Compositions and methods for treating cancer with fully human anti-CD20 / CD19 immunotherapy

Abstract

A chimeric antigen receptor comprising a fully human CD20 / CD19 antigen-binding domain is disclosed. Nucleic acids, recombinant expression vectors, host cells, antigen-binding fragments, and pharmaceutical compositions relating to the chimeric antigen receptor are also disclosed. Methods for treating or preventing cancer in a subject and methods for generating chimeric antigen receptor T cells are also disclosed.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 401,288, filed August 26, 2022, the entire contents of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically as an XML file named SL_ST26.xml. The XML file, created on August 17, 2023, is 174,928 bytes in size. The contents of the XML file are incorporated herein by reference in their entirety.

[0003] Field of the Disclosure The present application relates to the field of cancer, in particular to fully human CD20 and fully human CD19 antigen-binding domains (hereinafter referred to as "fully human CD20 / CD19"), as well as chimeric antigen receptors (CARs) comprising such fully human CD20 and fully human CD19 antigen-binding domains and methods of use thereof. [Background technology]

[0004] background Cancer is one of the most deadly threats to human health. In the United States alone, cancer affects nearly 1.3 million new patients each year, making it the second leading cause of death after cardiovascular disease, accounting for approximately one-quarter of all deaths. Solid tumors are responsible for most of these deaths. Although significant advances have been made in the medical treatment of certain cancers, the overall 5-year survival rate for all cancers has improved by only about 10% over the past 20 years. Cancer, or malignant tumors, metastasize and grow rapidly and uncontrolled, making treatment extremely difficult.

[0005] CD19 is an 85-95 kDa transmembrane cell surface glycoprotein receptor. It is a member of the immunoglobulin (Ig) superfamily of proteins and contains two extracellular Ig-like domains, a transmembrane domain, and an intracellular signaling domain (Tedder TF, Isaacs CM, 1989, J Immunol 143:712-171). CD19 modifies B cell receptor signaling, lowering the B cell receptor trigger threshold for antigen (Carter RH, and Fearon DT, 1992, Science 256:105-107), and cooperates with CD81 and CD21 to regulate this essential B cell signaling complex (Bradbury LE, Kansas GS, Levy S, Evans RL, Tedder TF, 1992, J Immunol 149:2841-50). During B cell ontogeny, CD19 can signal at pro-B, pre-pre-B, pre-B, and early B cell stages independently of antigen receptors, associates with Src family protein tyrosine kinases, becomes tyrosine phosphorylated, and induces both intracellular calcium mobilization and inositol phospholipid signaling (Uckun FM, Burkhardt AL, Jarvis L, Jun X, Stealy B, Dibirdik I, Myers DE, Tuel-Ahlgren L, Bolen JB 1983 J Biol Chem 268:21172-84). An important point relevant to the treatment of B cell malignancies is that CD19 is expressed in a tightly regulated manner on normal B cells and is restricted to early B cell precursors at the stage of IgH gene rearrangement, mature B cells, but not on hematopoietic stem cells or mature plasma cells (Anderson, KC, Bates, MP, Slaughenhout BL, Pinkus GS, Schlossman SF, Nadler LM, 1984, Blood 63:1424-1433).

[0006] CD20 (also known as LEU-16 or MS4A1) is a transmembrane 4A family protein that is expressed on the surface of B cells from the pro-B stage to the mature B cell stage and plays a role in B cell development and differentiation. The CD20 antigen is also expressed on various hematological tumors, and various monoclonal anti-CD20 antibodies have been used for the treatment of CD20-positive malignancies over the years (reviewed in Lim, Sean H. et al. "Anti-CD20 Monoclonal Antibodies: Historical and Future Perspectives," Haematologica 95.1 (2010): 135-143. PMC Web 31, July 2017). The anti-CD20 monoclonal antibody rituximab (Rituxan®) is widely used to treat B-cell lymphomas, such as follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL), as well as chronic lymphocytic leukemia (CLL) (Rituxan prescribing information).

[0007] Conventional treatment approaches for B-lineage leukemia and lymphoma may include chemotherapy, radiation therapy, and stem cell transplantation (see the world wide web at mayclinic.org). The high toxicity associated with these treatments and the risk of complications such as relapse, secondary malignancies, or GVHD have prompted a search for better treatment options. Expression of CD19 in both adult and pediatric (pre-B-ALL) B-cell malignancies has led to the exploitation of this target for both antibody and chimeric antigen receptor (CAR)-T cell-based therapies (Kochenderfer JN, Wilson WH, Janik JE, Dudley ME, Stetler-Stevenson M, Feldman SA, Maric I, Raffeld M, Nathan DA, Lanier BJ, Morgan RA, Rosenberg SA. 2010. Blood 116:4099-102; Lee DW, Kochenderfer JN, Stetler-Stevenson M, Cui YK, Delbrook C, Feldman SA, Orentas R, Sabatino M, Shah NN, Steinberg SM, Stroncek D, Tschernia N, Yuan C, Zhang H, Zhang L, Rosenberg SA, Wayne AS, Mackall CL. 2015. Lancet 385:517-28). Furthermore, the presence of the CD20 antigen in lymphomas (DLBCL, FL) and leukemias (CLL) makes it an attractive additional target for efficient tumor elimination and prevention of tumor antigen escape.

[0008] The current standard of care for B-lineage leukemias may consist of induction treatment with high-dose chemotherapy or radiation, followed by consolidation, optionally followed by stem cell transplantation and additional courses of chemotherapy (see cancer.gov on the world wide web). The high toxicity associated with these treatments and the risk of complications such as relapse, secondary malignancies, or GVHD have prompted research into better treatment options. Expression of CD19 in both adult and pediatric (pre-B-ALL) B-cell malignancies has led to the exploitation of this target for both antibody and chimeric antigen receptor (CAR)-T cell-based therapies (Kochenderfer JN, Wilson WH, Janik JE, Dudley ME, Stetler-Stevenson M, Feldman SA, Maric I, Raffeld M, Nathan DA, Lanier BJ, Morgan RA, Rosenberg SA. 2010. Blood 116:4099-102; Lee DW, Kochenderfer JN, Stetler-Stevenson M, Cui YK, Delbrook C, Feldman SA, Orentas R, Sabatino M, Shah NN, Steinberg SM, Stroncek D, Tschernia N, Yuan C, Zhang H, Zhang L, Rosenberg SA, Wayne AS, Mackall CL. 2015. Lancet 385:517-28).

[0009] Many novel approaches to treating B-cell leukemia and lymphoma have been developed, including bispecific antibodies that link anti-CD19 or anti-CD20 binding motifs to T-cell binding motifs (i.e., Blincyto®, blinatumomab, indicated for the treatment of Philadelphia chromosome-negative relapsed or refractory B-cell precursor acute lymphoblastic leukemia (ALL)). To date, most of the binding moieties for CD19 or CD20 employed in CAR constructs utilize domains derived from murine antibodies. Many of these products, including those developed by Novartis and Kite Pharmaceuticals, are currently under review for approval. In April 2017, Novartis announced that its CTL019 (tisagenlecleucel) received FDA Breakthrough Designation for the treatment of adult patients with refractory or relapsed (r / r) DLBCL (diffuse large B-cell lymphoma) who have failed two or more prior therapies, adding this designation to its designation for r / r B-cell acute lymphoblastic leukemia (ALL). These indications were based on the phase II JULIET study (NCT02445248) and ELIANA study (NCT02435849), respectively. The JULIET trial demonstrated a 45% overall response rate (ORR), with a 37% complete response rate (CR) and an 8% partial response rate (PR) at 3 months. In the ELIANA study, 82% of patients infused with the product achieved a CR or a CR with incomplete count recovery, and the recurrence-free survival rate at 6 months was 60%. Kite Pharmaceuticals' CAR-T product (KTE-C19, axicabtagene ciloleucel) was granted breakthrough designation for diffuse large B-cell lymphoma (DLBLC), transformed follicular lymphoma (TFL), and primary mediastinal B-cell lymphoma (PMBCL). The Kite ZUMA-3 Phase II trial of KTE-C19 in r / r ALL reported a 73% CR (over 2 months). Regardless of whether CAR-T antibody therapy is utilized, there remains a significant number of patients for whom these therapies do not work, leaving considerable room for improvement in treatment approaches.

[0010] Chimeric antigen receptors (CARs) are hybrid molecules containing three essential units: (1) an extracellular antigen-binding motif, (2) a linking / transmembrane motif, and (3) an intracellular T cell signaling motif (Long AH, Haso WM, Orentas RJ. Lessons learned from a highly active CD22-specific chimeric antigen receptor. Oncoimmunology. 2013;2(4):e23621). The antigen-binding motif of CARs is generally based on the single-chain fragment variable (scFv), the smallest binding domain of an immunoglobulin (Ig) molecule. Alternative antigen-binding motifs have also been engineered, for example, receptor ligands (i.e., IL-13 has been engineered to bind to tumor-expressed IL-13 receptors), intact immune receptors, library-derived peptides, and innate immune system effector molecules (e.g., NKG2D). Alternative cellular targets for CAR expression (e.g., NK or gamma-delta T cells) are also under development (Brown CE et al. Clin Cancer Res. 2012;18(8):2199-209; Lehner M et al. PLoS One. 2012;7(2):e31210). Considerable work remains to be done in defining the most active T cell populations for transduction with CAR vectors, determining optimal culture and expansion techniques, and defining the molecular details of the CAR protein structure itself.

[0011] The linking motif of the CAR can be a relatively stable structural domain, such as the constant domain of IgG, or can be designed as an extended, flexible linker. Structural motifs, such as those derived from the constant domain of IgG, can be used to extend the scFv binding domain away from the T cell membrane surface. This may be important for some tumor targets where the binding domain is particularly close to the tumor cell surface membrane (e.g., for disialoganglioside GD2; Orentas et al., unpublished observation). To date, the signaling motif used in CARs has always included the CD3-zeta chain, because this core motif is an important signal for T cell activation. The first reported second-generation CARs featured the CD28 signaling domain and CD28 transmembrane sequence. This motif was also used in third-generation CARs containing the CD137 (4-1BB) signaling motif (Zhao Y et al., J Immunol. 2009;183(9):5563-74). With the advancement of new technologies, activation of T cells by beads linked to anti-CD3 and anti-CD28 antibodies, and the presence of the canonical "signal 2" derived from CD28, no longer needs to be encoded by the CAR itself. Using bead activation, third-generation vectors were found to be no superior to second-generation vectors in in vitro assays and offered no clear benefit over second-generation vectors in mouse models of leukemia (Haso W, Lee DW, Shah NN, Stetler-Stevenson M, Yuan CM, Pastan IH, Dimitrov DS, Morgan RA, FitzGerald DJ, Barrett DM, Wayne AS, Mackall CL, Orentas RJ. Anti-CD22-chimeric antigen receptors targeting B cell precursor acute lymphoblastic leukemia. Blood. 2013;121(7):1165-74; Kochenderfer JN et al. Blood. 2012;119(12):2709-20).This is supported by the clinical success of second-generation CD28 / CD3-zeta (Lee DW et al. American Society of Hematology Annual Meeting. New Orleans, LA; December 7-10, 2013) and CD19-specific CARs with CD137 / CD3-zeta signaling (Porter DL et al. N Engl J Med. 2011;365(8):725-33). In addition to CD137, other tumor necrosis factor receptor superfamily members, such as OX40, can also provide important sustained signals in CAR-transduced T cells (Yvon E et al. Clin Cancer Res. 2009;15(18):5852-60). Equally important are the culture conditions under which the CAR T cell population is cultured, for example, the inclusion of cytokines IL-2, IL-7, and / or IL-15 (Kaiser AD et al. Cancer Gene Ther. 2015; 22(2):72-78).

[0012] Current challenges in broader and more effective application of CAR therapy against cancer relate to a lack of attractive targets. While generating binders to cell surface antigens is now readily achievable, discovering tumor-specific cell surface antigens while sparing normal tissue remains a challenging task. One possible way to confer greater target cell specificity to CAR-expressing T cells is to use a combinatorial CAR approach. In one system, the CD3-ζ and CD28 signaling units are split between two different CAR constructs expressed within the same cell; in another system, two CARs are expressed within the same T cell, but one has lower affinity and therefore requires the first CAR to be engaged for full activation of the other (Lanitis E et al. Cancer Immunol Res. 2013;1(1):43-53; Kloss CC et al. Nat Biotechnol. 2013;31(1):71-5). A second challenge in generating a single ScFv-based CAR as an immunotherapeutic agent is tumor cell heterogeneity. At least one group has developed a CAR strategy for glioblastoma, whereby an effector cell population simultaneously targets multiple antigens (HER2, IL-13Ra, EphA2) in hopes of avoiding expansion of target antigen-negative populations (Hegde M et al. Mol Ther. 2013;21(11):2087-101).

[0013] T cell-based immunotherapy is an emerging field in synthetic biology; multiple promoters and gene products are envisioned to direct these highly potent cells to the tumor microenvironment, where they can escape negative regulatory signals and mediate effective tumor killing. Elimination of unwanted T cells via drug-induced dimerization of an inducible caspase-9 construct with a chemical-based dimer, such as AP1903, demonstrates one way in which a powerful switch can be pharmacologically initiated to control T cell populations (Di Stasi A et al. N Engl J Med. 2011;365(18):1673-83). Creation of an effector T cell population immune to the negative regulatory effects of transforming growth factor-β by expression of a decoy receptor further demonstrates the extent to which effector T cells can be engineered for optimal antitumor activity (Foster AE et al. J Immunother. 2008;31(5):500-5). Thus, although CARs appear to be able to induce T cell activation in a manner similar to endogenous T cell receptors, the major obstacles to the clinical application of this technology to date have been the limited in vivo expansion of CAR T cells, the rapid loss of cells after infusion, and disappointing clinical activity, which may be due in part to the murine origin of some of the employed CAR sequences.

[0014] The use of brinotumomab (a bispecific anti-CD19 and anti-CD3 antibody) has shown excellent results in severely ill patients receiving this therapy. Despite this, the durable remission rate is less than 40%, and at best, only 50% of responders can be rescued by hematopoietic stem cell transplantation (HSCT) (see Gore et al., 2014, NCT01471782 and Von Stackelberg et al., 2014, NCT01471782, summarized in Benjamin, JE, Stein, AS, 2016, Therapeutic Advances in Hematology 7:142–156). The need for patients receiving either bispecific antibody therapy or CAR-T therapy to undergo subsequent HSCT to maintain a durable response remains an area of ​​active debate. High response rates have been reported in CD19 CAR-T trials, some exceeding 90%, but when the trials are reconsidered as "intent to treat" trials, the number may be closer to 70% (Davis KL, Mackall CL, 2016, Blood Advances 1:265-268). The best reported results at 12 months after CAR19 treatment show a 55% RFS and 79% OS in patients who were able to receive the T-cell product at the University of Pennsylvania (Maude SL, Teachey DT, Rheingold SR, Shaw PA, Aplenc R, Barrett DM, Barker CS, Callahan C, Frey NV, Farzana N, Lacey SF, Zheng A, Levine B, Melenhorst JJ, Motley L, Prter DL, June CH, Grupp SA. 2016. J Clin Oncol 34, No. 15 Suppl (May 2016) pp. 3011-3011). Summary of the Invention [Problem to be solved by the invention]

[0015] Thus, there is an urgent and long-felt need in the art to discover new compositions and methods for the treatment of B-ALL and other CD19- and / or CD20-expressing B-cell malignancies using approaches that can exhibit specific and effective anti-tumor effects without the drawbacks mentioned above. [Means for solving the problem]

[0016] The present invention addresses these needs by providing CAR compositions and therapeutic methods that can be used to treat cancer and other diseases and / or conditions. In particular, the invention disclosed and described herein provides CARs that can be used to treat diseases, disorders, or conditions associated with dysregulated expression of CD20 and / or CD19, and that have the following characteristics: i) high surface expression on transduced T cells; ii) high cytolysis of CD20- and / or CD19-expressing cells, with transduced T cells demonstrating in vivo proliferation and persistence; iii) multiple targeting to overcome antigen escape; iv) armor to overcome immunosuppression in the tumor microenvironment (TME); v) cytokine-stimulating elements that promote cytokine-autonomous T cell stimulation and enhance antitumor cytotoxicity, proliferation, memory formation, cytokine secretion, and persistence; and vi) the ability to overcome the physical barrier of the tumor stroma / extracellular matrix (ECM) and express CARs. and vii) a CAR comprising tandem fully human CD20 / CD19 antigen-binding domains that exhibit one or more of an on-switch or off-switch to control expression of the CAR, or a co-expressed functional "booster" element, and tumors that express a targetable antigen such as ROR1, mesothelin, HER2 / ERBB2, folate receptor I, PSMA, CD276, claudin 6, claudin 1.28, MUC1, MUC16, GD2, LRRC15, GPC3, FGFR4, FAP, IL-13Ra, EphA2, or other antigens, or any combination thereof. Therapeutic methods using such CARs are provided that can be used to treat solid tumors, including rheumatoid arthritis, lupus, celiac disease, Sjogren's syndrome, multiple sclerosis, polymyalgia rheumatica, ankylosing spondylitis, type 1 diabetes, alopecia areata, vasculitis, temporal arteritis, post-streptococcal autoimmune disorders, antineuronal antibody-mediated neuropsychiatric disorders, immune-mediated extrapyramidal movement disorders, Sydenham's chorea, autoimmune hemolytic disease, pulmonary fibrosis, systemic dermatosclerosis, or fibrotic diseases, including, for example, but not limited to, autoimmune, alloimmune, and autoaggressive diseases.

[0017] Summary of the Invention The present invention provides a novel tandem fully human CD20 and CD19 targeting antibody or its antigen-binding domain (hereinafter referred to as "fully human CD20 / CD19"), in which the CD19 targeting portion is located either before or after the CD20 targeting portion in the amino acid sequence, as well as a chimeric antigen receptor (tandem CAR) comprising such a CD20 and / or CD19 antigen-binding domain, as well as a host cell (e.g., T cell) expressing the receptor, and a nucleic acid molecule encoding the receptor.The CAR exhibits high surface expression on transduced T cells, high cytolysis, and transduced T cell proliferation and in vivo persistence.Methods of using the disclosed CAR, host cell, and nucleic acid molecule, for example, for treating cancer in a subject, are also provided.

[0018] In one aspect, an isolated nucleic acid molecule is provided encoding a tandem fully human CD20 / CD19 chimeric antigen receptor (CAR), comprising, from N-terminus to C-terminus, at least one fully human CD20 / CD19 antigen binding domain, at least one transmembrane domain, and at least one intracellular signaling domain, wherein the tandem fully human CD20 / CD19 CAR comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, and 48.

[0019] In one aspect, an isolated nucleic acid molecule is provided encoding a tandem fully human CD20 / CD19 chimeric antigen receptor (CAR), comprising, from N-terminus to C-terminus, at least one fully human CD20 / CD19 antigen binding domain, at least one transmembrane domain, and at least one intracellular signaling domain, wherein a tandem fully human CD20 / CD19 CAR encoded by an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, and 48 encodes a tandem fully human CD20 / CD19 CAR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, and 49.

[0020] In one embodiment, an isolated nucleic acid molecule is provided encoding a CAR, wherein the encoded extracellular fully human CD20 / CD19 antigen-binding domain comprises at least one single-chain variable fragment of an antibody that binds fully human CD20 / CD19.

[0021] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded extracellular fully human CD20 / CD19 antigen-binding domain comprises at least one heavy chain variable region of an antibody that binds fully human CD20 / CD19.

[0022] In yet another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded CAR extracellular fully human CD20 / CD19 antigen-binding domain further comprises at least one lipocalin-based antigen-binding antigen (anticalin) that binds to fully human CD20 / CD19.

[0023] In one embodiment, an isolated nucleic acid molecule is provided in which the encoded extracellular fully human CD20 / CD19 antigen-binding domain is connected to a transmembrane domain by a linker domain.

[0024] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded fully human CD20 / CD19 extracellular antigen-binding domain is preceded by a sequence encoding a leader or signal peptide.

[0025] In yet another embodiment, an isolated nucleic acid molecule is provided encoding a CAR comprising at least one fully human CD20 / CD19 antigen encoded by a nucleotide sequence comprising the fully human CD20 / CD19 nucleotide sequences contained within SEQ ID NOs: 1 and 3, respectively, wherein the CAR further encodes an extracellular antigen-binding domain that targets an antigen including, but not limited to, CD22, ROR1, mesothelin, CD33, CD38, CD123 (IL3RA), CD138, BCMA (CD269), GPC2, GPC3, FGFR4, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, TSLPR, NY-ESO-1 TCR, MAGE A3 TCR, or any combination thereof.

[0026] In yet another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, comprising at least one fully human CD20 / CD19 antigen binding domain encoded by a nucleotide sequence comprising the fully human CD20 / CD19 nucleotide sequence contained within SEQ ID NO: 48, wherein the CAR further encodes an extracellular antigen binding domain that targets an antigen including, but not limited to, CD22, ROR1, mesothelin, CD33, CD38, CD123 (IL3RA), CD138, BCMA (CD269), GPC2, GPC3, FGFR4, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, TSLPR, NY-ESO-1 TCR, MAGE A3 TCR, or any combination thereof.

[0027] In certain embodiments, the further encoded extracellular antigen-binding domain is an anti-CD22 scFv antigen-binding domain, an anti-ROR1 scFv antigen-binding domain, an anti-mesothelin scFv antigen-binding domain, an anti-CD33 scFv antigen-binding domain, an anti-CD38 scFv antigen-binding domain, an anti-CD123 (IL3RA) scFv antigen-binding domain, an anti-CD138 scFv antigen-binding domain, an anti-BCMA (CD269) scFv antigen-binding domain, an anti-GPC2 scFv antigen-binding domain, an anti-GPC3 scFv antigen-binding domain, an anti-FGFR4 scFv antigen-binding domain, an anti-TSLPR scFv antigen-binding domain, an anti-c-Met scFv antigen-binding domain, an anti-PMSA scFv antigen-binding domain, an anti-glycolipid F77 scFv antigen-binding domain, an anti-EGFRvIII scFv antigen-binding domain, an anti-GD-2 scFv antigen-binding domain, or an anti-NY-ESO-1 Provided is an isolated nucleic acid molecule encoding a CAR comprising a TCR scFv antigen-binding domain, an anti-MAGE A3 TCR scFv antigen-binding domain, or an amino acid sequence thereof having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, or any combination thereof.

[0028] In one embodiment, the CAR provided herein further comprises a linker or spacer domain.

[0029] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, in which an extracellular fully human CD20 / CD19 antigen-binding domain, an intracellular signaling domain, or both, is connected to a transmembrane domain by a linker or spacer domain.

[0030] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded linker domain is derived from the extracellular domain of CD8 or CD28 and is linked to a transmembrane domain.

[0031] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded CAR further comprises a transmembrane domain comprising a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD83, CD86, CD134, CD137, and CD154, or a combination thereof.

[0032] In yet another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded intracellular signaling domain further comprises a CD3 zeta intracellular domain.

[0033] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded intracellular signaling domain is positioned C-terminal to the CD3 zeta intracellular domain.

[0034] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded at least one intracellular signaling domain comprises a costimulatory domain, a primary signaling domain, or a combination thereof.

[0035] In a further embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded at least one costimulatory domain comprises a functional signaling domain of OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP12, and 4-1BB (CD137), or a combination thereof.

[0036] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, further comprising a leader sequence or signal peptide, wherein the nucleotide sequence of the leader or signal peptide comprises the nucleotide sequence of SEQ ID NO:11.

