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

Abstract

Disclosed are chimeric antigen receptors comprising a fully human CD20 / CD19 antigen binding domain. Nucleic acids, recombinant expression vectors, host cells, antigen binding fragments and pharmaceutical compositions relating to the chimeric antigen receptors are also disclosed. Also disclosed are methods of treating or preventing cancer in a subject, as well as methods of preparing chimeric antigen receptor T cells.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 401,288, filed on August 26, 2022, the entire contents of which are incorporated herein by reference in their entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which has been submitted electronically as an XML file named SL_ST26.xml. This XML file was created on August 17, 2023 and is 74,928 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety. Technical Field

[0005] The present application relates to the field of cancer, and in particular to fully human CD20 and fully human CD19 antigen binding domains (hereinafter referred to as "fully human CD20 / CD19") and chimeric antigen receptors (CARs) comprising such fully human CD20 and fully human CD19 antigen binding domains, and methods of using the same. Background Art

[0006] 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 and is the second leading cause of death after cardiovascular disease, accounting for about one-quarter of deaths. Solid tumors are responsible for most of these deaths. Despite significant advances in the medical treatment of some cancers, the overall 5-year survival rate for all cancers has only increased by about 10% in the past 20 years. Cancer or malignant tumors grow and metastasize rapidly in an uncontrolled manner, making treatment extremely difficult.

[0007] CD19 is a transmembrane cell surface glycoprotein receptor of 85 to 95 kDa. CD19 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, reduces the triggering threshold of B cell receptors to antigens (Carter, RH, and Fearon, DT, 1992, Science, 256: 105-107), and coordinates 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 is able to signal independently of antigen receptors at the pro-B cell, pre-pre-B cell, pre-B, and early B cell stages, and is associated with Src family protein tyrosine kinases, is 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). A key point relevant to the treatment of B-cell malignancies is that CD19 is expressed in a strictly regulated manner on normal B cells, restricted to early B-cell precursors and mature B cells at the stage of IgH gene rearrangement, 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).

[0008] CD20 (also known as LEU-16, MS4A1) is a transmembrane 4A family protein that is expressed on the surface of B cells from the progenitor B cell stage to the mature B cell stage and plays a role in B cell development and differentiation. The CD20 antigen is also expressed on a variety of hematological tumors, and a variety of monoclonal anti-CD20 antibodies have been used in the treatment of CD20-positive malignancies for many years (reviewed in Lim, Sean H. et al. "Anti-CD20 Monoclonal Antibodies: Historical and Future Perspectives." Haemaloglca 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) and chronic lymphocytic leukemia (CLL) (rituximab prescribing information).

[0009] Traditional treatments for B-lineage leukemias and lymphomas can include chemotherapy, radiotherapy, and stem cell transplantation (see mayclinic.org on the World Wide Web). The high toxicity associated with these treatments and the risk of complications (e.g., relapse, secondary malignancies, or GVHD) have prompted the search for better treatment alternatives. The expression of CD19 on both adult and pediatric (pre-B-ALL) B-cell malignancies has led to the use of this target for both antibody-based and chimeric antigen receptor (CAR)-T cell-based treatments.

[0010] (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, DelbrookC, Feldman SA, Orentas R, Sabatino M, Shah NN, Sternberg SM, Stroncek D, Tschemia N, Yuan C, Zhang H, Zhang L, Rosenberg SA, Wayne AS, Mackall CL, 2015, Lancet 385: 517-28).

[0011] Furthermore, the presence of the CD20 antigen on lymphomas (DLBCL, FL) and leukemias (CLL) makes it an attractive additional target for effective tumor clearance and prevention of tumor antigen escape.

[0012] The current standard of care for B-lineage leukemias may consist of remission induction therapy followed by consolidation by high-dose chemotherapy or radiation, and may feature stem cell transplantation and an additional course of chemotherapy as needed (see cancer.gov on the World Wide Web). The high toxicity associated with these treatments and the risk of complications (e.g., relapse, secondary malignancies, or GVHD) have prompted the search for better treatment options. The expression of CD19 on both adult and pediatric (pre-B-ALL) B-cell malignancies has led to the use of this target for both antibody-based and chimeric antigen receptor (CAR)-T cell-based therapies.

[0013] (Kochenderfer JN, Wilson WH, Jaik 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, DelbrookC, 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).

[0014] Many new approaches have been developed to treat B-cell leukemias and lymphomas, including bispecific antibodies that link anti-CD19 or anti-CD20 binding motifs to T-cell binding motifs (i.e., blinatumomab for the treatment of Philadelphia chromosome-negative relapsed or refractory B-cell precursor acute lymphoblastic leukemia (ALL), ). To date, many of the CD19 or CD20 binding moieties used in CAR constructs have utilized domains derived from murine antibodies. Many of these products are currently under consideration for approval, including those developed by Novartis and Kite Pharmaceuticals. In April 2017, Novartis announced that CTL019 (tisagenlecleuce1) 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 previous treatments, adding the designation to the treatment of r / r B-cell acute lymphoblastic leukemia (ALL). These indications are based on the Phase II JULIET study (NCT02445248) and the ELIANA study (NCT02435849), respectively. The JULIET trial showed an overall response rate (ORR) of 45%, with 37% complete responses (CR) and 8% partial responses (PR) at three months. In the ELIANA study, 82% of patients infused with the product achieved CR or CR with incomplete count recovery, and the relapse-free survival rate was 60% at 6 months. The CAR-T product (KTE-C19, axicabtagene ciloleucel) from Kite Pharmaceuticals was granted breakthrough designation for diffuse large B-cell lymphoma (DLBLC), transformed follicular lymphoma (transformed follicular lymphoma, TFL) and primary mediastinal B-cell lymphoma (primary mediastinal B-cell lymphoma, PMBCL). In the Kite ZUMA-3 Phase II trial of KTE-C19 in r / r ALL, 73% of CRs (at 2 months or longer) were reported. Regardless of whether antibodies for CAR-T therapy are used, there are still a significant number of patients who cannot be helped by these treatments, and there is considerable room for improved treatment methods.

[0015] Chimeric antigen receptor (CAR) is a hybrid molecule comprising three basic units: (1) extracellular antigen binding motif, (2) connection / transmembrane motif, and (3) 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 CAR is usually formed after the single chain variable fragment (ScFv) of the minimum binding domain of the immunoglobulin (Ig) molecule. Alternative antigen binding motifs have also been modified, such as receptor ligands (ie, IL-13 has been modified to bind to the IL-13 receptor expressed by the tumor), complete immune receptors, library-derived peptides, and innate immune system effector molecules (eg, NKG2D). Alternative cell targets for CAR expression (e.g., NK or γ-δ 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). There is still a lot of work to be done on defining the most active T cell populations for transduction with CAR vectors, determining the best culture and expansion techniques, and defining the molecular details of the CAR protein structure itself.

[0016] The connection motif of CAR can be a relatively stable structural domain, such as the constant domain of IgG, or a flexible joint designed to be extended. Structural motifs (such as those derived from IgG constant domains) can be used to extend the ScFv binding domain away from the T cell plasma membrane surface. This may be important for some tumor targets (such as disialoganglioside GD2; Orentas et al., unpublished observations) in which the binding domain is particularly close to the surface membrane of tumor cells. So far, the signaling motif used in CAR always includes the CD3-ζ chain because the core motif is a key signal for T cell activation. The second-generation CAR reported for the first time is characterized by the CD28 signaling domain and the CD28 transmembrane sequence. The motif is also used in the third-generation CAR containing the CD137 (4-1BB) signaling motif (Zhao Y et al. J Immunol. 2009; 183 (9): 5563-74). With the advent of new technologies that utilize beads linked to anti-CD3 and anti-CD28 antibodies to activate T cells, the presence of the classic “signal 2” from CD28 no longer needs to be encoded by the CAR itself. Using bead activation, third-generation vectors were found to be no better than second-generation vectors in in vitro assays, and they did not provide a clear benefit over second-generation vectors in mouse models of leukemia.

[0017] (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 acutelymphoblastic leukemia, Blood.2013;121(7):1165-74; Kochenderfer JN etal.Blood.2012;

[0018] 119 (12): 2709-20). The clinical success of CD19-specific CARs with second-generation CD28 / CD3-ζ (Lee DW et al. American Society of Hematology Annual Meeting. New Orleans, LA; December 7-10, 2013) and CD137 / CD3-ζ signaling formats (Porter DL et al. N Engl J Med. 2011; 365 (8): 725-33) confirmed this. In addition to CD137, other tumor necrosis factor receptor superfamily members (such as OX40) can also provide important persistent signals in CAR-transduced T cells (Yvon E et al. Clin Cancer Res. 2009; 15 (18): 5852-60). Also important are the culture conditions under which the CAR T cell population is cultured, such as the inclusion of cytokines IL-2, IL-7, and / or IL-15 (Kaiser AD et al. Cancer Gene Ther. 2015; 22(2): 72-78).

[0019] The current challenge in the wider and effective adaptation of CAR therapy for cancer is related to the lack of convincing targets. Establishing a binder for cell surface antigens is now easily achievable, but it is still a daunting challenge to find cell surface antigens that are specific to tumors while not harming normal tissues. A potential way to give CAR-expressing T cells greater target cell specificity is to use a combined CAR approach. In one system, CD3-ζ and CD28 signaling units are separated in two different CAR constructs expressed in the same cell; in another system, two CARs are expressed in the same T cell, but one has a lower affinity and therefore requires an alternative CAR to first engage so that the other has full activity (Lanitis E et al. Cancer Immunol Res. 2013; 1 (1): 43-53; Kloss CC et al. Nat Biotechnol. 2013; 31 (1): 71-5). The second challenge in producing a single ScFv-based CAR as an immunotherapeutic agent is the heterogeneity of tumor cells. At least one group has developed a CAR strategy for glioblastoma in which the effector cell population is simultaneously targeted to multiple antigens (HER2, IL-13Ra, EphA2) in the hope of avoiding the growth of the target antigen-negative population (Hegde M et al. Mol Ther. 2013; 21(11): 2087-101).

[0020] T cell-based immunotherapy has become a new frontier in synthetic biology; multiple promoters and gene products are considered to direct these highly potent cells to the tumor microenvironment, where they can both escape negative regulatory signals and mediate effective tumor killing. Elimination of unwanted T cells by drug-induced dimerization of inducible caspase 9 constructs with chemical-based dimerizers (e.g., API 903) shows a way in which a powerful switch that can control T cell populations can be pharmacologically activated (Di Stasi A et al. N Engl J Med. 2011; 365(18): 1673-83). The generation of effector T cell populations immune to the negative regulatory effects of transforming growth factor-β through the expression of decoy receptors further shows the extent to which effector T cells can be engineered to achieve optimal anti-tumor activity (Foster AE et al. J Immunother. 2008; 31(5): 500-5). Therefore, although CARs have been shown to trigger T cell activation in a manner similar to endogenous T cell receptors, the main obstacle to the clinical application of this technology to date has been limited to the in vivo expansion of CAR+ T cells, the rapid disappearance of cells after infusion, and disappointing clinical activity. This may be partly due to the murine origin of some of the CAR sequences used.

[0021] The use of blinotumomab (bispecific anti-CD19 and anti-CD3 antibodies) has shown impressive results for critically ill patients receiving this treatment. However, the durable remission rate is less than 40%, and hematopoietic stem cell transplant (HSCT) can only save 50% of responders at most (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 requirement for patients who have received bispecific antibodies or CAR-T therapy to subsequently undergo HSCT to maintain a durable response is still an area of ​​active debate. Although high responses have been reported for CD19 CAR-T trials, some even greater than 90%, this number may be closer to 70% if the trials are recast as “intent-to-treat” trials (Davis KL, Mackall CL, 2016, Blood Advances 1:265-268). At the University of Pennsylvania, the best results reported 12 months after CAR19 treatment showed an RFS of 55% and an OS of 79% in patients who were able to receive the T-cell product (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, no15_suppl (May 2016) 3011-3011).

[0022] Therefore, there is an urgent and long-felt need in the art to discover new compositions and methods for treating B-ALL and other B-cell malignancies expressing CD19 and / or CD20 using regimens that can exhibit specific and effective anti-tumor effects without the above-mentioned disadvantages.

