieces, 72 pieces, 73 pieces, 74 pieces, 75 pieces, 76 pieces , 77 pieces, 78 pieces, 79 pieces, 80 pieces, 81 pieces, 82 pieces, 83 pieces, 84 pieces, 85 pieces, 86 pieces, 87 pieces, 88 pieces, 89 pieces, 90 pieces, 91 pieces, 92 pieces, 93 pieces, 94 pieces, 95 pieces, 96 pieces, 97 pieces, 98 pieces, 99 pieces, 100 pieces, 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, 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 pieces, 902 pieces, 903 pieces, 904 pieces, 905 pieces, 906 pieces, 907 pieces, 908 pieces, 909 pieces, 910 pieces, 911 pieces, 912 pieces, 913 pieces, 91 4 pieces, 915 pieces, 916 pieces, 917 pieces, 918 pieces, 919 pieces, 920 pieces, 921 pieces, 922 pieces, 923 pieces, 924 pieces, 925 pieces, 926 pieces, 927 pieces, 928 pieces, 929 pieces, 930 pieces, 931 pieces, 932 pieces, 933 pieces, 934 pieces, 935 pieces, 936 pieces, 937 pieces, 938 pieces, 939 pieces, 940 pieces, 941 pieces, 942 pieces, 943 pieces, 944 pieces, 945 pieces, 946 pieces, 947 pieces, 948 pieces, 949 pieces, 950 pieces, 951 pieces, 952 pieces, 953 pieces, 954 pieces, 9 55 pieces, 956 pieces, 957 pieces, 958 pieces, 959 pieces, 960 pieces, 961 pieces, 962 pieces, 963 pieces, 964 pieces, 965 pieces, 966 pieces, 967 pieces, 968 pieces, 969 pieces, 970 pieces, 971 pieces, 972 pieces, 973 pieces, 974 pieces, 975 pieces, 976 pieces, 977 pieces, 978 pieces, 979 pieces, 980 pieces, 981 pieces, 9 82, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, or 1000 consecutive amino acids or nucleotides (or any range derivable therein). The nucleic acid molecule or polypeptide includes:

[0033] In some embodiments, the peptide, polypeptide, or nucleic acid has a sequence similar to one of SEQ ID NOs: 1-64. 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) similar, identical, or homologous; at most 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; Exactly 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range derivable therein) similar, identical, or homologous; SEQ ID NO: 1 to 64 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive amino acids or nucleotides (or any range derivable therein); at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive amino acids or nucleotides (or any range derivable therein); At most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive amino acids or nucleotides (or any range derivable therein). may include:

[0034] In some aspects, the present invention relates to a method for the preparation of a nucleic acid sequence comprising the steps of: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive amino acids or nucleotides (or any range derivable therein); at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive amino acids or nucleotides (or any range derivable therein); At most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive amino acids or nucleotides (or any range derivable therein). The peptide, polypeptide, or nucleic acid includes

[0035] The peptide, polypeptide, or nucleic acid is selected from the group consisting of SEQ ID NOs: 1-64, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid or nucleotide substitutions (or any range derivable therein); at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid or nucleotide substitutions (or any range derivable therein); At most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid or nucleotide substitutions (or any range derivable therein) The peptide, polypeptide, or nucleic acid may comprise: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid or nucleotide substitutions (or any range derivable therein); at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid or nucleotide substitutions (or any range derivable therein); At most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid or nucleotide substitutions (or any range derivable therein) and the substitution may be at 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, and / or 25 relative to SEQ ID NOs: 1-64. The substitution may be at any position of SEQ ID NOs: 1-64. In the case of amino acid substitutions, the substitution may be with alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine. In the case of nucleic acid substitutions, the substitution may be with guanine, cytosine, adenine, thymine, uracil, or other nucleotides.

[0036] Nucleotide sequences and protein sequences, polypeptide sequences and peptide sequences of various genes have been disclosed before and can be found in recognized computerized 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 as known to those skilled in the art.

[0037] It is contemplated that the compositions of the present disclosure will have from about 0.001 mg to about 10 mg of total polypeptide, peptide, and / or protein per ml. The concentration of protein in the composition may range from about 0.001 mg to about 10 mg per ml. about 0.001 mg / ml, 0.010 mg / ml, 0.050 mg / ml, 0.1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml, 1.0 mg / ml, 1.5 mg / ml, 2.0 mg / ml, 2.5 mg / ml, 3.0 mg / ml, 3.5 mg / ml, 4.0 mg / ml, 4.5 mg / ml, 5.0 mg / ml, 5.5 mg / ml, 6.0 mg / ml, 6.5 mg / ml, 7.0 mg / ml, 7.5 mg / ml, 8.0 mg / ml, 8.5 mg / ml, 9.0 mg / ml, 9.5 mg / ml, 10.0 mg / ml, or more (or any range derivable therein); at least about 0.001 mg / ml, 0.010 mg / ml, 0.050 mg / ml, 0.1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml, 1.0 mg / ml, 1.5 mg / ml, 2.0 mg / ml, 2.5 mg / ml, 3.0 mg / ml, 3.5 mg / ml, 4.0 mg / ml, 4.5 mg / ml, 5.0 mg / ml, 5.5 mg / ml, 6.0 mg / ml, 6.5 mg / ml, 7.0 mg / ml, 7.5 mg / ml, 8.0 mg / ml, 8.5 mg / ml, 9.0 mg / ml, 9.5 mg / ml, 10.0 mg / ml, or more (or any range derivable therein); or at most about 0.001 mg / ml, 0.010 mg / ml, 0.050 mg / ml, 0.1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml, 1.0 mg / ml, 1.5 mg / ml, 2.0 mg / ml, 2.5 mg / ml, 3.0 mg / ml, 3.5 mg / ml, 4.0 mg / ml, 4.5 mg / ml, 5.0 mg / ml, 5.5 mg / ml, 6.0 mg / ml, 6.5 mg / ml, 7.0 mg / ml, 7.5 mg / ml, 8.0 mg / ml, 8.5 mg / ml, 9.0 mg / ml, 9.5 mg / ml, 10.0 mg / ml, or more (or any range derivable therein); It could be.

[0038] A. Sequence The amino acid sequences of certain polypeptides, including chimeric antigen receptors and portions, regions, and domains thereof, are shown in Table 1.

[0039] [Table 1] TIFF2025508961000003.tif242169TIFF2025508961000004.tif136169

[0040] The sequences of certain anti-CD79b antibodies, such as those described in PCT Patent Application Publication WO2021 / 222944, that may be useful in generating the anti-CD79B CARs disclosed herein are provided below. TIFF2025508961000005.tif63160TIFF2025508961000006.tif245160TIFF2025508961000007.tif244160TIFF2025508961000008.tif238160

[0041] B. Variant Polypeptides The following is a discussion of the modification of protein amino acid subunits to create equivalent or even improved second generation variant polypeptides or peptides. For example, certain amino acids in a protein or polypeptide sequence can be replaced with other amino acids with or without appreciable loss of interactive binding ability with structures such as the antigen-binding region of an antibody or a binding site on a substrate molecule. Because it is the interactive ability and properties of a protein that define the functional activity of a 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 desirable properties. Thus, it is contemplated by the inventors that various changes can be made in the DNA sequence of a gene encoding a protein without appreciable loss of biological usefulness or activity.

[0042] The term "functionally equivalent codon" is used herein to refer to codons that code for the same amino acid, e.g., six different codons for arginine. "Neutral substitutions" or "neutral mutations," which refer to changes in codons that code for biologically equivalent amino acids, are also contemplated.

[0043] The amino acid sequence variants of the disclosure may be substitutional, insertional, or deletion variants. The changes in the polypeptides of the disclosure may affect 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more non-contiguous or contiguous amino acids of the protein or polypeptide compared to the wild type. Variants may include amino acid sequences that are at least 50%, 60%, 70%, 80%, or 90% identical to any of the sequences provided or referenced herein, including all values ​​and ranges therebetween. Variants 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.

[0044] It is also understood that the amino acid sequence and the nucleic acid sequence may contain additional residues, for example, additional N-terminal or C-terminal amino acids, i.e., 5' or 3' sequences, respectively, but are still essentially identical as shown in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of the biological protein activity to which protein expression pertains. The addition of terminal sequences applies in particular to nucleic acid sequences, which may contain, for example, various non-coding sequences adjacent to the 5' or 3' portion of the coding region.

[0045] Deletion variants typically lack one or more residues of the native or wild-type protein. Individual residues may be deleted, or multiple consecutive amino acids may be deleted. To generate truncated proteins, stop codons may be introduced (by substitution or insertion) into the coding nucleic acid sequence.

[0046] Insertional mutants typically involve the addition of amino acid residues at a non-terminal point in the polypeptide. This may include the insertion of one or more amino acid residues. Terminal additions may also be made and may include fusion proteins that are multimers or concatamers of one or more peptides or polypeptides described or referenced herein.

[0047] Substitution variants typically involve the exchange of one amino acid for another at one or more sites within the protein or polypeptide, and can be designed to modulate one or more properties of the polypeptide, with or without the loss of other functions or properties. Substitutions can be conservative substitutions, i.e., an amino acid is replaced with one of similar chemical properties. A "conservative amino acid substitution" can involve the exchange of a member of an amino acid class with another member of the same class. Conservative substitutions are well known in the art and include, for example, alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartic acid to glutamic acid; cysteine ​​to serine; glutamine to asparagine; glutamic acid to aspartic acid; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. Conservative amino acid substitutions may include unnatural amino acid residues, which are typically incorporated by chemical peptide synthesis rather than synthesis in biological systems. These include peptidomimetics or other reversed or inverted amino acid moieties.

[0048] Alternatively, the substitution may be a "non-conservative" substitution, such that the function or activity of the polypeptide is affected. Non-conservative changes typically involve replacing an amino acid residue with a chemically dissimilar amino acid residue, for example, replacing a polar or charged amino acid with a non-polar or uncharged amino acid, or vice versa. Non-conservative substitutions may involve exchanging a member of one amino acid class for a member of another class.

[0049] C. Chimeric antigen receptor In some aspects, chimeric antigen receptors (CARs) are disclosed. Generally, CARs comprise an extracellular antigen (or ligand) binding domain linked to one or more intracellular signaling components, in some aspects via a linker and / or a transmembrane domain. Such molecules typically mimic or approximate the signaling through natural antigen receptors, through such receptors in combination with co-stimulatory receptors, and / or through co-stimulatory receptors alone.

[0050] It is intended that chimeric constructs can be introduced into immune cells as naked DNA or in suitable vectors.Methods for stably transfecting cells with naked DNA by electroporation are known in the art.See, for example, U.S. Patent No. 6,410,319.Naked DNA generally refers to the DNA encoding chimeric receptor contained in a plasmid expression vector in the correct orientation for expression.

[0051] Alternatively, the chimeric CAR construct can be introduced into immune cells using a viral vector (e.g., retroviral vector, adenoviral vector, adeno-associated viral vector, or lentiviral vector).The vector suitable for use according to the method of the present disclosure does not replicate in immune cells.For example, a number of virus-based vectors are known, such as vectors based on HIV, SV40, EBV, HSV, or BPV, in which the copy number of the virus maintained in cells is low enough to maintain cell viability.

[0052] Certain aspects of the present disclosure relate to the use of nucleic acids, including nucleic acids encoding cancer antigen-specific CAR polypeptides, including CARs (hCARs) that are humanized to reduce immunogenicity, and that include at least one intracellular signaling domain, a transmembrane domain, and an extracellular domain that includes one or more signaling motifs. In certain aspects, 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 aspect, the specificity is derived from a peptide (e.g., a cytokine) that binds to a receptor.

[0053] It is contemplated that the CAR nucleic acid may be a human gene used to enhance cellular immunotherapy for human patients. In certain embodiments, the present disclosure includes a full-length CAR cDNA or coding region. The antigen-binding region or domain is a V of a single chain variable fragment (scFv) derived from a particular human monoclonal antibody (e.g., an anti-CD19 antibody, e.g., FMC63.3 and / or an anti-CD79b antibody, e.g., an anti-CD79b antibody described in PCT Patent Application Publication WO2021 / 222944). H Chain and V L The fragments may comprise fragments of the nucleotide chains of a human antigen-specific antibody. The fragments may also be any number of different antigen-binding domains of a human antigen-specific antibody. In a further particular embodiment, the fragment is a cancer antigen-specific scFv encoded by a sequence optimized for human codon usage for expression in human cells.