[0037] In yet another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded leader sequence comprises the amino acid sequence of SEQ ID NO:12.

[0038] In one aspect, provided herein is a chimeric antigen receptor (CAR) comprising, from N-terminus to C-terminus, at least one fully human CD20 / CD19 antigen-binding domain, at least one transmembrane domain, and at least one intracellular signaling domain.

[0039] In one embodiment, a CAR is provided, wherein the extracellular fully human CD20 / CD19 antigen-binding domain comprises at least one single-chain variable fragment of an antibody that binds to the antigen, or at least one heavy-chain variable region of an antibody that binds to the antigen, or a combination thereof.

[0040] In another embodiment, a CAR is provided, wherein at least one transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, TNFRSF19, or a combination thereof.

[0041] In some embodiments, CARs are provided wherein the CAR further encodes an extracellular antigen-binding domain comprising CD22, ROR1, mesothelin, CD33, CD38, CD123 (IL3RA), CD138, BCMA (CD269), GPC2, GPC3, FGFR4, TSLPR, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, TSLPR, NY-ESO-1 TCR, MAGE A3 TCR, or an amino acid sequence thereof having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity, or any combination thereof.

[0042] In one embodiment, the extracellular antigen-binding domain is an anti-CD22 scFv antigen-binding domain, an anti-ROR1 scFv antigen-binding domain, an anti-mesothelin scFv antigen-binding domain, an anti-CD33 scFv antigen-binding domain, an anti-CD38 scFv antigen-binding domain, an anti-CD123 (IL3RA) scFv antigen-binding domain, an anti-CD138 scFv antigen-binding domain, an anti-BCMA (CD269) scFv antigen-binding domain, an anti-GPC2 scFv antigen-binding domain, an anti-GPC3 scFv antigen-binding domain, an anti-FGFR4 scFv antigen-binding domain, an anti-TSLPR scFv antigen-binding domain, an anti-c-Met scFv antigen-binding domain, an anti-PMSA scFv antigen-binding domain, an anti-glycolipid F77 scFv antigen-binding domain, an anti-EGFRvIII scFv antigen-binding domain, an anti-GD-2 scFv antigen-binding domain, an anti-NY-ESO-1 TCR CARs are provided that include an scFv antigen-binding domain, an anti-MAGE A3 TCR scFv antigen-binding domain, or an amino acid sequence thereof having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, or any combination thereof.

[0043] In another embodiment, a CAR is provided wherein at least one intracellular signaling domain comprises a costimulatory domain and a primary signaling domain.

[0044] In yet another embodiment, a CAR is provided, wherein at least one intracellular signaling domain comprises a costimulatory domain that includes a functional signaling domain of a protein selected from the group consisting of OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP12 and 4-1BB (CD137), or a combination thereof.

[0045] In one embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 5, the nucleotide sequence of CAR D0144 (CD20_CD19 CD8 BBz).

[0046] In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 6 CAR D0144 (CD20_CD19 CD8 BBz).

[0047] In one embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 7, the nucleotide sequence of CAR D0255(CD20_CD19 CD28)CD28 BBz.

[0048] In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 8 CAR D0255 (CD20_CD19 CD28) CD28 BBz.

[0049] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 15, the nucleotide sequence of CAR D0258 (CD20 CD8 BBz_CD19 CD8 CD28z).

[0050] In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 16, CAR D0258 (CD20 CD8 BBz_CD19 CD8 CD28z).

[0051] In one embodiment, the CARs disclosed herein are modified to express or contain a detectable marker for use in diagnosis, monitoring and / or prediction of treatment outcome, such as progression-free survival in cancer patients, or to monitor the progress of such treatment.

[0052] In one embodiment, the nucleic acid molecule encoding the disclosed CAR can be contained in a vector, such as a viral vector, which can be a DNA vector, an RNA vector, a plasmid vector, a cosmid vector, a herpes virus vector, a measles virus vector, a lentivirus vector, an adenovirus vector, or a retrovirus vector, or a combination thereof.

[0053] In certain embodiments, the vector further comprises a promoter that is an inducible promoter, a tissue-specific promoter, a constitutive promoter, a suicide promoter, or any combination thereof.

[0054] In yet another embodiment, the CAR-expressing vector can be further modified to include one or more operable elements to control the expression of CAR T cells or to eliminate CAR-T cells by a suicide switch. The suicide switch can include, for example, an apoptosis-inducing signaling cascade or a drug that induces cell death. In a preferred embodiment, the CAR-expressing vector can be further modified to express an enzyme such as thymidine kinase (TK) or cytosine deaminase (CD).

[0055] In another aspect, a host cell comprising a nucleic acid molecule encoding a CAR is also provided. In some embodiments, the host cell is a T cell, for example, a primary T cell obtained from a subject. In one embodiment, the host cell is a CD8 + T cells.

[0056] In yet another aspect, a pharmaceutical composition is provided comprising an anti-tumor effective amount of a population of human T cells, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising the amino acid sequences of SEQ ID NOs: 2 and 4, wherein the CAR comprises at least one extracellular antigen-binding domain comprising a fully human CD20 / CD19 antigen-binding domain, at least one linker domain, at least one transmembrane domain, and at least one intracellular signaling domain, and the T cells are T cells of a human with cancer. The cancer includes, inter alia, hematological cancers such as leukemia (e.g., chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), or chronic myelogenous leukemia (CML)), lymphoma (e.g., mantle cell lymphoma, non-Hodgkin's lymphoma, or Hodgkin's lymphoma), or multiple myeloma, or a combination thereof.

[0057] In yet another embodiment, a pharmaceutical composition is provided comprising an anti-tumor effective amount of a population of human T cells, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising the amino acid sequence of SEQ ID NO: 49, wherein the CAR comprises at least one extracellular antigen-binding domain comprising a fully human CD20 / CD19 antigen-binding domain, at least one linker domain, at least one transmembrane domain, and at least one intracellular signaling domain, and the T cells are T cells of a human with cancer. The cancer includes, inter alia, hematological cancers such as leukemia (e.g., chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), or chronic myelogenous leukemia (CML)), lymphoma (e.g., mantle cell lymphoma, non-Hodgkin's lymphoma, or Hodgkin's lymphoma), or multiple myeloma, or a combination thereof.

[0058] In one embodiment, a pharmaceutical composition is provided, wherein at least one transmembrane domain of the CAR comprises a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, mesothelin, CD33, CD37, CD64, CD80, CD83, CD86, CD134, CD137, CD154, TNFRSF19, or a combination thereof.

[0059] In another embodiment, pharmaceutical compositions are provided wherein the human cancer comprises oral and pharyngeal cancer (tongue, mouth, pharynx, head and neck), digestive system cancer (esophagus, stomach, small intestine, colon, rectum, anus, liver, intrahepatic bile duct, gallbladder, pancreas), respiratory system cancer (larynx, lung and bronchus), bone and joint cancer, soft tissue cancer, skin cancer (melanoma, basal cell carcinoma and squamous cell carcinoma), childhood tumors (neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma), tumors of the central nervous system (brain tumor, astrocytoma, glioblastoma, glioma), and adult cancers including cancer of the breast, reproductive system (cervix, uterus, ovary, vulva, vagina, prostate, testicle, penis, endometrium), urinary system (bladder, kidney and renal pelvis, ureter), eye and orbit, endocrine system (thyroid), and brain and other nervous system, or any combination thereof.

[0060] In yet another embodiment, a pharmaceutical composition is provided comprising an antitumor-effective amount of a population of human T cells from a human having cancer, wherein the cancer is a refractory cancer that is unresponsive to one or more chemotherapeutic agents. The cancer includes hematopoietic cancer, myelodysplastic syndrome, pancreatic cancer, head and neck cancer, skin tumor, adult B-cell malignancies including acute lymphoblastic leukemia (ALL), minimal residual disease (MRD) in acute myeloid leukemia (AML), CLL (chronic lymphocytic leukemia), CML (chronic myeloid leukemia), non-Hodgkin's lymphoma (NHL), pediatric B-cell malignancies (including B-cell lineage ALL (acute lymphocytic leukemia)), multiple myeloma, lung cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, melanoma or other hematological cancers and solid tumors, or any combination thereof.

[0061] In another aspect, a method of generating CAR-containing T cells (hereinafter "CAR T cells") is provided, which method comprises transducing T cells with a vector or nucleic acid molecule encoding the disclosed CAR that specifically binds CD19 and / or CD20, thereby generating the CAR T cells.

[0062] In yet another aspect, a method of generating a population of RNA engineered cells is provided, comprising introducing in vitro transcribed or synthetic RNA of a nucleic acid molecule encoding a disclosed CAR into cells of a subject to generate CAR cells.

[0063] In one embodiment, the disease, disorder or condition associated with CD19 expression is cancer, including hematopoietic cancer, myelodysplastic syndrome, pancreatic cancer, head and neck cancer, skin tumor, adult B-cell malignancies including acute lymphoblastic leukemia (ALL), minimal residual disease (MRD) in acute myeloid leukemia (AML), CLL (chronic lymphocytic leukemia), CML (chronic myeloid leukemia), non-Hodgkin's lymphoma (NHL), pediatric B-cell malignancies (including B-cell lineage ALL (acute lymphocytic leukemia)), multiple myeloma, lung cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, melanoma or other hematological cancers and solid tumors, or any combination thereof.

[0064] In another embodiment, provided is a method of altering a tumor microenvironment to block T cell inhibition mediated by cells expressing CD19 and / or CD20 and inhibit tumor growth in a mammal, the method comprising administering to the mammal an effective amount of a composition comprising a CAR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, and 49. In one embodiment, the cells are selected from the group consisting of tumor cells expressing CD19 and / or CD20, tumor-associated macrophages, and any combination thereof.

[0065] In another embodiment, provided is a method of inhibiting, suppressing, or preventing immunosuppression of an anti-tumor or anti-cancer immune response in a mammal, comprising administering to the mammal an effective amount of a composition comprising a CAR selected from the group consisting of SEQ ID NOs: 2, 4, and 49. In one embodiment, the CAR inhibits an interaction between a first cell and a T cell, and the first cell is selected from the group consisting of a tumor cell expressing CD19 and / or CD20, a tumor-associated macrophage, and any combination thereof.

[0066] In another aspect, provided is a method of inducing anti-tumor immunity in a mammal, the method comprising administering to the mammal a therapeutically effective amount of T cells transduced with a vector or nucleic acid molecule encoding a disclosed CAR.

[0067] In another embodiment, a method of treating or preventing cancer in a mammal is provided, comprising administering to the mammal one or more of the disclosed CARs in an amount effective to treat or prevent cancer in the mammal. The method comprises administering to the subject a therapeutically effective amount of host cells expressing the disclosed CARs that specifically bind to CD19 and / or CD20 and / or one or more of the above-mentioned antigens under conditions sufficient to form an immune complex between the antigen-binding domain of the CAR and the extracellular domain of CD19 and / or CD20 and / or one or more of the above-mentioned antigens in the subject.

[0068] In yet another embodiment, a method is provided for treating a mammal having a disease, disorder, or condition associated with elevated expression of a tumor antigen, comprising administering to the subject an anti-tumor effective amount of a pharmaceutical composition comprising a population of T cells, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises at least one extracellular CD19 and / or CD20 antigen-binding domain, or any combination thereof, at least one linker or spacer domain, at least one transmembrane domain, at least one intracellular signaling domain, and wherein the T cells are from a subject with cancer.

[0069] In yet another embodiment, a method of treating cancer in a subject in need thereof is provided, comprising administering to the subject an anti-tumor effective amount of a pharmaceutical composition comprising a population of T cells, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), the CAR comprising the amino acid sequences of SEQ ID NOs: 2 and 4, or any combination thereof, at least one linker or spacer domain, at least one transmembrane domain, and at least one intracellular signaling domain, and the T cells are from a subject with cancer. In some embodiments of the aforementioned method, the at least one transmembrane domain comprises the transmembrane alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD19, CD22, mesothelin, CD33, CD37, CD64, CD80, CD83, CD86, CD134, CD137, CD154, TNFRSF16, TNFRSF19, or a combination thereof.

[0070] In yet another embodiment, a method of treating cancer in a subject in need thereof is provided, comprising administering to the subject an anti-tumor effective amount of a pharmaceutical composition comprising a population of T cells, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), the CAR comprising the amino acid sequence of SEQ ID NO: 49, at least one linker or spacer domain, at least one transmembrane domain, and at least one intracellular signaling domain, and the T cells are from a subject with cancer. In some embodiments of the above-described methods, the at least one transmembrane domain comprises a transmembrane alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD19, CD22, mesothelin, CD33, CD37, CD64, CD80, CD83, CD86, CD134, CD137, CD154, TNFRSF16, TNFRSF19, or a combination thereof.

[0071] In yet another embodiment, a method is provided for generating a persistent population of engineered T cells in a human diagnosed with cancer. In one embodiment, the method comprises administering to the human T cells engineered to express a CAR, wherein the CAR comprises the amino acid sequences of SEQ ID NOs: 2 and 4, or any combination thereof, at least one transmembrane domain, and at least one intracellular signaling domain, and wherein the persistent population of engineered T cells, or a population of progeny of the T cells, persists in the human for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, or 3 years after administration.

[0072] In yet another embodiment, a method is provided for generating a persistent population of engineered T cells in a human diagnosed with cancer. In one embodiment, the method comprises administering to the human T cells engineered to express a CAR, wherein the CAR comprises the amino acid sequence of SEQ ID NO: 49, at least one transmembrane domain, and at least one intracellular signaling domain, and wherein the persistent population of engineered T cells, or a population of progeny of the T cells, persists in the human for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, or 3 years after administration.

[0073] In one embodiment, the progeny T cells in the human comprise memory T cells. In another embodiment, the T cells are autologous T cells.

[0074] In all of the aspects and embodiments of the methods described herein, any of the above-mentioned cancers, diseases, disorders, or conditions associated with elevated expression of tumor antigens can be treated or prevented or ameliorated using one or more of the CARs disclosed herein.

[0075] In yet another aspect, there is provided a kit for generating the chimeric antigen receptor T cells as described above, or for preventing, treating, or ameliorating any of the cancers, diseases, disorders, or conditions associated with elevated expression of a tumor antigen in the subject as described above, the kit comprising a container containing any one of the nucleic acid molecules, vectors, host cells, or compositions disclosed above, or any combination thereof, and instructions for using the kit.

[0076] It is understood that the CARs, host cells, nucleic acids, and methods are useful beyond the specific aspects and embodiments described in detail herein. The foregoing features and advantages of the present disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying drawings. [Brief explanation of the drawings]

[0077] [Figure 1A] Figure 1A shows the structure and surface expression of CD20 and CD19 dual-targeting CARs in human primary T cells. CD20 and CD19 dual-targeting CARs were designed as tandem or bicistronic constructs. Tandem CAR constructs consisted of a fully human 20-19 tandem scFv targeting domain, hinge and transmembrane domain, a single 4-1BB or tandem CD28_4-1BB costimulatory domain, and a CD3ζ activation domain. The construct pLTG1497, containing a murine tandem scFv, served as a control. Duo CAR constructs included a mono-CD20 CAR followed by a 2A sequence and a mono-CD19 CAR with a different costimulatory or transmembrane domain. [Figure 1B] Figure 1B shows the structure and surface expression of a CD20 and CD19 dual-targeting CAR in human primary T cells. Primary T cells from healthy donors were activated by TransAct in the presence of IL-2 and transduced with a lentiviral vector encoding the CAR20_19 construct. Transduced T cells were assayed for CAR surface expression with anti-Fc-AF647 using CD19 Fc staining followed by flow cytometry. UTD - untransduced control. [Figure 2A] Figure 2. Cytotoxicity of the CAR20_19 construct in vitro. Luciferase-based cytotoxicity assays were performed using the CD19+ CD20+ tumor line (Figure 2A, Raji). All target lines were stably transduced with firefly luciferase. CAR T cells and tumor cells were cultured overnight at the indicated effector-to-target (E:T) ratios: 2.5:1, 5:1, or 10:1. The percentage of specific target lysis was assessed by luminometry. Data represent one independent experiment from two different donors. Mean ± SD of three technical replicates. One representative experiment from four separate donors is shown in the panel. [Figure 2B]Figure 2 shows the cytotoxicity of the CAR20_19 construct in vitro. Luciferase-based cytotoxicity assays were performed using the CD19+ CD20+ tumor line (Nalm-6, Figure 2B). All target lines were stably transduced with firefly luciferase. CAR T cells and tumor cells were cultured overnight at the indicated effector-to-target (E:T) ratios: 2.5:1, 5:1, or 10:1. The percentage of specific target lysis was assessed by luminometry. Data represent one independent experiment from two different donors. Mean ± SD of three technical replicates. One representative experiment from four separate donors is shown in the panel. [Figure 2C] Figure 2. Cytotoxicity of the CAR20_19 construct in vitro. Luciferase-based cytotoxicity assays were performed using a CD19+ CD20+ tumor line (Figure 2C) Reh and a CD19- CD20- tumor line. All target lines were stably transduced with firefly luciferase. CAR T cells and tumor cells were cultured overnight at the indicated effector-to-target (E:T) ratios: 2.5:1, 5:1, or 10:1. The percentage of specific target lysis was assessed by luminometry. Data represent one independent experiment from two different donors. Mean ± SD of three technical replicates. One representative experiment from four separate donors is shown in the panel. [Figure 2D] Figure 2. Cytotoxicity of the CAR20_19 construct in vitro. Luciferase-based cytotoxicity assays were performed using the CD19+ CD20+ tumor line 293T (Figure 2D). All target lines were stably transduced with firefly luciferase. CAR T cells and tumor cells were cultured overnight at the indicated effector-to-target (E:T) ratios: 2.5:1, 5:1, or 10:1. The percentage of specific target lysis was assessed by luminometry. Data represent one independent experiment from two different donors. Mean ± SD of three technical replicates. One representative experiment from four separate donors is shown in the panel. [Figure 3A]Figure 3A shows CAR T cytokine release in response to the Raji lymphoma cell line. CAR T cell culture supernatants were assessed alone or after overnight incubation with CD19+CD20+ Raji target cells at an E:T ratio of 10. (Figure 3A) Cytokine production of IL-2 was analyzed by ELISA. Mean ± SD of three technical replicates. Data represent one independent experiment from two separate donors. [Figure 3B] Figure 3B shows CAR T cytokine release in response to the Raji lymphoma cell line. CAR T cell culture supernatants were assessed alone or after overnight incubation with CD19+CD20+ Raji target cells at an E:T ratio of 10. (Figure 3B) Cytokine production of IFNγ was analyzed by ELISA. Mean ± SD of three technical replicates. Data represent one independent experiment from two separate donors. [Figure 3C] Figure 3C shows CAR T cytokine release in response to the Raji lymphoma cell line. CAR T cell culture supernatants were assessed alone or after overnight incubation with CD19+CD20+ Raji target cells at an E:T ratio of 10. (Figure 3C) Cytokine production of TNFα was analyzed by ELISA. Mean ± SD of three technical replicates. Data represent one independent experiment from two separate donors. [Figure 4A] Figure 4A) Schematic of long-term stimulation with CD19+CD20+ Raji lymphoma cells. In round 1, CAR T cells were co-cultured with Raji cells at an E:T ratio of 0.3:1 for 7 days. Residual CAR T cells and Raji cells were measured by flow cytometry and readjusted to an E:T ratio of 0.15:1 for round 2. [Figure 4B] Cytotoxicity of CAR20_19 constructs upon prolonged target cell stimulation. Figure 4B) The percentage of remaining Raji was analyzed using GFP by flow spectroscopy on days 4, 7, and 10. [Figure 4C]Figure 4C shows the cytotoxicity of the CAR20_19 construct upon long-term target cell stimulation. T cell numbers were measured by flow cytometry using CountBright™ Absolute Counting Beads. Fold expansion compared to input T cells was calculated and plotted. Arrows indicate the starting point of each round of co-culture. Data are representative results from one of two donors. [Figure 5A] FIG. 5A shows the experimental design and timeline for the in vivo challenge and re-challenge study of CAR T cells in murine NSG Raji lymphoma xenografts (FIG. 5A). [Figure 5B] Representative bioluminescence images (Figure 5B) of tumor progression in the mice under study. [Figure 5C] Summary of bioluminescence measurements depicting tumor progression kinetics over the course of the initial challenge and rechallenge study (FIG. 5C). DETAILED DESCRIPTION OF THE INVENTION

[0078] Detailed Description definition As used herein, the singular forms "a," "an," and "the" refer to both the singular and the plural unless the context clearly indicates otherwise. For example, the term "an antigen" includes single or multiple antigens and can be considered equivalent to the phrase "at least one antigen." As used herein, the term "comprises" means "includes." Thus, "comprising an antigen" means "including one antigen" without excluding other elements. The phrase "and / or" means "and" or "or." It should be further understood that any and all base or amino acid sizes and all molecular weight or molecular mass values ​​given for nucleic acids or polypeptides are approximate and provided for convenience unless otherwise specified. While many methods and materials similar or equivalent to those described herein can be used, particularly suitable methods and materials are described below. In case of conflict, the present specification, including explanations of terms, controls. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. In order to facilitate review of the various embodiments, the following explanations of terms are provided.

[0079] The term "about," when referring to a measurable value, e.g., amount, temporal duration, etc., is meant to encompass variations of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% from the specified value, where such variations are appropriate for practicing the disclosed methods.

[0080] Unless otherwise specified, the technical terms used in this specification follow conventional usage.The definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes VII, Oxford University Press, 1999; Kendrew et al. (ed.) The Encyclopedia of Molecular Biology, Blackwell Science Ltd., 1994; and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, VCH Publishers, Inc., 1995; and other similar references.

[0081] The present disclosure provides fully human CD20 / CD19 antibodies or fragments thereof, and chimeric antigen receptors (CARs) having such fully human CD20 / CD19 antigen-binding domains. Enhanced functional activity of CARs is directly related to enhanced functional activity of CAR-expressing T cells. As a result of these one or more modifications, CARs exhibit both high cytokine-induced cytolysis and cell surface expression on transduced T cells, along with increased in vivo T cell proliferation and persistence levels of transduced CAR-expressing T cells. The CARs of the present disclosure are advantageous in that a single CART lentiviral product can be utilized to treat multiple patient populations (i.e., CD19+, CD20+, or dual CD19+CD20+ cancer patients), providing flexibility in resource-limited settings.

[0082] The unique ability to combine functional moieties from different protein domains is a key and innovative feature of chimeric antigen receptors (CARs). The selection of each of these protein domains, as well as the specific combinations they employ, is a key design feature. Each design domain is an essential component that can be used to manipulate lymphocyte function in various CAR platforms. For example, the selection of an extracellular binding domain can enable an otherwise ineffective CAR.

[0083] The nonvariable framework components of immunoglobulin-derived protein sequences used to create the extracellular antigen-binding domain of CARs can be completely neutral or self-associate, rendering T cells metabolically exhausted and rendering therapeutic T cells expressing the CAR highly ineffective. This occurs regardless of the antigen-binding function of the CAR domain. Furthermore, the choice of intracellular signaling domain(s) can also determine the activity and durability of therapeutic lymphocyte populations used in immunotherapy. While the ability of these extracellular and intracellular domains to bind target antigens and deliver activation signals to T cells, respectively, are important aspects of CAR design, it has also become clear that the choice of source of the extracellular antigen-binding fragment can have a significant effect on CAR efficacy and therefore play a crucial role in CAR function and clinical utility.