[0023] The present invention addresses these needs by providing CAR compositions and treatment methods that can be used to treat cancer and other diseases and / or conditions. In particular, as disclosed and described herein, the present invention provides such CARs: the CARs can be used to treat diseases, disorders or conditions associated with dysregulated CD20 and / or CD19 expression, and the CARs contain a tandem full-human CD20 / CD19 antigen binding domain, which exhibits one or more of the following properties: i) high surface expression on transduced T cells; ii) high cell lysis of cells expressing CD20 and / or CD19, wherein the transduced T cells exhibit in vivo expansion and persistence; iii) multiple targeting to overcome antigen escape; iv) armor to overcome immunosuppression in the tumor microenvironment (tumor microenvironment, TME); v) cytokine stimulatory elements to promote autonomous T cell stimulation with the aid of cytokines, thereby enhancing anti-tumor cytotoxicity, expansion, memory formation, cytokine secretion, persistence; vi) digestive enzymes to overcome the physical barriers of the tumor stroma / extracellular matrix (extracellular matrix, ECM) and enable CAR T tumors can penetrate; and vii) turn on a switch (on-switch) or turn off a switch (off-switch) to control the expression of CAR or co-expressed functional "booster" elements, and treatment methods using such CARs, which can be used to treat solid tumors, including tumors expressing targeted antigens, 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, and other diseases and / or conditions expressing CAR-related targets, including, for example, but not limited to, autoimmune diseases, alloimmune diseases, and autoinvasive diseases, including rheumatoid arthritis, lupus, celiac disease, Sjögren's syndrome ( syndrome, multiple sclerosis, polymyalgia rheumatica, ankylosing spondylitis, type 1 diabetes mellitus, alopecia areata, vasculitis, temporal arteritis, poststreptococcal autoimmune disease, antineuronal antibody-mediated neuropsychiatric disorder, immune-mediated extrapyramidal movement disorder, Sydenham chorea, alloimmune hemolytic disease, pulmonary fibrosis, systemic sclerosis, or fibrotic disease. Summary of the invention

[0024] Provided herein are new tandem fully human CD20 and CD19 targeting antibodies or antigen binding domains thereof, wherein the CD19 targeting portion is located before or after the CD20 targeting portion in the amino acid sequence (hereinafter referred to as "fully human CD20 / CD19"), and chimeric antigen receptors (tandem CARs) containing such CD20 and / or CD19 antigen binding domains, and host cells (eg, T cells) expressing the receptors, and nucleic acid molecules encoding the receptors. CAR exhibits high surface expression on transduced T cells, with a high degree of cell lysis and transduced T cell in vivo expansion and persistence. Also provided are methods of using the disclosed CARs, host cells, and nucleic acid molecules, for example, to treat cancer in a subject.

[0025] In one aspect, an isolated nucleic acid molecule encoding a tandem fully human CD20 / CD19 chimeric antigen receptor (CAR) is provided, wherein the chimeric antigen receptor comprises 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 SEQ ID NO: 1, 3 and 48.

[0026] In one aspect, an isolated nucleic acid molecule encoding a tandem fully human CD20 / CD19 chimeric antigen receptor (CAR) is provided, wherein the chimeric antigen receptor comprises 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 encoded by a nucleic acid sequence selected from SEQ ID NO: 1, 3 and 48 encodes a tandem fully human CD20 / CD19 CAR comprising an amino acid sequence selected from SEQ ID NO. 2 4 and 49.

[0027] In one 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 single chain variable fragment of an antibody that binds to fully human CD20 / CD19.

[0028] 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 to fully human CD20 / CD19.

[0029] In 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.

[0030] In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular fully human CD20 / CD19 antigen binding domain is connected to a transmembrane domain via a linker domain.

[0031] 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 peptide or signal peptide.

[0032] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the CAR comprises at least one fully human CD20 / CD19 antigen binding domain, wherein the at least one fully human CD20 / CD19 antigen binding domain is encoded by a nucleotide sequence comprising the fully human CD20 / CD19 nucleotide sequences in SEQ ID No: 1 and 3, respectively, and wherein the CAR further encodes an extracellular antigen binding domain targeting 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.

[0033] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the CAR comprises at least one fully human CD20 / CD19 antigen binding domain encoded by such a nucleotide sequence: comprising a fully human CD20 / CD19 nucleotide sequence containing within SEQ ID No: 48, and wherein the CAR further encodes an extracellular antigen binding domain targeting an antigen, wherein the antigen is 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-1TCR, MAGE A3 TCR, or any combination thereof.

[0034] In certain embodiments, an isolated nucleic acid molecule encoding a CAR is provided, wherein the additionally encoded extracellular antigen binding domain comprises 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-PMSAScFv antigen binding domain, an anti-glycolipid F77 ScFv antigen binding domain, an anti-EGFRvIII ScFv antigen binding domain, anti-GD-2ScFv antigen binding domain, anti-NY-ESO-1TCR ScFv antigen binding domain, anti-MAGE A3 TCR ScFv antigen binding domain, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or any combination thereof.

[0035] In one aspect, a CAR provided herein further comprises a linker or spacer domain.

[0036] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the extracellular fully human CD20 / CD19 antigen binding domain, the intracellular signaling domain, or both are connected to the transmembrane domain via a linker or spacer domain.

[0037] 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 connected to the transmembrane domain.

[0038] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded CAR further comprises a transmembrane domain, wherein the transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of the alpha, beta or zeta chain of a T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD83, CD86, CD134, CD137 and CD154, or a combination thereof.

[0039] In 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.

[0040] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded intracellular signaling domain is arranged at the C-terminal side relative to the CD3ζ intracellular domain.

[0041] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded at least one intracellular signaling domain comprises a co-stimulatory domain, a primary signaling domain, or a combination thereof.

[0042] In other embodiments, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded at least one co-stimulatory 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.

[0043] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, which further comprises a leader sequence or a signal peptide, wherein the leader or signal peptide nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 11.

[0044] In another embodiment, an isolated nucleic acid molecule encoding CAR is provided, wherein the encoded leader sequence comprises the amino acid sequence of SEQ ID NO:12.

[0045] 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.

[0046] 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.

[0047] In another embodiment, a CAR is provided, wherein the at least one transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of the α, β or ζ chain of a T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, TNFRSF19, or a combination thereof.

[0048] In some embodiments, a CAR is 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 having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or any combination thereof.

[0049] In one embodiment, a CAR is provided, wherein the extracellular antigen binding domain comprises 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-PMSAScFv antigen binding domain, an anti-glycolipid F77ScFv antigen binding domain, an anti-EGFRvIII ScFv antigen binding domain, anti-GD-2ScFv antigen binding domain, anti-NY-ESO-1TCR ScFv antigen binding domain, anti-MAGE A3 TCR ScFv antigen binding domain, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or any combination thereof.

[0050] In another embodiment, a CAR is provided, wherein at least one intracellular signaling domain comprises a co-stimulatory domain and a primary signaling domain.

[0051] In another embodiment, a CAR is provided, wherein the at least one intracellular signaling domain comprises a co-stimulatory domain, wherein the co-stimulatory domain comprises 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.

[0052] In one embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 5, the nucleotide sequence of CAR D0144 (CD20_CD19 CD8 BBz).

[0053] In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO:6CAR D0144 (CD20_CD19 CD8BBz).

[0054] In one embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 7, the nucleotide sequence of CAR D0255 (CD20_CD19 CD28) CD28 BBz.

[0055] In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 8CAR D0255 (CD20_CD19 CD28) CD28 BBz.

[0056] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 15, the nucleotide sequence of CARD0258 (CD20 CD8 BBz_CD19 CD8 CD28z).

[0057] 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).

[0058] In one aspect, the CAR disclosed herein is modified to express or comprise a detectable marker for diagnosis, monitoring and / or prediction of treatment outcome (e.g., progression-free survival of a cancer patient) or for monitoring the progress of such treatment.

[0059] In one embodiment, the nucleic acid molecule encoding the disclosed CAR may be contained in a vector, such as a viral vector. The vector is a DNA vector, an RNA vector, a plasmid vector, a cosmid vector, a herpes virus vector, a measles virus vector, a lentiviral vector, an adenoviral vector, or a retroviral vector, or a combination thereof.

[0060] In certain embodiments, the vector further comprises a promoter, wherein the promoter is an inducible promoter, a tissue-specific promoter, a constitutive promoter, a suicide promoter, or any combination thereof.

[0061] In another embodiment, the vector expressing CAR may also be modified to include one or more control elements for controlling CAR T cell expression or removing CAR-T cells by suicide switch. The suicide switch may include, for example, a drug that induces apoptosis-inducing signaling cascade or induces cell death. In a preferred embodiment, the vector expressing CAR may also be modified to express an enzyme, such as thymidine kinase (thymidine kinase, TK) or cytosine deaminase (cytosinedeaminase, CD).

[0062] 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, such as a primary T cell obtained from a subject. In one embodiment, the host cell is a CD8 + T cells.

[0063] In another aspect, a pharmaceutical composition comprising an anti-tumor effective amount of a human T cell population is provided, wherein the T cell comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising an amino acid sequence of SEQ ID NO. 2 and 4, wherein the CAR comprises at least one extracellular antigen binding domain, at least one linker domain, at least one transmembrane domain and at least one intracellular signaling domain, and the extracellular antigen binding domain comprises a fully human CD20 / CD19 antigen binding domain, wherein the T cell is a T cell of a person suffering from cancer. The cancer particularly includes hematological cancer, 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.

[0064] In another aspect, a pharmaceutical composition comprising an anti-tumor effective amount of a human T cell population is provided, wherein the T cell comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising an amino acid sequence of SEQ ID NO. 49, wherein the CAR comprises at least one extracellular antigen binding domain, at least one linker domain, at least one transmembrane domain and at least one intracellular signaling domain, and the at least one extracellular antigen binding domain comprises a fully human CD20 / CD19 antigen binding domain, wherein the T cell is a T cell of a person suffering from cancer. The cancer particularly includes hematological cancers, such as leukemia (e.g., chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL) or chronic myeloid leukemia (CML), lymphoma (e.g., mantle cell lymphoma, non-Hodgkin's lymphoma or Hodgkin's lymphoma) or multiple myeloma, or a combination thereof.

[0065] 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 a T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, mesothelin, CD33, CD37, CD64, CD80, CD83, CD86, CD134, CD137, CD154, TNFRSF19, or a combination thereof.

[0066] In another embodiment, a pharmaceutical composition is provided wherein the human cancer comprises adult carcinoma, including: oral and pharynx 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 and squamous cell carcinoma), childhood tumors (neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma), tumors of the central nervous system (brain, astrocytoma, glioblastoma, glioma), as well as cancers of the breast, reproductive system (cervix, uterine body, ovary, vulva, vagina, prostate, testis, penis, endometrium), urinary system (bladder, kidney and renal pelvis, ureter), eye and orbit, endocrine system (thyroid), and brain and other nervous systems, or any combination thereof.

[0067] In another embodiment, a pharmaceutical composition comprising an anti-tumor effective amount of a human T cell population of a human suffering from cancer is provided, wherein the cancer is a refractory cancer that is not responsive to one or more chemotherapeutic agents. The cancer includes hematopoietic cancer, myelodysplastic syndrome, pancreatic cancer, head and neck cancer, skin tumors, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adult B-cell malignancies (including CLL (chronic lymphocytic leukemia), CML (chronic myeloid leukemia), non-Hodgkin's lymphoma (NHL)), minimal residual disease (MRD) in childhood B-cell malignancies (including B-lineage ALL (acute lymphoblastic 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.

[0068] In another aspect, a method for preparing a T cell comprising a CAR (hereinafter referred to as a "CAR-T cell") is provided. The method includes transducing a T cell with a vector or nucleic acid molecule encoding a disclosed CAR that specifically binds to CD19 and / or CD20, thereby preparing a CAR-T cell.

[0069] In another aspect, a method for producing an RNA-modified cell population is provided, comprising introducing in vitro transcribed RNA or synthetic RNA encoding a nucleic acid molecule of the disclosed CAR into cells of a subject, thereby producing CAR cells.

[0070] 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 tumors, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adult B-cell malignancies (including CLL (chronic lymphocytic leukemia), CML (chronic myeloid leukemia), non-Hodgkin lymphoma (NHL)), minimal residual disease (MRD) in childhood B-cell malignancies (including B-lineage ALL (acute lymphoblastic 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.

[0071] In another embodiment, a method of blocking T cell suppression mediated by cells expressing CD19 and / or CD20 and changing the tumor microenvironment to inhibit tumor growth in a mammal is provided, comprising administering to the mammal an effective amount of a composition comprising a CAR containing an amino acid sequence selected from SEQ ID NO: 2, 4, and 49. In one embodiment, the cell is selected from a tumor cell expressing CD19 and / or CD20, a tumor-associated macrophage, and any combination thereof.