[0054] The arrangement may be multimeric, such as a diabody or multimer. Multimers are most likely formed by cross-pairing the variable portions of the light and heavy chains into a diabody. The hinge portion of the construct may have multiple options, ranging from complete deletion, to maintaining the first cysteine, substituting proline instead of serine, to truncation up to the first cysteine. The Fc portion may be deleted. Any protein that is stable and / or dimerizes may serve this purpose. Only one of the Fc domains may be used, for example, either the CH2 or CH3 domain from a human immunoglobulin. The hinge, CH2, and CH3 regions of a human immunoglobulin that have been modified to improve dimerization may also be used. Only the hinge portion of an immunoglobulin may be used.

[0055] The sequence of the open reading frame encoding the chimeric receptor may be obtained from a genomic DNA source, a cDNA source, or may be synthesized (e.g., via PCR), or a combination thereof. Depending on the size of the genomic DNA and the number of introns, it may be desirable to use cDNA or a combination thereof, since introns have been found to stabilize mRNA. Similarly, it may be further advantageous to use endogenous or exogenous non-coding regions to stabilize the mRNA.

[0056] In some aspects, the antigen-specific binding (e.g., anti-CD19, anti-CD79b) or recognition component is linked to one or more transmembrane domains and intracellular signaling domains. In some embodiments, the CAR comprises a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, a transmembrane domain that is naturally associated with one of the domains in the CAR is used. In another embodiment, a transmembrane domain that is not naturally associated with one of the domains of the CAR is used. In some cases, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains with transmembrane domains of the same or different surface membrane proteins to minimize interaction with other members of the receptor complex. The transmembrane domain is, in some embodiments, derived from either natural or synthetic sources. If the source is natural, the domain is, in some aspects, derived from any membrane-bound or transmembrane protein. Transmembrane regions include (i.e., at least include; also include "from") transmembrane regions derived from the α, β, or ζ chains of the T cell receptor, CD28, DAP12, DAP10, NKG2D, CD3ζ, CD3ε, CD3γ, CD3δ, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, ICOS / CD278, etc. In some aspects, the transmembrane domain of the present disclosure is a transmembrane domain from CD8α.

[0057] In some embodiments, the 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 primary T cell activation signal, which may be initiated by, for example, CD3ζ and / or FcεRIγ, additional stimulatory signals may be utilized for immune effector cell proliferation and effector function after the chimeric receptor binds to the target antigen. For example, some or all of the human co-stimulatory receptors may be utilized to enhance cell activation, which may help improve in vivo persistence and improve the therapeutic success of adoptive immunotherapy. Examples include co-stimulatory domains from molecules such as DAP12, DAP10, NKG2D, CD2, CD28, CD27, 4-1BB, (CD137), OX40, ICOS, (CD278), CD30, HVEM, CD40, LFA-1 (CD11a / CD18), and ICAM-1, although in certain alternative embodiments, any one of these enumerations may be excluded from use in the CAR.

[0058] In certain embodiments, certain CAR molecules are encompassed herein. In some cases, the antigen-binding domain of the CAR is an scFv, and any scFv that binds to a cancer antigen can be utilized herein. When an scFv is utilized in the extracellular domain of the CAR, the variable heavy and variable light chains of the scFv can be in any order from N-terminus to C-terminus. For example, the variable heavy chain can be N-terminal to the variable light chain, or vice versa. The scFv and / or ligand that binds to the antigen in the CAR can be codon-optimized or non-optimized. In certain embodiments, the vector encodes a cancer antigen-specific CAR and also encodes one or more other molecules. For example, the vector can encode both a first CAR (e.g., an anti-CD19 CAR) and a second CAR (e.g., an anti-CD79b CAR).

[0059] On the same molecule, cancer antigen-specific CAR may comprise one or more antigen-specific extracellular domains, a specific hinge, a specific transmembrane domain, one or more specific costimulatory domains, and one or more specific activation signals.When multiple antigen-specific extracellular domains are utilized, for example, to target two different antigens, there may be a linker between the two antigen-specific extracellular domains.The examples of CARs contemplated herein include, but are not limited to, CD19-specific (also referred to as "anti-CD19") CAR and CD79b-specific (also referred to as "anti-CD79b") CAR.

[0060] In certain embodiments of certain CAR molecules, CARs may utilize DAP10, DAP12, 4-1BB, NKG2D, or other costimulatory domains (sometimes referred to herein as cytoplasmic domains). In some cases, CD3zeta is utilized without any costimulatory domain. In certain embodiments of certain CAR molecules, CARs may utilize any suitable transmembrane domain, for example, the transmembrane domain from DAP12, DAP10, 4-1BB, 2B4, OX40, CD27, NKG2D, CD8, CD28, IL12Rβ1, or IL12Rβ2.

[0061] 1. Signal peptide The polypeptide (e.g., CAR) of the present disclosure may include a signal peptide. "Signal peptide" refers to a peptide sequence that directs the transport and localization of a protein within a cell, for example, to a certain organelle (e.g., endoplasmic reticulum) and / or to the cell surface. In some embodiments, the signal peptide directs the nascent protein to the endoplasmic reticulum. This is essential if the receptor is glycosylated and anchored in the cell membrane.

[0062] In some embodiments, the signal peptide is cleaved after passing through the endoplasmic reticulum (ER), i.e., it is a cleavable signal peptide. In some aspects, the signal peptide has a restriction site at its carboxy terminus to facilitate cleavage.

[0063] In some aspects, a CAR of the present disclosure comprises a GM-CSFRα signal peptide. In some aspects, the GM-CSFRα signal peptide comprises SEQ ID NO:1. In some aspects, a CAR of the present disclosure comprises a CD8α signal peptide. In some aspects, the CD8α signal peptide comprises SEQ ID NO:10. In some aspects, an anti-CD19 CAR of the present disclosure comprises a GM-CSFRα signal peptide. In some aspects, an anti-CD79b CAR of the present disclosure comprises a CD8α signal peptide.

[0064] 2. Antigen-binding domain The polypeptides of the present disclosure may comprise one or more antigen-binding domains. An "antigen-binding domain" describes a polypeptide region 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., anti-CD19 antibody, anti-CD79b antibody). In some embodiments, the antigen-binding domain comprises a variable heavy (VH) region and a variable light (VL) region, where the VH region and the VL region are on the same polypeptide. In some embodiments, the antigen-binding domain comprises a linker between the VH region and the VL region. The use of the linker may allow the antigen-binding domain to form a structure that is desirable for antigen binding.

[0065] The variable regions of the antigen-binding domain of the polypeptides of the present disclosure can be modified by mutating amino acid residues in the VH and / or VL CDR1, CDR2, and / or CDR3 regions to improve one or more binding properties (e.g., affinity) of the antibody. The term "CDR" refers to the complementarity determining region based on the portions of the variable chains in immunoglobulins (antibodies) from which B cells arise and T cell receptors from which T cells arise, and these molecules bind to their specific antigens. Since most sequence variations associated with immunoglobulins and T cell receptors are found in the CDRs, these regions are sometimes referred to as hypervariable regions. Mutations can be introduced by site-directed mutagenesis or PCR-mediated mutagenesis, and the effect on antibody binding or other functional properties of interest can be evaluated in appropriate in vitro or in vivo assays. Preferably, conservative modifications are introduced, typically altering no more than one, two, three, four, or five residues in the CDR regions. Mutations can be amino acid substitutions, additions, or deletions.

[0066] Framework modifications can be made to the antibody to reduce immunogenicity, for example, by "backmutating" one or more framework residues to the corresponding germline sequence.

[0067] It is also contemplated that antigen-binding domains may be made multispecific and multivalent by multimerizing the antigen-binding domains using VH and VL region pairs that bind either the same antigen (multivalent) or different antigens (multispecificity).

[0068] The binding affinity of the antigen-binding region, e.g., the variable region (heavy chain variable region and / or light chain variable region) or the binding affinity of the CDRs is at least 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10-12 M, or 10 -13 In some embodiments, the KD of an antigen-binding region, e.g., a variable region (heavy chain variable region and / or light chain variable region) or the KD of a CDR is at least 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M or 10 -13 M (or any range that can be derived therein).

[0069] Binding affinity, K A , or K D can be determined by methods known in the art, for example, by surface plasmon resonance (SRP)-based biosensors, by equilibrium exclusion binding (KinExA), by polarization-modulated oblique-incidence reflectivity difference (OI-RD)-based optical scanners for microarray detection, or by ELISA.

[0070] In some aspects, a polypeptide comprising a humanized binding region has a binding affinity and / or expression level in a host cell that is equal to, better than, 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% as compared to a polypeptide comprising a non-humanized binding region, e.g., a binding region derived from a mouse.

[0071] In some embodiments, the framework regions of the human framework, e.g., FR1, FR2, FR3, and / or FR4, individually or collectively, are similar to those of the mouse framework. At least 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, 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, or 200 (or any range derivable therein) amino acid substitutions, consecutive amino acid additions, or consecutive amino acid deletions; At most 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, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 17 1, 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, or 200 (or any range derivable therein) amino acid substitutions, consecutive amino acid additions, or consecutive amino acid deletions, or Exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 102, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 5, 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, or 200 (or any range derivable therein) amino acid substitutions, consecutive amino acid additions, or consecutive amino acid deletions. may have the following structure:

[0072] In some embodiments, the framework regions of the mouse framework, e.g., FR1, FR2, FR3, and / or FR4, individually or collectively, are similar to those of the human framework. At least 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, 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, or 200 (or any range derivable therein) amino acid substitutions, consecutive amino acid additions, or consecutive amino acid deletions; At most 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, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 17 1, 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, or 200 (or any range derivable therein) amino acid substitutions, consecutive amino acid additions, or consecutive amino acid deletions, or Exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 102, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 5, 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, or 200 (or any range derivable therein) amino acid substitutions, consecutive amino acid additions, or consecutive amino acid deletions. may have the following structure:

[0073] The substitutions are at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 108, 109, 110, 111, 112, 13, 14, 15, 16, 17, 18, 19, 20, 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.

[0074] In some aspects, the antigen binding domain of the present disclosure is an anti-CD19 binding domain. The anti-CD19 binding domain may comprise a VH and a VL derived from an anti-CD19 antibody (e.g., FMC63.3). In some aspects, the anti-CD19 binding domain comprises a VL having SEQ ID NO:2. In some aspects, the anti-CD19 binding domain comprises a VH having SEQ ID NO:4. In some aspects, the VH and the VL are connected by a linker, for example, a linker having SEQ ID NO:3.

[0075] In some embodiments, the antigen binding domain of the present disclosure is an anti-CD79b binding domain. The anti-CD79b binding domain may comprise a VH and a VL derived from an anti-CD79b antibody (e.g., an anti-CD79b antibody described in PCT Patent Application Publication WO2021 / 222944, which is incorporated herein by reference). In some embodiments, the anti-CD79b binding domain comprises a VL having SEQ ID NO:11. In some embodiments, the anti-CD79b binding domain comprises a VH having SEQ ID NO:13. In some embodiments, the VL and the VH are connected by a linker, for example, a linker having SEQ ID NO:12.

[0076] 3. Peptide spacer A peptide spacer, e.g., an extracellular spacer, may link the antigen-binding domain to the transmembrane domain. In some embodiments, the peptide spacer is sufficiently flexible to allow the antigen-binding domain to orient in various directions to facilitate antigen binding. In one embodiment, the spacer comprises a hinge region derived from IgG. In some embodiments, the spacer comprises or further comprises a CH2CH3 region of an immunoglobulin and a portion of CD3. In some embodiments, the CH2CH3 region may have a L235E / N297Q or L235D / N297Q modification and may 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 to the CH2CH3 region. In some embodiments, the spacer is derived from IgG4. The extracellular spacer may comprise a hinge region.