[0084] The CARs disclosed herein are expressed at high levels in cells. Cells expressing the CARs have high proliferation rates in vivo, produce large amounts of cytokines, and have high cytotoxic activity against cells bearing the fully human CD20 / CD19 antigens to which the CARs bind. The use of an extracellular fully human CD20 / CD19 antigen-binding domain results in the generation of CARs that function better in vivo, while avoiding the induction of anti-CAR immunity in the host immune response and the death of the CAR T cell population. CARs expressing an extracellular fully human CD20 / CD19 ScFv antigen-binding domain exhibit superior activity / properties, including: i) prevention of the lack of CAR T persistence and function seen with mouse-derived binding sequences; ii) lack of effective local (i.e., intrapleural) delivery of the CAR; and iii) the ability to generate CAR T cell designs based on both high- and low-affinity binders to fully human CD20 / CD19. This last property allows researchers to better tune the efficacy and / or tissue specificity of CAR T products to toxicity, as tumors express more fully human CD20 / CD19 than normal tissues, allowing lower affinity binders to have greater specificity for tumors than normal tissues, thereby preventing non-on-target tumor toxicity and bystander cell killing.

[0085] Below is a detailed description of the CARs of the present invention, including a description of their extracellular fully human CD20 / CD19 antigen-binding domains, transmembrane domains, and intracellular domains, along with further description of CARs, antibodies and antigen-binding fragments thereof, conjugates, nucleotides, expression, vectors, and host cells, methods of treatment, compositions, and kits using the disclosed CARs.

[0086] In each of the foregoing aspects and embodiments above, scFv binders have been made against mesothelin, as disclosed, for example, in Applicant's issued U.S. Patent No. 10,183,993, entitled "Compositions and Methods for Treating Cancer with Anti-Mesothelin Immunotherapy," and assigned to Lentigen Technology Corporation under Docket No. LEN_017, and the nucleotide sequence scFv antigen of SEQ ID NO: 149 and the amino acid sequence of SEQ ID NO: 150, respectively, can be incorporated into a functional CAR, thereby incorporating the nucleotide sequence of SEQ ID NO: 39 and the amino acid sequence of SEQ ID NO: 40, respectively, into a DuoCAR therapy.

[0087] In each of the foregoing aspects and embodiments, in addition to scFv sequences, single-chain antigen binders (as opposed to scFvs) can be incorporated into single, tandem, DuoCAR, or multiple targeting CAR applications. For example, as disclosed in Applicant's issued U.S. Patent No. 10,426,797, entitled "Compositions and Methods For Treating Cancer With Anti-CD33 Immunotherapy" and assigned to Lentigen Technology, Inc. under application number LEN_018, CD33-specific heavy chain-only binders, the nucleotide sequence of SEQ ID NO: 41 and the amino acid sequence of SEQ ID NO: 42, respectively, can be incorporated into functional CARs, and LTG1906, the nucleotide sequence of SEQ ID NO: 43 and the amino acid sequence of SEQ ID NO: 44, respectively, targets malignant tumors that express CD33.

[0088] In each of the above aspects and embodiments, one example of a single, tandem, DuoCAR, or multiple targeting CAR therapeutic application is the treatment of leukemia that expresses CD19, CD20, and TSLPR antigens. In this case, LTG1496 or LTG1497 (SEQ ID NOs: 35 and 26, respectively) can be combined with the TSLPR-specific CAR (LTG1789) having the amino acid sequence of SEQ ID NO: 47 and SEQ ID NO: 48, respectively, which is made from the nucleotide sequence of SEQ ID NO: 45 and the TSLPR-specific scFV domain having the amino acid sequence of SEQ ID NO: 46.

[0089] In each of the aforementioned aspects and embodiments, another example of the application of single, tandem, DuoCAR, or multiple targeted CAR therapy is the treatment of cancers that express the CD38 antigen. For example, as disclosed in Applicant's issued U.S. Patent No. 11,103,533, filed November 30, 2018, entitled "Compositions and Methods For Treating Cancer With Anti-CD38 Immunotherapy," and assigned to Lentigen Technology, Inc. under application number LEN_026, a CD38-specific binder can be incorporated into one or more functional CARs that target malignant tumors that express CD38, as disclosed in Applicant's issued U.S. Patent Application No. 11,103,533, the entire contents of which are incorporated herein by reference.

[0090] In each of the aforementioned aspects and embodiments, another example of the application of single, tandem, duoCAR, or multiple targeted CAR therapy is the treatment of cancers that express the CD123 antigen. For example, the CD123-specific binder disclosed in Applicant's issued U.S. Patent No. 10,844,128, entitled "Compositions and Methods for Treating Cancer With Anti-CD123 Immunotherapy," filed on September 20, 2019, and assigned to Lentigen Technology, Inc. under application number LEN_024, which claims priority to Provisional Patent Application No. 62 / 734,106, filed on September 20, 2018, can be incorporated into one or more functional CARs that target malignant tumors that express CD123, as disclosed in Applicant's issued U.S. Patent Application No. 10,844,128, the entire contents of which are incorporated herein by reference.

[0091] In each of the aforementioned aspects and embodiments, another example of the application of single, tandem, DuoCAR, or multiple targeted CAR therapy is the treatment of cancers that express the CD123 antigen. For example, the CD123-specific binders disclosed in applicant's co-pending U.S. patent application Ser. No. 17 / 685,132, filed March 2, 2022, entitled "Compositions and Methods For Treating Cancer With Anti-CD123 Immunotherapy," and assigned to Lentigen Technology, Inc. under application number MBG_99, can be incorporated into one or more functional CARs that target malignant tumors that express CD123, as disclosed in applicant's co-pending U.S. patent application Ser. No. 17 / 685,132, the entire contents of which are incorporated herein by reference.

[0092] In each of the aforementioned aspects and embodiments, another example of an application of single, tandem, DuoCAR, or multiple targeted CAR therapy is the treatment of cancers that express the BCMA antigen. For example, the BCMA-specific binders disclosed in Applicant's issued U.S. Patent No. 11,052,112, filed May 30, 2019, entitled "Fully Human BCMA CAR T Cells for the Treatment of Multiple Myeloma and Other BCMA-Positive Malignancies," and assigned to Lentigen Technology, Inc. under application number MBG_13, can be incorporated into one or more functional CARs that target BCMA-expressing malignancies, as disclosed in Applicant's issued U.S. Patent No. 11,052,112, the entire contents of which are incorporated herein by reference.

[0093] In each of the aforementioned aspects and embodiments, another example of the application of single, tandem, DuoCAR, or multiple targeted CAR therapy is the treatment of solid tumor cancer. For example, the specific binders disclosed in applicant's co-pending U.S. Provisional Patent Application No. 63 / 393,088, filed July 28, 2022, entitled "Chimeric Antigen Receptor Therapies for Treating Solid Tumors," and assigned to Lentigen Technology, Inc. under application number MBG_106, can be incorporated into one or more functional CARs that target solid tumors, as disclosed in applicant's co-pending U.S. Provisional Patent Application No. 63 / 393,088, the entire contents of which are incorporated herein by reference.

[0094] In each of the aforementioned aspects and embodiments, an example of a tandem-CAR (comprising two scFv domains set forth in the nucleotide sequence of SEQ ID NO: 23 and the amino acid sequence of SEQ ID NO: 24) on which this technology is based includes CD20_CD19 CAR LTG1497, which has the nucleotide sequence of SEQ ID NO: 25 and the amino acid sequence of SEQ ID NO: 26. In some cases, better DuoCAR expression in target cells can be obtained by reversing the order of the two binders. Thus, both LTG1497, in which the CD19 scFv is more proximal as shown in the nucleotide sequence of SEQ ID NO: 25 and the amino acid sequence of SEQ ID NO: 26; and LTG1496, in which the CD19 scFv is more distal to the membrane as shown in the nucleotide sequence of SEQ ID NO: 33 and the amino acid sequence of SEQ ID NO: 34, can be used as one member of a DuoSet containing DuoCARs.

[0095] A. Chimeric Antigen Receptor (CAR) The CARs disclosed herein comprise at least one fully human CD20 / CD19 antigen-binding domain capable of binding to fully human CD20 / CD19, at least one transmembrane domain, and at least one intracellular domain.

[0096] Chimeric antigen receptors (CARs) are artificially constructed hybrid proteins or polypeptides containing an antibody antigen-binding domain (e.g., a single-chain variable fragment (scFv)) linked to a T cell signaling domain via a transmembrane domain. Characteristics of CARs include their ability to redirect T cell specificity and reactivity toward selected targets, leveraging the antigen-binding properties of monoclonal antibodies in a non-MHC-restricted manner. Non-MHC-restricted antigen recognition confers on CAR-expressing T cells the ability to recognize antigens independently of antigen processing, thus bypassing a major mechanism of tumor escape. Furthermore, when expressed in T cells, CARs advantageously do not dimerize with the alpha and beta chains of endogenous T cell receptors (TCRs).

[0097] As disclosed herein, the intracellular T cell signaling domain of a CAR can include, for example, a T cell receptor signaling domain, a T cell costimulatory signaling domain, or both. A T cell receptor signaling domain refers to a portion of a CAR that includes the intracellular domain of a T cell receptor, such as, but not limited to, the intracellular portion of the CD3 zeta protein. A costimulatory signaling domain refers to a portion of a CAR that includes the intracellular domain of a costimulatory molecule, which is a cell surface molecule other than an antigen receptor or its ligand, that is required for an efficient response of lymphocytes to antigens.

[0098] 1. Extracellular domain In one embodiment, CAR comprises target-specific binding element, otherwise called antigen-binding domain or part.The selection of domain depends on the type and number of ligands that define the surface of target cell.For example, antigen-binding domain can be selected to recognize the ligand that acts as a cell surface marker on target cells that is associated with specific disease state.Therefore, the examples of cell surface markers that can act as ligands for the antigen-binding domain in CAR include those associated with virus, bacteria and parasite infection, autoimmune disease and cancer cell.

[0099] In one embodiment, CAR can be engineered to target the tumor antigen of interest by engineering the desired antigen binding domain that specifically binds to the antigen on tumor cell.Tumor antigen is the protein produced by tumor cell that induces immune response, especially T cell-mediated immune response.The choice of antigen binding domain depends on the specific type of cancer to be treated. Tumor antigens include, for example, glioma-associated antigens, carcinoembryonic antigen (CEA), beta-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, Her2 / neu, survivin, and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, and fully human CD20 / CD19. The tumor antigens disclosed herein are included by way of example only; this list is not intended to be exhaustive, and further examples will be readily apparent to those of skill in the art.

[0100] In one embodiment, the tumor antigen comprises one or more antigenic cancer epitopes associated with malignant tumors. Malignant tumors express several proteins that can serve as target antigens for immune attack. These molecules include, but are not limited to, tissue-specific antigens, such as MART-1, tyrosinase, and GP100 in melanoma, and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation-related molecules, such as the oncogene HER-2 / Neu / ErbB-2. Yet another group of target antigens is carcinoembryonic antigens, such as carcinoembryonic antigen (CEA). In B-cell lymphomas, tumor-specific idiotypic immunoglobulins constitute truly tumor-specific immunoglobulin antigens unique to individual tumors. B-cell differentiation antigens, such as CD19, CD20, CD22, BCMA, ROR1, and CD37, are other candidate target antigens in B-cell lymphomas. Some of these antigens (CEA, HER-2, CD19, CD20, idiotype) have been used as targets for passive immunotherapy with monoclonal antibodies with limited success.

[0101] In a preferred embodiment, the tumor antigen is fully human CD20 / CD19, and tumors associated with fully human CD20 / CD19 expression include lung mesothelioma, ovarian and pancreatic cancer, or any combination thereof, which express high levels of the extracellular protein fully human CD20 / CD19.

[0102] The type of tumor antigen can also be a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). TSAs are unique to tumor cells and do not exist on other cells in the body. TAAs are not unique to tumor cells, but are instead expressed on normal cells under conditions that cannot induce a state of immunological tolerance to the antigen. The expression of an antigen on a tumor can occur under conditions that allow the immune system to respond to the antigen. A TAA can be an antigen that is expressed on normal cells during fetal development, when the immune system is immature and unable to respond, or it can be an antigen that is normally present at very low levels on normal cells but is expressed at a significantly higher level on tumor cells.

[0103] Non-limiting examples of TSAs or TAAs include differentiation antigens such as MART-1 / MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, HER-2 / neu; unique tumor antigens resulting from chromosomal translocations such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3 / CA 27.29 / BCAA, CA 195, CA These include 242, CA-50, CAM43, CD68 / P1, CO-029, FGF-5, G250, Ga733 / EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 / Mac-2 binding protein / cyclophilin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0104] In one embodiment, the antigen binding domain portion of the CAR targets an antigen including, but not limited to, CD19, CD20, CD22, ROR1, CD33, CD38, CD123, CD138, BCMA, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, FGFR4, TSLPR, NY-ESO-1 TCR, MAGE A3 TCR, etc.

[0105] In a preferred embodiment, the antigen binding domain portion of the CAR targets the extracellular CD20 / CD19 antigen.

[0106] In a preferred embodiment, the antigen binding domain portion of the CAR targets the extracellular CD20 antigen.

[0107] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD20 scFv antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 1, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD20 scFv antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 2.

[0108] In a preferred embodiment, the antigen binding domain portion of the CAR targets the extracellular CD19 antigen.

[0109] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD19 scFv antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 3, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD19 scFv antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 4.

[0110] In a preferred embodiment, the antigen binding domain portion of the CAR targets the extracellular CD20 and CD19 antigens.

[0111] In one preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD20 / CD19 scFv antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 48, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD20 / CD19 scFv antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 49, or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 49.

[0112] In various embodiments of fully human CD20 / CD19-specific multiple targeting, tandem, and bicistronic CARs disclosed herein, the general scheme is shown in Figure 1A and includes, from N-terminus to C-terminus, a CAR targeting domain, a transmembrane domain, a costimulatory domain, and a CD3 zeta activation domain. In tandem CARs, fully human tandem CD20 and CD19 targeting domains are included. In the case of bicistronic CARs, the first CD20-targeting CAR is composed of a fully human CD20-targeting domain, followed in frame by a second CD19-targeting CAR composed of a CD19-targeting domain, with a ribosome skip element inserted between the CD20 CAR and CD19 CAR sequences to facilitate coexpression.

[0113] In one embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 5 (D0144) (CD20_CD19 CD8 BBz), which encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 6.

[0114] In one embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 5, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, and encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 6, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity (D0144) (CD20_CD19 CD8 BBz).

[0115] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 7 (CAR D0255) (CD20_CD19 CD28 CD28 BBz), which encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 8 (CAR D0255) (CD20_CD19 CD28 CD28 BBz).

[0116] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 7, or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof, and encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 8, or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof (CAR D0255) (CD20_CD19 CD28 CD28 BBz).

[0117] In one embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 9 (CAR D0256) (CD20_CD19 CD8 CD28 BBz), which encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 10.

[0118] In one embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 9, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, and encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 10, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity (CAR D0256) (CD20_CD19 CD8 CD28 BBz).

[0119] In one embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 13 (CAR D0257) (CD20 CD8 CD28z_CD19 CD8 BBz), which encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 14.

[0120] In one embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 13, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, and encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 14, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity (CAR D0257) (CD20 CD8 CD28z_CD19 CD8 BBz).

[0121] In one embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 15 (CAR D0258) (CD20 CD8 BBz_CD19 CD8 CD28z), which encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 16.

[0122] In one embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 15, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, and encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 16, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity (CAR D0258) (CD20 CD8 BBz_CD19 CD8 CD28z).

[0123] In one embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 17 (CAR D0266) (CD20 CD8 OX40 OX40z_CD19 CD8 ICOSz), which encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0124] In one embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 17, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, and encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 18, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity (CAR D0266) (CD20 CD8 OX40 OX40z_CD19 CD8 ICOSz).

[0125] In one embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 19 (CAR LTG1497) (mCD20_CD19 CD8 BBz), which encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO:20.

[0126] In one embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 19, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, and encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 20, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity (CAR LTG1497) (mCD20_CD19 CD8 BBz).

[0127] Surface expression of anti-CD20 / CD19 CARs incorporating single-chain fragment variable (scFv) sequences reactive with fully human CD20 and CD19 antigens is shown in Example 1, Figure 1B, below. Expression levels of each ScFv-containing CAR were determined by flow cytometry analysis of LV-transduced T cells from healthy donors using CD19 Fc recombinant protein followed by anti-Fc AF647 (APC). The scFv-based anti-CD20 / CD19 CAR constructs D0144, D0255, D0256, D0257, D0258, and D0266, as well as a comparison construct, LTG1497, composed of a murine CD20 / CD19 scFv tandem targeting domain, were highly expressed in human primary T cells of both CD4+ and CD8+ T lineages (ranging from 49.7% to 78.6% of all T cells were CAR+), compared to untransduced T cell controls.

[0128] As shown in Example 1 and Figures 2A-2D, we demonstrated the high cytolytic activity of fully human CD20 / CD19 CARs. Primary human T cells were transduced with LVs encoding CAR constructs (D0144, D0255, D0256, D0257, D0258, D0266, and the comparative construct LTG1497; see Methods) and then incubated with Raji, NALM-6, REH, or 293T cell lines stably transduced with firefly luciferase for 18 hours for a luminescence-based in vitro killing assay. All leukemia lines tested express CD19 on their surface, except for the negative control 293T. CD20 expression differs among tumor lines. The Raji line is CD20 positive, while REH is CD20 negative, as is the control line 293T. The NALM-6 line expresses weak but detectable CD20.

[0129] All tumor lines expressing CD19 and / or CD20 were lysed by CAR T cells, but no lysis of the CD19-negative CD20-negative line 293T occurred.

[0130] The most potent specific lysis of Raji cells was mediated by CAR constructs D0255 and D0266, seen at the lowest effector to target ratio of 2.5:1 (Figure 2A). When combined with the target strains NALM 6 and Reh, potent and comparable lysis was achieved by all CARs (Figures 2B, 2C).

[0131] Next, we evaluated the ability of anti-CD20 / CD19 CAR T cells to secrete cytokines. Tumor cells were cocultured overnight with CAR T cells or control T cells at a target-to-effector ratio of 10:1 (+Raji group), and culture supernatants were analyzed by ELISA for IFN-gamma, TNF-alpha, and IL-2 (see Figures 3A–3C). Targetless CAR T cells were included as a negative control (-Raji group). An untransduced (UTD) negative T cell control was also included. All CAR T groups induced cytokines in response to tumor cells, whereas the negative control, -Raji, did not induce appreciable cytokines. Compared to the negative control, all CARs potently induced IFN-gamma, TNF-alpha, and IL-2.

[0132] The highest levels of TNF-alpha were produced by CARs D0255, D0257, D0258, and D0266, and the production of IFN-gamma and IL-2 followed a similar pattern (Figures 3A-3C).

[0133] To examine the performance of the fully human CD20 / CD19 CRA candidate upon prolonged exposure to antigen, we performed coculture experiments with Raji tumor cells. CAR T cells were placed in culture with Raji lymphoma cells at a low effector-to-target ratio of 0.3:1. After 7 days, CAR T cells were restimulated again with Raji cells at a target-to-effector ratio of 0.15:1 until study day 10 (Figure 4A). This setup created a very challenging environment for CAR T cell function, allowing even subtle functional differences between CAR T candidate constructs to be identified. Cell cultures were acquired by flow cytometry on days 0, 7, and 10 to assess the percentage of viable target cells remaining at the end of each co-incubation cycle and CAR T cell proliferation (Figures 4B-4C). At the end of the second co-culture cycle, the three CAR T cells that mediated the greatest reduction in the level of Raji target cells were D0255, D0257, and D0266 (Figure 4B). The best-proliferating CAR T cells were LTG1497, D0255, and D0144 (Figure 4C). Thus, we identified a number of CAR T cell candidates with potent cytotoxic and proliferation properties compared to the other constructs and LTG1497 comparators in this set.

[0134] To characterize the functional properties of CAR T cells in vivo, we treated NSG mice bearing established Raji xenograft tumors with 2 million low-dose fully human CD20 / CD19 CAR T cells D0255, D0256, D0266, D0257, or D0258, or mouse scFv CD20 / CD19 CAR comparator LTG1497 (Figure 5A). Mice treated with all CAR T constructs rejected tumors by day 13, and the mice remained in remission until day 55 (Figure 5B). In contrast, control tumor-only and UTD mice developed high tumor burdens (Figure 5B). On day 55, mice in all groups were re-challenged with 1 million additional Raji cells, and no additional CAR T treatment was administered. One mouse each in the LTG1497, D0256, and D0258 groups, and two mice each in the D0255 and D0266 groups, did not meet the criteria for rechallenge and were not enrolled. A new cohort of age-matched mice was also inoculated with Raji cells as a control at this time point. Tumor progression was followed until day 96. At day 96, all enrolled mice in CAR groups D0255 and D0258 were alive and tumor-free, while one mouse had died in each of the D0257, D0256, and LTG1497 groups, and three mice had died in group D0266. Thus, the ability to eliminate the initial xenograft tumor in vivo was demonstrated for all CAR constructs, and persistence of CAR T cell antitumor function upon tumor rechallenge was demonstrated for constructs D0255, D0258, and LTG1497 (Figure 5C). Thus, the new ranking of CAR T candidates for antitumor utility had to be determined empirically and could not be predicted based solely on the architecture of the CAR or the composition of the targeting domain.

[0135] Without intending to be limited to any particular mechanism of action, possible reasons for the enhanced therapeutic function associated with exemplary CARs of the present invention are believed to include, for example, but not by way of limitation, a) improved lateral movement within the plasma membrane, allowing for more efficient signaling; b) superior location within plasma membrane microdomains, such as lipid rafts, and a greater ability to interact with transmembrane signaling cascades associated with T cell activation; c) superior location within the plasma membrane due to preferential movement away from inhibitory or down-regulatory interactions, e.g., less proximity to or fewer interactions with phosphatases such as CD45; and d) superior assembly into the T cell receptor signaling complex (i.e., the immune synapse), or any combination thereof.

[0136] Although the present disclosure is exemplified using exemplary extracellular fully human CD20 / CD19 variable heavy chain only and scFv antigen-binding domains, other nucleotide and / or amino acid variants within the fully human CD20 / CD19 variable heavy chain only and scFv antigen-binding domains may be used to obtain fully human CD20 / CD19 antigen-binding domains for use in the CARs described herein.

[0137] Depending on the desired antigen to be targeted, the CAR can be further engineered to contain an appropriate antigen-binding domain specific for the desired antigen target. For example, if fully human CD20 / CD19 is the desired antigen to be targeted, an antibody against fully human CD20 / CD19 can be used as the antigen-binding domain incorporated into the CAR.

[0138] In one embodiment of the present invention, a CAR is provided that is capable of binding to a non-TSA or non-TAA, including, for example, but not limited to, an antigen derived from Retroviridae (e.g., human immunodeficiency viruses, e.g., HIV-1 and HIV-LP), Picornaviridae (e.g., poliovirus, hepatitis A virus, enterovirus, human coxsackievirus, rhinovirus, and echovirus), rubella virus, coronavirus, vesicular stomatitis virus, rabies virus, Ebola virus, parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus, influenza virus, hepatitis B virus, parvovirus, Adenoviridae, Herpesviridae (e.g., herpes simplex virus types 1 and 2 (HSV), varicella-zoster virus, cytomegalovirus (CMV), and herpes viruses), Poxviridae (e.g., smallpox virus, vaccinia virus, and poxvirus), or hepatitis C virus, or any combination thereof.