[0072] In another embodiment, a method of inhibiting, suppressing or preventing immunosuppression of an anti-tumor or anti-cancer immune response in a mammal is provided, comprising administering to the mammal an effective amount of a composition comprising a CAR selected from SEQ ID NOs: 2, 4 and 49. In one embodiment, the CAR inhibits the interaction between a first cell and a T cell, wherein the first cell is selected from a tumor cell expressing CD19 and / or CD20, a tumor-associated macrophage, and any combination thereof.

[0073] In another aspect, a method for inducing anti-tumor immunity in a mammal is provided, comprising administering to the mammal a therapeutically effective amount of T cells transduced with a vector or nucleic acid molecule encoding the disclosed CAR.

[0074] In another embodiment, a method for treating or preventing cancer in a mammal is provided, comprising administering one or more disclosed CARs to the mammal in an amount effective to treat or prevent cancer in the mammal. The method comprises administering to the subject a therapeutically effective amount of a host cell expressing a disclosed CAR that specifically binds to CD19 and / or CD20 and / or one or more of the foregoing antigens: the conditions are sufficient to form an immune complex of an antigen binding domain on the CAR with an extracellular domain of CD19 and / or CD20 and / or one or more of the foregoing antigens in the subject.

[0075] In another embodiment, a method for treating a mammal suffering from a disease, disorder or condition associated with elevated expression of a tumor antigen is provided, the method comprising administering to the subject a pharmaceutical composition comprising an anti-tumor effective amount of 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 T cells of a subject suffering from cancer.

[0076] In another embodiment, a method for treating cancer in a subject in need thereof is provided, comprising administering to the subject a pharmaceutical composition comprising an anti-tumor effective amount of a T cell population, wherein the T cell comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an amino acid sequence of SEQ ID NO: 2 and 4 or any combination thereof, at least one linker or spacer domain, at least one transmembrane domain, at least one intracellular signaling domain, wherein the T cell is a T cell of a subject suffering from cancer. In some embodiments of the aforementioned method, the at least one transmembrane domain comprises the following transmembrane: α, β or ζ chain of a T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD19, CD22, mesothelin, CD33, CD37, CD64, CD80, CD83, CD86, CD134, CD137, CD154, TNFRSF16, TNFRSF19, or a combination thereof.

[0077] In another embodiment, a method for treating cancer in a subject in need thereof is provided, comprising administering to the subject a pharmaceutical composition comprising an anti-tumor effective amount of a T cell population, wherein the T cell comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an amino acid sequence of SEQ ID NO.49, at least one linker or spacer domain, at least one transmembrane domain, at least one intracellular signaling domain, wherein the T cell is a T cell of a subject suffering from cancer. In some embodiments of the aforementioned method, the at least one transmembrane domain comprises the following transmembrane: α, β or ζ chain of a T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD19, CD22, mesothelin, CD33, CD37, CD64, CD80, CD83, CD86, CD134, CD137, CD154, TNFRSF16, TNFRSF19, or a combination thereof.

[0078] In another embodiment, a method for producing a persistent genetically modified T cell population in a person diagnosed with cancer is provided. In one embodiment, the method includes administering to a person a T cell genetically modified to express a CAR, wherein the CAR comprises SEQ ID NO: 2 and 4 amino acid sequences or any combination thereof, at least one transmembrane domain and at least one intracellular signaling domain, wherein after administration, the persistent genetically modified T cell population or the progeny population of the T cell continues in the person for at least one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, two years or three years.

[0079] In another embodiment, a method for producing a persistent genetically modified T cell population in a person diagnosed with cancer is provided. In one embodiment, the method includes administering to a person a T cell genetically modified to express a CAR, wherein the CAR comprises an amino acid sequence of SEQ ID NO: 49, at least one transmembrane domain, and at least one intracellular signaling domain, wherein after administration, the persistent genetically modified T cell population or the progeny population of the T cell continues in the person for at least one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, two years, or three years.

[0080] In one embodiment, the progeny T cells in the human comprise memory T cells. In another embodiment, the T cells are autologous T cells.

[0081] In all aspects and embodiments of the methods described herein, any of the aforementioned cancers, diseases, disorders or conditions associated with elevated expression of tumor antigens can be treated or prevented or ameliorated using one or more CARs disclosed herein.

[0082] In another aspect, a kit for preparing a chimeric antigen receptor T cell as described above or for preventing, treating or ameliorating any cancer, disease, disorder or condition associated with increased expression of a tumor antigen in a subject as described above is provided, 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.

[0083] It should be understood that CAR, host cells, nucleic acids and methods are available outside 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 with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figures 1A to 1B The CD20 and CD19 dual-targeting CAR structure and surface expression on human primary T cells are shown. Figure 1A )CD20 and CD19 dual-targeting CAR are designed as tandem or bicistronic constructs.Tandem CAR constructs include all-human 20-19 tandem scFv targeting domains, hinge domains and transmembrane domains, single 4-1BB or tandem CD28_4-1BB costimulatory domains and CD3ζ activation domains.Construct pLTG1497 with mouse tandem scFv is used as a control.Dual CAR (Duo CAR) constructs include single CD20 CAR, followed by 2A sequences, and single CD19 CAR with different costimulatory domains or transmembrane domains. Figure 1B) Primary T cells from healthy donors were activated with TransAct in the presence of IL-2 and transduced with a lentiviral vector encoding the CAR20_19 construct. CAR surface expression of transduced T cells was determined by staining with CD19 Fc followed by flow cytometry with anti-Fc-AF647. UTD-untransduced control.

[0085] Figures 2A to 2D The cytotoxicity of the CAR20_19 construct in vitro is shown. Luciferase-based cytotoxicity assay was performed using CD19+CD20+ tumor lines: Figure 2A )Raji, Figure 2B )Nalm-6, Figure 2C )Reh and CD19-CD20- tumor lines, Figure 2D )293T. All target lines were stably transduced with firefly luciferase. CAR T cells were co-cultured with tumor cells at a specified effector to target (E:T) ratio of 2.5:1, 5:1, or 10:1 overnight. The percentage of specific target cleavage was assessed by luminometry. Data represent an independent experiment from 2 different donors. Mean ± SD of three technical replicates. A representative experiment of 4 independent donors is shown in the figure.

[0086] Figures 3A to 3C CAR T cell factor release in response to Raji lymphoma cell line is shown. Culture supernatants of CAR T cells were evaluated after overnight incubation alone or with CD19+CD20+Raji target cells at an E:T ratio of 10. Figure 3A )IL-2, Figure 3B )IFNγ and Figure 3C ) Cytokine production of TNFα was analyzed by ELISA. Mean ± SD of three technical replicates. Data represent one independent experiment from two independent donors.

[0087] Figures 4A to 4C The cytotoxicity of the CAR20_19 construct under long-term target cell stimulation is shown. Figure 4A ) Long-term stimulation protocol for 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. The remaining CAR T cells and Raji cells were measured by flow cytometry and readjusted to an E:T ratio of 0.15:1 for the second round. Figure 4B ) The remaining Raji percentage was analyzed by flow cytometry using GFP on days 4, 7, and 10. Figure 4C )Use CountBright TMAbsolute counting beads were used to measure T cell numbers by flow cytometry. The expansion fold compared to the 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.

[0088] Figures 5A to 5C The experimental design and timeline for in vivo challenge and re-challenge studies of CAR T cells in mouse NSG Raji lymphoma xenografts are shown ( Figure 5A ), Representative bioluminescent images of tumor progression in mice in the study ( Figure 5B ), and profiles of bioluminescence measurements representing the kinetics of tumor progression during the initial challenge and re-challenge studies ( Figure 5C ). DETAILED DESCRIPTION

[0089] definition

[0090] Unless the context clearly indicates otherwise, the nouns used in this article without quantifier modification represent one / kind or more / kind. For example, the term "antigen" includes single / kind or multiple / kind antigens, and can be considered to be equivalent to the phrase "at least one / kind antigen". The term "comprising" used in this article means "including". Therefore, "comprising antigens" means "comprising antigens" without excluding other elements. The phrase "and / or" means "and" or "or / or". It should be further understood that, unless otherwise indicated, any and all base sizes or amino acid sizes and all molecular weights or molecular mass values ​​given for nucleic acids or polypeptides are approximate, and are provided for the purpose of description. Although many methods and materials similar or equivalent to those described herein can be used, specific suitable methods and materials are described below. In the event of conflict, this specification (including the explanation of terms) shall prevail. In addition, materials, methods and examples are only illustrative and are not intended to be restrictive. For ease of access to multiple embodiments, the following explanation of terms is provided:

[0091] The term "about" when referring to a measurable value (e.g., an amount, a 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 particular value, as such variations are suitable for performing the disclosed methods.

[0092] Unless otherwise indicated, technical terms herein are used according to conventional usage. Definitions of commonly used terms in molecular biology can be found in Benjamin Lewin, Genes VII, published by Oxford University Press, 1999; Kendrew et al. (ed.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994; and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995; and other similar references.

[0093] The present disclosure provides a full-human CD20 / CD19 antibody or its fragment and a chimeric antigen receptor (CAR) with such a full-human CD20 / CD19 antigen binding domain. The enhancement of the functional activity of CAR is directly related to the enhancement of the functional activity of the T cells expressing CAR. As a result of one or more of these modifications, CAR shows both high cytokine-induced cell lysis and cell surface expression on transduced T cells, as well as the persistence of the T cells expressing CAR in vivo T cell amplification level and transduction. The advantage of the CAR of the present disclosure is that a CART lentiviral product can be used to treat a variety of patient populations (i.e., CD19+, CD20+ or ​​dual CD19+CD20+ cancer patients), which provides flexibility in the case of limited resources.

[0094] The unique ability to combine functional parts derived from different protein domains is a key innovative feature of chimeric antigen receptors (CARs). The selection of each of these protein domains is a key design feature, as is the way in which its specificity is combined. Each design domain is an essential component that can be used between different CAR platforms to transform the function of lymphocytes. For example, the selection of extracellular binding domains can make otherwise ineffective CARs effective.

[0095] The constant framework components of the immunoglobulin-derived protein sequences used to establish the extracellular antigen binding domain of CAR can be completely neutral or can self-associate and drive T cells to a state of metabolic exhaustion, thereby making the effect of the therapeutic T cells expressing the CAR much lower. This occurs independently of the antigen binding function of the CAR domain. In addition, the selection of intracellular signaling domains can also control the activity and persistence of therapeutic lymphocyte populations for immunotherapy. Although the ability to bind to target antigens and transmit activation signals to T cells through these extracellular and intracellular domains, respectively, is an important CAR design aspect, it has also become apparent that the selection of the source of the extracellular antigen binding fragment can have a significant effect on the efficacy of CAR, and thus has a limiting effect on the function and clinical utility of CAR.

[0096] CAR disclosed herein is expressed at a high level in cells. Cells expressing CAR have a high in vivo proliferation rate, produce a large amount of cytokines, and have high cytotoxic activity against cells with full-human CD20 / CD19 antigens bound to CAR on their surface. The use of extracellular full-human CD20 / CD19 antigen binding domains leads to the production of CARs that work better in vivo, while avoiding the induction of anti-CAR immunity and killing CAR T cell populations in the host immune response. CARs expressing extracellular full-human CD20 / CD19 ScFv antigen binding domains show excellent activity / characteristics, including i) preventing poor CAR T persistence and function as seen in the case of mouse-derived binding sequences; ii) lack of regional (i.e., intrapleural) delivery of CAR can also be effective; and iii) can be based on both high-affinity and low-affinity binding agents for full-human CD20 / CD19 to produce CAR T cell designs. The latter property allows researchers to better tune the potency versus toxicity and / or tissue specificity of CAR T products, since lower affinity binders can have higher specificity for tumors compared to normal tissues due to higher expression of full human CD20 / CD19 on tumors compared to normal tissues, which can prevent on-target offtumor toxicity and bystander cell killing.

[0097] The following is a detailed description of the CAR of the present invention, including a description of its extracellular fully human CD20 / CD19 antigen binding domain, transmembrane domain and intracellular domain, as well as additional descriptions of CAR, antibodies and antigen binding fragments thereof, conjugates, nucleotides, expression, vectors and host cells, treatment methods, compositions and kits for using the disclosed CAR.

[0098] In each of the foregoing aspects and embodiments described above, for example, ScFv binders to mesothelin have been produced, as disclosed in U.S. Pat. No. 10,183,993, issued to the applicant, entitled “Compositions and Methods for Treating Cancer with Anti-Mesothelin Immunotherapy” and assigned to Lentigen Technology, Inc. (Event No. LEN_017), nucleotide sequence scFv antigen SEQ ID NO: 149 and amino acid sequence SEQ ID NO: 150, respectively, which can be incorporated into a functional CAR (which is nucleotide sequence SEQ ID NO: 39 and amino acid sequence SEQ ID NO: 40, respectively) and thus can be incorporated into DuoCAR therapy.