[0077] The term "hinge" as used herein refers to a flexible polypeptide connector region (also referred to herein as "hinge region") that provides structural flexibility and spacing for adjacent polypeptide regions, and may be composed of natural or synthetic polypeptides. A "hinge" derived from an immunoglobulin (e.g., IgG1) is generally defined as the section from Glu216 to Pro230 of human IgG1 (Burton (1985) Molec. Immunol., 22: 161-206). Hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues that form inter-heavy chain disulfide (SS) bonds in the same positions. The hinge region may be a naturally occurring or non-naturally occurring hinge region, including but not limited to altered hinge regions as described in U.S. Pat. No. 5,677,425, which is incorporated herein by reference. The hinge region may comprise a complete hinge region derived from an antibody of a class or subclass different from that of the CH1 domain. The term "hinge" may also include regions derived from CD8 and other receptors which provide similar functions in providing flexibility and spacing for adjacent regions.

[0078] The extracellular spacer is At least 4, 5, 6, 7, 8, 9, 10, 12, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 75, 100, 110, 119, 120, 130, 140, 150, 160, 170, 180, 190, 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, 17, 218, 219, 220, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 260, 270, 280, 290, 300, 325, 350, or 400 amino acids (or any range derivable therein), At most 4, 5, 6, 7, 8, 9, 10, 12, 15, 16, 17, 18, 19, 20, 20, 25, 30, 35, 40, 45, 50, 75, 100, 110, 119, 120, 130, 140, 150, 160, 170, 180, 190, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217 , 218, 219, 220, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 260, 270, 280, 290, 300, 325, 350, or 400 amino acids (or any range derivable therein); Exactly 4, 5, 6, 7, 8, 9, 10, 12, 15, 16, 17, 18, 19, 20, 20, 25, 30, 35, 40, 45, 50, 75, 100, 110, 119, 120, 130, 140, 150, 160, 170, 180, 190, 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, 7, 218, 219, 220, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 260, 270, 280, 290, 300, 325, 350, or 400 amino acids (or any range derivable therein). In some embodiments, the extracellular spacer consists of or comprises a hinge region derived from an immunoglobulin (e.g., IgG). The amino acid sequence of an immunoglobulin hinge region is known in the art. See, for example, Tan et al. (1990) Proc. Natl. Acad. Sci. USA 87: 162; and Huck et al. (1986) Nucl. Acids Res.

[0079] The length of the extracellular spacer can affect the signaling activity of the CAR and / or the expansion properties of the CAR-T cells in response to antigen-stimulated CAR signaling. In some embodiments, short spacers are used, e.g., less than 50, 45, 40, 30, 35, 30, 25, 20, 15, 14, 13, 12, 11, or 10 amino acids. In some embodiments, long spacers are used, e.g., at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268 Spacers of 27, 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 may be advantageous in increasing expansion in vivo or in vitro.

[0080] When the extracellular spacer comprises multiple moieties, there may be anywhere from 0 to 50 amino acids between the various moieties, for example, between the hinge and the CH2 or CH3 region, or between the CH2 and CH3 regions when both are present. at least 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, or 50 amino acids (or any range derivable therein); at most 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, or 50 amino acids (or any range derivable therein); or Exactly 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, or 50 amino acids (or any range derivable therein) In some embodiments, the extracellular spacer consists essentially of the hinge, CH2 region, and / or CH3 region, meaning that the hinge, CH2 region, and / or CH3 region are the only identifiable regions present, to the exclusion of all other domains or regions, although there may be additional amino acids present that are not part of the identifiable regions.

[0081] In some aspects, a CAR of the present disclosure comprises a CD8α hinge. In some aspects, the hinge comprises SEQ ID NO:5.

[0082] 4. Transmembrane domain The polypeptides of the present disclosure may include a transmembrane domain. In some aspects, the transmembrane domain is a hydrophobic alpha helix that spans the membrane. Different transmembrane domains may confer different receptor stabilities.

[0083] In some embodiments, the transmembrane domain is inserted between the extracellular spacer and the cytoplasmic region. In some embodiments, the transmembrane domain is inserted between the extracellular spacer and one or more costimulatory regions. In some embodiments, a linker is between the transmembrane domain and one or more costimulatory regions.

[0084] Any transmembrane domain that inserts 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 CD28, CD8, CD4, CD3zeta, CD134, or CD7.

[0085] In some aspects, the CAR of the present disclosure comprises a CD8α transmembrane domain. In some aspects, the CD8α transmembrane domain comprises SEQ ID NO:6.

[0086] 5.Cytoplasmic region After antigen recognition, the receptor of the present disclosure can be clustered and a signal can be transmitted to the cell through the cytoplasmic region. In some embodiments, the co-stimulatory domain described herein is a part of the cytoplasmic region. In some embodiments, the cytoplasmic region comprises an intracellular signaling domain. The intracellular signaling domain may comprise a primary signaling domain and one or more co-stimulatory domains.

[0087] Cytoplasmic and / or costimulatiory regions suitable for use in the polypeptides of the present disclosure include any desired signaling domain that generates a distinct and detectable signal in response to activation via binding of an antigen to the antigen-binding domain (e.g., increasing the production of one or more cytokines by the cell; altering the transcription of a target gene; altering the activity of a protein; altering cell behavior, e.g., cell death; cell proliferation; cell differentiation; cell survival; modulating cell signaling responses, etc.). In some embodiments, the cytoplasmic region comprises at least one (e.g., 1, 2, 3, 4, 5, 6, etc.) ITAM motif as described herein. In some embodiments, the cytoplasmic region comprises a DAP10 / CD28-type signaling chain.

[0088] Cytoplasmic regions suitable for use in the polypeptides of the present disclosure include intracellular signaling polypeptides that contain an immunoreceptor tyrosine-based activation motif (ITAM). The ITAM motif is YX1X2(L / I), where X1 and X2 are independently any amino acid. In some cases, the cytoplasmic region contains one, two, three, four, or five ITAM motifs. In some cases, the ITAM motif is repeated twice in the endodomain, with the first and second instances of the ITAM motif separated from each other by 6-8 amino acids. For example, (YX1X2(L / I))(X3)n(YX1X2(L / I)), where n is an integer between 6 and 8, and each of the 6-8 X3 can be any amino acid.

[0089] A suitable cytoplasmic region may be an ITAM motif-containing portion derived from a polypeptide containing an ITAM motif. For example, a suitable cytoplasmic region may be an ITAM motif-containing domain derived from any ITAM motif-containing protein. Thus, a suitable endodomain does not need to contain the entire sequence of the entire protein from which it originates. Examples of suitable ITAM motif-containing polypeptides include, but are not limited to, DAP12, DAP10, FCER1G (Fcε receptor Iγ chain); CD3D (CD3δ); CD3E (CD3ε); CD3G (CD3γ); CD3ζ; and CD79A (antigen receptor complex-associated protein α chain).

[0090] In some embodiments, a suitable cytoplasmic region may comprise an ITAM motif-containing portion of the full-length DAP12 amino acid sequence. In some embodiments, the cytoplasmic region is derived from FCER1G (also known as FCRG; Fcε receptor Iγ chain; Fc receptor γ chain; fc-εRl-γ; fcRγ; fceRIγ; high affinity immunoglobulin ε receptor subunit γ; immunoglobulin E receptor, high affinity, γ chain, etc.). In some embodiments, a suitable cytoplasmic region may comprise an ITAM motif-containing portion of the full-length FCER1G amino acid sequence.

[0091] In some embodiments, the cytoplasmic region is derived from the T cell surface glycoprotein CD3 delta chain (also known as CD3D; CD3-DELTA; T3D; CD3 antigen, delta subunit; CD3 delta; CD3 delta; CD3d antigen, delta polypeptide (TiT3 complex); OKT3, delta chain; T cell receptor T3 delta chain; T cell surface glycoprotein CD3 delta chain, etc.). In some embodiments, a suitable cytoplasmic region may comprise an ITAM motif-containing portion of the full-length CD3 delta amino acid sequence. In some embodiments, the cytoplasmic region is derived from the T cell surface glycoprotein CD3 epsilon chain (also known as CD3e, CD3 epsilon; T cell surface antigen T3 / Leu-4 epsilon chain, T cell surface glycoprotein CD3 epsilon chain, AI504783, CD3, CD3-epsilon, T3e, etc.). In some embodiments, a suitable cytoplasmic region may comprise an ITAM motif-containing portion of the full-length CD3 epsilon amino acid sequence. In some embodiments, the cytoplasmic region is derived from the T cell surface glycoprotein CD3 gamma chain (also known as CD3G, CD3 gamma, T cell receptor T3 gamma chain, CD3-GAMMA, T3G, gamma polypeptide (TiT3 complex), etc.). In some embodiments, a suitable cytoplasmic region may comprise an ITAM motif-containing portion of the full-length CD3 gamma amino acid sequence. In some embodiments, the cytoplasmic region is derived from the T cell surface glycoprotein CD3 zeta chain (also known as CD3Z, CD3 zeta, T cell receptor T3 zeta chain, CD247, CD3-ZETA, CD3H, CD3Q, T3Z, TCRZ, etc.). In some embodiments, a suitable cytoplasmic region may comprise an ITAM motif-containing portion of the full-length CD3 zeta amino acid sequence.

[0092] 6. Auxiliary stimulation area Non-limiting examples of suitable costimulatory regions, e.g., costimulatory regions contained in the cytoplasmic domain, include, but are not limited to, polypeptides derived from 4-lBB (CD137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, and HVEM.

[0093] A costimulatory region may have a length of at least 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, or 300 amino acids, or any range derivable therein, at most 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, or 300 amino acids, or any range derivable therein, or exactly 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, or 300 amino acids, or any range derivable therein. In some embodiments, the costimulatory region is derived from the intracellular portion of the transmembrane protein 4-1BB (also known as TNFRSF9; CD137; CDwl37; ILA, etc.). In some aspects, the costimulatory region is derived from the intracellular portion of the transmembrane protein CD28 (also known as Tp44). In some aspects, the costimulatory region is derived from the intracellular portion of the transmembrane protein ICOS (also known as AILIM, CD278, and CVID1). In some aspects, 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 aspects, the costimulatory region is derived from the intracellular portion of the transmembrane protein BTLA (also known as BTLA1 and CD272). In some aspects, the costimulatory region is derived from the intracellular portion of the transmembrane protein CD27 (also known as S152, T14, TNFRSF7, and Tp55). In some aspects, the costimulatory region is derived from the intracellular portion of the transmembrane protein CD30 (also known as TNFRSF8, D1S166E, and Ki-1). In some embodiments, the costimulatory region is derived from the intracellular portion of the transmembrane protein GITR (also known as TNFRSF18, 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 known as TNFRSF14, RP3-395M20.6, ATAR, CD270, HVEA, HVEM, LIGHTR, and TR2).

[0094] In some aspects, the CAR of the present disclosure comprises a 4-1BB costimulatory domain. In some aspects, the 4-1BB costimulatory domain comprises SEQ ID NO:7. In some aspects, the CAR of the present disclosure comprises an OX40 costimulatory domain. In some aspects, the OX40 costimulatory domain comprises SEQ ID NO:14. In some aspects, the anti-CD19 CAR of the present disclosure comprises a 4-1BB costimulatory domain. In some aspects, the anti-CD79b CAR of the present disclosure comprises an OX40 costimulatory domain.

[0095] 7. Peptide Linker In some aspects, the polypeptides of the present disclosure include a peptide linker (sometimes referred to as a linker). Peptide linkers may be used to separate any of the peptide domains / regions described herein. As an example, the linker may be between the signal peptide and the antigen binding domain, between the VH and VL of the antigen binding domain, between the antigen binding domain and the peptide spacer, between the peptide spacer and the transmembrane domain, adjacent to the costimulatory domain, on the N region or C region of the costimulatory domain, and / or between the transmembrane domain and the endodomain. The peptide linker may have any of a variety of amino acid sequences. The domains and regions can usually be joined by flexible peptide linkers, although other chemical bonds are not excluded. The linker may be a peptide of about 6 to about 40 amino acids in length, or about 6 to about 25 amino acids in length. These linkers can be generated by using synthetic oligonucleotides that code for the linker to join the proteins.