[0139] In another aspect of the present invention, a CAR capable of binding to an antigen derived from a bacterial strain of Staphylococci, Streptococcus, Escherichia coli, Pseudomonas, or Salmonella is provided. In particular, a CAR capable of binding to an antigen derived from an infectious bacterium, such as Helicobacter pyloris, Legionella pneumophilia, a bacterial strain of Mycobacteria sp. (e.g., M. tuberculosis, M. avium, M. intracellulare, M. kansaii, or M. gordonea), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitides, Listeria monocytogenes, Streptococcus pyogenes, Group A Streptococcus, Group B Streptococcus (Streptococcus agalactiae), Streptococcus pneumoniae, or Clostridium tetani, or a combination thereof, is provided.

[0140] 2. Transmembrane domain With respect to the transmembrane domain, the CAR comprises one or more transmembrane domains fused to the extracellular CD20 / CD19 antigen-binding domain of the CAR.

[0141] Transmembrane domains can be derived from either natural or synthetic sources. If the source is natural, the domain can be derived from any membrane-bound or transmembrane protein.

[0142] The transmembrane region particularly used in the CAR described herein can be derived from (i.e., can include at least one of) the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD28, mesothelin, CD33, CD37, CD64, CD80, CD83, CD86, CD134, CD137, CD154, TNFRSF16, or TNFRSF19. Alternatively, the transmembrane domain can be synthetic, in which case it predominantly contains hydrophobic residues such as leucine and valine. Preferably, a triad of phenylalanine, tryptophan, and valine is found at each end of the synthetic transmembrane domain. Optionally, a short oligopeptide or polypeptide linker, preferably between 2 and 10 amino acids in length, can form the link between the transmembrane domain and the cytoplasmic signaling domain of the CAR. A glycine-serine dyad provides a particularly suitable linker.

[0143] In one embodiment, a transmembrane domain naturally associated with one of the domains in the CAR is used in addition to the transmembrane domain.

[0144] In some cases, transmembrane domains may be selected by amino acid substitution or otherwise to avoid binding of such domains to transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

[0145] In one embodiment, the transmembrane domain in the CAR of the present invention is a CD8 transmembrane domain. In one embodiment, the CD8 transmembrane domain comprises the nucleic acid sequence of SEQ ID NO: 35. In one embodiment, the CD8 transmembrane domain comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 36. In another embodiment, the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 36.

[0146] In one embodiment, the encoded transmembrane domain comprises an amino acid sequence having at least one, two, or three alterations (e.g., substitutions) but not more than 20, 10, or 5 alterations (e.g., substitutions) of the amino acid sequence of SEQ ID NO: 34, 36, or 48, or a sequence having 95-99% identity to the amino acid sequence of SEQ ID NO: 34, 36, or 48.

[0147] In some examples, the transmembrane domain of the CAR comprises a CD8 alpha hinge domain. In one embodiment, the CD8 hinge domain comprises the nucleic acid sequence of SEQ ID NO: 37. In one embodiment, the CD8 hinge domain comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 38. In another embodiment, the CD8 hinge domain comprises the amino acid sequence of SEQ ID NO: 38, or a sequence thereof with 95-99% identity.

[0148] In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded linker domain is derived from the extracellular domain of CD8 and is linked to a transmembrane CD8 domain, a transmembrane CD28 domain, or a combination thereof.

[0149] In one embodiment, the transmembrane domain in the CAR of the present invention is a CD28 transmembrane domain. In one embodiment, the CD28 transmembrane domain comprises the nucleic acid sequence of SEQ ID NO: 47. In one embodiment, the CD28 transmembrane domain comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 48. In another embodiment, the CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 48.

[0150] In one embodiment, the encoded transmembrane domain comprises an amino acid sequence having at least one, two, or three alterations (e.g., substitutions) but not more than 20, 10, or 5 alterations (e.g., substitutions) of the amino acid sequence of SEQ ID NO: 47, or a sequence having 95-99% identity to the amino acid sequence of SEQ ID NO: 48.

[0151] 3. Spacer domain In CARs, a spacer domain can be located between the extracellular domain and the transmembrane domain, or between the intracellular domain and the transmembrane domain. A spacer domain refers to any oligopeptide or polypeptide that functions to link the transmembrane domain to the extracellular domain and / or the transmembrane domain to the intracellular domain. A spacer domain contains up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids.

[0152] In some embodiments, the linker may include a spacer element, which, if present, increases the size of the linker, thereby increasing the distance between the effector molecule or detectable marker and the antibody or antigen-binding fragment. Exemplary spacers are known to those of skill in the art, including those described in U.S. Patent Nos. 7,964,566, 7,498,298, 6,884,869, 6,323,315, 6,239,104, 6,034,065, 5,780,588, 5,665,860, 5,663,149, 5,635,483, 5,599,902, 5,554,725, 5,530,097, 5,521 Nos. 5,284, 5,504,191, 5,410,024, 5,138,036, 5,076,973, 4,986,988, 4,978,744, 4,879,278, 4,816,444 and 4,486,414, as well as those listed in U.S. Patent Application Publication Nos. 20110212088 and 20110070248, each of which is incorporated herein by reference in its entirety.

[0153] The spacer domain preferably has a sequence that promotes the binding of CAR to the antigen and enhances signal transduction into the cell. Examples of amino acids that are predicted to promote binding include cysteine, charged amino acids, and serine and threonine in potential glycosylation sites, and these amino acids can be used as amino acids that constitute the spacer domain.

[0154] The spacer domain may be the entire or a portion of the hinge region of CD8 alpha (NCBI RefSeq: NP_001759.3), amino acids 137-206 (SEQ ID NO: 39), CD8 beta (GenBank: AAA35664.1), amino acids 315-396 of CD4 (NCBI RefSeq: NP_000607.1), or amino acids 137-152 of CD28 (NCBI RefSeq: NP_006130.1). Alternatively, a portion of the constant region of an antibody heavy or light chain may be used. Furthermore, the spacer domain may be an artificially synthesized sequence.

[0155] In some examples, the transmembrane domain of the CAR comprises a CD28 hinge domain. In one embodiment, the CD28 hinge domain comprises the nucleic acid sequence of SEQ ID NO: 31. In one embodiment, the CD28 hinge domain comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 32. In another embodiment, the CD28 hinge domain comprises the amino acid sequence of SEQ ID NO: 32, or a sequence thereof with 95-99% identity.

[0156] In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded linker domain is derived from the extracellular domain of CD28 and is linked to a transmembrane CD28 domain, a transmembrane CD28 domain, or a combination thereof.

[0157] Furthermore, a signal peptide sequence may be linked to the N-terminus of a CAR. Signal peptide sequences are present at the N-terminus of many secretory proteins and membrane proteins and have a length of 15 to 30 amino acids. Many of the protein molecules referred to above as intracellular domains have signal peptide sequences, and these signal peptides may be used as signal peptides for a CAR. In one embodiment, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 12.

[0158] 4. Intracellular domain The cytoplasmic domain or other intracellular signaling domain of a CAR is responsible for activating at least one of the normal effector functions of the immune cell in which the CAR is placed. The term "effector function" refers to a specialized function of a cell. For example, the effector function of a T cell can be cytolytic activity or helper activity, including cytokine secretion. Thus, the term "intracellular signaling domain" refers to the portion of a protein that transmits an effector function signal and instructs the cell to perform a specialized function. While the entire intracellular signaling domain can usually be used, it is often not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the intact chain, as long as it transmits the effector function signal. Thus, the term intracellular signaling domain is meant to include any truncated portion of the intracellular signaling domain sufficient to transmit the effector function signal.

[0159] Preferred examples of intracellular signaling domains for use in CARs include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivatives or variants of these sequences, and any synthetic sequences with the same functional capability.

[0160] It is known that signals generated through the TCR alone are insufficient for full activation of T cells, and that secondary or costimulatory signals are also required. Thus, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences) and those that act antigen-dependently to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences).

[0161] Primary cytoplasmic signaling sequences regulate the primary activation of the TCR complex in either a stimulatory or inhibitory manner. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs, or ITAMs.

[0162] Examples of ITAMs containing primary cytoplasmic signaling sequences of particular use in the CARs disclosed herein include those derived from TCR zeta (CD3 zeta), FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. Specific, non-limiting examples of ITAMs include amino acids 51-164 of CD3 zeta (NCBI RefSeq: NP_932170.1), amino acids 45-86 of Fc epsilon RI gamma (NCBI RefSeq: NP_004097.1), amino acids 201-244 of Fc epsilon RI beta (NCBI RefSeq: NP_000130.1), amino acids 139-182 of CD3 gamma (NCBI RefSeq: NP_000064.1), amino acids 128-171 of CD3 delta (NCBI RefSeq: NP_000723.1), amino acids 153-207 of CD3 epsilon (NCBI RefSeq: NP_000724.1), and CD5 (NCBI RefSeq: NP_000724.1). These peptides include those with amino acids 402-495 of CD79a (NCBI RefSeq:NP_055022.2), 707-847 of CD79a (NCBI RefSeq:NP_001762.2), 166-226 of CD79a (NCBI RefSeq:NP_001774.1), 182-229 of CD79b (NCBI RefSeq:NP_000617.1), and 177-252 of CD66d (NCBI RefSeq:NP_001806.2), as well as variants thereof with the same functions as these peptides. The amino acid numbers based on the NCBI RefSeq IDs or GenBank amino acid sequence information described herein are numbered based on the full length of the precursor of each protein (including the signal peptide sequence, etc.). In one embodiment, the cytoplasmic signaling molecule in the CAR comprises a cytoplasmic signaling sequence derived from CD3 zeta.

[0163] In a preferred embodiment, the intracellular domain of the CAR can be designed to include a CD3-zeta signaling domain, either alone or in combination with any other desired cytoplasmic domain(s) useful in the context of the CAR. For example, the intracellular domain of the CAR can include a CD3 zeta chain portion and a costimulatory signaling region. The costimulatory signaling region refers to a portion of the CAR that includes the intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for efficient lymphocyte response to antigens. Examples of such costimulatory molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83. Specific, non-limiting examples of such costimulatory molecules include amino acids 236-351 of CD2 (NCBI RefSeq: NP_001758.2), amino acids 421-458 of CD4 (NCBI RefSeq: NP_000607.1), amino acids 402-495 of CD5 (NCBI RefSeq: NP_055022.2), amino acids 207-235 of CD8 alpha (NCBI RefSeq: NP_001759.3), amino acids 196-210 of CD83 (GenBank: AAA35664.1), amino acids 181-220 of CD28 (NCBI RefSeq: NP_006130.1), and CD137 (4-1BB, NCBI The present disclosure primarily exemplifies 4-1BB as a costimulatory signaling element, but other costimulatory elements are within the scope of the present disclosure.

[0164] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR can be linked to each other randomly or in a specific order. Optionally, a short oligopeptide or polypeptide linker, preferably between 2 and 10 amino acids in length, can form the linkage. Glycine-serine duplexes provide particularly suitable linkers.

[0165] In one embodiment, the intracellular domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of CD28. In another embodiment, the intracellular domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of 4-1BB. In yet another embodiment, the intracellular domain is designed to comprise the signaling domain of CD3-zeta and the signaling domains of CD28 and 4-1BB.

[0166] In one embodiment, the intracellular domain in the CAR is designed to comprise the signaling domain of 4-1BB and the signaling domain of CD3-zeta, wherein the signaling domain of 4-1BB comprises the nucleic acid sequence set forth in SEQ ID NO: 42, 44, or 45, and the signaling domain of CD3-zeta comprises the nucleic acid sequence set forth in SEQ ID NO: 43.

[0167] In another embodiment, the intracellular domain in the disclosed CAR is designed to comprise the signaling domain of CD28 and the signaling domain of CD3-zeta, wherein the signaling domain of CD28 comprises the nucleic acid sequence set forth in SEQ ID NO: 21, and the signaling domain of CD3-zeta comprises the nucleic acid sequence set forth in SEQ ID NO: 42, 45, or 46.

[0168] In another embodiment, the intracellular domain in the CAR is designed to comprise the binding signaling domain of CD28_4-1BB and the signaling domain of CD3-zeta, wherein the signaling domain of CD28_4-1BB comprises the nucleic acid sequence set forth in SEQ ID NO: 25, and the signaling domain of CD3-zeta comprises the nucleic acid sequence set forth in SEQ ID NO: 42, 45, or 46.

[0169] 5. Further description of CAR The functional portion of the CAR disclosed herein is also expressly included within the scope of the present invention. The term "functional portion", when used in reference to a CAR, refers to any one or more parts or fragments of the CAR disclosed herein, which part or fragment retains the biological activity of the CAR (parent CAR) of which it is a part. A functional portion includes, for example, a part of a CAR that retains the ability to recognize target cells or detect, treat or prevent disease to a similar degree, the same degree, or a higher degree than the parent CAR. With respect to a parent CAR, a functional portion can, for example, comprise about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95% or more of the parent CAR.

[0170] The functional portion can comprise additional amino acids at the amino or carboxy end of the portion, or at both ends, which additional amino acids are not found in the amino acid sequence of the parent CAR. Preferably, the additional amino acids do not interfere with the biological function of the functional portion, such as, for example, recognizing target cells, detecting cancer, treating or preventing cancer, etc. More preferably, the additional amino acids enhance the biological activity of the functional portion compared to the biological activity of the parent CAR.

[0171] The functional variants of the CAR disclosed herein are included within the scope of this disclosure.The term "functional variant" as used herein refers to a CAR, polypeptide or protein that has substantial or significant sequence identity or similarity with the parent CAR, and this functional variant retains the biological activity of the CAR it is a variant of.Functional variants include, for example, variants of the CAR (parent CAR) described herein that retain the ability to recognize target cells to a similar degree, the same degree, or a higher degree than the parent CAR.With respect to the parent CAR, functional variants can be, for example, at least about 30%, 50%, 75%, 80%, 90%, 98% or more identical in amino acid sequence to the parent CAR.

[0172] A functional variant can, for example, comprise the amino acid sequence of a parent CAR with at least one conservative amino acid substitution. Alternatively, or in addition, a functional variant can comprise the amino acid sequence of a parent CAR with at least one non-conservative amino acid substitution. In this case, it is preferred that the non-conservative amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. The non-conservative amino acid substitution can enhance the biological activity of the functional variant, so that the biological activity of the functional variant is increased compared to the parent CAR.

[0173] The amino acid substitutions in the CAR are preferably conservative amino acid substitutions. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid with certain physical and / or chemical properties is replaced with another amino acid with the same or similar chemical or physical properties. For example, a conservative amino acid substitution can be an acidic / negatively charged polar amino acid substituting another acidic / negatively charged polar amino acid (e.g., Asp or Glu), an amino acid having a nonpolar side chain substituting another amino acid having a nonpolar side chain (e.g., Ala, Gly, Val, He, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), a basic / positively charged polar amino acid substituting another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.), an uncharged amino acid with a polar side chain substituting another uncharged amino acid with a polar side chain (e.g., Asn, Gin, Ser, Thr, Tyr, etc.), an amino acid with a beta-branched side chain substituting another amino acid with a beta-branched side chain (e.g., He, Thr, and Val), an amino acid with an aromatic side chain substituting another amino acid with an aromatic side chain (e.g., His, Phe, Trp, and Tyr), etc.

[0174] A CAR can consist essentially of the specified amino acid sequence(s) described herein, such that other components, e.g., other amino acids, do not significantly alter the biological activity of the functional variant.

[0175] CARs (including functional portions and functional variants) can be of any length, i.e., comprise any number of amino acids, provided that the CAR (or functional portion or variant thereof) retains its biological activity, e.g., the ability to specifically bind to an antigen, detect diseased cells in a mammal, or treat or prevent a disease in a mammal, etc. For example, a CAR can be about 50 to about 5,000 amino acids in length, e.g., 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1,000 or more amino acids in length.

[0176] CARs (including functional portions and functional variants of the invention) can contain synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, -amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3-hydroxyproline and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydrobenzoic ... Examples of suitable hydroxybenzoates include 2-amino-2-methyl-2-propanol, ...

[0177] CARs (including functional portions and functional variants) can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized, e.g., via disulfide bridges, or converted into acid addition salts and / or optionally dimerized or polymerized, or conjugated.

[0178] CAR (including its functional part and functional variant) can be obtained by methods known in the art.CAR can be produced by any suitable method of producing polypeptide or protein.Suitable methods for de novo synthesis of polypeptide and protein are described in references such as Chan et al., Fmoc Solid Phase Peptide Synthesis, Oxford University Press, Oxford, United Kingdom, 2000; Peptide and Protein Drug Analysis, Reid, R. (ed.), Marcel Dekker, Inc., 2000; Epitope Mapping, Westwood et al. (ed.), Oxford University Press, Oxford, United Kingdom, 2001; and U.S. Patent No. 5,449,752.Polypeptide and protein can also be produced recombinantly using the nucleic acid described herein by using standard recombinant methods. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. Furthermore, some CARs (including functional portions and functional variants thereof) can be isolated and / or purified from sources such as plants, bacteria, insects, mammals, e.g., rats, humans, etc. Isolation and purification methods are well known in the art. Alternatively, the CARs described herein (including functional portions and functional variants thereof) can be commercially synthesized by companies. In this regard, CARs can be synthetic, recombinant, isolated, and / or purified.

[0179] B. Antibodies and Antigen-Binding Fragments One embodiment further provides a CAR, a T cell expressing a CAR, an antibody that specifically binds to one or more of the antigens disclosed herein, or an antigen-binding domain or portion thereof. As used herein, "a T cell expressing a CAR" or "CAR T cell" refers to a T cell that expresses a CAR, e.g., has antigen specificity determined by the antibody-derived targeting domain of the CAR.

[0180] As used herein, an "antigen-binding domain" can include antibodies and antigen-binding fragments thereof. The term "antibody" is used herein in the broadest sense and encompasses various antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antigen-binding fragments thereof, so long as they exhibit the desired antigen-binding activity. Non-limiting examples of antibodies include, for example, intact immunoglobulins known in the art, as well as variants and fragments thereof that retain binding affinity to antigens.

[0181] A "monoclonal antibody" is an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and directed against a single antigenic epitope. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. In some examples, a monoclonal antibody is an antibody produced by a single clone of B lymphocytes, or an antibody produced by a cell transfected with nucleic acid encoding the antibody light and heavy chain variable regions of a single antibody (or antigen-binding fragment thereof), or its progeny. In some examples, a monoclonal antibody is isolated from a subject. A monoclonal antibody may have conservative amino acid substitutions that have substantially no effect on antigen binding or other immunoglobulin functions. Exemplary methods for producing monoclonal antibodies are known, see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual, 2nd ed. Cold Spring Harbor Publications, New York (2013).

[0182] Typically, immunoglobulins have heavy (H) chains and light (L) chains interconnected by disulfide bonds. Immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable domain genes. There are two types of light chains, lambda (λ) and kappa (κ). There are five major heavy chain classes (or isotypes): IgM, IgD, IgG, IgA, and IgE, which determine the functional activity of antibody molecules.

[0183] Each heavy and light chain contains a constant region (or constant domain) and a variable region (or variable domain; see, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p. 91 (2007)). In some embodiments, the variable regions of the heavy and light chains combine to specifically bind to an antigen. In further embodiments, only the heavy chain variable region is required. For example, naturally occurring camelid antibodies consisting only of heavy chains are functional and stable in the absence of light chains (see, e.g., Hamers-Casterman et al., Nature, 363:446-448, 1993; Sheriff et al., Nat. Struct. Biol., 3:733-736, 1996). References to "VH" or "VH" refer to the variable region of an antibody heavy chain, including antigen-binding fragments such as Fv, scFv, dsFv, or Fab. References to "VL" or "VL" refer to the variable domain of an antibody light chain, including that of an Fv, scFv, dsFv or Fab.

[0184] The variable regions of light and heavy chains contain a "framework" region interrupted by three hypervariable regions, also called "complementarity-determining regions" or "CDRs" (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991). The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, which is the combined framework regions of the constituent light and heavy chains, functions to position and align the CDRs in three-dimensional space.

[0185] CDRs are primarily responsible for binding to the epitope of an antigen. The amino acid sequence boundaries of a given CDR can be easily determined using any of several well-known schemes, including those described in Kabat et al. ("Sequences of Proteins of Immunological Interest", 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991; "Kabat" numbering scheme), Al-Lazikani et al. (JMB 273, 927-948, 1997; "Chothia" numbering scheme), and Lefranc et al. ("IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains", Dev. Comp. Immunol., 27:55-77, 2003; "IMGT" numbering scheme). The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3 (N-terminus to C-terminus) and are also typically identified by the chain in which the particular CDR is located. Thus, a VH CDR3 is the CDR3 from the variable domain of the heavy chain of the antibody in which it is found, while a VL CDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found. The light chain CDRs are sometimes referred to as LCDR1, LCDR2, and LCDR3. The heavy chain CDRs are sometimes referred to as LCDR1, LCDR2, and LCDR3.

[0186] "Antigen-binding fragments" are portions of full-length antibodies that retain the ability to specifically recognize their cognate antigen, as well as various combinations of such portions. Non-limiting examples of antigen-binding fragments include Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. Antibody fragments include antigen-binding fragments produced by engineering whole antibodies or those synthesized de novo using recombinant DNA methodologies (see, e.g., Kontermann and Dubel (eds.), Antibody Engineering, Vols. 1-2, 2nd Edition, Springer Press, 2010).

[0187] Single-chain antibodies (scFvs) are genetically engineered molecules containing the VH and VL domains of one or more antibodies (or antibodies) linked by a suitable polypeptide linker as a genetically fused single-chain molecule (see, e.g., Bird et al., Science, 242:423-426, 1988; Huston et al., Proc. Natl. Acad. Sci., 85:5879-5883, 1988; Ahmad et al., Clin. Dev. Immunol., 2012, doi:10.1155 / 2012 / 980250; Marbry, IDrugs, 13:543-549, 2010). The intramolecular orientation of the VH and VL domains in scFvs is typically not critical for scFvs. Thus, scFvs with both possible configurations (VH domain-linker domain-VL domain; VL domain-linker domain-VH domain) can be used.

[0188] In dsFv, the heavy and light variable chains are mutated to introduce disulfide bonds to stabilize the association of the chains. Also included are diabodies, which are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain but use a linker that is too short to allow pairing between the two domains on the same chain, thereby pairing the domains with complementary domains on another chain to create two antigen-binding sites (see, for example, Holliger et al., Proc. Natl. Acad. Sci., 90:6444-6448, 1993; Poljak et al., Structure, 2:1121-1123, 1994).

[0189] Antibodies also include genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies) and heteroconjugate antibodies (e.g., bispecific antibodies). See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rd ed., W.H. Freeman & Co., New York, 1997.

[0190] Non-naturally occurring antibodies can be constructed using solid-phase peptide synthesis, can be produced recombinantly, or can be obtained by screening combinatorial libraries consisting of variable heavy and light chains, for example, as described in Huse et al., Science 246:1275-1281 (1989), which is incorporated herein by reference. These and other methods of producing, for example, chimeric, humanized, CDR-grafted, single-chain, and bifunctional antibodies are well known to those of skill in the art (Winter and Harris, Immunol. Today 14:243-246 (1993); Ward et al., Nature 341:544-546 (1989); Harlow and Lane, supra, 1988; Hilyard et al., Protein Engineering: A practical approach (IRL Press 1992); Borrabeck, Antibody Engineering, 2nd Edition (Oxford University Press 1995); each of which is incorporated herein by reference).

[0191] An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competition assay; conversely, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competition assay. Antibody competition assays are known, and exemplary competition assays are provided herein.