[0099] In each of the aforementioned aspects and embodiments described above, in addition to the scFv sequence, a single-chain antigen binder (as opposed to scFv) can be incorporated into a single, tandem, DuoCAR or multi-targeted CAR application. For example, as disclosed in U.S. Pat. No. 10,426,797 entitled "Compositions and Methods For Treating Cancer With Anti-CD33 Immunotherapy" and assigned to the applicant of Lentigen Technology, Inc. (Event No. LEN_018), a CD33-specific heavy chain-only binder (nucleotide sequence SEQ ID NO: 41 and amino acid sequence SEQ ID NO: 42, respectively) can be incorporated into a functional CAR targeting a malignant disease expressing CD33, LTG1906 (which is a nucleotide sequence SEQ ID NO: 43 and an amino acid sequence SEQ ID NO: 44, respectively).

[0100] In each of the aforementioned aspects and embodiments described above, an example of a single, tandem, DuoCAR or multi-targeted CAR therapeutic application is the treatment of leukemias expressing CD19, CD20 and TSLPR antigens. In this case, LTG1496 or LTGI497 (SEQ ID NO: 35, 26, respectively) can be combined with TSLPR-specific CAR (LTG1789) (SEQ ID NO: 47 and amino acid sequence SEQ ID NO: 48, respectively), which is produced by TSLPR-specific scFV domains (nucleotide sequence SEQ ID NO: 45 and amino acid sequence SEQ ID NO: 46).

[0101] In each of the aforementioned aspects and embodiments described above, another example of a single, tandem, DuoCAR or multi-targeted CAR therapeutic application is to treat cancer expressing CD38 antigen. For example, as disclosed in the authorized U.S. Patent No. 11,103,533 of the applicant entitled "Compositions and Methods For Treating Cancer With Anti-CD38 Immunotherapy" filed on November 30, 2018 and transferred to Lentigen Technology, Inc. (Event No. LEN_026), the CD38-specific binding agent can be incorporated into one or more functional CARs targeting malignancies expressing CD38, as disclosed in the authorized U.S. Patent No. 11,103,533 of the applicant, the entire contents of which are incorporated herein by reference.

[0102] In each of the aforementioned aspects and embodiments described above, another example of single, tandem, DuoCAR or multi-targeted CAR therapeutic applications is to treat cancer expressing CD123 antigens. For example, as entitled "Compositions and Methods For Treating Cancer With Anti-CD123 Immunotherapy" submitted on September 20, 2019 and transferred to Lentigen Technology, Inc. (Event Number LEN 024) and requiring the provisional patent application No. 62 / 734,106 filed on September 20, 2018 The applicant's authorized U.S. Patent No. 10,844,128 of the priority of the applicant may be incorporated into one or more functional CARs of malignant diseases targeting expression of CD123, as disclosed in the authorized U.S. Patent No. 10,844,128 of the applicant, the entire contents of which are incorporated herein by reference.

[0103] In each of the aforementioned aspects and embodiments described above, another example of a single, tandem, DuoCAR or multi-targeted CAR therapeutic application is to treat cancer expressing CD 123 antigens. For example, as disclosed in the U.S. co-pending patent application No. 17 / 685,132 of the applicant entitled "Compositions and Methods For Treating Cancer With Anti-CD123 Immunotherapy" filed on March 2, 2022 and transferred to Lentigen Technology, Inc. (Event No. MBG 99), CD123 specific binding agents may be incorporated into one or more functional CARs targeting malignancies expressing CD123, as disclosed in the applicant's co-pending U.S. patent application No. 17 / 685,132, the entire contents of which are incorporated herein by reference.

[0104] In each of the aforementioned aspects and embodiments described above, another example of a single, tandem, DuoCAR or multi-targeted CAR therapeutic application is to treat cancers expressing BCMA antigens. For example, as disclosed in the U.S. Patent No. 11,052,112 of the applicant's authorization, entitled "Fully Human BCMACAR T Cells for the Treatment of Multiple Myeloma and Other BCMA-Positive Malignancies" filed on May 30, 2019 and transferred to Lentigen Technology, Inc. (Event No. MBG_13), the BCMA-specific binding agent can be incorporated into one or more functional CARs targeting malignancies expressing BCMA, as disclosed in the U.S. Patent No. 11,052,112 of the applicant's authorization, the entire contents of which are incorporated herein by reference.

[0105] In each of the aforementioned aspects and embodiments described above, another example of a single, tandem, DuoCAR or multi-targeted CAR therapeutic application is the treatment of solid tumor cancer. For example, as disclosed in the co-pending U.S. provisional patent application No. 63 / 393,088 of the applicant entitled "ChimericAntigen Receptor Therapies for Treating Solid Tumors" and assigned to LentigenTechnology, Inc (Event No. MBG_106) filed on July 28, 2022, specific binding agents may be incorporated into one or more functional CARs targeting solid tumors, as disclosed in the co-pending U.S. provisional patent application No. 63 / 393,088 of the applicant, the entire contents of which are incorporated herein by reference.

[0106] In each of the aforementioned aspects and embodiments described above, the tandem CAR (comprising 2 scFv domains, such as described in nucleotide sequence SEQ ID NO: 23 and amino acid sequence SEQ ID NO: 24) on which the technology is based includes CD20_CD19 CAR LTG1497, which is a nucleotide sequence SEQ ID NO: 25 and an amino acid sequence SEQ ID NO: 26. In some cases, reversing the order of the two binding agents can provide better DuoCAR expression in target cells. Therefore, LTG1497 as shown in nucleotide sequence SEQ ID NO: 25 and amino acid sequence SEQ ID NO: 26 (wherein CD19 scFv is closer to the membrane) and LTG1496 as shown in nucleotide sequence SEQ ID NO: 33 and amino acid sequence SEQ ID NO: 34 (wherein CD19 scFV is further away from the membrane) can both be used as one of the members of the DuoSet comprising DuoCAR.

[0107] A. Chimeric Antigen Receptor (CAR)

[0108] The CAR disclosed herein comprises 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.

[0109] Chimeric antigen receptor (CAR) is an artificially constructed hybrid protein or polypeptide, which comprises an antigen binding domain (e.g., single-chain variable fragment (ScFv)) of an antibody connected to a T cell signaling domain via a transmembrane domain. The characteristics of CAR include its ability to redirect T cell specificity and reactivity toward a selected target in a non-MHC restricted manner, as well as the antigen binding properties of monoclonal antibodies. Non-MHC restricted antigen recognition gives CAR-expressing T cells the ability to recognize antigens independently of antigen processing, thus bypassing the main tumor escape mechanism. In addition, when expressed in T cells, CAR advantageously does not dimerize with endogenous T cell receptor (TCR) α and β chains.

[0110] As disclosed herein, the intracellular T cell signaling domain of a CAR may include, for example, a T cell receptor signaling domain, a T cell co-stimulatory signaling domain, or both. A T cell receptor signaling domain refers to a portion of a CAR comprising an intracellular domain of a T cell receptor (e.g., such as, but not limited to, an intracellular portion of a CD3 ζ protein). A co-stimulatory signaling domain refers to a portion of a CAR comprising an intracellular domain of a co-stimulatory molecule, which is a cell surface molecule other than an antigen receptor or its ligand required for an effective response of a lymphocyte to an antigen.

[0111] 1. Extracellular domain

[0112] In one embodiment, CAR comprises a target-specific binding element, which is also referred to as an antigen binding domain or portion. The selection of the domain depends on the type and number of ligands that define the surface of the target cell. For example, an antigen binding domain can be selected to identify ligands that act as cell surface markers on target cells associated with a specific disease state. Therefore, some examples of cell surface markers that can act as ligands for antigen binding domains in CAR include those associated with viruses, bacteria and parasitic infections, autoimmune diseases and cancer cells.

[0113] In one embodiment, CAR can be modified to target tumor antigens by modifying the desired antigen binding domain that specifically binds to the antigen on the tumor cell. Tumor antigens are proteins produced by tumor cells that cause immune responses (especially T cell-mediated immune responses). The choice of antigen binding domain will depend on the specific type of cancer to be treated. Tumor antigens include, for example, glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostate enzyme, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, Her2 / neu, survivin and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF-1), prostate-specific antigen (PSA ... In some embodiments, the present invention relates to tumor antigens, such as glioma cell lineage, ...

[0114] In one embodiment, tumor antigens include one or more antigenic cancer epitopes associated with malignant tumors. Malignant tumors express many 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 oncogenes HER-2 / Neu / ErbB-2. Another group of target antigens is onco-fetal antigen (onco-fetal antigen), such as carcinoembryonic antigen (CEA). In B cell lymphoma, tumor-specific idiotypic immunoglobulins constitute the unique real tumor-specific immunoglobulin antigens of individual tumors. B cell differentiation antigens (such as CD19, CD20, CD22, BCMA, ROR1 and CD37) are other candidates for target antigens in B cell lymphoma. Some of these antigens (CEA, HER-2, CD19, CD20, idiotype) have been used as targets for passive immunotherapy with monoclonal antibodies with limited success.

[0115] 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 cancer and pancreatic cancer expressing high levels of extracellular protein fully human CD20 / CD19, or any combination thereof.

[0116] The type of tumor antigen may also be a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). TSAs are unique to tumor cells and do not appear on other cells in the body. TAAs are not unique to tumor cells and are instead expressed on normal cells under conditions where a state of immune tolerance to the antigen cannot be induced. Expression of an antigen on a tumor may occur under conditions that enable the immune system to respond to the antigen. A TAA may be an antigen that is expressed on normal cells during fetal development when the immune system is immature and unable to respond, or it may be an antigen that is normally present at very low levels on normal cells but is expressed at much higher levels on tumor cells.

[0117] Some non-limiting examples of TSAs or TAAs include the following: differentiation antigens, such as MART-1 / MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2; and tumor-specific multi-lineage 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 caused by 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, CA19-9, CA72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, α-fetoprotein, β-HCG, BCA225, BTAA, CA125, CA15-3\CA27.29\BCAA, CA195, CA242, 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.

[0118] In one embodiment, the antigen binding domain portion of CAR targets antigens including but not limited to the following: CD19, CD20, CD22, ROR1, CD33, CD38, CD123, CD138, BCMA, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, FGFR4, TSLPR, NY-ESO-1TCR, MAGE A3 TCR, etc.

[0119] In a preferred embodiment, the antigen binding domain portion of the CAR targets the extracellular CD20 / CD19 antigen.

[0120] In a preferred embodiment, the antigen binding domain portion of the CAR targets the extracellular CD20 antigen.

[0121] 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 having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto. 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 having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0122] In a preferred embodiment, the antigen binding domain portion of the CAR targets the extracellular CD19 antigen.

[0123] 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 having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto. 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 having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0124] In a preferred embodiment, the antigen binding domain portion of the CAR targets the extracellular CD20 antigen and the CD19 antigen.

[0125] In a 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 having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto. 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 having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0126] In the various embodiments of the fully human CD20 / CD19-specific multi-targeted, tandem and bicistronic CARs disclosed herein, the general scheme is as follows Figure 1AAs shown, and from the N-terminus to the C-terminus, it contains a CAR targeting domain, a transmembrane domain, a co-stimulatory domain, and a CD3ζ activation domain. In the tandem CAR, all-human tandem CD20 and CD19 targeting domains are included. For the bicistronic CAR, the first CD20 targeting CAR contains a full-human CD20 targeting domain, and next is the second CD19 targeting CAR in the frame, which consists of a CD19 targeting domain, and a ribosomal skipping element is inserted between the CD20 CAR and CD19 CAR sequences to promote co-expression.

[0127] In one embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO:5(D0144)(CD20_CD19CD8BBz), and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO:6.

[0128] In one embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 5, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and encodes a CAR (D0144) (CD20_CD19 CD8 BBz) comprising an amino acid sequence as shown in SEQ ID NO: 6, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0129] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO:7 (CAR D0255) (CD20_CD19 CD28 CD28 BBz), and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO:8 (CAR D0255) (CD20_CD19CD28 CD28 BBz).

[0130] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO:7, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof, and encodes a CAR (CAR D0255) (CD20_CD19 CD28 CD28 BBz) comprising the amino acid sequence shown in SEQ ID NO:8, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof.

[0131] In one embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO:9 (CAR D0256) (CD20_CD19 CD8 CD28 BBz), and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO:10.

[0132] In one embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 9, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and encodes a CAR (CAR D0256) (CD20_CD19 CD8 CD28 BBz) comprising the amino acid sequence as shown in SEQ ID NO: 10, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0133] In one embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 13 (CAR D0257) (CD20CD8 CD28z_CD19 CD8 BBz), and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO: 14.