[0096] A peptide linker with some degree of flexibility can be used. The peptide linker can have virtually any amino acid sequence, keeping in mind that suitable peptide linkers have sequences that generally result in flexible peptides. The use of small amino acids such as glycine and alanine is useful in creating flexible peptides. The creation of such sequences is routine for those skilled in the art.

[0097] A suitable linker can be readily selected and may be of any suitable length, for example, a linker of 1 amino acid (e.g., Gly) to 20 amino acids, 2 to 15 amino acids, 3 to 12 amino acids, including 4 to 10 amino acids, 5 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids, or may be 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, or 7 amino acids.

[0098] Suitable linkers can be readily selected and can be of any suitable different length, for example, a linker of 1 amino acid (e.g., Gly) to 20 amino acids, 2 to 15 amino acids, 3 to 12 amino acids, including 4 to 10 amino acids, 5 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids, or can be 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, or 7 amino acids.

[0099] II. Nucleic acids In certain embodiments, nucleic acid sequences may be present in various cases, such as recombinant vectors of isolated segments and integrated sequences or recombinant polynucleotides encoding one or both chains of an antibody, or fragments, derivatives, muteins, or variants thereof, polynucleotides sufficient for use as hybridization probes, PCR or sequencing primers for identifying, analyzing, mutating, or amplifying polynucleotides encoding polypeptides, antisense nucleic acids for inhibiting polynucleotide expression, and complementary sequences of the foregoing described herein. Nucleic acids encoding epitopes for some of the antibodies provided herein are also provided. Nucleic acids encoding fusion proteins comprising these peptides are also provided. Nucleic acids may be single-stranded or double-stranded and may include RNA and / or DNA nucleotides and artificial variants thereof (e.g., peptide nucleic acids).

[0100] The term "polynucleotide" refers to a nucleic acid molecule that is recombinant or isolated from total genomic nucleic acid. The term "polynucleotide" includes oligonucleotides (nucleic acids of 100 residues or less in length), recombinant vectors including, for example, plasmids, cosmids, phages, viruses, and the like. A polynucleotide, in certain aspects, includes a regulatory sequence that is substantially isolated from the naturally occurring gene or protein coding sequence of the regulatory sequence. A polynucleotide may be single-stranded (coding or antisense) or double-stranded, and may be RNA, DNA (genomic, cDNA, or synthetic), analogs thereof, or combinations thereof. Additional coding or non-coding sequences may be present in a polynucleotide, but need not be present in the polynucleotide.

[0101] In this regard, the terms "gene", "polynucleotide", or "nucleic acid" are used to refer to a nucleic acid that encodes a protein, polypeptide, or peptide (including any sequences necessary for correct transcription, post-translational modification, or localization). As will be understood by those skilled in the art, this term encompasses genomic sequences, expression cassettes, cDNA sequences, and even smaller engineered nucleic acid segments that express or can be adapted to express proteins, polypeptides, domains, peptides, fusion proteins, and variants. A nucleic acid that encodes all or a portion of a polypeptide may contain a contiguous nucleic acid sequence that encodes all or a portion of such a polypeptide. It is also intended that a particular polypeptide may be encoded by a nucleic acid that contains variations having slightly different nucleic acid sequences, but nonetheless encodes the same or substantially similar protein.

[0102] In certain embodiments, polynucleotide variants have substantial identity to the sequences disclosed herein; polynucleotide variants include at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity, including all values ​​and ranges between at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more, when comparing the polynucleotide sequences provided herein using the methods described herein (e.g., BLAST analysis using standard parameters).In certain aspects, the isolated polynucleotide comprises a nucleotide sequence that encodes a polypeptide having at least 90%, preferably 95% or more identity to the amino acid sequence described herein over the entire length of the sequence, or a nucleotide sequence that is complementary to the isolated polynucleotide.

[0103] As the total length of a nucleic acid segment can vary considerably, the nucleic acid segment can be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, etc., regardless of the length of the coding sequence itself. The nucleic acid can be of any length. The nucleic acid 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 in length, and / or can include one or more additional sequences, such as regulatory sequences, and / or can be part of a larger nucleic acid, such as a vector. Thus, it is contemplated that nucleic acid fragments of almost any length can be used, with the total length being preferably limited by the ease of preparation and use in the intended recombinant nucleic acid protocol. In some cases, the nucleic acid sequence may code for a polypeptide sequence with additional heterologous coding sequences, such as heterologous coding sequences that allow purification, transport, secretion, post-translational modification of the polypeptide, or that allow therapeutic benefits such as targeting or efficacy. As mentioned above, tags or other heterologous polypeptides may be added to the modified polypeptide coding sequence. Here, "heterologous" refers to a polypeptide that is not identical to the modified polypeptide.

[0104] A. Vector The polypeptide of the present disclosure, including the dual CAR of the present disclosure, can be delivered to recipient immune cells by any suitable vector, including viral vector or non-viral vector.Examples of viral vectors include at least retrovirus, lentivirus, adenovirus, or adeno-associated virus vector.Examples of non-viral vectors include at least plasmid, transposon, lipid, nanoparticle, etc.

[0105] When vectors encoding two types of antigen-targeting CAR cells are transduced into immune cells, the CARs may or may not be included on the same vector or with the same vector. In some cases, the CARs are expressed from the same vector molecule, e.g., the same viral vector molecule. In such cases, the expression of the CARs may or may not be regulated by the same regulatory element. When the CARs are on the same vector, they may or may not be expressed as separate polypeptides. When the CARs are expressed as separate polypeptides, they may be separated, for example, by a 2A element or an IRES element on the vector (or both species may be used once or multiple times on the same vector). In some aspects, the dual CAR expressing vector of the present disclosure is a multicistronic (e.g., bicistronic) vector expressing an anti-CD19 CAR and an anti-CD79b CAR separated by a 2A element. In some aspects, the 2A element is a T2A element. In some aspects, the 2A element is a P2A element. In some aspects, the 2A element is an E2A element.

[0106] B. Sequence Certain nucleotide sequences of the polynucleotides, including polynucleotides expressing chimeric antigen receptors and portions and regions thereof, are set forth in Table 2.

[0107] [Table 2] TIFF2025508961000010.tif240169TIFF2025508961000011.tif234169TIFF2025508961 000012.tif243169TIFF2025508961000013.tif243169TIFF2025508961000014.tif96169

[0108] III. Cancer Therapy In some aspects, the methods of the disclosure include administering a cancer therapy to the subject. In some aspects, the cancer therapy includes a local cancer therapy. In some aspects, the cancer therapy excludes a systemic cancer therapy. In some aspects, the cancer therapy excludes a local therapy. In some aspects, the cancer therapy includes a local cancer therapy without administration of a systemic cancer therapy. In some aspects, the cancer therapy includes an immunotherapy, which may be an immune checkpoint therapy. Any of these cancer therapies may also be excluded. A combination of these therapies may also be administered.

[0109] The term "cancer" as used herein may be used to describe solid tumors, metastatic cancers, or non-metastatic cancers. In certain embodiments, cancer may occur in the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, colon, rectum, anus, gums, head, kidney, liver, lung, nasopharynx, cervix, ovary, pancreas, prostate, skin, stomach, testicle, tongue, or uterus. In some embodiments, cancer is recurrent cancer. In some embodiments, cancer is stage I cancer. In some embodiments, cancer is stage II cancer. In some embodiments, cancer is stage III cancer. In some embodiments, cancer is stage IV cancer.

[0110] The cancer may specifically be of the following histological types, but is not limited to: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; mixed hepatocellular and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenomatous intrapolypoid adenocarcinoma; adenocarcinoma, familial polyposis coli; solid tumors; carcinoid tumor, malignant; bronchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenocortical carcinoma; endometroid carcinoma; cutaneous adnexal carcinoma; apocrine adenocarcinoma; adenocarcinoma);mucoepidermoid carcinoma;cystadenocarcinoma;papillary cystadenocarcinoma;papillary serous cystadenocarcinoma;mucinous cystadenocarcinoma;mucinous adenocarcinoma;signet ring cell carcinoma;invasive ductal carcinoma;medullary carcinoma;lobular carcinoma;inflammatory carcinoma;Paget's disease, breast;acinic cell carcinoma;adenosquamous carcinoma;adenocarcinoma with squamous metaplasia;thymoma, malignant;ovarian stromal tumor, malignant;theca cell tumor, malignant;granulosa cell tumor, malignant;androblastoma, malignant;Sertoli cell tumor;Leydig cell tumor, malignant;lipid cell tumor, malignant;paraganglioma, malignant;extra-mammary paraganglioma, malignant;pheochromocytoma;angioangiosarcoma;malignant melanoma;amelanotic melanoma;superficial spreading melanoma;malignant melanoma in giant pigmented nevus melanoma in giant pigmented nevus);epithelioid cell melanoma;blue nevus, malignant;sarcoma;fibrosarcoma;fibrous histiocytoma, malignant;myxosarcoma;liposarcoma;leiomyosarcoma;rhabdomyosarcoma;embryonal rhabdomyosarcoma;alveolar rhabdomyosarcoma;stromal sarcoma;mixed tumor, malignant;Mullerian mixed tumor;nephroblastoma;hepatoblastoma;Carcinosarcoma;Mesenchymoma, malignant;Brenner tumor, malignant;Phyllodes tumor, malignant;Synovial sarcoma;Mesothelioma, malignant;Dysgerminoma;Embryonal carcinoma;Teratoma, malignant;Ovarian goiter, malignant;Choriocarcinoma;Mesonephroma, malignant;Hemangiosarcoma;Hemangioendothelioma, malignant;Kaposi's sarcoma;Hemangiopericytoma, malignant;Lymphangiosarcoma;Osteosarcoma;Parocytoma;Chondrosarcoma;Chondrosarcoma, malignant;Mesenchymal chondrosarcoma;Giant cell tumor of bone;Ewing's sarcoma;Odontogenic tumor, malignant;Ameloblastic odontosarcoma;Enamel epithelium tumor, malignant;ameloblastic fibrosarcoma;pinealoma, malignant;chordoma;glioma, malignant;ependymoma;astrocytoma;protoplasmic astrocytoma;fibrous astrocytoma;astroblastoma;glioblastoma;oligodendroglioma;oligodendroglioblastoma;anaplastic neuroectodermal;cerebellar sarcoma;ganglionucleoblastoma;neuroblastoma;retinoblastoma;olfactory nerve tumor;meningioma, malignant;neurofibrosarcoma;schwannoma, malignant;granular cell tumor, malignant;malignant lymphoma;Hodgkin's disease disease);Hodgkin's disease;lateral granuloma;malignant lymphoma, small lymphocytic;malignant lymphoma, large cell diffuse;malignant lymphoma, follicular;mycosis fungoides;other non-Hodgkin's lymphomas specified;malignant histiocytosis;multiple myeloma;mast cell sarcoma;immunoproliferative small intestinal disease;leukemia;lymphocytic leukemia;plasma cell leukemia;erythroleukemia;lymphocytic leukemia;myeloid leukemia;basophilic leukemia;eosinophilic leukemia;monocytic leukemia;mast cell leukemia;megakaryoblastic leukemia;myeloid sarcoma;and hairy cell leukemia.;

[0111] In some embodiments, the present disclosure provides a method for immunotherapy, comprising administering an effective amount of a composition comprising the CAR of the present disclosure. In one embodiment, a medical disease or disorder is treated by administering a dual CAR expressing cell population that induces an immune response. In certain embodiments of the present disclosure, cancer is treated by administering a dual CAR immune cell population that induces an immune response. Provided herein is a method for treating cancer or delaying the progression of cancer in an individual, comprising administering an effective amount of an antigen-specific cell therapy to the individual. The method can be applied to the treatment of immune disorders, solid cancers, and hematological cancers, as examples. Specifically, the 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's lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia, and chronic lymphocytic leukemia.

[0112] Certain embodiments relate to methods of treating leukemia. Leukemia is a cancer of the blood or bone marrow, characterized by abnormal proliferation (production by proliferation) of blood cells, usually white blood cells (leukocytes). Leukemia is part of a broad collection of diseases called hematological malignancies. Leukemia is a broad term that covers a range of diseases. Leukemia is clinically and pathologically divided into its acute and chronic forms.