[0192] A "humanized" antibody or antigen-binding fragment comprises a human framework region and one or more CDRs derived from a non-human (e.g., mouse, rat, or synthetic) antibody or antigen-binding fragment. The non-human antibody or antigen-binding fragment providing the CDRs is referred to as the "donor," and the human antibody or antigen-binding fragment providing the framework is referred to as the "acceptor." In one embodiment, all CDRs are derived from the donor immunoglobulin in the humanized immunoglobulin. Constant regions need not be present, but if present, can be substantially identical to human immunoglobulin constant regions, e.g., at least about 85-90%, e.g., about 95% or more identical. Thus, all parts of a humanized antibody or antigen-binding fragment, except possibly for the CDRs, are substantially identical to the corresponding parts of a natural human antibody sequence.

[0193] A "chimeric antibody" is an antibody that contains sequences from two different antibodies, typically from different species. In some instances, a chimeric antibody contains one or more CDRs and / or framework regions from one human antibody and CDRs and / or framework regions from another human antibody.

[0194] A "fully human antibody" or "human antibody" is an antibody that contains sequences derived from the human genome but does not contain sequences derived from another species. In some embodiments, a human antibody contains CDRs, framework regions, and (if present) an Fc region derived from the human genome. Human antibodies can be identified and isolated using technology to create sequences based on sequences derived from the human genome, for example, by phage display, or using transgenic animals (see, e.g., Barbas et al., Phage display: A Laboratory Manuel. 1st ed. New York: Cold Spring Harbor Laboratory Press, 2004 Print.; Lonberg, Nat. Biotech., 23:1117-1125, 2005; Lonenberg, Curr. Opin. Immunol., 20:450-459, 2008).

[0195] An antibody can have one or more binding sites. If more than one binding site is present, these binding sites may be identical to one another or different. For example, naturally occurring immunoglobulins have two identical binding sites, single-chain antibodies or Fab fragments have one binding site, while bispecific or bifunctional antibodies have two different binding sites.

[0196] Methods for testing antibodies for the ability to bind to any functional portion of a CAR are known in the art and include any antibody-antigen binding assay, such as radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation, and competitive inhibition assays (see, e.g., Janeway et al., infra, U.S. Patent Application Publication No. 2002 / 0197266 A1, and U.S. Patent No. 7,338,929).

[0197] CARs, T cells expressing CARs, antibodies or antigen-binding portions thereof can also be modified to include detectable labels, such as radioisotopes, fluorophores (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), enzymes (e.g., alkaline phosphatase, horseradish peroxidase), and elemental particles (e.g., gold particles).

[0198] C. Conjugate CARs, T cells expressing CARs, or monoclonal antibodies or antigen-binding fragments thereof specific for one or more of the antigens disclosed herein can be conjugated to agents such as effector molecules or detectable markers using several means known to those skilled in the art. Both covalent and non-covalent binding means can be used. Conjugates include, but are not limited to, molecules in which an effector molecule or detectable marker is covalently linked to an antibody or antigen-binding fragment that specifically binds to one or more of the antigens disclosed herein. Those skilled in the art will recognize that the conjugation of an effector molecule or detectable marker to an antibody or antigen-binding fragment that specifically binds to one or more of the antigens disclosed herein can be used to conjugate an effector molecule or detectable marker to an antibody or antigen-binding fragment that specifically binds to one or more of the antigens disclosed herein. 125 I, 32 P, 14 C. 3 H and 35 It will be understood that a variety of effector molecules and detectable markers may be used, including, but not limited to, S, as well as other labels, targeting moieties, ligands, and the like.

[0199] The choice of a particular effector molecule or detectable marker will depend on the particular target molecule or cell and the desired biological effect. Thus, for example, the effector molecule can be a cytotoxin used to bring about the death of a particular target cell (e.g., a tumor cell).

[0200] The procedure for attaching an effector molecule or detectable marker to an antibody or antigen-binding fragment varies depending on the chemical structure of the effector. Polypeptides typically contain various functional groups, such as carboxylic acid (COOH), free amine (-NH), or sulfhydryl (-SH) groups, which are available for reaction with appropriate functional groups on an antibody to attach an effector molecule or detectable marker. Alternatively, the antibody or antigen-binding fragment can be derivatized to expose or attach additional reactive functional groups. Derivatization can include attachment of any of several known linker molecules, such as those available from Pierce Chemical Company, Rockford, IL. The linker can be any molecule used to attach an antibody or antigen-binding fragment to an effector molecule or detectable marker. The linker can form covalent bonds to both the antibody or antigen-binding fragment and the effector molecule or detectable marker. Suitable linkers are well known to those skilled in the art and include, but are not limited to, straight-chain or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. When the antibody or antigen-binding fragment and the effector molecule or detectable marker are polypeptides, the linkers can be attached to the constituent amino acids through their side groups (e.g., via a disulfide linkage to cysteine) or to the amino and carboxyl groups of the alpha carbon of the terminal amino acid.

[0201] In some embodiments, the linker may include a spacer element, which, if present, increases the size of the linker, thereby increasing the distance between the effector molecule or detectable marker and the antibody or antigen-binding fragment. Exemplary spacers are known to those of skill in the art, including those described in U.S. Patent Nos. 7,964,566, 7,498,298, 6,884,869, 6,323,315, 6,239,104, 6,034,065, 5,780,588, 5,665,860, 5,663,149, 5,635,483, 5,599,902, 5,554,725, 5,530,097, 5,521 Nos. 5,284, 5,504,191, 5,410,024, 5,138,036, 5,076,973, 4,986,988, 4,978,744, 4,879,278, 4,816,444 and 4,486,414, as well as those listed in U.S. Patent Application Publication Nos. 20110212088 and 20110070248, each of which is incorporated herein by reference in its entirety.

[0202] In some embodiments, the linker is cleavable under intracellular conditions, such that cleavage of the linker releases the effector molecule or detectable marker from the antibody or antigen-binding fragment in the intracellular environment. In yet other embodiments, the linker is non-cleavable, and the effector molecule or detectable marker is released, for example, by antibody degradation. In some embodiments, the linker is cleavable by a cleaving agent present in the intracellular environment (e.g., within a lysosome, endosome, or caveolae). The linker can be, for example, a peptide linker that is cleaved by an intracellular peptidase or protease enzyme, including, but not limited to, a lysosomal or endosomal protease. In some embodiments, the peptide linker is at least two amino acids long or at least three amino acids long. However, the linker can also be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids long, e.g., 1-2, 1-3, 2-5, 3-10, 3-15, 1-5, 1-10, or 1-15 amino acids long. Proteases can include cathepsins B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives, resulting in the release of active drugs inside target cells (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). For example, a peptide linker cleavable by the thiol-dependent protease cathepsin B can be used (e.g., a phenylalanine-leucine or glycine-phenylalanine-leucine-glycine linker). Other examples of such linkers are described, for example, in U.S. Pat. No. 6,214,345, which is incorporated herein by reference. In a specific embodiment, the peptide linker cleavable by intracellular proteases is a valine-citrulline linker or a phenylalanine-lysine linker (see, e.g., U.S. Pat. No. 6,214,345, which describes the synthesis of doxorubicin using a valine-citrulline linker).

[0203] In other embodiments, the cleavable linker is pH-sensitive, i.e., sensitive to hydrolysis at a certain pH value. Typically, pH-sensitive linker is hydrolyzable under acidic conditions. For example, acid-labile linkers (e.g., hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal, etc.) that are hydrolyzable in lysosomes can be used (see, for example, U.S. Patent No. 5,122,368; U.S. Patent No. 5,824,805; U.S. Patent No. 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661). Such linkers are relatively stable under neutral pH conditions, such as in blood, but are unstable at pHs below 5.5 or 5.0, which is approximately the pH of the lysosome. In certain embodiments, the hydrolyzable linker is a thioether linker (e.g., a thioether attached to a therapeutic agent via an acylhydrazone bond) (see, e.g., U.S. Pat. No. 5,622,929).

[0204] In other embodiments, the linker is cleavable under reducing conditions (eg, a disulfide linker). A variety of disulfide linkers are known in the art, including, for example, those that can be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene)-, SPDB, and SMPT (see, e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (C.W. Vogel, ed., Oxford University Press, 1987); Phillips et al., Cancer Res. 68:9280-9290, 2008). See also U.S. Patent No. 4,880,935.

[0205] In yet other specific embodiments, the linker is a malonate linker (Johnson et al., 1995, Anticancer Res. 15:1387-93), a maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1299-1304), or a 3'-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1305-12).

[0206] In still other embodiments, the linker is non-cleavable and the effector molecule or detectable marker is released by antibody degradation (see U.S. Patent Application Publication No. 2005 / 0238649, the entire contents of which are incorporated herein by reference).

[0207] In some embodiments, the linker is resistant to cleavage in an extracellular environment. For example, when the conjugate is present in an extracellular environment (e.g., in plasma), about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 3% or less, or about 1% or less of the linkers in the sample of the conjugate are cleaved. Whether a linker is resistant to cleavage in an extracellular environment can be determined, for example, by incubating the conjugate containing the desired linker with plasma for a predetermined period (e.g., 2, 4, 8, 16, or 24 hours), and then quantifying the amount of free effector molecule or detectable marker present in the plasma. Various exemplary linkers that can be used in the conjugates are described in WO2004-010957, U.S. Patent Application Publication No. 2006 / 0074008, U.S. Patent Application Publication No. 20050238649, and U.S. Patent Application Publication No. 2006 / 0024317, each of which is incorporated herein by reference in its entirety.

[0208] In some embodiments, conjugates of a CAR, a T cell expressing a CAR, an antibody or antigen-binding portion thereof, and one or more small molecule toxins, such as calicheamicin, maytansinoids, dolastatins, auristatins, trichothecines, and CC1065, and derivatives of these toxins that have toxin activity, are provided.

[0209] Maytansine compounds suitable for use as maytansinoid toxin moieties are well known in the art and can be isolated from natural sources according to known methods, produced using genetic engineering techniques (see Yu et al. (2002) PNAS 99:7968-7973), or synthetically prepared maytansinol and maytansinol analogs according to known methods. Maytansinoids are mitotic inhibitors that act by inhibiting tubulin polymerization. Maytansine was first isolated from the East African shrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it was discovered that certain microorganisms also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042). Synthetic maytansinol and its derivatives and analogs are described, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428; and 4,311,140. Nos. 3,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533, each of which is incorporated herein by reference. Maytansinoid-containing conjugates, methods for making same, and their therapeutic uses are disclosed, for example, in U.S. Patent Nos. 5,208,020; 5,416,064; 6,441,163 and European Patent EP 0 425 235 B1, the disclosures of which are expressly incorporated herein by reference.

[0210] Additional toxins can be used with CARs, CAR-expressing T cells, antibodies, or antigen-binding portions thereof. Exemplary toxins include Pseudomonas exotoxin (PE), ricinus toxin, abrin, diphtheria toxin and its subunits, ribotoxin, ribonuclease, saporin and calicheamicin, and botulinum toxins A-F. These toxins are well known in the art, and many are readily available from commercial sources (e.g., Sigma Chemical Company, St. Louis, MO). Contemplated toxins also include variants of these toxins (see, e.g., U.S. Patent Nos. 5,079,163 and 4,689,401).

[0211] Saporin is a toxin derived from Saponaria officinalis that disrupts protein synthesis by inactivating the 60S portion of the ribosomal complex (Stirpe et al., Bio / Technology, 10:405-412, 1992). However, this toxin does not have a mechanism for specific entry into cells and therefore requires conjugation to an antibody or antigen-binding fragment that recognizes an internalized cell surface protein in order to be efficiently taken up by cells.

[0212] Diphtheria toxin is isolated from Corynebacterium diphtheriae. Typically, diphtheria toxin for use in immunotoxins is mutated to reduce or eliminate nonspecific toxicity. A mutant known as CRM107, which has full enzymatic activity but significantly reduced nonspecific toxicity, has been known since the 1970s (Laird and Groman, J. Virol. 19:220, 1976) and has been used in human clinical trials. See U.S. Patent Nos. 5,792,458 and 5,208,021.

[0213] Castor toxin is the lectin RCA60 from Ricinus communis (castor bean). For examples of castor toxins, see U.S. Patent Nos. 5,079,163 and 4,689,401. Ricinus communis agglutinin (RCA) is a lectin derived from the plant Ricinus communis (castor bean), according to its molecular weight of approximately 65 kD and 120 kD, respectively. 60 and RCA 120 The toxin exists in two forms, termed the A chain and the B chain (Nicholson and Blaustein, J. Biochim. Biophys. Acta 266:543, 1972). The A chain is responsible for inactivating protein synthesis and killing the cell. The B chain binds the toxin to cell surface galactose residues and facilitates transport of the A chain into the cytosol (Olsnes et al., Nature 249:627-631, 1974 and U.S. Pat. No. 3,060,165).

[0214] Ribonucleases have also been conjugated to targeting molecules for use as immunotoxins (see Suzuki et al., Nat. Biotech. 17:265-70, 1999). Exemplary ribotoxins, such as α-sarcin and restrictocin, are discussed, for example, in Rathore et al., Gene 190:31-5, 1997; and Goyal and Batra, Biochem. 345(2):247-54, 2000. Calicheamicin, originally isolated from Micromonospora echinospora, generates double-strand breaks in DNA that lead to apoptosis and is a member of the enediyne antitumor antibiotic family (see, for example, Lee et al., J. Antibiot. 42:1070-87, 1989). This drug is the toxic moiety of an immunotoxin in clinical trials (see, eg, Gillespie et al., Ann. Oncol. 11:735-41, 2000).

[0215] Abrin includes toxic lectins from Abrus precatorius. The toxicants, abrins a, b, c, and d, have molecular weights of approximately 63 kD and 67 kD and are composed of two disulfide-linked polypeptide chains, A and B. The A chain inhibits protein synthesis; the B chain (abrin-b) binds to D-galactose residues (see Funatsu et al., Agr. Biol. Chem. 52:1095, 1988; and Olsnes, Methods Enzymol. 50:330-335, 1978).

[0216] CARs, CAR-expressing T cells, monoclonal antibodies specific for one or more of the antigens disclosed herein, and antigen-binding fragments thereof can also be conjugated to a detectable marker; for example, a detectable marker detectable by ELISA, spectrophotometry, flow cytometry, microscopy, or imaging techniques (e.g., computed tomography (CT), computed axial tomography (CAT) scan, magnetic resonance imaging (MRI), nuclear magnetic resonance imaging (NMRI), magnetic resonance tomography (MTR), ultrasound, fiber optic testing, and laparoscopic testing). Specific non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzymatic linkages, radioactive isotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI). For example, useful detectable markers include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, and the like. Bioluminescent markers, such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP), are also used. CARs, CAR-expressing T cells, antibodies, or antigen-binding portions thereof, can also be conjugated to enzymes useful for detection, such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, glucose oxidase, and the like. When a CAR, CAR-expressing T cells, antibodies, or antigen-binding portions thereof, is conjugated to a detectable enzyme, it can be detected by adding an additional reagent that the enzyme uses to produce a discernible reaction product. For example, when the agent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine results in a visually detectable colored reaction product. CARs, CAR-expressing T cells, antibodies, or antigen-binding portions thereof, can also be conjugated with biotin and detected via indirect measurement of avidin or streptavidin binding. Note that avidin itself can be conjugated with an enzyme or fluorescent label.

[0217] CAR, CAR-expressing T cells, antibodies, or their antigen-binding portions can be conjugated with paramagnetic agents such as gadolinium. Paramagnetic agents such as superparamagnetic iron oxide can also be used as labels. Antibodies can also be conjugated with lanthanides (e.g., europium and dysprosium) and manganese. Antibodies or antigen-binding fragments can also be labeled with a predetermined polypeptide epitope recognized by a secondary reporter (e.g., leucine zipper pair sequence, binding site for secondary antibody, metal binding domain, epitope tag).

[0218] CAR, CAR-expressing T cells, antibodies, or antigen-binding portions thereof can also be conjugated with radiolabeled amino acids.Radiolabels can be used for both diagnostic and therapeutic purposes.For example, radiolabels can be used to detect one or more of the antigens and antigen-expressing cells disclosed herein by x-ray, emission spectroscopy, or other diagnostic techniques.In addition, radiolabels can be used therapeutically as toxins for treating tumors in subjects, for example, for treating neuroblastoma.Examples of labels for polypeptides include, but are not limited to, the following radioisotopes or radionucleotides: 3 H, 14 C. 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I.

[0219] Means for detecting such detectable markers are well known to those skilled in the art. Thus, for example, radiolabels can be detected using photographic film or scintillation counters, fluorescent markers can be detected using a photodetector to detect emitted illumination, enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and chromogenic labels are detected by simply visualizing the colored label.

[0220] D. Nucleotides, Expression, Vectors and Host Cells Further provided by one embodiment of the present invention is a nucleic acid comprising a nucleotide sequence encoding any of the CARs, antibodies, or antigen-binding portions thereof described herein (including functional portions and functional variants thereof). The nucleic acids of the present invention may comprise a nucleotide sequence encoding any of the leader sequences, antigen-binding domains, transmembrane domains, and / or intracellular T cell signaling domains described herein.

[0221] In some embodiments, nucleotide sequence can be codon modified.Without being bound by any particular theory, it is believed that codon optimization of nucleotide sequence can increase the translation efficiency of mRNA transcript.The codon optimization of nucleotide sequence can include replacing native codon with another codon that encodes the same amino acid but can be translated by tRNA that is more readily available in cells, thus increasing translation efficiency.The optimization of nucleotide sequence can also reduce the secondary mRNA structure that interferes with translation, thus increasing translation efficiency.

[0222] In embodiments of the invention, a nucleic acid can comprise a codon-modified nucleotide sequence encoding the antigen-binding domain of a CAR of the invention. In another embodiment of the invention, a nucleic acid can comprise a codon-modified nucleotide sequence encoding any of the CARs described herein (including functional portions and functional variants thereof).

[0223] "Nucleic acid," as used herein, includes "polynucleotides," "oligonucleotides," and "nucleic acid molecules," and generally refers to a polymer of DNA or RNA that can be single- or double-stranded, synthetic or obtained from natural sources (e.g., isolated and / or purified), and can contain natural, non-natural, or modified nucleotides, and can contain natural, non-natural, or modified internucleotide linkages, e.g., phosphoramidate or phosphorothioate linkages, in place of the phosphodiesters found between nucleotides in unmodified oligonucleotides. In some embodiments, a nucleic acid does not contain any insertions, deletions, inversions, and / or substitutions. However, in some cases, as discussed herein, it may be appropriate for a nucleic acid to contain one or more insertions, deletions, inversions, and / or substitutions.

[0224] Recombinant nucleic acids can have sequences that do not occur in nature or that are created by the artificial combination of two otherwise separate segments of sequence. This artificial combination is often achieved by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acid, for example, by genetic engineering techniques such as those described in Sambrook et al., supra. Nucleic acids can be constructed based on chemical synthesis and / or enzymatic ligation reactions using procedures known in the art. See, for example, Sambrook et al., supra and Ausubel et al., supra. For example, nucleic acids can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides (e.g., phosphorothioate derivatives and acridine-substituted nucleotides) designed to increase the biological stability of the molecule or to increase the physical stability of the duplex formed upon hybridization.Examples of modified nucleotides that can be used to generate nucleic acids include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-substituted adenines, 7-methylguanine, and the like. Examples of nucleic acids include, but are not limited to, uracil-5-oxyacetic acid (v), ubutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleic acids of the present invention can be purchased from a company such as Integrated DNA Technologies (Coralville, IA, USA).

[0225] The nucleic acid can comprise any isolated or purified nucleotide sequence encoding a CAR or any of its functional portions or variants. Alternatively, the nucleotide sequence can comprise a nucleotide sequence that is degenerate to any of the sequences, or a combination of degenerate sequences.

[0226] One embodiment also provides an isolated or purified nucleic acid comprising a nucleotide sequence that is complementary to or that hybridizes under stringent conditions to the nucleotide sequence of any of the nucleic acids described herein.

[0227] Nucleotide sequences that hybridize under stringent conditions may also hybridize under high stringency conditions. "High stringency conditions" means that a nucleotide sequence specifically hybridizes to a target sequence (any nucleotide sequence of a nucleic acid described herein) in an amount detectably stronger than nonspecific hybridization. High stringency conditions include conditions that distinguish polynucleotides with exact complementary sequences, or polynucleotides containing only a few scattered mismatches, from random sequences that happen to have several small regions (e.g., 3-10 bases) that match the nucleotide sequence. Such small regions of complementarity are more easily melted than full-length complements of 14-17 or more bases, and high stringency hybridization makes them readily distinguishable. Relatively high stringency conditions include low salt and / or high temperature conditions, such as those provided by about 0.02-0.1 M NaCl or equivalent at temperatures of about 50-70°C. Such highly stringent conditions tolerate little, if any, mismatch between the nucleotide sequence and the template or target strand and are particularly suitable for detecting expression of any of the CARs of the present invention. It is generally understood that conditions can be made more stringent by the addition of increasing amounts of formamide.

[0228] Also provided are nucleic acids comprising a nucleotide sequence that is at least about 70% or more, e.g., about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to any of the nucleic acids described herein.

[0229] In one embodiment, the nucleic acid may be incorporated into a recombinant expression vector. In this regard, one embodiment provides a recombinant expression vector comprising any of the nucleic acids. For purposes of this specification, the term "recombinant expression vector" refers to a genetically engineered oligonucleotide or polynucleotide construct that allows a host cell to express an mRNA, protein, polypeptide, or peptide when the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide, and when the vector is contacted with a cell under conditions sufficient for the mRNA, protein, polypeptide, or peptide to be expressed in the cell. The vector as a whole does not exist in nature.

[0230] However, portions of the vector may be naturally occurring. Recombinant expression vectors may be single-stranded or double-stranded, synthetic or derived from partially natural sources, and may contain any type of nucleotide, including, but not limited to, DNA and RNA, which may contain natural, non-natural, or modified nucleotides. Recombinant expression vectors may contain naturally occurring or non-naturally occurring internucleotide linkages, or both types of linkages. Preferably, the non-naturally occurring or modified nucleotides or internucleotide linkages do not interfere with the transcription or replication of the vector.

[0231] In one embodiment, the recombinant expression vector may be any suitable recombinant expression vector and may be used to transform or transfect any suitable host cell. Suitable vectors include those designed for propagation and propagation or for expression, or both, such as plasmids and viruses. The vector may be selected from the group consisting of the pUC series (Fermentas Life Sciences, Glen Burnie, MD), the pBluescript series (Stratagene, LaJolla, CA), the pET series (Novagen, Madison, WI), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, CA).

[0232] Bacteriophage vectors, such as λυTΙO, λυTΙ 1, λZapII (Stratagene), EMBL4, and λΝΜΙ 149, can also be used. Examples of plant expression vectors include pBI01, pBI101.2, pBHO1.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). The recombinant expression vector can be a viral vector, such as a retroviral or lentiviral vector. Lentiviral vectors are vectors derived from at least a portion of the lentiviral genome, including, in particular, self-inactivating lentiviral vectors such as those provided in Milone et al., Mol. Ther. 17(8):1453-1464 (2009). Other examples of lentiviral vectors that may be used in the clinic include, for example, but not limited to, Oxford BioMedica plc's LENTIVECTOR® gene delivery technology, Lentigen's LENTIMAX™ vector system, etc. Non-clinical forms of lentiviral vectors are also available and known to those skilled in the art.

[0233] Several transfection techniques are generally known in the art (see, e.g., Graham et al., Virology 52:456-467 (1973); Sambrook et al., supra; Davis et al., Basic Methods in Molecular Biology, Elsevier (1986); and Chu et al., Gene 13:97 (1981)).