[0134] In one embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 13, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof, and encodes a CAR (CAR D0257) (CD20 CD8 CD28z_CD19 CD8 BBz) comprising the amino acid sequence as shown in SEQ ID NO: 14, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof.

[0135] In one embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 15 (CAR D0258) (CD20CD8 BBz_CD19 CD8 CD28z), and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO: 16.

[0136] In one embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 15, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof, and encodes a CAR (CAR D0258) (CD20 CD8 BBz_CD19 CD8 CD28z) comprising the amino acid sequence as shown in SEQ ID NO: 16, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof.

[0137] In one embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 17 (CAR D0266) (CD20CD8 OX40 OX40z_CD19 CD8 ICOSz), and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO: 18.

[0138] In one embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 17, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof, and encodes a CAR (CAR D0266) (CD20 CD8 OX40 OX40z_CD19 CD8 ICOSz) comprising the amino acid sequence as shown in SEQ ID NO: 18, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof.

[0139] In one embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 19 (CAR LTG1497) (mCD20_CD19 CD8 BBz), and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO: 20.

[0140] In one embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 19, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and encodes a CAR (CAR LTG1497) (mCD20_CD19 CD8 BBz) comprising the amino acid sequence as shown in SEQ ID NO: 20, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0141] Surface expression of anti-CD20 / CD19 CARs incorporating single-chain fragment variable (ScFv) sequences reactive with fully human CD20 and CD19 antigens is described in Example 1 below. Figure 1BShown in. The expression level of each ScFv-containing CAR was determined by using CD19 Fc recombinant protein, followed by flow cytometry analysis of LV-transduced T cells from healthy donors using anti-Fc AF647 (APC). Compared to untransduced T cell controls, ScFv-based anti-CD20 / CD19 CAR constructs D0144, D0255, D0256, D0257, D0258, D0266 and comparative constructs LTG1497 containing mouse CD20 / CD19 scFv tandem targeting domains were highly expressed in human primary T cells (in both CD4+T and CD8+T lineages) (ranging from 49.7% to 78.6% CAR+ of total T cells).

[0142] As in Example 1 and Figures 2A to 2D As shown, it is shown that the high cell lysis activity of all-human CD20 / CD19 CAR.Human primary T cells are transduced with LV encoding CAR constructs (D0144, D0255, D0256, D0257, D0258, D0266 and comparison constructs LTG1497, see method), and then incubated with Raji, NALM-6, REH or 293T cell lines stably transduced with firefly luciferase for 18 hours for in vitro killing assays based on luminescence. All leukemia lines tested express CD19 on their surfaces, while negative control 293T is not. CD20 expression varies between tumor lines. Raji is CD20 positive, while REH is CD20 negative, as is control line 293T. NALM-6 has weak but detectable CD20 expression.

[0143] All tumor lines expressing CD19 and / or CD20 were lysed by CAR T cells, whereas the CD19-negative CD20-negative line 293T did not undergo lysis.

[0144] The most efficient and specific lysis of Raji cells was mediated by CAR constructs D0255 and D0266, as observed by lysis at the lowest effector to target ratio of 2.5:1 ( Figure 2A When combined with the target lines NALM 6 and Reh, all CARs achieved potent and comparable lysis ( Figure 2B , 2C).

[0145] The cytokine secretion capacity of anti-CD20 / CD19 CAR T cells was then evaluated. Tumor cells were co-incubated with CAR T cells or control T cells at an effector to target ratio of 10:1 overnight (+Raji group), and the culture supernatants were analyzed for IFNγ, TNFα, and IL-2 by ELISA (see Figures 3A to 3C). In the absence of target, 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, while the negative control -Raji did not produce significant cytokine induction. All CARs strongly induced IFNγ, TNFα, and IL-2 compared to the negative control.

[0146] The highest levels of TNF-α were produced by CARs D0255, D0257, D0258, and D0266, and the production of IFN-γ and IL-2 followed a similar pattern ( Figures 3A to 3C ).

[0147] To determine how the fully human CD20 / CD19 CRA candidate performs during chronic antigen exposure, we performed co-culture 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, and after 7 days, CAR T cells were restimulated with Raji cells at an effector to target ratio of 0.15:1 until study day 10 ( Figure 4A This setting established an extremely challenging environment for CAR T cell function in order to pinpoint even the smallest functional differences between CAR T candidate constructs. Cell cultures were acquired by flow cytometry on days 0, 7, and 10 to evaluate the percentage of viable target cells remaining at the end of each co-incubation period, as well as CAR T cell expansion ( Figures 4B to 4C The three CAR T cells that mediated the greatest reduction in Raji target cell levels at the end of the second co-incubation cycle were D0255, D0257, and D0266 ( Figure 4B ). The CAR T cells with the best expansion were LTG1497, D0255, and D0144 ( Figure 4C ). Thus, we have identified a number of CAR T cell candidates with potent cytotoxic and expansion properties compared to other constructs in this panel and the LTG1497 comparator.

[0148] To characterize the functional properties of CAR T cells in vivo, we treated NSG mice bearing established Raji xenograft tumors with a low dose of two million fully human CD20 / CD19 CAR T cells D0255, D0256, D0266, D0257, or D0258, or the murine scFv CD20 / CD19 CAR comparator LTGI497. Figure 5A In mice treated with all CAR t constructs, tumors were rejected by day 13, and mice remained in remission by day 55 ( Figure 5BIn contrast, mice in the control group with only tumors and UTD developed high tumor burden ( Figure 5B ). On the 55th day, mice in all groups were re-attacked with one million additional Raji cells, and no additional CAR T treatment was applied. One mouse in each group of LTG1497, D0256, D0258 and two mice in each group of D0255 and D0266 did not meet the re-attack criteria and were not recruited. At this time, a new, age-matched group of mice was also inoculated with Raji cells as a control. Tumor progression was followed up until the 96th day. On the 96th day, in CAR groups D0255 and D0258, all recruited mice survived and had no tumors, lost one mouse in each group of D0257, D0256, LTG1497, and lost three mice in group D0266. Therefore, for all CAR constructs, the ability to clear the initial xenograft tumor in vivo was demonstrated, and for constructs D0255, D0258 and LTG1497, the persistence of CAR T cell anti-tumor function after tumor re-attack was demonstrated ( Figure 5C ). Therefore, the emerging ranking of CAR T candidates in terms of their antitumor utility must be determined empirically and cannot be predicted based solely on CAR structure or targeting domain composition.

[0149] Without intending to be limited to any particular mechanism of action, possible reasons for the enhanced therapeutic function associated with the exemplary CARs of the present invention are believed to include, for example, but not limited to: a) improved lateral movement within the plasma membrane allowing more efficient signal transduction, b) superior location within the plasma membrane microdomains (e.g., lipid rafts), and greater ability to interact with transmembrane signaling cascades associated with T cell activation, c) superior location within the plasma membrane by preferentially moving away from inhibitory (dampening) or downregulatory interactions (e.g., lower proximity or interaction with phosphatases (e.g., CD45)), and d) superior assembly into T cell receptor signaling complexes (i.e., immune synapses), or any combination thereof.

[0150] Although the present disclosure has been exemplified with 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 can be used to derive the fully human CD20 / CD19 antigen binding domains used in the CARs described herein.

[0151] Depending on the desired antigen to be targeted, CAR can be additionally engineered to include a suitable antigen binding domain specific to the desired antigen target. For example, if full human CD20 / CD19 is the desired antigen to be targeted, antibodies against full human CD20 / CD19 can be used as the antigen binding domain incorporated into CAR.

[0152] In one aspect of the present invention, a CAR capable of binding to a non-TSA or non-TAA is provided, wherein the non-TSA or non-TAA includes, for example, but not limited to, an antigen derived from the following: Retroviridae (e.g., human immunodeficiency virus, such as 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, Herpes Viridae [e.g., Herpes simplex virus type 1 and type 2 (HSV), varicella-zoster virus, cytomegalovirus (CMV) and herpes virus], Poxviridae (e.g., smallpox virus, vaccinia virus and poxvirus) or Hepatitis C virus, or any combination thereof.

[0153] In another aspect of the present invention, a CAR is provided that is capable of binding to an antigen derived from the following bacterial strains: Staphylococci, Streptococcus, Escherichia coli, Pseudomonas, or Salmonella. In particular, CARs are provided that can bind to antigens derived from infectious bacteria, such as Helicobacter pyloris, Legionella pneumophilia, strains 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 pyogenes, and Streptococcus pyogenes. agalactiae), Streptococcus pneumoniae or Clostridium tetani, or a combination thereof.

[0154] 2. Transmembrane domain

[0155] With respect to transmembrane domains, the CAR comprises one or more transmembrane domains fused to the extracellular CD19 / CD20 antigen binding domain of the CAR.

[0156] The transmembrane domain may be derived from natural or synthetic sources. When the source is natural, the domain may be derived from any membrane-bound or transmembrane protein.

[0157] The transmembrane region particularly used for CAR described herein may be derived from the following (ie, at least comprising the following transmembrane region): α, β or ζ chain of T cell receptor, CD28, CD3ε, 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 may be synthetic, in which case it will mainly comprise hydrophobic residues, such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan and valine will be present at each end of the synthetic transmembrane domain. Optionally, a short oligopeptide linker or polypeptide linker (preferably 2 to 10 amino acids in length) may form a connection between the transmembrane domain of CAR and the cytoplasmic signaling domain. Glycine-serine doublets provide particularly suitable linkers.

[0158] In one embodiment, in addition to the above-mentioned transmembrane domains, a transmembrane domain that is naturally associated with one of the domains in the CAR is used.

[0159] In some cases, transmembrane domains may be selected or amino acid substitutions may be made to prevent such domains from binding to transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

[0160] 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 a nucleic acid sequence of SEQ ID NO: 35. In one embodiment, the CD8 transmembrane domain comprises a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO: 36. In another embodiment, the CD8 transmembrane domain comprises an amino acid sequence of SEQ ID NO: 36.

[0161] In one embodiment, the encoded transmembrane domain comprises an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 20, 10 or 5 modifications (e.g., substitutions) of an amino acid sequence of SEQ ID NO: 34, 36 or 48, or a sequence with 95% to 99% identity to an amino acid sequence of SEQ ID NO: 34, 36 or 48.

[0162] In some cases, the transmembrane domain of CAR comprises a CD8.α. hinge domain. In one embodiment, the CD8 hinge domain comprises a nucleic acid sequence of SEQ ID NO:37. In one embodiment, the CD8 hinge domain comprises a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO:38. In another embodiment, the CD8 hinge domain comprises an amino acid sequence of SEQ ID NO:38, or a sequence having 95% to 99% identity thereto.

[0163] 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.

[0164] 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 a nucleic acid sequence of SEQ ID NO: 47. In one embodiment, the CD28 transmembrane domain comprises a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO: 48. In another embodiment, the CD28 transmembrane domain comprises an amino acid sequence of SEQ ID NO: 48.

[0165] In one embodiment, the encoded transmembrane domain comprises an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 20, 10 or 5 modifications (e.g., substitutions) of an amino acid sequence of SEQ ID NO: 47, or a sequence having 95% to 99% identity to an amino acid sequence of SEQ ID NO: 48.

[0166] 3. Spacer domain

[0167] In CAR, a spacer domain may be arranged between the extracellular domain and the transmembrane domain, or between the intracellular domain and the transmembrane domain. The spacer domain means any oligopeptide or polypeptide for connecting the transmembrane domain to the extracellular domain and / or the transmembrane domain to the intracellular domain. The spacer domain comprises up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids.

[0168] In several embodiments, the linker may include a spacer element that, when present, increases the size of the linker such that the distance between the effector molecule or detectable marker and the antibody or antigen-binding fragment is increased. Exemplary spacers are known to those of ordinary skill and include those in U.S. Pat. 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 , 5,521,284, 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, and those listed in U.S. Patent Publication Nos. 20110212088 and 20110070248, each of which is incorporated herein by reference in its entirety.

[0169] The spacer domain preferably has a sequence that promotes CAR binding to the antigen and enhances signal transduction into the cell. Some examples of amino acids that are expected 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 constituting the spacer domain.

[0170] As the spacer domain, all or part of the following can be used: amino acids 137 to 206 (which is the hinge region) of CD8.α (NCBI RefSeq: NP.sub.--001759.3) (SEQ ID NO: 39), amino acids 135 to 195 of CD8.β (GenBank: AAA35664.1), amino acids 315 to 396 of CD4 (NCBI RefSeq: NP.sub.--000607.1), or amino acids 137 to 152 of CD28 (NCBI RefSeq: NP.sub.--006130.1). In addition, as the spacer domain, a part of the constant region of the antibody H chain or L chain can be used. In addition, the spacer domain can be an artificially synthesized sequence.