[0113] In some embodiments of the method of the present disclosure, the activated CD4 T cells and / or CD8 T cells in the individual are characterized by CD4 T cells and / or CD8 T cells producing γ-IFN and / or enhanced cytolytic activity compared to before administration of the combination. γ-IFN can be measured by any means known in the art, including, for example, intracellular cytokine staining (ICS) involving cell fixation, permeabilization, and staining with an antibody against γ-IFN. Cytolytic activity can be measured by any means known in the art, for example, using a mixed effector and target cell cell killing assay.

[0114] In some embodiments, the subject may be administered nonmyeloablative lymphodepleting chemotherapy prior to T cell therapy. The nonmyeloablative lymphodepleting chemotherapy may be any suitable therapy that may be administered by any suitable route. In particular, if the cancer is melanoma that may metastasize, the nonmyeloablative lymphodepleting chemotherapy may include, for example, administration of cyclophosphamide and fludarabine. An exemplary administration route for administering cyclophosphamide and fludarabine is intravenous administration. Similarly, any suitable dose of cyclophosphamide and fludarabine may be administered. In certain aspects, the dose is about 25 mg / m 2 Fludarabine is administered for 5 days, followed by cyclophosphamide at approximately 60 mg / kg for 2 days.

[0115] In certain embodiments, a T cell growth factor that promotes the proliferation and activation of autologous T cells is administered to the subject simultaneously with or after the autologous T cells. The T cell growth factor can be any suitable growth factor that promotes the proliferation and activation of autologous T cells. Examples of suitable T cell growth factors include interleukin (IL)-2, IL-7, IL-15, and / or IL-12, which can be used alone or in various combinations, such as IL-2 and IL-7, IL-2 and IL-15, IL-7 and IL-15, IL-2, IL-7 and IL-15, IL-12 and IL-7, IL-12 and IL-15, or IL-12 and IL2.

[0116] Therapeutically effective amounts of immune cells can be administered by a number of routes, including parenteral administration, for example, intravenous, intraperitoneal, intramuscular, intrasternal, or intraarticular injection, or infusion.

[0117] For isolated, accessible solid tumors, intratumoral injection or injection into the tumor vasculature is specifically contemplated. Local, regional, or systemic administration may also be appropriate. For tumors >4 cm, the volume administered is about 4-10 ml (particularly 10 ml), whereas for tumors <4 cm, a volume of about 1-3 ml (particularly 3 ml) is used. Multiple injections delivered as a single dose comprise a volume of about 0.1 to about 0.5 ml.

[0118] The T cell population can be administered in a treatment regimen consistent with the disease, for example, in a single or multiple doses over one to several days to remit the disease state, or in regular doses over an extended period to inhibit disease progression and prevent disease recurrence. The exact dose employed in the formulation will also depend on the route of administration and the severity of the disease or disorder, and must be determined according to the professional judgment and circumstances of each patient. The therapeutically effective amount of T cells will depend on the subject being treated, the severity and type of affliction, and the method of administration. In some embodiments, a dose that can be used in the treatment of a human subject is at least 3.8×10 4 , at least 3.8 × 10 5 , at least 3.8 × 10 6 , at least 3.8 × 10 7 , at least 3.8 × 10 8 , at least 3.8 × 10 9 , or at least 3.8 × 10 10 T cells / m 2 In certain embodiments, the dose used in treating a human subject is about 3.8×10 9 ~Approx. 3.8×10 10 T cells / m 2 In a further embodiment, the therapeutically effective amount of T cells is about 5×10 6 cells / kg body weight~approx. 7.5×10 8 Cells / kg body weight, e.g., about 2×10 7 Cells ~ approx. 5 x 10 8 cells / kg body weight, or approximately 5 x 10 7 cells ~ approx. 2 x 10 8The amount of T cells may be cells / kg body weight.The exact amount of T cells can be easily determined by those skilled in the art based on the age, weight, sex and physiological condition of the subject.Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0119] In certain embodiments of the present disclosure, an effective amount of dual CAR expressing immune cells is delivered to an individual in need thereof, for example, an individual with cancer.The cells then enhance the individual's immune system to attack cancer cells.In some cases, the individual is provided with one or more doses of immune cells.When an individual is provided with two or more doses of immune cells, the period between administrations must be sufficient to allow time for proliferation in the individual.In certain embodiments, the period between doses is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or more.

[0120] In certain embodiments, the cells engineered to express dual CARs are administered in a therapeutically effective amount (10 3 ~10 10 A therapeutically effective amount is provided to an individual in the range of 10 3 ~10 10 , 10 3 ~10 9 , 10 3 ~10 8 , 10 3 ~10 7 , 10 3 ~10 6 , 10 3 ~10 5 , 10 3 ~10 4 , 10 4 ~10 10 , 10 4 ~10 9 , 10 4 ~10 8 , 10 4 ~10 7 , 10 4 ~10 6 , 10 4 ~10 5 , 10 5 ~10 10 , 105 ~10 9 , 10 5 ~10 8 , 10 5 ~10 7 , 10 5 ~10 6 , 10 6 ~10 10 , 10 6 ~10 9 , 10 6 ~10 8 , 10 6 ~10 7 , 10 7 ~10 10 , 10 7 ~10 9 , 10 7 ~10 8 , 10 8 ~10 10 , 10 8 ~10 9 , or 10 9 ~10 10 Thus, in certain embodiments, an individual with a particular cancer is provided with a therapeutically effective amount of the dual CAR-expressing cells one or more times.

[0121] IV. Infinite immune cells Certain aspects of the present disclosure relate to immune cells engineered to express one or more genes. Expressing one or more genes directly or indirectly increases the life span of the cell compared to cells that do not express the one or more genes. In certain aspects, the cell is engineered to express one or more genes, including one or more heterologous genes. In other cases, the cell is engineered to have upregulation of expression of one or more genes endogenous to the cell, for example, by manipulating one or more regulatory elements of one or more endogenous genes to the cell.

[0122] In certain embodiments, immune cells are engineered to express BCL6 and one or more pro-survival or anti-apoptotic or cell survival genes (there may or may not be overlap in genes classified as pro-survival or anti-apoptotic or cell survival). As used herein, pro-survival genes refer to nucleic acid polymers that can exert anti-apoptotic functions or promote survival by any mechanism. The nucleic acid polymers that can exert anti-apoptotic functions may 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 polymers that can exert anti-apoptotic functions may be one or more of the inhibitor of apoptosis protein (IAP) family genes, such as XIAP, c-IAPl, C-IAP2, NAIP, and survivin. The nucleic acid polymer capable of exerting anti-apoptotic function may 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. The nucleic acid polymer for knockdown or knockout may be an shRNA expression cassette. Alternatively, these caspase genes can also be knocked out by gene editing methods (CRISPR, TALEN, zinc finger method, etc.). The nucleic acid polymer capable of exerting anti-apoptotic function may 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 capable of exerting anti-apoptotic functions may have anti-apoptotic effects, for example, insulin-like growth factor (IGF-1), Hsp70, Hsp27, cFLIP, BNIP3, FADD, Akt, and NF-κB, Raf-1 and MEK1, p90Rsk, C-Jun, BNIP2, BAG1, HSPA9, HSP90B1, miRNA21, miR-106b-25, miR-206, miR-221 / 222, miR-17-92, miR-133, miR-143, miR-145, miR-155, miR-330, etc.

[0123] Infinite T cells can be generated using either wild-type or mutant BCL6. We have confirmed that infinite T cells can be generated using either wild-type BCL6 or mutant BCL6 with one specific nucleotide difference. In wild-type BCL6, the codon for the amino acid at position 395 is CCT (encoding proline / P), and in mutant BCL6, the codon for the amino acid at position 395 is CTT (encoding leucine / L). The nucleotide and amino acid sequences of the two BCL6 genes are shown below (mutations in the wild-type sequence are underlined):

[0124] Wild type BCL6 aa sequence: TIFF2025508961000015.tif92159

[0125] Nucleotide sequence of wild-type BCL6 (mutation codons in the wild-type sequence are underlined): TIFF2025508961000016.tif180160

[0126] The aa sequence of mutant BCL6 (leucine mutations underlined): TIFF2025508961000017.tif92159

[0127] Nucleotide sequence of mutant BCL6 (leucine codons underlined): TIFF2025508961000018.tif180160

[0128] Immune cells include T cells (e.g. regulatory T cells, CD4 + T cells, CD8 + The immune cells may be any type of immune cell, including T cells, αβT cells, γ-δT cells, or mixtures thereof), NK cells, invariant NKT cells, NKT cells, innate lymphocytes, or mixtures thereof. The immune cells may be virus-specific, express a CAR, and / or express a TCR. In some embodiments, the cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells (DCs), mast cells, eosinophils, and / or basophils. Also provided herein are methods of generating and manipulating immune cells, as well as methods of using and administering the cells for adoptive cell therapy, where the cells may be autologous or allogeneic. Thus, the immune cells may be used as immunotherapy, e.g., to target cancer cells. These immune cells may be used for therapy as a single cell type or as a combination of multiple immune cell types. In certain embodiments, the immune cells are CD3+, CD4+, CD8+, CD16+, or mixtures thereof.

[0129] Immune cells can be isolated from a subject, particularly a human subject. Immune cells can be obtained from a subject of interest, for example, a subject suspected of having a particular disease or condition, a subject suspected of having a predisposition to a particular disease or condition, or a subject undergoing therapy for a particular disease or condition. Immune cells can be collected from any location in a subject where immune cells are found, including, but not limited to, blood, umbilical cord blood, spleen, thymus, lymph nodes, and bone marrow. Immune cells isolated can be used directly or can be stored for a period of time, for example, by freezing.

[0130] Immune cells can be enriched / purified from any tissue in which they are found, including, but not limited to, blood (including blood collected by blood bank or umbilical cord blood bank), spleen, bone marrow, tissue removed and / or exposed during surgical procedures, and tissue obtained via biopsy procedures. The tissue / organ from which immune cells are enriched, isolated, and / or purified can be isolated from both living and non-living subjects, with the non-living subject being an organ donor. In certain embodiments, immune cells are isolated from blood, such as peripheral blood or umbilical cord blood. In some aspects, immune cells isolated from umbilical cord blood have increased immunoregulatory capacity, for example, as measured by suppression of CD4+ T cells or CD8+ T cells. In certain aspects, immune cells are isolated from pooled blood, particularly pooled umbilical cord blood, for increased immunoregulatory capacity. Pooled blood may be from two or more sources, for example, three, four, five, six, seven, eight, nine, ten, or more sources (eg, donor subjects).

[0131] The immune cell population can be obtained from a subject who needs a therapy associated with decreased immune cell activity or suffers from a disease associated with decreased immune cell activity. Thus, the cells can be autologous to the subject who needs the therapy. Alternatively, the immune cell population can be obtained from a donor, such as a partially or fully histocompatible donor, or a fully histocompatible donor. The immune cell population can be taken from peripheral blood, umbilical cord blood, bone marrow, spleen, or any other organ / tissue in which the subject or donor has immune cells. The immune cells can be isolated from a pool of subjects and / or donors, such as pooled umbilical cord blood.

[0132] When the immune cell population is obtained from a donor different from the subject, the donor may be allogeneic, so long as the obtained cells are compatible with the subject in that they can be introduced into the subject. Allogeneic donor cells may or may not be human leukocyte antigen (HLA)-compatible.

[0133] Further methods and compositions related to Infinite Immune Cells are described in PCT Patent Application Publication No. WO / 2021 / 034982, which is incorporated by reference in its entirety.

[0134] AT cells In some embodiments, the immune cell is a T cell. In the last two decades, several basic approaches have been described for inducing, activating and expanding functional anti-tumor effector cells. These include autologous cells, such as tumor-infiltrating lymphocytes (TILs); T cells activated ex vivo with autologous DCs or PBMCs, lymphocytes, artificial antigen-presenting cells (APCs), or beads coated with T cell ligands and activating antibodies, or cells isolated by target cell membrane capture; allogeneic cells that naturally express anti-host tumor T cell receptors (TCRs); and non-tumor-specific autologous or allogeneic cells that are genetically reprogrammed or "redirected" to express tumor-reactive TCRs or chimeric TCR molecules that exhibit antibody-like tumor recognition capabilities, known as "T-bodies". These approaches have led to a large number of protocols for T cell preparation and immunization that can be used in the methods described herein.