[0234] Transfection methods include calcium phosphate coprecipitation (see, e.g., Graham et al., supra), direct microinjection into cultured cells (see, e.g., Capecchi, Cell 22:479-488 (1980)), electroporation (see, e.g., Shigekawa et al., BioTechniques 6:742-751 (1988)), liposome-mediated gene transfer (see, e.g., Mannino et al., BioTechniques 6:682-690 (1988)), lipid-mediated transduction (see, e.g., Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987)), and high velocity micropropellants. Microprojectile delivery (see, eg, Klein et al., Nature, 327:70-73 (1987)) is also included.

[0235] In one embodiment, recombinant expression vectors can be prepared using standard recombinant DNA techniques, for example, as described in Sambrook et al., supra, and Ausubel et al., supra. Circular or linear expression vector constructs can be prepared to contain a replication system that is functional in prokaryotic or eukaryotic host cells. Replication systems can be derived from, for example, ColEl, 2μ plasmid, λ, SV40, bovine papilloma virus, etc.

[0236] Recombinant expression vectors, taking into account whether the vector is DNA- or RNA-based, can optionally include regulatory sequences, e.g., transcriptional and translational initiation and termination codons, specific to the type of host cell (e.g., bacterial, fungal, plant, or animal) into which the vector will be introduced. Recombinant expression vectors can include restriction sites to facilitate cloning.

[0237] The recombinant expression vector may contain one or more marker genes to allow for the selection of transformed or transfected host cells. Marker genes include biocide resistance, resistance to, e.g., antibiotics, heavy metals, etc., complementation in auxotrophic hosts to provide prototrophy, etc. Suitable marker genes for the expression vectors of the present invention include, for example, the neomycin / G418 resistance gene, the hygromycin resistance gene, the histidinol resistance gene, the tetracycline resistance gene, and the ampicillin resistance gene.

[0238] The recombinant expression vector may comprise a native or non-native promoter operably linked to the nucleotide sequence encoding the CAR (including its functional portion and functional variant), or to a nucleotide sequence complementary to or hybridizing with the nucleotide sequence encoding the CAR. The selection of a promoter, for example, strong, weak, inducible, tissue-specific, and developmentally specific, is within the skill of those skilled in the art. Similarly, combining a nucleotide sequence with a promoter is also within the skill of those skilled in the art. The promoter may be a non-viral promoter or a viral promoter, such as a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, or a promoter found in the long terminal repeat of murine stem cell virus.

[0239] Recombinant expression vectors can be designed for transient expression, stable expression, or both, and can be made for constitutive or inducible expression.

[0240] Furthermore, recombinant expression vectors can be made to contain suicide genes. As used herein, the term "suicide gene" refers to a gene that causes cells that express the suicide gene to die. A suicide gene can be a gene that confers sensitivity to a drug or other agent to the cell in which the gene is expressed, or a gene that causes the cell to die when contacted with or exposed to a drug. Suicide genes are known in the art (see, for example, Suicide Gene Therapy: Methods and Reviews, Springer, Caroline J. (Cancer Research UK Centre for Cancer Therapeutics at the Institute of Cancer Research, Sutton, Surrey, UK), Humana Press, 2004), and include, for example, herpes simplex virus (HSV) thymidine kinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase, and nitroreductase.

[0241] One embodiment further provides a host cell containing any of the recombinant expression vectors described herein. As used herein, the term "host cell" refers to any type of cell that can contain a recombinant expression vector of the present invention. The host cell can be a eukaryotic cell, such as a plant, animal, fungus, or algae, or a prokaryotic cell, such as a bacterium or protist. The host cell can be a cultured cell or a primary cell, i.e., directly isolated from an organism such as a human. The host cell can be an adherent cell or a suspension cell, i.e., a cell that grows in suspension. Suitable host cells are known in the art and include, for example, DH5a E. coli cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, etc. For the purpose of amplifying or replicating a recombinant expression vector, the host cell can be a prokaryotic cell, such as a DH5a cell. For the purpose of producing a recombinant CAR, the host cell can be a mammalian cell. The host cell can be a human cell. The host cell can be of any cell type, originate from any type of tissue, and be at any stage of development, but the host cell can be a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC). The host cell can be a T cell.

[0242] For purposes herein, T cells can be any T cells, e.g., cultured T cells, e.g., primary T cells, or T cells from cultured T cell lines, e.g., Jurkat, SupTl, etc., or T cells obtained from a mammal. When obtained from a mammal, T cells can be obtained from a number of sources, including, but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. T cells can also be enriched or purified. T cells can be human T cells. T cells can be T cells isolated from a human. T cells can be any type of T cell and at any developmental stage, including, but not limited to, CD4+ / CD8+ double-positive T cells, CD4+ helper T cells, e.g., Th1 and Th2 cells, CD8+ T cells (e.g., cytotoxic T cells), tumor-infiltrating cells, memory T cells, memory stem cells, i.e., Tscm naive T cells, etc. T cells can be CD8+ T cells or CD4+ T cells.

[0243] In one embodiment, the CARs described herein can be used in suitable non-T cells, such as cells with immune effector function, such as NK cells and T-like cells generated from pluripotent stem cells.

[0244] Also provided by one embodiment is a population of cells comprising at least one host cell described herein. The population of cells can be a heterogeneous population comprising host cells comprising any of the described recombinant expression vectors in addition to at least one other cell, e.g., a host cell (e.g., a T cell), that does not comprise any recombinant expression vector, or a cell other than a T cell, e.g., a B cell, macrophage, neutrophil, erythrocyte, hepatocyte, endothelial cell, epithelial cell, muscle cell, brain cell, etc. Alternatively, the population of cells can be a substantially homogeneous population, wherein the population primarily comprises host cells comprising (e.g., consisting essentially of) the recombinant expression vector. The population can also be a clonal population of cells, wherein all cells in the population are clones of a single host cell comprising the recombinant expression vector, such that all cells in the population comprise that recombinant expression vector. In one embodiment of the present invention, the population of cells is a clonal population comprising host cells comprising the recombinant expression vector described herein.

[0245] CARs (including functional portions and variants thereof), nucleic acids, recombinant expression vectors, host cells (including populations thereof), and antibodies (including antigen-binding portions thereof) can be isolated and / or purified. For example, a purified (or isolated) host cell preparation is one in which the host cells are more pure than the cells in their natural environment in the body. Such host cells can be produced, for example, by standard purification techniques. In some embodiments, a host cell preparation is purified such that the host cells represent at least about 50%, e.g., at least about 70%, of the total cell content of the preparation. For example, the purity can be at least about 50%, greater than about 60%, about 70%, or about 80%, or can be about 100%.

[0246] E. Treatment Method It is contemplated that the CARs disclosed herein can be used in methods for treating or preventing disease in mammals. In this regard, one embodiment provides a method for treating or preventing cancer in a mammal, comprising administering to the mammal a CAR, a nucleic acid, a recombinant expression vector, a host cell, a population of cells, an antibody and / or an antigen-binding portion thereof, and / or a pharmaceutical composition in an amount effective to treat or prevent cancer in the mammal.

[0247] One embodiment further comprises lymphodepleting the mammal prior to administering a CAR disclosed herein. Examples of lymphodepletion can include, but are not limited to, non-myeloablative lymphodepleting chemotherapy, myeloablative lymphodepleting chemotherapy, total body irradiation, etc.

[0248] For the purpose of the method of administering host cells or a group of cells, the cells can be allogeneic or autologous to the mammal.Preferably, the cells are autologous to the mammal.As used herein, allogeneic refers to any material derived from a different animal of the same species as the individual into which the material is introduced.Two or more individuals are said to be allogeneic to each other if the genes are not identical at one or more loci.In some embodiments, allogeneic materials from individuals of the same species can be genetically sufficiently different to interact antigenically.As used herein, "autologous" refers to any material derived from the same individual that is subsequently reintroduced into the individual.

[0249] The mammal referred to herein may be any mammal. As used herein, the term "mammal" refers to any mammal, including, but not limited to, rodent mammals, e.g., mice and hamsters, and logomorph mammals, e.g., rabbits. The mammal may be from the carnivora order, including felines (cats) and canines (dogs). The mammal may be from the artiodactyla order, including bovines (cows) and swines (pigs), or from the perissodactyla order, including equines (horses). The mammal may be from the primate order, ceboids or simoids (monkeys), or anthropoids (humans and apes). Preferably, the mammal is a human.

[0250] For these methods, the cancer may be acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer (e.g., bladder carcinoma), bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, cancer of the anus, anal canal, or anorectum, eye cancer, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid carcinoma The cancer may be any cancer, including any of the following: colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, head and neck cancer (e.g., head and neck squamous cell carcinoma), Hodgkin's lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, leukemia, liquid tumors, liver cancer, lung cancer (e.g., non-small cell lung cancer and lung adenocarcinoma), lymphoma, mesothelioma, mast cell tumor, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin's lymphoma, B-chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia (ALL) and Burkitt's lymphoma, ovarian cancer, pancreatic cancer, peritoneal, omental and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumor, synovial sarcoma, gastric cancer, testicular cancer, thyroid cancer, and ureteral cancer.

[0251] The terms "treat" and "prevent," and words derived therefrom, as used herein, do not necessarily mean 100% or complete treatment or prevention. Rather, there are various degrees of treatment or prevention that one of skill in the art would recognize as having a potential beneficial or therapeutic effect. In this regard, the method may provide any amount or level of treatment or prevention of cancer in a mammal.

[0252] Furthermore, the treatment or prevention provided by this method can include treatment or prevention of one or more conditions or symptoms of the disease, such as cancer, being treated or prevented. Also, for purposes herein, "prevention" can include delaying the onset of the disease, or its symptoms or conditions.

[0253] Another embodiment provides a method of detecting the presence of cancer in a mammal, the method comprising: (a) contacting a sample comprising one or more cells from the mammal with a CAR, a nucleic acid, a recombinant expression vector, a host cell, a population of cells, an antibody and / or antigen-binding portion thereof, or a pharmaceutical composition, thereby forming a complex; and (b) detecting the complex, wherein detection of the complex indicates the presence of cancer in the mammal.

[0254] The sample can be obtained by any suitable method, for example, biopsy or autopsy.Biopsy is the removal of tissue and / or cells from an individual.Such removal can be the collection of tissue and / or cells from an individual in order to perform experiments on the removed tissue and / or cells.This experiment can include experiments to determine whether the individual has a certain condition or disease state and / or whether they are suffering from a certain condition or disease state.The condition or disease can be, for example, cancer.

[0255] For embodiments of methods for detecting the presence of a proliferation disorder, e.g., cancer, in a mammal, the sample containing mammalian cells can be a sample containing whole cells, a lysate thereof, or a fraction of a whole cell lysate, e.g., a nuclear or cytoplasmic fraction, a whole protein fraction, or a nucleic acid fraction. When the sample contains whole cells, these cells can be any cells of a mammal, e.g., cells of any organ or tissue, including blood cells or endothelial cells.

[0256] The contacting can occur in vitro or in vivo with respect to a mammal. Preferably, the contacting is in vitro.

[0257] Also, detection of complexes can be carried out by many methods known in the art.For example, the CAR disclosed herein, polypeptide, protein, nucleic acid, recombinant expression vector, host cell, cell population, or antibody or its antigen-binding portion can be labeled with detectable label, such as the radioisotope disclosed above, fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), enzyme (e.g., alkaline phosphatase, horseradish peroxidase) and element particle (e.g., gold particle), etc.

[0258] The method of testing CAR for its ability to recognize target cells and antigen specificity is known in the art.For example, Clay et al., J. Immunol, vol. 163: 507-513 (1999) teaches a method for measuring the release of cytokines (e.g., interferon-γ, granulocyte / monocyte colony stimulating factor (GM-CSF), tumor necrosis factor α (TNF-α) or interleukin 2 (IL-2)).In addition, CAR function can be evaluated by measuring cytotoxicity, as described in Zhao et al., J. Immunol. vol. 174: 4415-4423 (2005).

[0259] Another embodiment provides the use of the CARs, nucleic acids, recombinant expression vectors, host cells, populations of cells, antibodies or antigen-binding portions thereof, and / or pharmaceutical compositions of the invention to treat or prevent a proliferative disorder, such as cancer, in a mammal. The cancer can be any of the cancers described herein.

[0260] Any administration method, including local and systemic administration, can be used for the disclosed therapeutic agents. For example, topical, oral, intravascular (e.g., intravenous), intramuscular, intraperitoneal, intranasal, intradermal, intrathecal, and subcutaneous administration can be used. The specific administration mode and dosing regimen will be selected by the attending clinician, taking into account the characteristics of the case (e.g., the subject, the disease, the disease state involved, and whether the treatment is preventive). When more than one agent or composition is administered, one or more administration routes can be used; for example, the chemotherapeutic agent can be administered orally, and the antibody or antigen-binding fragment or conjugate or composition can be administered intravenously. Administration methods include injection, in which the CAR, CAR T cell, conjugate, antibody, antigen-binding fragment, or composition is provided in a non-toxic pharmaceutically acceptable carrier, such as water, saline, Ringer's solution, dextrose solution, 5% human serum albumin, solid oil, ethyl oleate, or liposome. In some embodiments, local administration of the disclosed compounds can be used, for example, by applying an antibody or antigen-binding fragment to an area of ​​tissue from which a tumor has been removed or to an area suspected of being prone to tumor development. In some embodiments, sustained intratumoral (or near-tumoral) release of a pharmaceutical preparation containing a therapeutically effective amount of an antibody or antigen-binding fragment can be beneficial. In other examples, the conjugate is applied topically to the cornea as eye drops or intravitreally to the eye.

[0261] The disclosed therapeutic agents can be formulated in unit dosage forms suitable for individual administration of precise dosage amounts. Furthermore, the disclosed therapeutic agents can be administered in a single dose or in a multiple-dose schedule. A multiple-dose schedule is one in which the main course of treatment may involve more than one discrete dose, e.g., 1 to 10 doses, followed by other doses given at subsequent time intervals as needed to maintain or enhance the effect of the composition. Treatment may involve a daily dose or multiple daily doses of the compound(s) over a period of several days to several months or even years. Thus, the dosing regime will also be determined, at least in part, based on the specific needs of the subject being treated and will be dependent on the judgment of the administering practitioner.

[0262] Typical dosages of antibodies or conjugates can range from about 0.01 to about 30 mg / kg, for example, from about 0.1 to about 10 mg / kg.

[0263] In particular examples, the subject is administered a therapeutic composition comprising one or more of the conjugate, antibody, composition, CAR, CAR T cell, or additional agent in a multiple daily dosing schedule, e.g., at least 2 consecutive days, 10 consecutive days, etc., for a period of, e.g., weeks, months, or years. In one example, the subject is administered the conjugate, antibody, composition, or additional agent for a period of at least 30 days, e.g., at least 2 months, at least 4 months, at least 6 months, at least 12 months, at least 24 months, or at least 36 months.

[0264] In some embodiments, the disclosed methods include providing a subject with surgery, radiation therapy, and / or chemotherapy in combination with the disclosed antibodies, antigen-binding fragments, conjugates, CARs, or CAR-expressing T cells (e.g., sequentially, substantially simultaneously, or simultaneously). Such agents and treatment methods and therapeutic dosages are known to those skilled in the art and can be determined by a skilled clinician. Preparation and dosing schedules for additional agents can be used according to manufacturer's instructions or can be as empirically determined by one skilled in the art. Preparation and dosing schedules for such chemotherapy are also described in Chemotherapy Service, (1992) MC Perry (ed.), Williams & Wilkins, Baltimore, Md.

[0265] In some embodiments, the combination therapy may include administering to the subject a therapeutically effective amount of an additional cancer inhibitor.Non-limiting examples of additional therapeutic agents that can be used in the combination therapy include microtubule binding agents, DNA intercalators or crosslinkers, DNA synthesis inhibitors, DNA and RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, gene regulators, and angiogenesis inhibitors.These agents (administered in therapeutically effective amounts) and treatments can be used alone or in combination.For example, any suitable anti-cancer or anti-angiogenic agent can be administered in combination with the CAR, CAR-T cells, antibodies, antigen-binding fragments, or conjugates disclosed herein.The methods and therapeutic dosages of such agents are known to those skilled in the art and can be determined by skilled clinicians.

[0266] Additional chemotherapeutic agents include alkylating agents such as nitrogen mustards (e.g., chlorambucil, chlormethine, cyclophosphamide, ifosfamide, and melphalan), nitrosoureas (e.g., carmustine, fotemustine, lomustine, and streptozocin), platinum compounds (e.g., carboplatin, cisplatin, oxaliplatin, and BBR3464), busulfan, dacarbazine, mechlorethamine, procarbazine, temozolomide, thiotepa, and uramustine; antimetabolites such as folates (e.g., methotrexate, pemetrexed, and raltitrexed), purines (e.g., cladribine, clofarabine, fludarabine, mercaptopurine, and thioguanine), pyrimidines (e.g., capecitabine), cytarabine, fluorouracil, and gemcitabine; plant alkaloids, such as For example, podophyllum (e.g., etoposide and teniposide), taxanes (e.g., docetaxel and paclitaxel), vincas (e.g., vinblastine, vincristine, vindesine, and vinorelbine); cytotoxic / antitumor antibiotics, such as members of the anthracycline family (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, and valrubicin), bleomycin, rifampicin, hydroxyurea, and mitomycin; topoisomerase inhibitors, such as topotecan and irinotecan; monoclonal antibodies, such as alemtuzumab, bevacizumab, cetuximab, gemtuzumab, rituximab, panitumumab, pertuzumab, and trastuzumab; tumor-affinity photosensitizers, such as aminolevulinic acid, methyl aminolevulinate, porfimer sodium, and verteporfin;and other drugs, such as alitretinoin, altretamine, amsacrine, anagrelide, arsenic trioxide, asparaginase, axitinib, bexarotene, bevacizumab, bortezomib, celecoxib, denileukin diftitox, erlotinib, estramustine, gefitinib, hydroxycarbamide, imatinib, lapatinib, pazopanib, pentostatin, masoprocol, mitotane, pegaspargase, tamoxifen, sorafenib, sunitinib, vemurafenib, vandetanib, and tretinoin. The selection and therapeutic dosage of such drugs are known to those skilled in the art and can be determined by a skilled clinician.

[0267] Combination therapy can provide synergistic effects and can be proven to be synergistic, that is, the effect achieved when active ingredients are used together is greater than the sum of the effects that can be obtained from using these compounds separately.Synergistic effects can be achieved when active ingredients are (1) co-formulated and administered or delivered simultaneously as a combined unit dosage formulation; (2) delivered alternately or in parallel as separate formulations; or (3) by some other regimen.When delivered alternately, synergistic effects can be achieved when these compounds are administered or delivered sequentially, for example, by different injections in separate syringes.Generally, during alternation, each active ingredient in an effective dosage is administered continuously, that is, sequentially, whereas in combination therapy, two or more active ingredients in an effective dosage are administered together.

[0268] In one embodiment, an effective amount of an antibody or antigen-binding fragment or conjugate thereof that specifically binds to one or more of the antigens disclosed herein is administered to a subject with a tumor after anti-cancer treatment. After a sufficient amount of time has passed for the administered antibody or antigen-binding fragment or conjugate to form an immune complex with the antigen expressed on each cancer cell, the immune complex is detected. The presence (or absence) of the immune complex indicates the effectiveness of the treatment. For example, an increase in immune complexes compared to a control taken before treatment indicates that the treatment is ineffective, and a decrease in immune complexes compared to a control taken before treatment indicates that the treatment is effective.

[0269] F. Biopharmaceutical Compositions Provided herein are biopharmaceutical or biologic compositions (hereinafter "compositions") for use in gene therapy, immunotherapy, and / or cell therapy, comprising one or more of the disclosed CARs, or CAR-expressing T cells, antibodies, antigen-binding fragments, conjugates, CARs, or T cells expressing CARs that specifically bind to one or more antigens disclosed herein, in a carrier (e.g., a pharmaceutically acceptable carrier). These compositions can be prepared in unit dosage forms for administration to a subject. The amount and timing of administration to achieve a desired outcome is at the discretion of the treating clinician. These compositions can be formulated for systemic (e.g., intravenous) or local (e.g., intratumoral) administration. In one example, the disclosed CARs, or CAR-expressing T cells, antibodies, antigen-binding fragments, conjugates, are formulated for parenteral administration, such as intravenous administration. Compositions comprising the disclosed CARs, or CAR-expressing T cells, conjugates, antibodies, or antigen-binding fragments, are used for, e.g., treatment and detection of tumors, such as, but not limited to, neuroblastoma. In some examples, these compositions are useful for treating or detecting cancer. Compositions comprising the CARs disclosed herein, or T cells, conjugates, antibodies, or antigen-binding fragments expressing the CARs, are also used, for example, to detect pathological angiogenesis.

[0270] Compositions for administration can include a solution of CAR, or CAR-expressing T cells, conjugates, antibodies, or antigen-binding fragments, dissolved in a pharmaceutically acceptable carrier, such as an aqueous carrier. A variety of aqueous carriers, such as buffered saline, can be used. These solutions are sterile and generally free of undesirable substances. The compositions can be sterilized by conventional, well-known sterilization techniques. The compositions can contain pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, adjuvant drugs, etc., such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of CAR, or CAR-expressing T cells, antibodies, or antigen-binding fragments, or conjugates in these formulations can vary widely and is selected primarily based on fluid volume, viscosity, body weight, etc., according to the particular mode of administration selected and the needs of the subject. Actual methods for preparing such dosage forms for use in gene therapy, immunotherapy, and / or cell therapy are known or will become apparent to those skilled in the art.

[0271] A typical composition for intravenous administration contains about 0.01 to about 30 mg / kg of antibody or antigen-binding fragment or conjugate (or a corresponding dose of CAR, or T cells expressing a CAR, or a conjugate comprising the antibody or antigen-binding fragment) per subject per day. Actual methods for preparing administrable compositions will be known or apparent to those of skill in the art and are described in more detail in publications such as Remington's Pharmaceutical Science, 19th Edition, Mack Publishing Company, Easton, PA (1995).

[0272] CARs, or CAR-expressing T cells, antibodies, antigen-binding fragments, or conjugates, can be provided in lyophilized form and rehydrated with sterile water before administration, but they can also be provided in sterile solutions of known concentrations. The CAR, or CAR-expressing T cells, antibodies, or antigen-binding fragments, or conjugates, solution is then added to an infusion bag containing 0.9% sodium chloride, USP, and in some cases administered at a dosage of 0.5 to 15 mg / kg body weight. Considerable experience is available in the field for administering antibody or antigen-binding fragment and conjugate drugs; for example, antibody drugs have been commercially available in the United States since the approval of Rituxan® in 1997. CARs, or CAR-expressing T cells, antibodies, antigen-binding fragments, and their conjugates, can be administered by slow infusion rather than intravenous injection or intravenous bolus. In one example, a higher loading dose is administered with subsequent maintenance doses administered at lower levels. For example, an initial loading dose of 4 mg / kg of the antibody or antigen-binding fragment (or a corresponding dose of a conjugate comprising the antibody or antigen-binding fragment) can be infused over a period of approximately 90 minutes, followed by weekly maintenance doses of 2 mg / kg for 4-8 weeks infused over a period of 30 minutes if the previous dose was well tolerated.