[0171] In some cases, the transmembrane domain of the CAR comprises a CD28 hinge domain. In one embodiment, the CD28 hinge domain comprises a nucleic acid sequence of SEQ ID NO: 31. In one embodiment, the CD28 hinge domain comprises a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO: 32. In another embodiment, the CD28 hinge domain comprises an amino acid sequence of SEQ ID NO: 32 or a sequence having 95% to 99% identity thereto.

[0172] 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 the transmembrane CD28 domain, the transmembrane CD28 domain, or a combination thereof.

[0173] In addition, in CAR, the signal peptide sequence can be connected to the N-terminus. The signal peptide sequence is present at the N-terminus of many secretory proteins and membrane proteins and is 15 to 30 amino acids in length. Since many of the protein molecules mentioned above as intracellular domains have signal peptide sequences, these signal peptides can be used as signal peptides of CAR. In one embodiment, the signal peptide comprises the amino acid sequence shown in SEQ ID NO: 12.

[0174] 4. Intracellular domain

[0175] The cytoplasmic domain or other intracellular signaling domain of the CAR is responsible for activating at least one normal effector function of the immune cell into which the CAR has been placed. The term "effector function" refers to a specialized function of a cell. For example, the effector function of a T cell may be a cell lytic activity or an auxiliary activity, including the secretion of cytokines. Therefore, the term "intracellular signaling domain" refers to a portion of a protein that transduces an effector function signal and directs the cell to perform a specialized function. Although the entire intracellular signaling domain can usually be used, the entire chain does not have to be used in many cases. In terms of using a truncated portion of an intracellular signaling domain, such a truncated portion can be used instead of a complete chain as long as it transduces an effector function signal. Therefore, the term intracellular signaling domain is intended to include any truncated portion of an intracellular signaling domain sufficient to transduce an effector function signal.

[0176] Some preferred examples of intracellular signaling domains for CAR include cytoplasmic sequences of T cell receptors (TCRs) and co-receptors, which function together to initiate signal transduction after antigen receptor engagement; and any derivatives or variants of these sequences and any synthetic sequences having the same functional capabilities.

[0177] It is known that the signal generated by TCR alone is insufficient to fully activate T cells and that secondary or co-stimulatory signals are also required. Therefore, it can be said that T cell activation is mediated by two different types of cytoplasmic signaling sequences: those that trigger antigen-dependent primary activation through TCR (primary cytoplasmic signaling sequences) and those that act in an antigen-independent manner to provide secondary or co-stimulatory signals (secondary cytoplasmic signaling sequences).

[0178] The primary cytoplasmic signaling sequence regulates the primary activation of the TCR complex in a stimulatory manner or in an inhibitory manner. The primary cytoplasmic signaling sequence that acts in a stimulatory manner may comprise a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM.

[0179] Some examples of ITAMs comprising primary cytoplasmic signaling sequences that are particularly useful in the CARs disclosed herein include those derived from: TCRζ (CD3ζ), FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. Some specific non-limiting examples of ITAMs include peptides having the following sequences: amino acids 51 to 164 of CD3.ζ. (NCBI RefSeq: NP.sub.--932170.1), amino acids 45 to 86 of Fc.ε.RI.γ. (NCBI RefSeq: NP.sub.--004097.1), amino acids 201 to 244 of Fc.ε.RI.β. (NCBI RefSeq: NP.sub.--000130.1), amino acids 139 to 182 of CD3.γ. (NCBI RefSeq: NP.sub.--000064.1), amino acids 128 to 171 of CD3.δ. (NCBI RefSeq: NP.sub.--000723.1), amino acids 140 to 145 of CD3.ε. (NCBI RefSeq: NP.sub.--00064.1). RefSeq: NP.sub.--000724.1), amino acids 153 to 207 of CD5 (NCBI RefSeq: NP.sub.--055022.2), amino acids 707 to 847 of 0022 (NCBI RefSeq: NP.sub.--001762.2), amino acids 166 to 226 of CD79a (NCBI RefSeq: NP.sub.--001774.1), amino acids 182 to 229 of CD79b (NCBI RefSeq: NP.sub.--000617.1), and amino acids 177 to 252 of CD66d (NCBI RefSeq: NP.sub.--001806.2), and variants thereof having the same functions as these peptides. The amino acid number based on the amino acid sequence information of the NCBI RefSeq ID or GenBank described herein is 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 CD3ζ.

[0180] In a preferred embodiment, the intracellular domain of CAR can be designed to include the CD3-ζ signaling domain itself, or it can be combined with any other desired cytoplasmic domain available in the case of CAR. For example, the intracellular domain of CAR may include a CD3ζ chain portion and a costimulatory signaling region. The costimulatory signaling region refers to a part of a CAR that includes an intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands required for the effective response of lymphocytes to antigens. Some examples of such costimulatory molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (lymphocyte function-associated antigen-1, LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83, etc. Some specific non-limiting examples of such costimulatory molecules include peptides having the following sequences: amino acids 236 to 351 of CD2 (NCBI RefSeq: NP.sub.--001758.2), amino acids 421 to 458 of CD4 (NCBI RefSeq: NP.sub.--000607.1), amino acids 402 to 495 of CD5 (NCBI RefSeq: NP.sub.--055022.2), amino acids 207 to 235 of CD8.α. (NCBI RefSeq: NP.sub.--001759.3), amino acids 196 to 210 of CD83 (GenBank: AAA35664.1), amino acids 181 to 220 of CD28 (NCBI RefSeq: NP.sub.--006130.1), amino acids 181 to 220 of CD137 (4-1BB, NCBI RefSeq: NP.sub.--001552.2) amino acids 214 to 255, amino acids 241 to 277 of CD134 (OX40, NCBI RefSeq: NP.sub.--003318.1) and amino acids 166 to 199 of ICOS (NCBI RefSeq: NP.sub.--036224.1), and variants thereof having the same functions as these peptides. Therefore, although the disclosure herein is mainly illustrated by 4-1BB as a costimulatory signaling element, other costimulatory elements are also within the scope of the disclosure.

[0181] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of CAR can be interconnected in a random or specific order. Optionally, a short oligopeptide linker or polypeptide linker (preferably 2 to 10 amino acids in length) can form a connection. Glycine-serine doublets provide particularly suitable linkers.

[0182] In one embodiment, the intracellular domain is designed to include the signaling domain of CD3-ζ and the signaling domain of CD28. In another embodiment, the intracellular domain is designed to include the signaling domain of CD3-ζ and the signaling domain of 4-1BB. In another embodiment, the intracellular domain is designed to include the signaling domain of CD3-ζ and the signaling domain of CD28 and 4-1BB.

[0183] In one embodiment, the intracellular domain in the CAR is designed to include the signaling domain of 4-1BB and the signaling domain of CD3-ζ, wherein the signaling domain of 4-1BB includes the nucleic acid sequence shown in SEQ ID NO:42, 44 or 45 and the signaling domain of CD3-ζ includes the nucleic acid sequence shown in SEQ ID NO:43.

[0184] 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-ζ, wherein the signaling domain of CD28 comprises the nucleic acid sequence shown in SEQ ID NO:21, and the signaling domain of CD3-ζ comprises the nucleic acid sequence shown in SEQ ID NO:42, 45 or 46.

[0185] In another embodiment, the intracellular domain in the CAR is designed to include a linked signaling domain of CD28_4-1BB and a signaling domain of CD3-ζ, wherein the signaling domain of CD28_4-1BB comprises the nucleic acid sequence shown in SEQ ID NO: 25, and the signaling domain of CD3-ζ comprises the nucleic acid sequence shown in SEQ ID NO: 42, 45 or 46.

[0186] 5. Additional description of CAR

[0187] The functional part of CAR disclosed herein is also clearly included in the scope of the present invention.When referring to CAR use, the term "functional part" refers to any part or fragment of one or more CAR disclosed herein, and the part or fragment retains the biological activity of the CAR (parent CAR) as part thereof. The functional part covers, for example, those CARs that retain the ability to recognize target cells or detect, treat or prevent diseases to a similar degree or to the same degree or to a higher degree as the parent CAR. Depending on the parent CAR, the functional part may include, for example, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95% or more of the parent CAR.

[0188] The functional part may include additional amino acids at the amino or carboxyl end of the part or at both ends, and the additional amino acids are not present in the amino acid sequence of the parent CAR. Desirably, the additional amino acids do not interfere with the biological function of the functional part, such as identifying target cells, detecting cancer, treating or preventing cancer, etc. More desirably, the additional amino acids enhance the biological activity compared to the biological activity of the parent CAR.

[0189] The scope of the present disclosure includes functional variants of CAR disclosed herein. The term "functional variant" used herein refers to a CAR, polypeptide or protein having substantial or significant sequence identity or similarity with a parent CAR, and the functional variant retains the biological activity of the CAR as a variant. Functional variants encompass, for example, those 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 as the parent CAR. According to the parent CAR, the functional variant may, for example, have at least about 30%, 50%, 75%, 80%, 90%, 98% or more identity with the parent CAR in terms of amino acid sequence.

[0190] Functional variants may, for example, comprise the amino acid sequence of a parent CAR having at least one conservative amino acid substitution. As an alternative or supplement, functional variants may comprise the amino acid sequence of a parent CAR having at least one non-conservative amino acid substitution. In this case, it is preferred that non-conservative amino acid substitutions do not interfere with or inhibit the biological activity of functional variants. Non-conservative amino acid substitutions can enhance the biological activity of functional variants so that the biological activity of functional variants is improved compared to parent CAR.

[0191] The amino acid substitutions of 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 having certain physical and / or chemical properties is exchanged for another amino acid having the same or similar chemical or physical properties. For example, conservative amino acid substitutions can be acidic / negatively charged polar amino acids replacing another acidic / negatively charged polar amino acid (e.g., Asp or Glu), amino acids having non-polar side chains replacing another amino acid having non-polar side chains (e.g., Ala, Gly, Val, He, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), basic / positively charged polar amino acids replacing another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.), uncharged amino acids having polar side chains replacing another uncharged amino acids having polar side chains (e.g., Asn, Gin, Ser, Thr, Tyr, etc.), amino acids having β-branched side chains replacing another amino acid having β-branched side chains (e.g., He, Thr and Val), amino acids having aromatic side chains replacing another amino acid having aromatic side chains (e.g., His, Phe, Trp and Tyr), etc.

[0192] The CAR may consist essentially of one or more specific amino acid sequences described herein, such that other components (e.g., other amino acids) do not substantially alter the biological activity of the functional variant.

[0193] CAR (including functional parts and functional variants) can have any length, that is, can contain any number of amino acids, provided that CAR (or its functional part or functional variant) retains its biological activity, for example, 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, the length of CAR can be about 50 to about 5000 amino acids, for example, 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more amino acids in length.

[0194] CAR (including functional parts and functional variants of the present invention) may include synthetic amino acids to replace 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-carboxylphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, Indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, -aminocyclopentanecarboxylic acid, a-aminocyclohexanecarboxylic acid, a-aminocycloheptanecarboxylic acid, a-(2-amino-2-norbornane)-carboxylic acid, gamma-diaminobutyric acid, beta-diaminopropionic acid, homophenylalanine and a-tert-butylglycine.

[0195] CAR (including functional parts and functional variants) can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized (via, for example, a disulfide bridge) or converted into an acid addition salt and / or optionally dimerized or multimerized or conjugated.

[0196] CAR (including functional parts and functional variants thereof) can be obtained by methods known in the art. CAR can be prepared by any suitable method for preparing a polypeptide or protein. Suitable methods for synthesizing polypeptides and proteins from scratch 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, ed. Reid, R., Marcel Dekker, Inc., 2000; Epitope Mapping, ed. Westwood et al., Oxford University Press, Oxford, United Kingdom, 2001; and U.S. Patent No. 5,449,752. In addition, the nucleic acids described herein can be used to recombinantly produce polypeptides and proteins using standard recombinant methods. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. In addition, some CARs (including functional parts and functional variants thereof) can be separated and / or purified from sources such as plants, bacteria, insects, mammals (e.g., rats, humans, etc.). Methods for separation and purification are well known in the art. Alternatively, the CARs described herein (including functional parts and functional variants thereof) can be commercially synthesized by a company. In this regard, CARs can be synthetic, recombinant, separated and / or purified.

[0197] B. Antibodies and Antigen-binding Fragments

[0198] One embodiment also provides a CAR, a T cell expressing CAR, an antibody, or an antigen binding domain or part thereof that specifically binds to one or more antigens disclosed herein. "CAR-expressing T cell" or "CART cell" used herein means a T cell expressing CAR, and has an antigen specificity determined by a targeting domain derived from, for example, an antibody of CAR.