[0135] In some embodiments, the T cells are derived from blood, bone marrow, lymph, umbilical cord, or lymphoid organs. In some aspects, the cells are human cells. The cells are typically primary cells, e.g., cells isolated directly from a subject and / or cells isolated from a subject and frozen. In some embodiments, the cells include one or more subsets of T cells or other cell types, e.g., the total T cell population, CD4 + cells, CD8 +The cells and subpopulations thereof include those defined by, for example, function, activation state, maturity, differentiation potential, expansion, recirculation, localization, and / or persistence ability, antigen specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and / or degree of differentiation. The cells may be allogeneic and / or autologous with respect to the subject to be treated. In some aspects, for example, for off-the-shelf technology, the cells exhibit pluripotency and / or multipotency, such as stem cells, e.g., induced pluripotent stem cells (iPSCs). In some embodiments, the method includes isolating cells from a subject, preparing, treating, culturing, and / or manipulating the cells as described herein, and reintroducing the cells into the same patient before and after cryopreservation.

[0136] T cell subtypes and subpopulations (e.g., CD4 + and / or CD8 + Among the naive T (T N ) cells, effector T cells (T EFF ), memory T cells and their subtypes, such as stem cell memory T (TSC M ), Central Memory T (TC M ), Effector Memory T (T EM ), or terminally differentiated effector memory T cells, tumor infiltrating lymphocytes (TILs), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal-associated invariant T (MAIT) cells, natural 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 / β T cells, and γ / δ T cells.

[0137] In some embodiments, one or more of the T cell populations are enriched or depleted for cells that are positive for a particular marker, e.g., 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 populations of T cells (e.g., non-memory cells) but present or expressed at relatively high levels on certain other populations of T cells (e.g., memory cells).

[0138] In some embodiments, T cells are separated from the PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other leukocytes, e.g., CD14. + Selection step or CD8 + The selection process is CD4 + Helper and CD8 + It is used to separate cytotoxic T cells. + Populations and CD8 + The population can be further sorted into subpopulations by positive or negative selection against markers expressed, or expressed to a relatively high extent, on one or more naive, memory, and / or effector T cell subpopulations.

[0139] In some embodiments, CD8 + T cells are further enriched or depleted for naive, central memory, effector memory, and / or central memory stem cells, e.g., by positive or negative selection based on surface antigens associated with each subpopulation. In some embodiments, central memory T (T CM ) cells or stem cell memory cells are enriched to enhance efficacy, e.g., to improve long-term survival, expansion, and / or engraftment following administration, which in some aspects is particularly robust in such subpopulations.

[0140] In some embodiments, the T cells are autologous T cells. In this method, a tumor sample is obtained from a patient and a single cell suspension is obtained. The single cell suspension can be obtained in any suitable manner, for example, mechanically (e.g., by disaggregating the tumor using a gentleMACS™ Dissociator, Miltenyi Biotec, Auburn, Calif.) or enzymatically (e.g., collagenase or DNase). The single cell suspension of the product of the enzymatic digestion of the tumor is cultured in interleukin-2 (IL-2) or other growth factors.

[0141] The cultured T cells can be pooled and rapidly expanded. Rapid expansion results in at least about a 50-fold (e.g., 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold or more) increase in the number of antigen-specific T cells over a period of about 10 to about 14 days. More preferably, rapid expansion results in at least about a 200-fold (e.g., 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold or more) increase in the number of antigen-specific T cells over a period of about 10 to about 14 days.

[0142] Expansion can be accomplished by any of a number of methods known in the art. For example, T cells can be rapidly expanded using non-specific T cell receptor stimulation in the presence of feeder lymphocytes and either interleukin-2 (IL-2) or interleukin-15 (IL-15), with IL-2 being preferred. Non-specific T cell receptor stimulation can include about 30 ng / ml OKT3, a mouse monoclonal anti-CD3 antibody (available from Ortho-McNeil®, Raritan, NJ). Alternatively, T cells can be rapidly expanded in vitro by stimulating peripheral blood mononuclear cells (PBMCs) with one or more antigens of the cancer (including antigenic portions thereof, e.g., epitopes, or cells), optionally 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, in the presence of a T cell growth factor, e.g., 300 IU / ml IL-2 or IL-15, with IL-2 being preferred. In vitro induced T cells are rapidly expanded by restimulation with the same antigen of the cancer pulsed onto HLA-A2 expressing antigen presenting cells or onto antigen presenting cells expressing other HLA molecules. In vitro induced T cells can also be expanded in the absence of antigen presenting cells.

[0143] Autologous T cells can be modified to express T cell growth or differentiation factors that promote the proliferation, differentiation, and activation of autologous T cells. Suitable T cell growth factors include, for example, interleukin (IL)-2, IL-7, IL-15, IL-18, IL-21, and IL-12. Suitable modification methods are known in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3 rded., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. In certain aspects, the modified autologous T cells express high levels of T cell growth factor. T cell growth factor coding sequences, such as the coding sequence for IL-12, are readily available in the art, as are promoters that facilitate high level expression when operably linked to the coding sequence for T cell growth factor.

[0144] B.NK cells In some embodiments, the immune cell is a natural killer (NK) cell. NK cells are a subpopulation of lymphocytes that have spontaneous cytotoxicity against various tumor cells, virus-infected cells, and some normal cells in bone marrow and thymus. NK cells differentiate and mature in bone marrow, lymph nodes, spleen, tonsils, and thymus. NK cells can be detected by specific surface markers such as CD16, CD56, and / or CD8 in humans. NK cells do not express T cell antigen receptor, pan T marker CD3, or surface immunoglobulin B cell receptor.

[0145] In certain embodiments, the NK cells are derived from human peripheral blood mononuclear cells (PBMCs), unstimulated leukapheresis products (PBSCs), human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs), bone marrow, tissue, or umbilical cord blood by methods well known in the art.

[0146] C.NKT cells Natural killer T (NKT) cells are a heterogeneous group of T cells that share properties with T cells and natural killer cells. Many of these cells recognize the nonpolymorphic CD1d molecule, an antigen-presenting molecule that binds self and foreign lipids and glycolipids. Natural killer T (NKT) cells constitute only about 0.1% of all peripheral blood T cells. NKT cells are a T cell subset that co-express the αβ T cell receptor, but also express various molecular markers typically associated with NK cells, such as NK1.1. Invariant natural killer T (iNKT) cells express high levels and depend on the transcription factor promyelocytic leukemia zinc finger for their development. There are currently five major distinct iNKT cell subsets. These subset cells produce a distinct set of cytokines upon activation. The subtypes iNKT1, iNKT2, and iNKT17 mirror Th cell subsets in cytokine production. In addition, there are subtypes that are specialized for T follicular helper-like function and IL-10-dependent regulatory function.

[0147] D. Innate lymphocytes Innate lymphoid cells (ILCs) are a collection of innate immune cells that originate from common lymphoid progenitors (CLPs) and belong to the lymphoid lineage. These cells are defined by the absence of antigen-specific B-cell or T-cell receptors due to the lack of recombination activating genes (RAGs). ILCs do not express myeloid or dendritic cell markers. ILCs play a role in the regulation of protective immunity as well as homeostasis and inflammation, and thus, dysregulation of ILCs can result in immune conditions such as allergy, bronchial asthma, and autoimmune diseases. ILCs can be divided based on the cytokines they can produce, as well as the transcription factors that regulate their development and function.

[0148] V. Cell Formulation and Culture In certain embodiments, the cells of the present disclosure may be specially formulated and / or cultured in a specific medium. The cells may be formulated in such a way that they are suitable for delivery to a recipient without adverse effects.

[0149] In a particular aspect, the medium can be prepared using, as its basal medium, any of media used in culturing animal cells, such as AIM V, X-VIVO-15, NeuroBasal, EGM2, TeSR, BME, BGJb, CMRL 1066, Glasgow MEM, Improved MEM Zinc Option, IMDM, Medium 199, Eagle MEM, αMEM, DMEM, Ham, RPMI-1640, and Fischer's media, and any combination thereof, but the medium may not be particularly limited thereto as long as it can be used for culturing animal cells. In particular, the medium may be xeno-free or chemically defined.

[0150] The medium may be a serum-containing medium or a serum-free medium, or may be a xeno-free medium. In order to prevent contamination with heterogeneous animal-derived components, the serum may be derived from the same animal as the serum of the stem cells. Serum-free medium refers to a medium that does not have raw serum or unpurified serum, and therefore can include a medium with purified blood-derived components or animal tissue-derived components (e.g., growth factors).

[0151] The medium may or may not contain any substitute for serum. The substitute for serum may include albumin (e.g., lipid-rich albumin, bovine albumin, albumin substitutes such as recombinant or humanized albumin, vegetable starch, dextran, and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolgiycerol, or materials that suitably contain equivalents thereof. The substitute for serum may be prepared, for example, by the method disclosed in International Publication No. 98 / 30679 (incorporated herein in its entirety). Alternatively, any commercially available material may be used for added convenience. Commercially available materials include knockout serum replacement (KSR), Chemically-defined lipid concentrated (Gibco), and Glutamax (Gibco).

[0152] In certain embodiments, the medium contains the following: vitamins, e.g., biotin; DL-alpha tocopherol acetate; DL-alpha-tocopherol; vitamin A (acetate); proteins, e.g., BSA (bovine serum albumin) or human albumin, fatty acid free fraction V (FAFV), and / or acetylcholinesterase inhibitors. 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 diHCl; sodium selenite; and / or T3 (triodo-I-thyronine). In certain embodiments, one or more of these may be explicitly excluded.

[0153] In some embodiments, the medium further comprises vitamins. In some embodiments, the medium comprises one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or thirteen of the following (and any range derivable therein): biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, nicotinamide folate, pyridoxine, riboflavin, thiamine, inositol, vitamin B12, or the medium comprises combinations thereof or salts thereof. In some embodiments, the medium comprises or consists essentially of biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, nicotinamide folate, pyridoxine, riboflavin, thiamine, inositol, and vitamin B12. In some embodiments, the vitamin comprises or consists essentially of biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, or a combination or salt thereof. In some embodiments, the medium further comprises a protein. In some embodiments, the protein comprises albumin or bovine serum albumin, a fraction of BSA, catalase, insulin, transferrin, superoxide dismutase, or a combination thereof. In some embodiments, the medium further comprises one or more of the following: corticosterone, D-galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triiodo-I-thyronine, or a combination thereof. In some embodiments, the medium comprises one or more of the following: B-27® supplement, Xenofree B-27® supplement, GS21™ supplement, or a combination thereof. In some embodiments, the medium comprises or further comprises amino acids, simple sugars, inorganic ions, hi some embodiments, the amino acids comprise arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine, or combinations thereof.In some embodiments, the inorganic ions include sodium, potassium, calcium, magnesium, nitrogen, or phosphorus, or combinations or salts thereof. In some embodiments, the medium further includes one or more of the following: molybdenum, vanadium, iron, zinc, selenium, copper, or manganese, or combinations thereof. In certain embodiments, the medium comprises or consists essentially of one or more vitamins discussed herein, and / or one or more proteins discussed herein, and / or one or more of the following: corticosterone, D-galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triiodo-I-thyronine, B-27® supplement, XenoFree B-27® supplement, GS21™ supplement, amino acids (e.g., arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine), monosaccharides, inorganic ions (e.g., sodium, potassium, calcium, magnesium, nitrogen, and / or phosphorus) or salts thereof, and / or molybdenum, vanadium, iron, zinc, selenium, copper, or manganese. In certain embodiments, one or more of these may be explicitly excluded.

[0154] The medium may also contain one or more exogenously 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, one or more of which may be explicitly excluded in certain embodiments.

[0155] One or more of the medium components may be may be added at a concentration of at least 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; may be added at a concentration of at most 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; or It may be added at a concentration of 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.