[0273] Controlled-release parenteral formulations can be prepared as implants, oily injections, or granular systems. For a broad review of protein delivery systems, see Banga, AJ, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Technomic Publishing Company, Inc., Lancaster, PA (1995). Granular systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain a therapeutic protein, such as a cytotoxin or drug, as a central core. In microspheres, the therapeutic agent is dispersed throughout the particle. Particles smaller than about 1 μm, microspheres, and microcapsules, are commonly referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Capillaries have a diameter of approximately 5 μm, so only nanoparticles can be administered intravenously. Microparticles are typically approximately 100 μm in diameter and are administered subcutaneously or intramuscularly. See, e.g., Kreuter, J., Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, NY, pp. 219-342 (1994); and Tice and Tabibi, Treatise on Controlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, NY, pp. 315-339 (1992).

[0274] The polymers can be used for ion-controlled release of the CARs disclosed herein, or T cells, antibodies or antigen-binding fragments, or conjugate compositions expressing the CARs. Various degradable and non-degradable polymer matrices used for controlled drug delivery are known in the art (Langer, Accounts Chem. Res. 26:537-542, 1993). For example, the block copolymer polaxamer 407 exists as a viscous but still mobile liquid at low temperatures, but forms a semi-fluid gel at body temperature. It has been shown to be an effective vehicle for the formulation and sustained delivery of recombinant interleukin-2 and urease (Johnston et al., Pharm. Res. 9:425-434, 1992; and Pec et al., J. Parent. Sci. Tech. 44(2):58-65, 1990). Alternatively, hydroxyapatite has been used as a microcarrier for the controlled release of proteins (Ijntema et al., Int. J. Pharm. 112:215-224, 1994). In yet another embodiment, liposomes are used for the controlled release and drug targeting of lipid-encapsulated drugs (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA (1993)). Numerous additional systems for controlled delivery of therapeutic proteins are known (see U.S. Patent Nos. 5,055,303; 5,188,837; 4,235,871; 4,501,728; 4,837,028; 4,957,735; 5,019,369; 5,055,303; 5,514,670; 5,413,797; 5,268,164; 5,004,697; 4,902,505; 5,506,206; 5,271,961; 5,254,342 and 5,534,496).

[0275] G.Kit In one embodiment, also provided is a kit that uses the CAR disclosed herein.For example, a kit for treating tumor in a subject or a kit for producing CAR T cells that express one or more of the CARs disclosed herein.The kit typically includes the disclosed antibody, antigen-binding fragment, conjugate, nucleic acid molecule, CAR or CAR-expressing T cells disclosed herein.More than one of the disclosed antibody, antigen-binding fragment, conjugate, nucleic acid molecule, CAR or CAR-expressing T cells can be included in the kit.

[0276] The kit may include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container may be formed from a variety of materials, such as glass or plastic. The container typically holds a composition comprising one or more of the disclosed antibodies, antigen-binding fragments, conjugates, nucleic acid molecules, CARs, or CAR-expressing T cells. In some embodiments, the container may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle). The label or package insert indicates that the composition is used to treat a specific condition.

[0277] The label or package insert typically further includes instructions for using the disclosed antibody, antigen-binding fragment, conjugate, nucleic acid molecule, CAR, or CAR-expressing T cell, for example, in a method for treating or preventing tumors or in a method for generating CAR T cells. The package insert typically includes instructions customarily included in the commercial packaging of a therapeutic product, including information about indications, usage, dosage, administration, contraindications, and / or warnings regarding the use of the therapeutic product. The instructional material can be written in electronic form (e.g., a computer diskette or compact disc) or visual (e.g., a video file). The kit can also include additional components to facilitate the specific application for which the kit is designed. Thus, for example, the kit can further include a means for detecting the label (e.g., an enzyme substrate for an enzymatic label, a filter set for detecting a fluorescent label, an appropriate secondary label such as a secondary antibody, etc.). The kit can further include buffers and other reagents routinely used for the implementation of a particular method. Such kits and their appropriate contents are well known to those skilled in the art.

[0278] Example The present invention is further illustrated by the following examples, which should not be construed as imposing limitations on the scope of the present invention in any way. On the contrary, it is readily understood that recourse must be had to various other embodiments, modifications, and equivalents thereof, which may occur to those skilled in the art after reading the description herein without departing from the spirit of the present invention and / or the scope of the appended claims.

[0279] Example 1 Development of a CD19 and CD20 dual-targeting CAR T construct Adoptive immunotherapy for cancer using genetically engineered autologous human T cells is currently being evaluated in many centers. One common approach to generating cell populations for adoptive immunotherapy is to isolate T cells from patients by apheresis and transduce them ex vivo with retroviral or lentiviral vectors that integrate into the host genome and express chimeric antigen receptors (CARs), as reviewed in [1]. Chimeric antigen receptors are created by linking functional sequence domains from different subunits of immunologically active proteins. For example, an scFv domain created from the VH and VL domains of an anti-CD19 or anti-CD20 antibody may be linked to a transmembrane sequence derived from CD28 or CD8, which may then be linked to the CD3-zeta chain and intracellular signaling domains derived from CD28 or CD137 [2, 3]. CARs thus confer both the binding domain derived from the scFv and the linked signaling domains in a single transmembrane protein, enabling activation of vector-transduced T cells. Here, this transformed T cell population (CAR-T) can functionally target cells bearing its cognate antigen for destruction by active cytolysis as well as by indirect immune effector mechanisms mobilized by the production of cytokines such as interferon-gamma (IFNγ), interleukin-2 (IL-2), and tumor necrosis factor-alpha (TNFα). Adoptive immunotherapy using chimeric antigen receptor-modified T cells specifically targeting CD19 has proven effective against pre-B ALL in children [4, 5]. The efficacy of CAR-modified T cells in adult hematologic malignancies is more heterogeneous.

[0280] While the University of Pennsylvania's experience with anti-CD19 CAR-T therapy in three CLL patients appears to have demonstrated universally positive responses, the National Cancer Institute's Department of Surgery reported a mix of partial responses, stable disease, and one complete response in a diverse collection of eight patients with adult B-cell malignancies [6, 7]. Thus, anti-CD19 CARs are not universally effective and could benefit from further enhancement of their anti-tumor targeting potential. The Thomas-Tikhonenko laboratory has elegantly described escape mechanisms employed by B-ALL during anti-CD19 CAR-T therapy, including alternative splicing, frameshift mutations, and missense mutations in CD19 [8]. One way to broaden the targeting range of CAR-T products while simultaneously more effectively targeting malignancies is to include two binding domains in a single CAR structure. Malignancies that express tandem CD19 and CD20 include chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), mantle cell lymphoma (MCL), prolymphocytic leukemia (PLL), and splenic lymphoma with hairy lymphocytes (SLVL).[9] A single CAR vector targeting both antigens has the potential to target a broader variety of hematological malignancies and target them more effectively.

[0281] In this example, we describe the development of fully human CAR T cells that simultaneously target Cd19 and CD20 tumor antigens to prevent tumor antigen escape. We also describe the optimal architecture of the CAR T and the selection of the most effective hinge and transmembrane CAR domains.

[0282] material and method cell line The Burkitt lymphoma cell line Raji was purchased from the American Tissue Culture Collection (ATCC, Manassas, VA). The REH and NALM-6 leukemia lines were purchased from the Leibniz Institute DSMZ (Braunschweig, Germany). Cells were cultured in RPMI-1640 medium (Corning, NY) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Hyclone, Logan, UT). Single-cell clones of luciferase- and GFP-expressing cell lines were generated by stably transducing wild-type tumor lines with a lentiviral vector encoding firefly luciferase and GFP linked to the 2A peptide (Lentigen Technology, Gaithersburg, MD), followed by selection of luciferase-positive clones. The human embryonic kidney line 293T was purchased from ATCC and cultured in Dynamis medium (Gibco / Thermo Fisher Scientific, Grand Island, NY) supplemented with 4 mM L-glutamine (Lonza, Morristown, NJ). The luciferase-expressing 293T cell line was generated by stably transducing a wild-type tumor line with a lentiviral vector encoding firefly luciferase (Lentigen Technology, Gaithersburg, MD), followed by selection for luciferase-positive cells.

[0283] Generation of CAR constructs and production of lentiviral vectors Human anti-CD19 and CD20 chimeric antigen receptor (CAR) constructs were designed as tandem or bicistronic CARs. Various single-chain variable fragment (scFv) sequences targeting the extracellular domain of human CD19 were identified in-house, and the scFv targeting CD20 was derived from ofatumumab. For tandem CARs, the anti-CD20 scFv was linked to the anti-CD19 scFv via a G4S linker, followed by a CD8 or CD28 hinge transmembrane domain, a 4-1BB or CD28_4-1BB costimulatory domain, and a CD3-ζ activation domain sequence. The bicistronic CAR consisted of a CD20-targeting monoCAR and a CD19-targeting monoCAR linked with a P2A ribosomal skipping element. The mono-CAR constructs used the CD8 hinge region, CD8 or OX40 transmembrane domains, costimulatory domains derived from human 4-1BB, CD28, ICOS, and OX40 proteins, and the CD3-ζ activation domain. A previously developed tandem CAR20_19 (LTG1497) carrying mouse CD19 and CD20 scFvs was included as a control construct (D. Schneider et al., "A tandem CD19 / CD20 CAR lentiviral vector drives on-target and off-target antigen modulation in leukemia cell lines." Journal for immunotherapy of cancer, Vol. 5, No. 42, 2017). The CAR sequence was cloned into a lentiviral vector (LV) expression cassette under the control of the human EF-1α promoter (Lentigen Technology, Gaithersburg, MD). Lentiviral particles were generated by transient transfection of HEK 293T cells, pelleted by centrifugation, and stored at -80°C until transduction.

[0284] Preparation and transduction of primary T cells Primary T cells from healthy donors were isolated from leukapheresis collections (AllCells, Alameda, CA) or processed buffy coats (Oklahoma Blood Institute, Tulsa, OK) with written consent from the donors. CD4+ and CD8+ human T cells were purified by positive selection using a 1:1 mixture of CD4 and CD8 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's protocol. Purified T cells were activated with CD3 / CD28 MACS® GMP T Cell TransAct Reagent (Miltenyi Biotec) and 1 × 10 6 Activated T cells were cultured at a density of 0.5 × 10 cells / ml in serum-free TexMACS medium supplemented with 30 IU / ml IL-2. Furthermore, activated T cells were transduced with lentiviral vector particles encoding the CAR construct at an MOI of 80 on day 1. On day 3, transduced T cells were washed and 0.5 × 10 6 The cells were resuspended in 0.5 ml of TexMACS medium containing 30 IU / ml IL-2 and continued to grow. Cultures were then replenished with fresh TexMACS medium containing 30 IU / ml IL-2 every 2–3 days and harvested on days 8–10.

[0285] Flow cytometry analysis of CAR surface expression 0.5 million CAR T cells were washed with chilled AutoMACS buffer supplemented with 0.5% bovine serum albumin (Miltenyi Biotec, Bergisch Gladbach, Germany) and stained with CD19-Fc peptide (R&D System, Minneapolis, MN) followed by anti-Fc-AF647 conjugate (Jackson ImmunoResearch, West Grove, PA). 7-aminoactinomycin D staining (7-AAD, BD Biosciences, San Jose, CA) was added to exclude dead cells. CD4 and CD8 populations were separated using a CD4 antibody labeled with Vioblue fluorescent dye. Untransduced cells (UTD) were used as a negative control. Cells were washed twice, resuspended in 200 μl of running buffer, and acquired by flow cytometry. Flow cytometry analysis was performed on a MACSQuant® 10 Analyzer (Miltenyi Biotec) and data plots were generated using FlowJo software (Ashland, OR).

[0286] CAR T cell cytotoxicity and cytokine assays To assess CAR T cell-mediated cytotoxicity, 5 x 10 cells stably transduced with firefly luciferase were used. 3 Tumor target cells were combined with CAR T cells at the indicated effector-to-target ratios and incubated overnight at 37°C with 5% CO2. SteadyGlo reagent (Promega, Madison WI) was added to each well, and the resulting luminescence was quantified as counts per second (sample CPS). Target-only wells (maximum CPS) and target-only wells with 1% Tween-20 (minimum CPS) were used to determine the assay range. Percent specific lysis was calculated as (1 - (sample CPS - minimum CPS) / (maximum CPS - minimum CPS)). For cytokine release analysis, 5 x 10 4 Effector and 5x10 3Targets were co-cultured overnight, and supernatants were removed from the co-cultures and analyzed for IFNγ, TNFα, and IL-2 concentrations by ELISA (eBioscience, San Diego, CA). Three technical replicates were performed for each condition, and each experiment was repeated using CAR T cells generated from a different healthy donor.

[0287] Long-term CAR T and tumor cell co-culture assay CD19 and CD20 dual-targeting CAR T cells or non-transduced control T cells (1 × 10 6 ) to 1×10 6 The cells were co-cultured with Raji lymphoma cells at a target-to-effector ratio of 0.3:1 for 7 days, with fresh medium added as needed. On days 4 and 7, the cell mixtures were analyzed by flow cytometry. CountBright™ absolute counting beads (Thermofisher, Eugene, OR) were included in the cell mixtures to obtain absolute cell counts. After excluding dead cells by 7-AAD staining, live T (CD3 + ) Counting number and tumor cell (GFP + ) was recorded. On day 7, when the % Raji cells in most cell mixtures was less than 10%, fresh Raji cells were added to the co-culture mixture to readjust the E:T ratio to 0.15:1. On day 10, the culture products were re-evaluated by flow cytometry to assess the cytotoxicity of each CAR T cell.

[0288] result Example 1 describes the generation and in vitro evaluation of CAR T cells targeting both CD20 and CD19 antigens for the treatment of B-cell malignancies.

[0289] A schematic diagram of a bispecific CAR construct targeting both CD19 and CD20 antigens is shown in Figure 1A. The fully human binder for CAR19 was developed in-house, and the fully human scFv targeting CD20 is derived from ofatumumab. Tandem CARs were designed so that a CD20 scFv was linked to a CD19 scFv via a G4S linker, followed in frame by a CD8 or CD28 hinge and transmembrane domain, a 4-1BB or CD28_4-1BB costimulatory domain, and a CD3ζ activation domain. Duo CAR constructs were composed of a mono-CD20 CAR and a mono-CD19 CAR separated by a P2A sequence. The mono-CAR consisted of a CD19 or CD20 scFv, a CD8 hinge, a transmembrane domain derived from CD8 or OX40, a costimulatory domain derived from 4-1BB, CD28, OX40, or ICOS, and a CD3ζ activation domain. The CAR sequence was further incorporated into a third-generation lentiviral vector and transduced into human primary T cells at an MOI of 80 to generate CD20_19 CAR T cells under the control of the mammalian EF-1α promoter. The previously evaluated murine binder-containing CAR construct LTG1497 was also included as a positive control, while untransduced T cells (UTD) from the same donor were used as a negative control. CAR19 surface expression on transduced T cells was measured by flow cytometry using CD19-Fc followed by staining with anti-Fc Alexa Flour 647. CAR20 expression was not pursued due to lack of detection reagents. Different CD20_19 CAR constructs showed higher surface expression than the positive control LTG1497 (n = 4 donors). Flow plots (CD4 vs. CAR19) from one representative donor are shown in Figure 1B. For this donor, LTG1497 showed the lowest surface expression (49.7%), followed by D0144 (58.0%), while other CAR20_19 constructs ranged from 60% to 80%, demonstrating robust transduction efficiency and CAR expression.

[0290] To evaluate the target-specific cytotoxicity of the CD20_19 CAR in vitro, we used CD19 + CD20+ Leukemia or lymphoma lines (Raji, Nalm-6, and Reh) and CD19 - CD20 - The non-leukemia line 293T was selected as the target line. CAR-T cells were co-cultured with the target tumor cell line at effector-to-target ratios of 2.5:1, 5:1, and 10:1. After overnight co-culture, the cytotoxicity of the CAR was analyzed by a luminescence-based in vitro killing assay (Figure 2A-C). CD19 + In the cell lines Raji, Nalm-6, and Reh, the CAR20_19 construct demonstrated comparable or superior killing ability compared to CAR LTG1497 at all E:T ratios tested. In the Raji and Reh lines, no cytotoxicity was observed in the UTD group, and some killing was observed (background killing) when UTD cells were cocultured with NALM-6 at high E:T ratios. Furthermore, no or limited background killing of the CD19- and CD20-negative 293T cell line was observed (Figure 2D), demonstrating the robust target-specific cytotoxicity of all designed CD20_19 CAR constructs.

[0291] The production of T cell homeostatic and proinflammatory cytokines IL-2, IFNγ, and TNFα by the fully human CD20_19 CAR and the control construct LTG1497 was examined by ELISA. Culture supernatants after overnight co-incubation of CAR T cells with the Raji target line at an E:T ratio of 10 (Figures 4A-4C) were collected for measurement of specific cytokine release. The fully human CAR20_19 construct demonstrated significant cytokine responses compared with the UTD or CAR-alone controls, demonstrating robust target-specific CAR T cell cytokine responses. The intensity of cytokine release for each CAR20_19 construct was comparable to or greater than that of the control construct LTG1497.

[0292] To further characterize the CAR20_19 construct, we designed a long-term coculture assay. CAR T cells were cocultured with Raji cells at a low E:T ratio of 0.3:1, and UTD cells were included as a negative control. The culture mixture of CAR T cells and Raji cells was analyzed by flow cytometry to examine the remaining percentage of Raji cells and the fold-expansion of T cells during coculture (Figures 4B and 4C). On day 4, the percentage of Raji cells decreased in the coculture product of Raji and CAR T cells, and CAR T cell proliferation was observed. In contrast, the coculture of Raji and UTD cells increased the percentage of Raji cells, and T cells showed no proliferation. Furthermore, CARs D0256 and D0266 showed the highest Raji clearance. On day 7, all Raji cells, except those in the UTD group, were killed by T cells. The CD20_19 CAR showed a nearly fold-expansion increase at the end of the first round of coculture compared to the control CAR LTG1497. Raji cells were then added to the coculture, and the ET ratio was readjusted to 0.15:1. By day 10, the killing potency between the groups could be distinguished. Notably, CAR D0266 exhibited the highest killing potency during the second round of coculture, followed by D0255, D0257, D0258, and D0256, and the control LTG1497. The D0144 CAR had almost no killing potency (Figure 4B). CAR T cells continued to proliferate during the second round of coculture. CARs LTG1497, D0256, and D0144 showed slightly more proliferation than the other CARs 20_19. A similar trend was observed across different donors, with most CARs 20_19 maintaining higher killing potency than the control CAR1497, except for CAR D0144 (data not shown).

[0293] Among all CAR20_19 constructs, D0144 exhibited the lowest transduction efficiency and poor cytotoxicity during long-term coculture. Compared to LTG1497, all other CAR20_19 constructs demonstrated high CAR transduction efficiency, comparable or stronger cytotoxicity in overnight or long-term killing assays, and specific cytokine induction in vitro. In conclusion, all CAR20_19 constructs except D0144 were evaluated for in vivo tumor clearance capacity.