[0199] As used herein, "antigen binding domain" may include antibodies and antigen binding fragments thereof. The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) and antigen binding fragments thereof, as long as they exhibit the desired antigen binding activity. Some non-limiting examples of antibodies include, for example, intact immunoglobulins and variants and fragments thereof known in the art that retain binding affinity for an antigen.

[0200] "Monoclonal antibody" is an antibody obtained from a group of substantially homogeneous antibodies, that is, except for naturally occurring mutations that may be present in small amounts, the individual antibodies comprising the group are identical. Monoclonal antibodies are highly specific, directed against a single antigenic epitope. The modifier "monoclonal" represents the characteristics of an antibody such as obtained from a substantially homogeneous antibody group, and is not interpreted as requiring the production of antibodies by any particular method. In some instances, a monoclonal antibody is an antibody produced by a monoclonal clone of a B lymphocyte or a cell or its progeny transfected with nucleic acid encoding a light chain and a heavy chain variable region of an antibody (or its antigen-binding fragment). In some instances, a monoclonal antibody is isolated from an object. A monoclonal antibody may have a conservative amino acid substitution that has substantially no effect on antigen binding or other immunoglobulin functions. Exemplary methods for producing monoclonal antibodies are known, for example, referring to Harlow & Lane, Antibodies, A Laboratory Manual, 2nd edition. Cold Spring Harbor Publications, New York (2013).

[0201] Generally speaking, immunoglobulins have heavy chains (H) and light chains (L) interconnected by disulfide bonds. Immunoglobulin genes include κ, λ, α, γ, δ, ε and μ constant region genes and numerous immunoglobulin variable domain genes. There are two types of light chains, lambda (λ) and kappa (κ). There are five major heavy chain species (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA and IgE.

[0202] Each heavy chain and light chain comprises a constant region (or constant domain) and a variable region (or variable domain; see, e.g., Kindt et al. Kuby Immunology, 6th edition, WH Freeman and Co., page 91 (2007)). In several embodiments, the heavy chain and light chain variable regions combine to specifically bind to an antigen. In some other 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). Reference to "VH" or "VH" refers to the variable region of an antibody heavy chain, including an antigen-binding fragment, such as a variable region of an Fv, ScFv, dsFv or Fab. References to "VL" or "VL" refer to the variable domain of an antibody light chain, including the variable domain of an Fv, ScFv, dsFv or Fab.

[0203] The light and heavy chain variable regions 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, USDepartment of Health and Human Services, 1991). The sequences of the framework regions of different light or heavy chains are relatively conserved within species. The framework region of an antibody (i.e., the combined framework regions of the component light and heavy chains) is used to position and align the CDRs in three-dimensional space.

[0204] The CDR is primarily responsible for binding to the epitope of the antigen. The amino acid sequence boundaries of a given CDR can be readily determined using any of a number of well-known schemes, including those described by Kabat et al. ("Sequences of Proteins of Immunological Interest," 5th Edition. 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 usually referred to as CDR1, CDR2, and CDR3 (from N-terminus to C-terminus), and are also usually identified by the chain in which the specific CDR is located. Thus, VH CDR3 is the CDR3 of the variable domain of the heavy chain of the antibody in which it is present, and VLCDR1 is the CDR1 of the variable domain of the light chain of the antibody in which it is present. Light chain CDRs are sometimes referred to as LCDR1, LCDR2, and LCDR3. Heavy chain CDRs are sometimes referred to as LCDR1, LCDR2, and LCDR3.

[0205] "Antigen binding fragments" are portions of full-length antibodies that retain the ability to specifically recognize a cognate antigen, as well as various combinations of such portions. Some 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 modifying intact antibodies or those synthesized de novo using recombinant DNA methods (see, e.g., Kontermann and Dubel (eds.), Antibody Engineering, Vols. 1-2, 2nd ed., Springer Press, 2010).

[0206] Single-chain antibodies (ScFv) are genetically engineered molecules comprising the VH and VL domains of one or more antibodies connected 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, ID Rugs, 13: 543-549, 2010). The intramolecular orientation of the VH and VL domains in ScFv is generally not critical for ScFv. Therefore, ScFvs with both possible arrangements (VH domain-linker domain-VL domain; VL domain-linker domain-VH domain) can be used.

[0207] In dsFv, the heavy and light variable chains have been 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 a linker that is too short to allow pairing between the two domains on the same chain is used, thereby forcing the domains to pair with the complementary domains of another chain and generate two antigen binding sites (see, e.g., Holliger et al., Proc. Natl. Acad. Sci., 90: 6444 6448, 1993; Poljak et al., Structure, 2: 1121 1123, 1994).

[0208] 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., WH Freeman & Co., New York, 1997.

[0209] Non-naturally occurring antibodies can be constructed using solid phase peptide synthesis, can be recombinantly produced, or can be obtained, for example, by screening a combinatorial library consisting of a variable heavy chain and a variable light chain as described in Huse et al., Science 246: 1275-1281 (1989), which is incorporated herein by reference. These and other methods for preparing, for example, chimeric, humanized, CDR-grafted, single-chain and bifunctional antibodies are well known to those skilled 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 ed. (Oxford University Press 1995); each of which is incorporated herein by reference).

[0210] 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, and vice versa, 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.

[0211] "Humanized" antibodies or antigen-binding fragments include human framework regions and one or more CDRs from non-human (e.g., mouse, rat, or synthetic) antibodies or antigen-binding fragments. The non-human antibody or antigen-binding fragment providing the CDR is referred to as a "donor," and the human antibody or antigen-binding fragment providing the framework is referred to as an "acceptor." In one embodiment, in a humanized immunoglobulin, all CDRs are from a donor immunoglobulin. The constant region does not need to be present, but if present, it may be substantially identical to a human immunoglobulin constant region, e.g., at least about 85% to 90%, e.g., about 95% or higher identity. Therefore, except for possible CDRs, all parts of a humanized antibody or antigen-binding fragment are substantially identical to corresponding parts of a natural human antibody sequence.

[0212] A "chimeric antibody" is an antibody comprising sequences derived from two different antibodies (which are generally from different species). In some instances, a chimeric antibody comprises one or more CDRs and / or framework regions from one human antibody and CDRs and / or framework regions from another human antibody.

[0213] "Fully human antibodies" or "human antibodies" are antibodies that contain sequences from (or derived from) the human genome, but do not contain sequences from other species. In some embodiments, human antibodies contain CDRs, framework regions, and Fc regions (if present) from (or derived from) the human genome. Human antibodies can be identified and isolated using techniques for producing antibodies based on sequences derived from the human genome, such as by phage display or using transgenic animals (see, e.g., Barbas et al. Phage display: A Laboratory Manuel. 1st edition. New York: Cold Spring Harbor Laboratory Press, 2004. Print.; Lonberg, Nat. Biotech., 23: 1117-1125, 2005; Lonenberg, Curr. Opin. Immunol., 20: 450-459, 2008).

[0214] An antibody may have one or more binding sites. If there is more than one binding site, the binding sites may be identical to each other or may be different. For example, a naturally occurring immunoglobulin has two identical binding sites, a single-chain antibody or Fab fragment has one binding site, and a bispecific or bifunctional antibody has two different binding sites.

[0215] Methods for testing the ability of an antibody to bind to any functional portion of a CAR are known in the art and include any antibody-antigen binding assay, such as, for example, radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation, and competitive inhibition assays (see, e.g., Janeway et al., infra, U.S. Patent Application Publication No. 2002 / 0197266A1 and U.S. Patent No. 7,338,929).

[0216] In addition, CAR, CAR-expressing T cells, antibodies, or antigen-binding portions thereof can 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).

[0217] C. Conjugates

[0218] CARs, T cells expressing CARs, or monoclonal antibodies or antigen-binding fragments thereof that are specific for one or more antigens disclosed herein can be conjugated to agents such as effector molecules or detectable markers using any number of methods known to those skilled in the art. Both covalent and non-covalent attachment methods can be used. Conjugates include, but are not limited to, molecules in which there are effector molecules or detectable markers covalently attached to antibodies or antigen-binding fragments that specifically bind to one or more antigens disclosed herein. Those skilled in the art will appreciate that a variety of effector molecules and detectable markers can be used, including but not limited to chemotherapeutic agents, anti-angiogenic agents, toxins, radioactive agents (e.g. 125 I. 32 P. 14 C. 3 H and 35 S) and other labels, target moieties and ligands, etc.

[0219] The choice of a particular effector molecule or detectable marker depends on the particular target molecule or cell and the desired biological effect. Thus, for example, the effector molecule can be a cytotoxin used to cause the death of a particular target cell (e.g., a tumor cell).

[0220] The method for attaching an effector molecule or a detectable marker to an antibody or Fab varies according to the chemical structure of the effector. Polypeptides generally contain a variety of functional groups; for example, carboxylic acid (COOH), free amine (-NH2) or sulfhydryl (-SH) groups, which can be used to react with suitable functional groups on the antibody to cause the binding of effector molecules or detectable markers. Alternatively, the antibody or Fab is derivatized to expose or attach additional reactive functional groups. Derivatization may involve attaching any of many known linker molecules (e.g., those available from Pierce Chemical Company, Rockford, IL). A linker can be any molecule for connecting an antibody or Fab to an effector molecule or a detectable marker. A linker can form a covalent bond with both an antibody or Fab and an effector molecule or a detectable marker. Suitable linkers are well known to those skilled in the art, and include, but are not limited to, straight or branched 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 linker can be linked to the constituent amino acids via its side groups (e.g., to cysteine ​​via a disulfide bond) or to the alpha carbon amino and carboxyl groups of the terminal amino acids.

[0221] In several embodiments, the linker may include a spacer element that, when present, increases the size of the linker such that the distance between the effector molecule or detectable marker and the antibody or antigen-binding fragment is increased. Exemplary spacers are known to those of ordinary skill in the art and include those listed in U.S. Pat. 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, 5,521,284 , 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, and U.S. Patent Nos. 20110212088 and 20110070248, each of which is incorporated herein by reference in its entirety.

[0222] In some embodiments, the joint is cleavable under intracellular conditions, so that the cutting of the joint in the intracellular environment releases the effector molecule or detectable marker from the antibody or Fab. In other embodiments, the joint is not cleavable, and the effector molecule or detectable marker is released, for example, by antibody degradation. In some embodiments, the joint can be cut by a cutting agent present in the intracellular environment (for example, in a lysosome or endosome or caveolea). The joint can be, for example, a peptide joint, which is cut by an intracellular peptidase or protease (including but not limited to a lysosomal protease or an endosomal protease). In some embodiments, the peptide joint is at least two amino acids long or at least three amino acids long. However, the joint can be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids long, for example, 1 to 2, 1 to 3, 2 to 5, 3 to 10, 3 to 15, 1 to 5, 1 to 10, 1 to 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 in target cells (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83: 67-123). For example, a peptide linker (e.g., phenylalanine-leucine or glycine-phenylalanine-leucine-glycine linker) that can be cleaved by the sulfhydryl-dependent protease cathepsin B can be used. Other examples of such linkers are described in, e.g., U.S. Patent No 6,214,345, which is incorporated herein by reference. In a specific embodiment, the peptide linker that can be cleaved by an intracellular protease is a valine-citrulline linker or a phenylalanine-lysine linker (see, e.g., U.S. Patent No 6,214,345, which describes the synthesis of doxorubicin with a valine-citrulline linker).

[0223] In other embodiments, the cleavable linker is pH sensitive, i.e., sensitive to hydrolysis at certain pH values. Typically, pH sensitive linkers are hydrolyzable under acidic conditions. For example, acid-labile linkers (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitamides, orthoesters, acetals, ketals, etc.) that are hydrolyzable in lysosomes can be used (see, e.g., U.S. Pat. Nos. 5,122,368, 5,824,805, 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 (e.g., conditions in blood), but are unstable below pH 5.5 or 5.0 (the approximate pH of lysosomes). In certain embodiments, the hydrolyzable linker is a thioether linker (e.g., a thioether attached to the therapeutic agent via an acylhydrazone bond (see, e.g., U.S. Pat. No. 5,622,929).

[0224] In other embodiments, the linker is cleavable under reducing conditions (e.g., 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-α-methyl-α-(2-pyridyl-dithio) toluene)-, SPDB, and SMPT. (See, e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (CW Vogeled., Oxford U. Press, 1987); Phillips et al., Cancer Res. 68:9280-9290, 2008). See also U.S. Pat. No. 4,880,935).

[0225] In 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).