[0156] In certain embodiments, the cells of the present disclosure are specially formulated. The cells of the present disclosure may or may not be formulated as a cell suspension. In certain cases, the cells of the present disclosure are formulated in a single dose form. The cells of the present disclosure may be formulated for systemic administration or for local administration. In some cases, the cells are formulated for storage prior to use, and the cell formulation may include one or more cryopreservation agents, such as DMSO (e.g., dissolved in 5% DMSO). The cell formulation may include albumin, including human albumin, and certain formulations may include 2.5% human albumin. The cells may be specially formulated for intravenous administration. For example, the cells are formulated for intravenous administration for less than one hour. In certain embodiments, the cells are in a formulated cell suspension that is stable at room temperature for 1 hour, 2 hours, 3 hours, or 4 hours or more from the time of thawing.

[0157] A. Cell Certain embodiments relate to cells that contain the polypeptide or nucleic acid of the present disclosure. In some embodiments, the cells are immune cells or T cells. "T cells" include all types of CD3-expressing immune cells, including T-helper cells, invariant natural killer T (iNKT) cells, cytotoxic T cells, regulatory T cells (Treg), gamma-delta T cells, natural killer (NK) cells, and neutrophils. T cells may refer to CD4+ T cells or CD8+ T cells.

[0158] Suitable mammalian cells include primary cells and immortalized cell lines. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, and the like. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), human embryonic kidney (HEK) 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 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.

[0159] In some cases, the cell is not an immortalized cell line, but rather 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. In one example, the cell is a T lymphocyte obtained from an individual. In another example, the cell is a cytotoxic cell obtained from an individual. In another example, the cell is a stem cell (e.g., a peripheral blood stem cell) or progenitor cell obtained from an individual.

[0160] VI. General Pharmaceutical Compositions In some embodiments, the pharmaceutical composition is administered to the subject. Various aspects may include administering an effective amount of the composition to the subject. In some embodiments, the cell therapy (e.g., dual CAR T cells) is administered to the subject to protect against or treat a condition (e.g., cancer). Furthermore, such compositions can be administered in combination with additional therapeutic agents (e.g., chemotherapeutic agents, immunotherapeutic agents, biotherapeutic agents, etc.). Such compositions are generally dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

[0161] The phrase "pharmacologically acceptable" or "pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other adverse reactions when administered to animals or humans. As used herein, "pharmacologically acceptable carriers" include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmacoactive substances is well known in the art. Except where any conventional media or agent is incompatible with the active ingredient, its use in immunogenic and therapeutic compositions is contemplated. Supplementary active ingredients, such as other anti-infective agents and vaccines, can also be incorporated into the composition.

[0162] The active compound can be formulated for parenteral administration, for example, for injection via intravenous, intramuscular, subcutaneous or intraperitoneal route.Typically, such compositions can be prepared as liquid solutions or suspensions.Solid forms can also be prepared that are suitable for use in preparing solutions or suspensions by adding liquid before injection.Preparations can also be emulsified.

[0163] Pharmaceutical forms suitable for injection 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 dosage form must be sterile and must be fluid to the extent that it can be easily injected. The dosage form must also be stable under the conditions of manufacture and storage, and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

[0164] Proteinaceous compositions may be formulated in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of the protein), acid addition salts formed with inorganic acids such as, for example, hydrochloric acid or phosphoric acid, or acid addition salts formed with organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, and the like. Salts formed with free carboxyl groups can also be derived from inorganic bases such as, for example, sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, and the like.

[0165] The pharmaceutical compositions may contain, for example, a solvent or dispersion medium containing water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants. The activity of microorganisms can be prevented by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include an isotonic agent, for example, sugar or sodium chloride. Prolonged absorption of the injectable compositions can be achieved by using an absorption-delaying agent, for example, aluminum monostearate and gelatin, in the composition.

[0166] Sterile injectable solutions are prepared by incorporating the required amount of active compound into a suitable solvent with various other ingredients as required above, followed by sterilization by filtration, or by an equivalent procedure.Generally, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle that contains the basic dispersion medium and other necessary ingredients from those listed above.For sterile powders for the preparation of sterile injectable solutions, the preferred preparation method is vacuum drying and freeze-drying, which produce a powder of the active ingredient and any additional desired ingredients from a previously sterilized solution that has been sterilized by filtration.

[0167] Administration of the composition is typically via any common route, including, but not limited to, oral or intravenous administration. Alternatively, administration may be orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, or intranasal. Such compositions will usually be administered as pharma- ceutically acceptable compositions that include physiologically acceptable carriers, buffers, or other excipients.

[0168] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation, and in such amount as is therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the injectable types described above.

[0169] References The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. TIFF2025508961000019.tif121160TIFF2025508961000020.tif62160 EXAMPLES

[0170] The following examples are included to demonstrate certain aspects of the invention. It should be understood by those of skill in the art that the techniques disclosed in the following examples are techniques discovered by the inventors to work well in the practice of the invention, and therefore can be considered to constitute certain aspects for practicing the invention. However, in light of this disclosure, it should be understood by those of skill in the art that many changes can be made to the specific aspects disclosed and still obtain the same or similar results without departing from the spirit and scope of the invention.

[0171] Example 1 - Design and use of dual CD19-CD79b CAR T cells method Generation of dual CD19-CD79b CAR construct The dual CD19-CD79b CAR described in this embodiment is composed of two second generation CARs, anti-CD19 CAR (mouse anti-CD19 FMC63) and anti-CD79b CAR (mouse anti-CD79b 28B clone), linked by T2A self-cleaving peptide. CD19 CAR contains CD8α hinge and transmembrane domain, 4-1BB costimulatory domain and CD3ζ intracellular domain. CD79b CAR contains CD8α hinge and transmembrane domain, OX40 costimulatory domain and CD3ζ activation domain. The two CAR constructs were cloned into a bicistronic third generation lentiviral expression vector under the control of human EF1α promoter. See Figure 1A.

[0172] Dual CD19-CD79b CAR-containing lentiviral transduction in primary T cells Primary T cells were enriched from peripheral blood mononuclear cells (PBMCs) obtained from normal donors. Purified T cells were activated with antibodies against CD3, CD28, and CD2 and cultured with IL-2 for 60–72 h. Activated T cells were transduced with RD114 pesudeotyped lentivirus containing dual CD19-CD79b CARs. Expression of CD19 CAR and CD79b CAR was assessed by flow cytometry by staining with CD19 protein-conjugated FITC and CD79b protein-conjugated PE.

[0173] Degranulation assay Non-transduced T cells, CD19 CAR T cells, and dual CD19-CD79b CAR T cells were co-cultured with tumor cells derived from Daudi, SUDHL6, PDX203, PDX300, NALM6, Daudi-CD19KO, SUDHL6-CD19KO, and K562 cell lines, respectively, for 5–6 h. Monensin (0.7 μL / mL) and APC-conjugated anti-CD107a antibody were added to the culture medium at the beginning of the co-culture. Degranulation was assessed by confirming CD107a expression in T cells at the end of the culture period by flow cytometry.

[0174] Cytotoxicity and proliferation assays Untransduced T, CD19 CAR T, and dual CD19-CD79b CAR T were labeled with Cell Trace™ Far Red, and tumor cell lines (Daudi, SUDHL6, PDX203, PDX300, Daudi-CD19KO, SUDHL6-CD19KO, NALM6, and K562) were labeled with Cell Trace™ Violet. Effector cells were co-cultured with tumor cells at effector:target ratios of 1:2, 1:1, and 2:1. Untransduced T alone and tumor cells alone were used as controls. At four time points (days 0, 1, 2, and 4), the percentage of live and dead cells and T cell proliferation were analyzed by flow cytometry after staining with Aqua-live / dead, PerCP-Cy5.5-CD4, PE-CF594-CD8, CD19 protein-bound FITC, and CD79b protein-bound PE. Absolute numbers of viable tumor cells were calculated using CountBright™ absolute counting beads (ThermoFisher Scientific).

[0175] result Generation of dual CD19-CD79b CAR T cells We created a bicistronic CD19-CD79b CAR construct that can be used to target CD19 and CD79b antigens by co-expressing two CARs on the surface of primary T cells (Figure 1A). Approximately 3 days after lentiviral transduction, high expression of both CD19 and CD79b CARs was demonstrated on the T cell surface by staining with CD19 protein-conjugated FITC and CD79b protein-conjugated PE, indicating high transduction efficiency (Figure 1B).

[0176] Dual CD19-CD79b CAR T cells degranulate in response to B cell lymphoma and leukemia cell lines in a CD19- or CD79b-dependent manner Degranulation is an indirect measure of perforin-granzyme-mediated target cell killing by T cells. High CD107a expression was observed in dual CD19-CD79b CAR T cells when they were co-cultured with B cell lymphoma / leukemia tumor cell lines as fresh T cells (Figures 2A-2E) or cryopreserved and thawed T cells (Figures 2F-2G). Importantly, dual CAR T cells recognized CD19+CD79b+, CD19-CD79b+, and CD19+CD79b- tumor cells, but not CD19-CD79b- tumor cells. This suggests that both CARs expressed on the cell surface were functional and that expression of one or the other target antigen was required for degranulation.

[0177] Dual CD19-CD79b CAR T cells exhibit cytotoxicity against B-cell leukemia and lymphoma cell lines in a CD19- or CD79b-dependent manner Similar to the degranulation assay, dual CD19-CD79b CAR T cells specifically recognized and induced lysis of CD19+CD79b+ (Daudi, SUDHL6, PDX203, and PDX300) (Figure 3A), CD19+CD79b- (NALM6) (Figure 3B), and CD19-CD79b+ (Daudi-CD19KO and SUDHL6-CD19KO) tumor cells (Figure 3C), but not CD19-CD79b- tumor cells (K562) (Figure 3D). Percent specific lysis by CAR T cells for each tumor cell line was calculated as follows, normalized to non-transduced T cells: % specific lysis = 1 - [target viable cells with CAR T cells / target viable cells with non-transduced T cells] (Figures 3E-3I).

[0178] Dual CD19-CD79b CAR T cells proliferate in response to B-cell leukemia and lymphoma cell lines in a CD19- or CD79b-dependent manner After 4 days of co-culture, dual CD19-CD79b CAR T cells were observed to proliferate when co-cultured with B-cell leukemia and lymphoma tumor cell lines (Daudi, SUDHL6, PDX203, PDX300, Daudi-CD19KO, SUDHL6-CD19KO, and NALM6).

[0179] Example 2 - Selection of dual CAR constructs Several different designs of dual CD19-CD79b CAR constructs were generated with various permutations of promoters (MSCV or EF1a), self-cleaving peptides (E2A, P2A, or T2A), order of CD19 CAR vs. CD79b CAR, hinge / transmembrane (H / TM) domains (CD8a or CD28), and costimulatory domains (CD28, 4-1BB, or OX-40). All constructs had CD3 zeta (ζ) as the signaling domain. Dual CAR constructs were then introduced into 293T cells by transfection or transduced into primary T cells by lentivirus. Transfection or transduction efficiency was assessed by flow cytometry after 48 hours for 293T cells and after 7 or 8 days for primary T cells, respectively. The percentage of cells that were positive for CD19 CAR or CD79b CAR or both are shown in Figures 5A and 5B. The data show that most of the dual CAR constructs, except for pX2K and pX2L, resulted in unequal expression of one or the other CAR. pX2K and pX2L had comparable expression of their respective CARs, and the transduction efficiency of pX2K was higher than that of pX2L. Based on these results, the pX2K dual CAR construct was selected for further development. Results represent at least two experiments of various constructs.

[0180] Example 3 - CD19-CD79b dual CAR T cells produced effector cytokines in response to B cell lymphoma and leukemia cell lines in a CD19- or CD79b-dependent manner Dual CD19-CD79b CAR T cells specifically recognized and released IFN-γ (Figure 6A (where UTD is zero for all lines), IL-2 (Figure 6B), and TNF-α (Figure 6C) in response to the CD19+CD79b+ B cell lymphoma cell lines Daudi, SUDHL6, PDX203-5D4, and PDX300-5E6, the CD19+CD79b- B cell leukemia cell line NALM6, and the CD19-CD79b+ Daudi-CD19KO and SUDHL6-CD19KO lymphoma cell lines, but not in response to CD19-CD79b-K562 tumor cells. CD19 CAR T cells were used as a control.