[0294] References for Example 1 1. Lee, DW, et al., The future is now: chimeric antigen receptors as new targeted therapies for childhood cancer. Clinical Cancer Research, 2012. 18(10): p. 2780-2790. 2. Kochenderfer, JN, et al., Construction and pre-clinical evaluation of an anti-CD19 chimeric antigen receptor. Journal of immunotherapy (Hagerstown, Md.: 1997), 2009. 32(7): p. 689. 3. Jensen, M., et al., CD20 is a molecular target for scFvFc: zeta receptor redirected T cells: implications for cellular immunotherapy of CD20+ malignancy. Biology of Blood and Marrow Transplantation, 1998. 4(2): p. 75-83. 4. Lee, D.W., et al., T cells expressing CD19 chimeric antigen receptors for acute lymphoblastic leukaemia in children and young adults: a phase 1 dose-escalation trial. The Lancet, 2015. 385(9967): p. 517-528. 5. Grupp, S.A., et al., Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. New England Journal of Medicine, 2013. 368(16): p. 1509-1518. 6. Porter, D.L., et al., Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia. New England Journal of Medicine, 2011. 365(8): p. 725-733. 7. Kochenderfer, J.N., et al., B-cell depletion and remissions of malignancy along with cytokine-associated toxicity in a clinical trial of anti-CD19 chimeric-antigen-receptor-transduced T cells. Blood, 2012. 119(12): p. 2709-2720. 8. Sotillo, E., et al., Convergence of acquired mutations and alternative splicing of CD19 enables resistance to CART-19 immunotherapy. Cancer discovery, 2015. 5(12): p. 1282-1295. 9. Ginaldi, L., et al., Levels of expression of CD19 and CD20 in chronic B cell leukaemias. Journal of clinical pathology, 1998. 51(5): p. 364-369. Sequences of the present disclosure The nucleic acid and amino acid sequences listed below are shown using standard abbreviations for nucleotide bases and three-letter codes for amino acids as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the represented strand. In the accompanying sequence listing: SEQ ID NO: 1 Nucleotide sequence of CD20-reactive scFv1 binding domain GAAATTGTGTTGACTCAGTCACCGGCAACTCTTAGTCTCTCCCCTGGAGAGAGAGCAACACTGAGCTGCAGAGCATCTCAAAGTGTTTCCAGCTATCTGGCCTGGTACCAGCAAAAGCCAGGCCAGGCACCACGACTTCTGATTTACGACGCAAGTAACAGGGCGACTGGCATCCCCGCCCGCTTTTCCGGATCAGGCAGTGGCACAGACTTTACTTTGACGATCAGTTCTTTGGAGCCAGAGGACTTTGCTGTTTACTACTGTCAGCAGAGATCCAATTGGCCGATTACCTTCGGCCAAGGTACCAGGCTGGAGATAAAGGGTGGCGGCGGCTCTGGTGGAGGTGGTTCCGGTGGGGGAGGTAGCGAAGTGCAATTGGTGGAAAGCGGTGGTGGCTTGGTTCAACCTGGGCGGTCCTTGAGATTGTCATGCGCCGCATCCGGCTTCACCTTCAACGACTATGCAATGCACTGGGTGCGGCAAGCTCCAGGCAAGGGCCTTGAGTGGGTCAGCACTATTAGCTGGAACTCCGGGAGTATCGGATACGCTGATTCAGTCAAAGGTCGATTTACCATTTCCCGCGATAATGCCAAAAAAAGCTTGTATCTCCAAATGAACTCACTCAGGGCTGAAGACACCGCGCTGTATTACTGTGCTAAAGACATCCAATATGGCAATTACTATTACGGGATGGATGTATGGGGGCAAGGGACAACGGTCACTGTCTCATCC Amino acid sequence of the CD20-reactive scFv1-binding domain, SEQ ID NO: 2 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIKGGGGSGGGGSGGGS EVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSS SEQ ID NO: 3 Nucleotide sequence of CD19-reactive scFv2 binding domain GAGGTCCAGCTGGTACAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCAGCTACTATATGCATTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATTAATCAACCCTAGTGGTGGTAGCACAAGCTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTACTATTGTGCGAGATCGGATCGGGGAATTACCGCCACGGACGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAGGCGGTGGAGGCTCAGGTGGAGGAGGTAGCGGAGGCGGTGGTTCCCAGTCTGTGCTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGGCGGATGGCCAAGATTACCTGTGGGGGAAGTGACATTGGAAATAAAAATGTCCACTGGTATCAGCAGAAGCCTGGCCAGGCTCCTGTCCTGGTTGTCTATGATGATTACGACCGGCCCTCAGGGATCCCTGAGCGATTTTCAGGCTCCAACTCTGGGGACGCGGCCACCCTGACGATCAGCACGGTCGAAGTCGGGGATGAGGCCGACTATTTCTGTCAGGTGTGGGACGGTAGTGGTGATCCTTATTGGATGTTCGGCGGAGGGACCCAGCTCACCGTTTTAGGT Amino acid sequence of the CD19-reactive scFv2-binding domain, SEQ ID NO: 4 EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGLINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSDRGITATDAFDIWGQGTMVTVSSG GGGSGGGGSGGGGSQSVLTQPPSVSVAPGRMAKITCGGSDIGNKNVHWYQQKPGQAPVLVVYDDYDRPSGIPERFSGSNSGDAATLTISTVEVGDEADYFCQVWDGSGDPYWMFGGGTQLTVLG SEQ ID NO: 5 Nucleotide sequence of CAR D0144 (CD20_CD19 CD8 BBz) SEQ ID NO: 6: Amino acid sequence of CAR D0144 (CD20_CD19 CD8 BBz) MLLLVTSLLLCELPHPAFLLIPEIVLTQSPATLLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIKGGGGSGGGGSGGGSEVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEW VSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSSGGGGGSGGGGSGGGGGGGSGGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGLINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDT AVYYCARSDRGITATDAFDIWGQGTMVTVSSGGGGSGGGGSGGGSQSVLTQPPSVSVAPGRMAKITCGGSDIGNKNVHWYQQKPGQAPVLVVYDDYDRPSGIPERFSGSNSGDAATLTISTVEVGDEADYFCQVWDGSGDPYWMFGGGTQLTVLGAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAG GAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 7 Nucleotide sequence of CAR D0255 (CD20_CD19 CD28) CD28 BBz SEQ ID NO: 8 Amino acid sequence of CAR D0255 (CD20_CD19 CD28) CD28 BBz MLLLVTSLLLCELPHPAFLLIPEIVLTQSPATLLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIKGGGGSGGGGSGGGSEVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSG SIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSGGGSEVQLVQSGA EVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGLINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSDRGITATDAFD IWGQGTMVTVSSGGGGSGGGSGGGGSQSVLTQPPSVSVAPGRMAKITCGGSDIGNKNVHWYQQKPGQAPVLVVYDDYDRPSGIPERFSGSNSGDAATLTISTVEVGDEADYFCQVWDGSGDPYWMFGGGTQLTVLGAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVA FIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 9 Nucleotide sequence of CAR D0256 (CD20_CD19 CD8 CD28 BBz) SEQ ID NO: 10 Amino acid sequence of CAR D0256 (CD20_CD19 CD8 CD28 BBz) MLLLVTSLLLCELPHPAFLLIPEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIKGGGGSGGGGSGGGSEVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGS IGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSGGGSEVQLVQSGAE VKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGLINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSDRGITATDAFDI WGQGTMVTVSSGGGGSGGGGSGGGGSQSVLTQPPSVSVAPGRMAKITCGSDIGNKNVHWYQQKPGQAPVLVVYDDYDRPSGIPERFSGSNSGDAATLTISTVEVGDEADYFCQVWDGSGDPYWMFGGGTQLTVLGAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLS LVITLYCRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAP AYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 11 Nucleotide sequence of leader / signal peptide sequence (LP) atgctgctgctggtgaccagcctgctgctgtgcgaactgccgcatccggcgtttctgctgattccg SEQ ID NO: 12: Amino acid sequence of leader / signal peptide sequence (LP) MLLLVTSLLLCELPHPAFLLIP SEQ ID NO: 13 Nucleotide sequence of CAR D0257 (CD20 CD8 CD28z_CD19 CD8 BBz) SEQ ID NO: 14 Amino acid sequence of CAR D0257 (CD20 CD8 CD28z_CD19 CD8 BBz) SEQ ID NO: 15 Nucleotide sequence of CAR D0258 (CD20 CD8 BBz_CD19 CD8 CD28z) SEQ ID NO: 16 Amino acid sequence of CAR D0258 (CD20 CD8 BBz_CD19 CD8 CD28z) SEQ ID NO: 17 Nucleotide sequence of CAR D0266 (CD20 CD8 OX40 OX40z_CD19 CD8 ICOSz) SEQ ID NO: 18 Amino acid sequence of CAR D0266 (CD20 CD8 OX40 OX40z_CD19 CD8 ICOSz) SEQ ID NO: 19 Nucleotide sequence of CAR LTG1497 (mCD20_CD19 CD8 BBz) SEQ ID NO: 20 Amino acid sequence of CAR LTG1497 (mCD20_CD19 CD8 BBz) MLLLVTSLLLCELPHPAFLLIPEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIVLTQSPAILSASPGEKVTMTCRASSSVNY MDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKGGGGSGGGGSGGGGSGGGGSGGGSDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQ GNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAG GAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 21 Nucleotide sequence of CD28 costimulatory domain AGGAGTAAACGCAGCCGCCTGCTGCATTCAGACTACATGAACATGACCCCACGGCGGCCCGGCCCAACGCGCAAACACTACCAACCTTACGCCCCACCGCGAGACTTTGCCGCCTACAGATCC SEQ ID NO: 22 Amino acid sequence of CD28 costimulatory domain RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS SEQ ID NO: 23 Nucleotide sequence of 4-1BB costimulatory domain AAGCGCGGACGGAAGAAACTCTTGTACATCTTCAAGCAGCCGTTCATGCGCCCTGTGCAAACCACCCAAGAAGAGGACGGGTGCTCCTGCCGGTTCCCGGAAGAGGAAGAGGGCGGCTGCGAACTG SEQ ID NO: 24: Amino acid sequence of 4-1BB costimulatory domain KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL SEQ ID NO: 25 Nucleotide sequence of tandem CD28_4-1BB costimulatory domain AGGAGTAAACGCAGCCGCCTGCTGCATTCAGACTACATGAACATGACCCCACGGCGGCCCGGCCCAACGCGCAAACACTACCAACCTTACGCCCCGCGAGACTTTGCCGCCTACAGATCCAAGCGGACGGAAGAAACTCTTGTACATCTTCAAGCAGCCGTTCATGCGCCCTGTGCAAACCACCCAAGAAGAGGACGGGTGCTCCTGCCGGTTCCCGGAAGAGGAAGAGGGCGGCTGCGAACTG SEQ ID NO: 26: Amino acid sequence of tandem CD28_4-1BB costimulatory domain RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL SEQ ID NO: 27 Nucleotide sequence of OX40 costimulatory domain GTGGCGGCAATTCTCGGCCTGGACTTGTCCTTGGTCTGCTTGGTCCGCTCGCAATACTTCTGGCCTTGTACCTGCTCCGCAGAGACCAAAGACTTCCGCCCGACGCCCACAAGCCCCCAGGAGGAGGTTCCTTCAGAACGCCTATACAAGAAGAACAAGCAGATGCCCACTCTACCCTGGCTAAAATC SEQ ID NO: 28: Amino acid sequence of OX40 costimulatory domain VAAILGLGLVLGLLGPLAILLALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI SEQ ID NO: 29 Nucleotide sequence of ICOS costimulatory domain TGGCTGACAAAAAAGAAGTATTCATCTAGTGTACATGATCCGAACGGTGAATACATGTTCATGCGCGCGGTGAACACGGCCAAGAAGAGCAGACTGACCGACGTAACCCTT SEQ ID NO: 30 Amino acid sequence of ICOS costimulatory domain WLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL SEQ ID NO: 31 Nucleotide sequence of CD28 hinge domain ATCGAAGTGATGTATCCACCTCCGTACCTCGATAACGAGAAATCAAATGGAACGATCATTCATGTGAAAGGGAAACATCTGTGCCCAAGCCCATTGTTCCCAGGTCCGTCAAAACCA SEQ ID NO: 32 Amino acid sequence of CD28 hinge domain IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP SEQ ID NO: 33 Nucleotide sequence of the OX40 transmembrane domain GTGGCGGCAATTCTCGGCCTGGGACTTGTCCTTGGTCTGCTTGGTCCGCTCGCAATACTTCTG SEQ ID NO: 34: Amino acid sequence of the OX40 transmembrane domain VAAILGLGLVLGLLGPLAILL SEQ ID NO: 35 DNA CD8 transmembrane domain nucleotide sequence atttgggccccgctggccggcacttgcggcgtgctcctgctgtcgctggtcatcaccctt tactgc SEQ ID NO: 36: Amino acid sequence of CD8 transmembrane domain Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys SEQ ID NO: 37 DNA CD8 hinge domain nucleotide sequence actaccacccctgcccctcggccgccgactccggccccaaccatcgcaagccaacccctc tccttgcgccccgaagcttgccgcccggccgcgggtggagccgtgcatacccgggggctg gactttgcctgcgatatctac SEQ ID NO: 38: Amino acid sequence of CD8 hinge domain Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr SEQ ID NO: 39: Amino acid sequence of the hinge and transmembrane region of CD8 alpha from amino acids 137 to 206 (NCBI RefSeq: NP.sub.--001759.3) Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys SEQ ID NO: 40 Nucleotide sequence of the DNA signaling domain of 4-1BB aagaggggccggaagaagctgctttacatcttcaagcagccgttcatgcggcccgtgcag acgactcaggaagaggacggatgctcgtgcagattccctgaggaggaagaggggggatgc gaactg SEQ ID NO: 41 Amino acid sequence of the signaling domain of 4-1BB Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu SEQ ID NO: 42 Nucleotide sequence of the DNA signaling domain of CD3-zeta CGCGTGAAGTTTTCCCGGTCCGCCGACGCTCCGGCGTACCAGCAGGGGCAAAACCAGCTGTACAACGAACTTAACCTCGGTCGCCGGGAAGAATATGACGTGCTGGACAAGCGGCGGGGAAGAGATCCCGAGATGGGTGGAAAGCCGCGGCGGAAGAACCCTCAGGAG GGCTTGTACAACGAGCTGCAAAAGGACAAAATGGCCGAAGCCTACTCCGAGATTGGCATGAAGGGAGAGCGCAGACGCGGGAAGGGACACGATGGACTGTACCAGGGACTGTCAACCGCGACTAAGGACACTTACGACGCCCTGCACATGCAGGCCCTGCCCCCGCGC SEQ ID NO: 43: Amino acid sequence of CD3 zeta RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 44 Alternative nucleotide sequence of CD3zeta_1 AGGGTGAAGTTTAGCCGCTCAGCCGATGCACCGGCCTACCAGCAGGGACAGAACCAGCTCTACAACGAGCTCAACCTGGGTCGGCGGGAAGAATATGACGTGCTGGACAAACGGCGCGGCAGAGATCCGGAGATGGGGGGAAAGCCGAGGAGGAAGAACCCTCAAGAG GGCCTGTACAACGAACTGCAGAAGGACAAGATGGCGGAAGCCTACTCCGAGATCGGCATGAAGGGAGAACGCCGGAGAGGGAAGGGTCATGACGGACTGTACCAGGGCCTGTCAACTGCCACTAAGGACACTTACGATGCGCTCCATATGCAAGCTTTGCCCCCGCGG SEQ ID NO: 45 Alternative nucleotide sequence of CD3zeta_2 AGAGTCAAATTTTCCAGGTCCGCAGATGCCCCCGCGTACCAGCAAGGCCAGAACCAACTTTACAACGAACTGAACCTGGGTCGCCGGGAGGAATATGATGTGCTGGATAAACGAAGGGGGAGGGACCCTGAGATGGGAGGGAACCTCGCAGGAAAAACCCGCAGGAA GGTTTGTACAACGAGTTGCAGAAGGATAAGATGGCTGAGGCTTACTCTGAAATAGGGATGAAGGGAGAGAGACGGAGAGGAAAAGGCCATGATGGCCTTTACCAGGGCTTGAGCACAGCAACAAAGGATACTTACGACGCTCTTCACATGCAAGCTCTGCCACCACGG SEQ ID NO: 46 Nucleotide sequence of CD28 transmembrane domain TTCTGGGTGCTTGTCGTTGTTGGGGGTGTACTCGCATGTTATTCTTTGCTGGTGACTGTGGCGTTTATCATCTTCTGGGTA SEQ ID NO: 47: Amino acid sequence of CD28 transmembrane domain FWVLVVVGGVLACYSLLVTVAFIIFWV SEQ ID NO: 48 Nucleotide sequence of CD20 / CD19 reactive scFv binding domain SEQ ID NO: 49 Amino acid sequence of CD20 / CD19 reactive scFv binding domain EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIKGGGGSGGGGSGGGSEVQLVES GGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGG SGGGGSGGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGLINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSDRGITATDAFDIWGQGTMV TVSSGGGGSGGGGSGGGGSQSVLTQPPSVSVAPGRMAKITCGGSDIGNKNVHWYQQKPGQAPVLVVYDDYDRPSGIPERFSGSNSGDAATLTISTVEVGDEADYFCQVWDGSGDPYWMFGGGTQLTVLG

Claims

1. 1. An isolated nucleic acid molecule encoding a fully human CD20 / CD19 tandem chimeric antigen receptor (CAR), comprising at least one extracellular antigen-binding domain comprising a fully human CD20 / CD19 antigen-binding domain, at least one transmembrane domain, and at least one intracellular signaling domain, wherein the fully human CD20 / CD19 tandem chimeric antigen receptor (CAR) is encoded by a nucleotide sequence comprising SEQ ID NO: 5, 7, 9, 13, 15, 17, or 19.

2. The isolated nucleic acid molecule of claim 1, wherein the encoded at least one fully human CD20 / CD19 antigen-binding domain comprises at least one single-chain variable fragment of an antibody that binds to CD20 or CD19.

3. The isolated nucleic acid molecule of claim 1, wherein the encoded at least one fully human CD20 / CD19 antigen-binding domain comprises at least one heavy chain variable region of an antibody that binds to CD20 or CD19.

4. The isolated nucleic acid molecule of claim 1, wherein the encoded at least one fully human CD20 / CD19 antigen-binding domain, the at least one intracellular signaling domain, or both, are connected to the transmembrane domain by a linker or spacer domain.

5. 5. The isolated nucleic acid molecule of claim 4, wherein the encoded linker or spacer domain is derived from the extracellular domain of CD8 or CD28 and is linked to a transmembrane domain.

6. The isolated nucleic acid molecule of claim 1, wherein the encoded extracellular completely human CD20 / CD19 antigen-binding domain is preceded by a leader nucleotide sequence encoding a leader peptide.

7. 7. The isolated nucleic acid molecule of claim 6, wherein the leader nucleotide sequence comprises a nucleotide sequence comprising SEQ ID NO: 11, which encodes the leader amino acid sequence of SEQ ID NO:

12.

8. 2. The isolated nucleic acid molecule of claim 1, wherein the transmembrane domain comprises a transmembrane domain of a protein comprising the alpha, beta, or zeta chain of the T-cell receptor, CD8, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD83, CD86, CD134, CD137, CD154, and TNFRSF19, an IgG hinge, CD2, CH3 domain, or any combination thereof.

9. 2. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid sequence encoding the fully human CD20 / CD19 tandem chimeric antigen receptor (CAR) is encoded by a nucleotide sequence comprising SEQ ID NO: 5, 7, 9, 13, 15, 17, or 19, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.

10. 2. The isolated nucleic acid molecule of claim 1, wherein the encoded at least one intracellular signaling domain further comprises a CD3 zeta intracellular domain.

11. 11. The isolated nucleic acid molecule of claim 10, wherein the encoded at least one intracellular signaling domain is located C-terminal to the CD3 zeta intracellular domain.

12. 2. The isolated nucleic acid molecule of claim 1, wherein the encoded at least one intracellular signaling domain comprises a costimulatory domain, a primary signaling domain, or any combination thereof.

13. 13. The isolated nucleic acid molecule of claim 12, wherein the encoded at least one costimulatory domain comprises a functional signaling domain of OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP12, and 4-1BB (CD137), or any combination thereof.

14. A chimeric antigen receptor (CAR) encoded by the isolated nucleic acid molecule of claim 1.

15. The CAR of claim 14, comprising at least one extracellular antigen-binding domain comprising a fully human CD20 / CD19 antigen-binding domain comprising the amino acid sequence of SEQ ID NO: 2, 4, or 49, at least one transmembrane domain, and at least one intracellular signaling domain.

16. The CAR of claim 15, wherein the fully human CD20 / CD19 antigen-binding domain comprises at least one single-chain variable fragment of an antibody that binds to CD20 or CD19.

17. The CAR of claim 15, wherein the fully human CD20 / CD19 antigen-binding domain comprises at least one heavy chain variable region of an antibody that binds to CD20 or CD19.

18. 16. The CAR of claim 15, wherein the transmembrane domain comprises a transmembrane domain of a protein comprising the alpha, beta, or zeta chain of the T-cell receptor, CD8, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, and TNFRSF19, or any combination thereof.

19. 19. The CAR of claim 18, wherein the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 36, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:

36.

20. The CAR of claim 15, wherein the at least one extracellular antigen-binding domain comprising a fully human CD20 / CD19 antigen-binding domain comprising the amino acid sequence of SEQ ID NO: 2, 4, or 49, and the at least one intracellular signaling domain, or both, are connected to the transmembrane domain by a linker or spacer domain.

21. The CAR of claim 20, wherein the linker or spacer domain is derived from the extracellular domain of CD8 or CD28 and is linked to a transmembrane domain.

22. The CAR of claim 17, wherein the at least one intracellular signaling domain comprises a costimulatory domain and a primary signaling domain.

23. The CAR of claim 22, wherein the at least one intracellular signaling domain comprises a costimulatory domain comprising a functional signaling domain of a protein selected from the group consisting of OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP12, and 4-1BB (CD137), or a combination thereof.

24. A vector comprising the nucleic acid molecule of claim 1.

25. 25. The vector of claim 24, selected from the group consisting of a DNA vector, an RNA vector, a plasmid vector, a cosmid vector, a herpes virus vector, a measles virus vector, a lentivirus vector, an adenovirus vector, or a retrovirus vector, or a combination thereof.

26. 25. The vector of claim 24, further comprising a promoter.

27. 27. The vector of claim 26, wherein the promoter is an inducible promoter, a constitutive promoter, a tissue-specific promoter, a suicide promoter, or any combination thereof.

28. A cell comprising the vector of claim 24.

29. 29. The cell of claim 28, which is a T cell.

30. 29. The cell of claim 28, wherein the T cell is a CD8+ T cell or a CD4+ T cell.

31. 29. The cell of claim 28, which is a human cell.

32. 25. A method of producing a cell comprising transducing a T cell with the vector of claim 24.

33. 10. A method for generating a population of RNA-engineered cells, comprising introducing in vitro transcribed or synthetic RNA into cells, wherein the RNA comprises the nucleic acid molecule of claim 1.

34. 30. A method of producing anti-tumor immunity in a mammal, comprising administering to said mammal an effective amount of the cells of claim 28.

35. A method for treating or preventing cancer in a mammal, comprising administering to the mammal the CAR of claim 15 in an amount effective to treat or prevent cancer in the mammal.

36. 1. A pharmaceutical composition comprising an anti-tumor effective amount of a population of human T cells, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), the CAR comprising at least one extracellular antigen-binding domain comprising a fully human CD20 / CD19 antigen-binding domain comprising the amino acid sequence of SEQ ID NO: 2, 4, or 49, at least one linker domain, at least one transmembrane domain, and at least one intracellular signaling domain, and the T cells are from a human with cancer.

37. 37. The pharmaceutical composition of claim 36, wherein the at least one transmembrane domain comprises a transmembrane domain of a protein comprising the alpha, beta, or zeta chain of the T-cell receptor, CD8, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, and TNFRSF19, or any combination thereof.

38. 37. The pharmaceutical composition of claim 36, wherein the T cells are T cells of a human with a hematological cancer.

39. 39. The pharmaceutical composition of claim 38, wherein the hematological cancer is leukemia or lymphoma.

40. 40. The pharmaceutical composition of claim 39, wherein the leukemia is chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), or chronic myelogenous leukemia (CML).

41. 40. The pharmaceutical composition of claim 39, wherein the lymphoma is mantle cell lymphoma, non-Hodgkin's lymphoma, or Hodgkin's lymphoma.

42. 39. The pharmaceutical composition of claim 38, wherein the hematological cancer is multiple myeloma.

43. 37. The pharmaceutical composition of claim 36, wherein the human cancer comprises oral and pharyngeal cancer (tongue, mouth, pharynx, head and neck), digestive system cancer (esophagus, stomach, small intestine, colon, rectum, anus, liver, intrahepatic bile duct, gallbladder, pancreas), respiratory system cancer (larynx, lung and bronchus), bone and joint cancer, soft tissue cancer, skin cancer (melanoma, basal cell carcinoma and squamous cell carcinoma), childhood tumors (neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma), tumors of the central nervous system (brain tumor, astrocytoma, glioblastoma, glioma), and adult cancers including cancer of the breast, reproductive system (cervix, uterus, ovary, vulva, vagina, prostate, testicle, penis, endometrium), urinary system (bladder, kidney and renal pelvis, ureter), eye and orbit, endocrine system (thyroid), and brain and other nervous system, or any combination thereof.

44. 1. A method of treating a mammal having a disease, disorder, or condition associated with elevated expression of a tumor antigen, comprising administering to the subject an anti-tumor effective amount of a pharmaceutical composition comprising a population of T cells, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), the CAR comprising at least one extracellular antigen-binding domain comprising a fully human CD20 / CD19 antigen-binding domain comprising the amino acid sequence of SEQ ID NO: 2, 4, or 49, at least one linker or spacer domain, at least one transmembrane domain, and at least one intracellular signaling domain, and the T cells are T cells of the subject with cancer.

45. 1. A method of treating cancer in a subject in need thereof, comprising administering to the subject an anti-tumor effective amount of a pharmaceutical composition comprising a population of T cells, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), the CAR comprising at least one extracellular antigen-binding domain comprising a fully human CD20 / CD19 antigen-binding domain comprising the amino acid sequence of SEQ ID NO: 2, 4, or 49, at least one linker or spacer domain, at least one transmembrane domain, and at least one intracellular signaling domain, and wherein the T cells are from the subject with cancer.

46. 46. ​​The method of claim 44 or 45, wherein the at least one transmembrane domain comprises a transmembrane domain of a protein comprising the alpha, beta, or zeta chain of the T-cell receptor, CD8, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154, or any combination thereof.

47. 10. A method for producing a chimeric antigen receptor-expressing cell, comprising introducing the isolated nucleic acid of claim 1 into a cell.

48. 48. A method for producing a chimeric antigen receptor-expressing cell according to claim 47, wherein the cell is a T cell or a cell population comprising a T cell.

49. 1. A pharmaceutical composition comprising an anti-allergic, anti-autoimmune, anti-alloimmune, or anti-autoaggressive effective amount of a population of human T cells, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), the CAR comprising at least one extracellular antigen-binding domain comprising a fully human CD20 / CD19 antigen-binding domain comprising the amino acid sequence of SEQ ID NO: 2, 4, or 49, at least one linker domain, at least one transmembrane domain, and at least one intracellular signaling domain, and the T cells are from a human having a disease.

50. 50. The pharmaceutical composition of claim 49, wherein the disease comprises an allergic disease, including asthma, atopic eczema, rhinitis, skin allergies, type I diabetes, or any combination thereof.

51. 50. The pharmaceutical composition of claim 49, wherein the disease comprises autoimmune, alloimmune, and autoaggressive diseases, including rheumatoid arthritis, lupus, celiac disease, Sjogren's syndrome, multiple sclerosis, polymyalgia rheumatica, ankylosing spondylitis, type 1 diabetes, alopecia areata, vasculitis, temporal arteritis, post-streptococcal autoimmune disorders, antineuronal antibody-mediated neuropsychiatric disorders, immune-mediated extrapyramidal movement disorders, Sydenham's chorea, autoimmune hemolytic disease, pulmonary fibrosis, systemic dermatosclerosis, or fibrotic diseases, or any combination thereof.

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