[0181] Example 4 - CD19-CD79b dual CAR T cells have potent anti-tumor activity in vivo in the NALM6 CD19+CD79b- cell line xenograft model NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ(NSG) mice were treated with 0.2x10 6 Firefly luciferase-expressing NALM6 tumor cells were injected intravenously into each mouse. Four days later, 5x10 6 CAR+ cells CD19 CAR T or CD19-CD79b dual CAR T cells, and the corresponding non-transduced T cells were injected into tumor-bearing mice via tail vein injection. Tumor burden was measured by bioluminescence imaging on the indicated days (Figure 7A) and quantified by total flux (n=5 mice / group, significance between treatment groups determined by Mann-Whitney test, p=ns, **, p<0.01) (Figure 7B). Kaplan-Meier survival curves of mice treated with non-transduced T, CD19 CAR T, and CD19-CD79b dual CAR T (n=5 mice / group). Significance between treatment groups was determined by log-rank (Mantel-Cox) test (p=ns, **, p<0.01) (Figure 7C). Anti-tumor activity was comparable to CD19 CAR T cells, indicating that the CD19-targeted CAR in the dual CAR T cells was functional.

[0182] Example 5 - CD19-CD79b dual CAR T cells have potent anti-tumor activity in vivo in a patient-derived xenograft model with PDX203-5D4 CD19KO, which is CD19-CD79b+ NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ(NSG) mice, 0.2x10 6 Firefly luciferase-expressing PDX203-5D4 CD19KO cells were injected intravenously. After 15 days, 3x10 6 CAR+ cells CD19 CAR T, CD79b CAR T, or CD19-CD79b dual CAR T cells and corresponding non-transduced T cells were injected into tumor-bearing mice via the tail vein. Tumor burden was assessed by bioluminescence imaging on days 0, 7, 14, and 21.

[0183] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. Although the compositions and methods of the present invention have been described in terms of certain specific embodiments, it will be apparent to those skilled in the art that modifications can be made to the methods described herein and to the steps or sequence of steps of the methods without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are chemically and physiologically related can be substituted for the agents described herein while still achieving the same or similar results. All such similar substitutions and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Claims

1. (a) a first sequence encoding an anti-CD19 chimeric antigen receptor (CAR), the anti-CD19 CAR comprising: (i) GM-CSF receptor alpha signal peptide; (ii) a CD19-binding domain; (iii) the CD8α hinge region; (iv) CD8α transmembrane domain; (v) the 4-1BB signaling domain; and (vi) CD3ζ signaling domain and (b) a second sequence encoding an anti-CD79b CAR, the anti-CD79b CAR comprising: (i) CD8α signal peptide; (ii) the CD79b-binding domain; (iii) the CD8α hinge region; (iv) CD8α transmembrane domain; (v) the OX40 signaling domain; and (vi) CD3ζ signaling domain and the second sequence comprising A polynucleotide comprising:

2. The polynucleotide of claim 1, wherein the first sequence is 5' to the second sequence.

3. 3. The polynucleotide of claim 1 or 2, further comprising a third sequence encoding a T2A peptide.

4. The polynucleotide of claim 3, wherein the third sequence is 3' to the first sequence and 5' to the second sequence.

5. The polynucleotide of claim 3, wherein the T2A peptide comprises SEQ ID NO:

9.

6. The polynucleotide of any one of claims 1 to 5, wherein the GM-CSF receptor signal peptide comprises SEQ ID NO:

1.

7. The polynucleotide of any one of claims 1 to 6, wherein the CD19 binding domain is an scFv of an anti-CD19 antibody.

8. The polynucleotide of claim 7, wherein the CD19 binding domain comprises a VL having SEQ ID NO:2 and a VH having SEQ ID NO:

4.

9. The polynucleotide of claim 8, wherein the CD19 binding domain further comprises a linker connecting the VL and VH.

10. The polynucleotide of claim 9, wherein the linker comprises SEQ ID NO:

3.

11. The polynucleotide of any one of claims 1 to 10, wherein the CD8α hinge region of (a) comprises SEQ ID NO:

5.

12. The polynucleotide of any one of claims 1 to 11, wherein the CD8α transmembrane domain of (a) comprises SEQ ID NO:

6.

13. The polynucleotide of any one of claims 1 to 12, wherein the 4-1BB signaling domain comprises SEQ ID NO:

7.

14. The polynucleotide of any one of claims 1 to 13, wherein the CD3 zeta signaling domain of (a) comprises SEQ ID NO:

8.

15. The polynucleotide of any one of claims 1 to 14, wherein the CD8α signal peptide comprises SEQ ID NO:

10.

16. The polynucleotide of any one of claims 1 to 15, wherein the CD79b binding domain is an scFv of an anti-CD79 antibody.

17. The polynucleotide of claim 16, wherein the CD79b binding domain comprises a VL having SEQ ID NO:11 and a VH having SEQ ID NO:

13.

18. The polynucleotide of claim 17, wherein the CD79b binding domain further comprises a linker connecting the VL and VH.

19. The polynucleotide of claim 18, wherein said CD79b binding domain comprises a linker having SEQ ID NO:

12.

20. The polynucleotide of any one of claims 1 to 19, wherein the CD8α hinge region of (b) comprises SEQ ID NO:

5.

21. The polynucleotide of any one of claims 1 to 20, wherein the CD8α transmembrane domain of (b) comprises SEQ ID NO:

6.

22. 22. The polynucleotide of any one of claims 1 to 21, wherein the OX40 signaling domain comprises SEQ ID NO:

14.

23. The polynucleotide of any one of claims 1 to 22, wherein the CD3 zeta signaling domain of (b) comprises SEQ ID NO:

8.

24. 24. The polynucleotide of any one of claims 1 to 23, comprising a sequence encoding a polypeptide having SEQ ID NO:

15.

25. 25. The polynucleotide of claim 24, comprising a sequence having at least 90% identity to SEQ ID NO:

30.

26. 26. The polynucleotide of any one of claims 1 to 25, further comprising a promoter.

27. 27. The polynucleotide of claim 26, wherein the promoter is an EF1α promoter.

28. A vector comprising the polynucleotide of any one of claims 1 to 27.

29. A polypeptide encoded by the polynucleotide of any one of claims 1 to 25 or the vector of claim 28.

30. 30. A method for producing an engineered cell, comprising the step of introducing into a cell the polynucleotide of any one of claims 1 to 27, the vector of claim 28, or the polypeptide of claim 29.

31. 29. An engineered cell comprising the polynucleotide of any one of claims 1 to 25 or the vector of claim 28.

32. 32. The engineered cell of claim 31 , which is a T cell.

33. 32. The engineered cell of claim 31 , which is a natural killer (NK) cell.

34. 32. The engineered cell of claim 31, which is a macrophage.

35. A population of engineered cells comprising the engineered cell of any one of claims 31-33.

36. The population of engineered cells of claim 35, wherein at least 20% of the engineered cells in the population express both an anti-CD19 CAR and an anti-CD79b CAR.

37. 37. The population of engineered cells of claim 35 or claim 36, wherein the population of engineered cells comprises a subset that expresses either an anti-CD19 CAR or an anti-CD79b CAR, and at least 80% of the subset expresses both an anti-CD19 CAR and an anti-CD79b CAR.

38. 38. A pharmaceutical composition comprising the population of engineered cells of any one of claims 35 to 37 and an excipient.

39. 40. A method for treating a subject for cancer, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 38.

40. 40. The method of claim 39, wherein the subject has a B cell malignancy.

41. 41. The method of claim 40, wherein the subject has 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, or chronic lymphocytic leukemia.

42. The subject is + CD79b + The method of any one of claims 39 to 41, wherein the patient has cancer.

43. The subject is - CD79b + The method of any one of claims 39 to 41, wherein the patient has cancer.

44. The subject is + CD79b - The method of any one of claims 39 to 41, wherein the patient has cancer.

45. In the order 5' to 3', (a) a first sequence encoding an anti-CD19 chimeric antigen receptor (CAR), the anti-CD19 CAR comprising: (i) the GM-CSF receptor signal peptide comprising SEQ ID NO:1; (ii) a CD19 binding domain comprising SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4; (iii) the CD8α hinge region comprising SEQ ID NO:5; (iv) the CD8α transmembrane domain comprising SEQ ID NO:6; (v) a 4-1BB signaling domain comprising SEQ ID NO:7; and (vi) a CD3 ζ signaling domain comprising SEQ ID NO:8 and (b) a second sequence encoding a T2A peptide comprising SEQ ID NO:9; and (c) a third sequence encoding an anti-CD79b CAR, wherein the anti-CD79b CAR comprises: (i) the CD8α signal peptide comprising SEQ ID NO:1-; (ii) the CD79b binding domain comprising SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13; (iii) the CD8α hinge region comprising SEQ ID NO:5; (iv) the CD8α transmembrane domain comprising SEQ ID NO:6; (v) an OX40 signaling domain comprising SEQ ID NO:14; and (vi) a CD3 ζ signaling domain comprising SEQ ID NO:8 and the third sequence comprising A polynucleotide comprising:

46. 46. ​​The polynucleotide of claim 45, comprising a sequence encoding a polypeptide having SEQ ID NO:

15.

47. 47. The polynucleotide of claim 46, comprising a sequence having at least 90% identity to SEQ ID NO:

30.

48. 48. The polynucleotide of claim 47, comprising SEQ ID NO:

30.

49. 49. The polynucleotide of any one of claims 45 to 48, further comprising a promoter.

50. 50. The polynucleotide of claim 49, wherein the promoter is an EF1α promoter.

51. A vector comprising the polynucleotide of any one of claims 45 to 48.

52. 52. A polypeptide encoded by the polynucleotide of any one of claims 45 to 48 or the vector of claim 51.

53. 52. A method of producing an engineered cell, comprising the step of introducing into a cell the polynucleotide of any one of claims 45 to 48, the vector of claim 51, or the polypeptide of claim 52.

54. 52. An engineered cell comprising the polynucleotide of any one of claims 45 to 48, the vector of claim 51, or the polypeptide of claim 52.

55. 55. The engineered cell of claim 54, which is a T cell.

56. 55. The engineered cell of claim 54, which is a natural killer (NK) cell.

57. 57. A population of engineered cells comprising the engineered cell of any one of claims 54-56.

58. 58. A pharmaceutical composition comprising the population of engineered cells of claim 57 and an excipient.

59. 60. A method for treating a subject having cancer, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 58.

60. 60. The method of claim 59, wherein the subject has a B cell malignancy.

61. 61. The method of claim 60, wherein the subject has 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, or chronic lymphocytic leukemia.

62. The subject is + CD79b + The method of any one of claims 59 to 61, wherein the patient has cancer.

63. The subject is - CD79b + The method of any one of claims 59 to 61, wherein the patient has cancer.

64. The subject is + CD79b - The method of any one of claims 59 to 61, wherein the patient has cancer.

65. A polynucleotide encoding a polypeptide having SEQ ID NO:

15.

66. 66. The polynucleotide of claim 65, comprising a sequence having at least 90% identity to SEQ ID NO:

30.

67. The polynucleotide of claim 66, comprising SEQ ID NO:

30.

68. A vector comprising the polynucleotide of any one of claims 65 to 67.

69. 69. An engineered cell comprising the polynucleotide of any one of claims 65 to 67 or the vector of claim 68.

70. 70. The engineered cell of claim 69, which is a T cell.

71. 70. The engineered cell of claim 69, which is a natural killer (NK) cell.

72. 70. The engineered cell of claim 69, which is a macrophage.

73. 72. A population of engineered cells comprising the engineered cell of any one of claims 69-71.

74. 74. A pharmaceutical composition comprising the population of engineered cells of claim 73 and an excipient.

75. 75. A method for treating a subject having cancer, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 74.

76. 76. The method of claim 75, wherein the subject has a B cell malignancy.

77. 77. The method of claim 76, wherein the subject has 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, or chronic lymphocytic leukemia.

78. The subject is + CD79b + The method of any one of claims 75 to 77, wherein the patient has cancer.

79. The subject is - CD79b + The method of any one of claims 75 to 77, wherein the patient has cancer.

80. The subject is + CD79b - The method of any one of claims 75 to 77, wherein the patient has cancer.

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