[0226] In other embodiments, the linker is not cleavable and the effector molecule or detectable marker is released by degradation of the antibody (see U.S. Publication No. 2005 / 0238649, which is incorporated herein by reference in its entirety).

[0227] In several embodiments, the linker is resistant to cleavage in the extracellular environment. For example, when the conjugate is present in the extracellular environment (e.g., in plasma), no more than about 20%, no more than about 15%, no more than about 10%, no more than about 5%, no more than about 3%, or no more than about 1% of the linker is cleaved in a sample of the conjugate. It can be determined, for example, by incubating the conjugate containing the linker of interest with plasma for a predetermined period of time (e.g., 2, 4, 8, 16, or 24 hours) and then quantifying the amount of free effector molecules or detectable markers present in the plasma to determine whether the linker is resistant to cleavage in the extracellular environment. A variety of exemplary linkers that can be used in the conjugate are described in WO 2004-010957, U.S. Publication No 2006 / 0074008, U.S. Publication No 20050238649, and U.S. Publication No 2006 / 0024317, each of which is incorporated herein by reference in its entirety.

[0228] In several embodiments, conjugates of CAR, CAR-expressing T cells, antibodies, or antigen-binding portions thereof, and one or more small molecule toxins (e.g., calicheamicin, maytansinoid, dolastatin, auristatin, trichothecene, and CC1065), and derivatives of these toxins having toxin activity, are provided.

[0229] Maytansine compounds suitable for use as maytansine compounds toxin part are well known in the art, and can be separated from natural sources according to known methods, produced using genetic engineering techniques (see Yu et al. (2002) PNAS 99: 7968-7973), or maytansinol and maytansinol analogs are prepared synthetically according to known methods. Maytansine compounds are mitotic inhibitors that play a role by inhibiting tubulin polymerization. Maytansine was first separated from the East African shrub Maytenus serrata (USP No 3,896,111). Subsequently, it was found that some microorganisms also produced maytansine compounds, such as maytansinol and C-3 maytansinol esters (USP No 4,151,042). and 4,371,533, each of which is incorporated herein by reference. Conjugates comprising maytansine compounds, methods for their preparation and therapeutic uses thereof are disclosed, for example, in US Pat. 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.

[0230] Other toxins can be used with CAR, T cells expressing CAR, antibodies or their antigen binding portions.Exemplary toxins include Pseudomonas exotoxin (PE), ricin, abrin, diphtheria toxin and its subunits, nuclear toxins (ribotoxin), ribonucleases, saporin (saporin) and calicheamicin and botulinum toxin A to F. These toxins are well known in the art, and many can be easily obtained from commercial sources (such as Sigma Chemical Company, St.Louis, MO).Contemplated toxins also include variants of toxins (see, for example, referring to U.S. Patent No 5,079,163 and 4,689,401).

[0231] Saponin is a toxin derived from soapwort (Saponaria officinalis) that disrupts protein synthesis by inactivating the 60S portion of the ribosome complex (Stirpe et al., Bio / Technology, 10:405-412, 1992). However, the toxin does not have a mechanism for specific entry into cells and therefore needs to be conjugated to an antibody or antigen-binding fragment that recognizes a cell surface protein that is internalized in order to be effectively taken up by cells.

[0232] Diphtheria toxin is isolated from Corynebacterium diphtheriae. Typically, diphtheria toxins used for immunotoxins are mutated to reduce or eliminate nonspecific toxicity. A mutant known as CRM107 with full enzyme 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 No 5,792,458 and U.S. Patent No 5,208,021.

[0233] Ricin is a lectin RCA60 from Ricinus communis (castor bean). For some examples of ricin, see U.S. Pat. No. 5,079,163 and U.S. Pat. No. 4,689,401. Ricinus communis agglutinin (RCA) exists in two forms, which are denoted RCA and RCA according to their molecular weights of about 65 kD and 120 kD, respectively. 60 and RCA 120 (Nicholson & Blaustein, J. Biochim. Biophys. Acta 266:543, 1972). The A chain is responsible for inactivating protein synthesis and killing cells. The B chain binds ricin to galactose residues on the cell surface and helps the A chain to be transported into the cytosol (Olsnes et al., Nature 249:627-631, 1974 and U.S. Patent No 3,060,165).

[0234] Ribonucleases are also conjugated to targeting molecules for use as immunotoxins (see Suzuki et al., Nat. Biotech. 17:265-70, 1999). Some exemplary ribotoxins (e.g., α-sarcin and restrictocin) are discussed, for example, in Rathore et al., Gene 190:31-5, 1997; and Goyal and Batra, Biochem. 345 Pt 2:247-54, 2000. Calicheamicin was first isolated from Micromonospora echinospora and is a member of the enediyne family of antitumor antibiotics that cause double-strand breaks in DNA, leading to apoptosis (see, e.g., Lee et al., J. Antibiot. 42:1070-87, 1989). This drug is the toxic portion of an immunotoxin in clinical trials (see, eg, Gillespie et al., Ann. Oncol. 11:735-41, 2000).

[0235] Abrin comprises toxic lectins from Abrus precatorius. The toxin components abrin a, b, c and d have a molecular weight of approximately 63 kD to 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).

[0236] CARs specific for one or more antigens disclosed herein, T cells expressing CARs, monoclonal antibodies, antigen-binding fragments thereof, may also be conjugated to detectable markers; for example, detectable markers that can be detected by ELISA, spectrophotometry, flow cytometry, microscopy, or diagnostic imaging techniques (e.g., computed tomography (CT), computed axial tomography (CAT), magnetic resonance imaging (MRI), nuclear magnetic resonance imaging (NMRI), magnetic resonance tomography (MTR), ultrasound, fiber optics, and laparoscopy). Some specific non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzyme conjugates, radioactive isotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI). For example, available detectable markers include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, etc. Bioluminescent markers such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP) are also used. CAR, T cells expressing CAR, antibodies, or antigen-binding portions thereof may also be conjugated with enzymes (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, glucose oxidase, etc.) that can be used for detection. When CAR, T cells expressing CAR, antibodies, or antigen-binding portions thereof are conjugated with a detectable enzyme, it can be detected by adding additional reagents, and the enzyme uses the reagent to produce a distinguishable reaction product. For example, when the reagent horseradish peroxidase is present, hydrogen peroxide and diaminobenzidine are added to produce a visually detectable colored reaction product. CAR, CAR-expressing T cells, antibodies, or antigen-binding portions thereof, can also be conjugated to biotin and detected by indirect measurement of avidin or streptavidin binding. It should be noted that avidin itself can be conjugated to an enzyme or fluorescent label.

[0237] CAR, T cells expressing CAR, antibodies or antigen-binding portions thereof can be conjugated with paramagnetic agents (e.g., gadolinium). Paramagnetic agents (e.g., 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 (e.g., a leucine zipper pair sequence, a binding site of a second antibody, a metal binding domain, an epitope tag) recognized by a second reporter.

[0238] CAR, T cells expressing CAR, antibodies or antigen-binding portions thereof may also be conjugated to radiolabeled amino acids. Radiolabels can be used for both diagnostic and therapeutic purposes. For example, radiolabels can be used to detect one or more antigens and cells expressing antigens disclosed herein by x-rays, emission spectroscopy or other diagnostic techniques. In addition, radiolabels can be used therapeutically as toxins for treating tumors in subjects, such as for treating neuroblastoma. Some 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.

[0239] Methods for detecting such detectable markers are well known to those skilled in the art. Thus, for example, radioactive labels can be detected using photographic film or scintillation counters, and fluorescent markers can be detected using photodetectors to detect emitted illumination. Enzyme labels are usually detected by providing a substrate to the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label.

[0240] D. Nucleotides, expression, vectors and host cells

[0241] One embodiment of the present invention also provides a nucleic acid comprising a nucleotide sequence encoding any CAR, antibody, or antigen binding portion thereof described herein (including functional portions and functional variants thereof). The nucleic acid of the present invention may include a nucleotide sequence encoding any leader sequence, antigen binding domain, transmembrane domain, and / or intracellular T cell signaling domain described herein.

[0242] In some embodiments, the nucleotide sequence can be codon-modified. Without being bound by a particular theory, it is believed that codon optimization of the nucleotide sequence improves the translation efficiency of the mRNA transcript. Codon optimization of the nucleotide sequence may involve replacing a natural codon with another codon that encodes the same amino acid but can be translated by tRNA that is more easily available in the cell, thereby improving translation efficiency. Optimization of the nucleotide sequence can also reduce secondary mRNA structures that interfere with translation, thereby improving translation efficiency.

[0243] In one embodiment of the invention, nucleic acid may include a codon-modified nucleotide sequence encoding the antigen binding domains of the CAR of the present invention. In another embodiment of the invention, nucleic acid may include a codon-modified nucleotide sequence encoding any CAR described herein (including its functional parts and functional variants).

[0244] "Nucleic acid" as used herein includes "polynucleotide", "oligonucleotide" and "nucleic acid molecule", and generally means a polymer of DNA or RNA, which can be single-stranded or double-stranded, synthesized or obtained (e.g., isolated and / or purified) from a natural source, which can contain natural, non-natural or altered nucleotides, and which can contain natural, non-natural or altered internucleotide connections, such as phosphoramidate connections or phosphorothioate connections, rather than phosphodiester between nucleotides present in unmodified oligonucleotides. In some embodiments, nucleic acid does not include any insertion, deletion, inversion and / or replacement. However, as discussed herein, in some cases for nucleic acid, it may be appropriate to include one or more insertions, deletions, inversions and / or replacements.

[0245] Recombinant nucleic acid can be a nucleic acid having a non-natural sequence or having a sequence prepared by artificially combining two otherwise separated sequence segments. This artificial combination is usually accomplished by chemical synthesis, or more commonly by artificial manipulation of separated nucleic acid segments, such as by genetic engineering techniques (such as those described in Sambrook et al. (ibid)). Nucleic acids can be constructed based on chemical synthesis and / or enzymatic ligation using methods known in the art. See, for example, Sambrook et al. (ibid) and Ausubel et al. (ibid). For example, naturally occurring nucleotides or a variety of modified nucleotides can be used to chemically synthesize nucleic acids, which are designed to improve the biological stability of the molecule or improve the physical stability of the duplex formed after hybridization (such as nucleotides substituted with thiophosphate derivatives and acridine). Some examples of modified nucleotides that can be used to generate nucleic acids include, but are not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-substituted Adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosyl quercetin, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyl adenine, uracil-5-hydroxyacetic acid (v), wybutoxosine, pseudouracil, quercetin, 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 nucleic acids of the present invention can be purchased from a company such as Integrated DNA Technologies (Coralville, IA, USA).

[0246] The nucleic acid may comprise any isolated or purified nucleotide sequence encoding any CAR or a functional part or functional variant thereof. Alternatively, the nucleotide sequence may comprise a nucleotide sequence that is degenerate to any sequence or a combination of degenerate sequences.

[0247] One embodiment also provides an isolated or purified nucleic acid comprising a nucleotide sequence that is complementary to the nucleotide sequence of any nucleic acid described herein or a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of any nucleic acid described herein.

[0248] Nucleotide sequences hybridized under stringent conditions can hybridize under high stringency conditions. "High stringency conditions" means that the nucleotide sequence specifically hybridizes with a target sequence (nucleotide sequence of any nucleic acid described herein) in an amount that is detectably stronger than non-specific hybridization. High stringency conditions include conditions that distinguish polynucleotides with precise complementary sequences or polynucleotides containing only a few scattered mismatches from random sequences that happen to have a few small regions (e.g., 3 to 10 bases) that match the nucleotide sequence. Such complementary small regions are more easily melted than the full-length complements of 14 to 17 or more bases, and highly stringent hybridization makes them easy to distinguish. Relatively high stringency conditions include, for example, low salt and / or high temperature conditions, such as provided by about 0.02M to 0.1M NaCl or equivalents at a temperature of about 50°C to 70°C. Such high stringency conditions allow small (if any) mismatches between nucleotide sequences and templates or target chains, and are particularly suitable for detecting the expression of any CAR of the present invention. It is generally recognized that conditions can be made more stringent by adding an increased amount of formamide.

[0249] 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.

[0250] In one embodiment, nucleic acid can be incorporated into a recombinant expression vector. In this regard, one embodiment provides a recombinant expression vector comprising any nucleic acid. For purposes herein, the term "recombinant expression vector" means a genetically modified oligonucleotide or polynucleotide construct that allows host cells to express mRNA, protein, polypeptide or peptide when the construct comprises a nucleotide sequence encoding mRNA, protein, polypeptide or peptide and when the vector is contacted with a cell under conditions sufficient to allow mRNA, protein, polypeptide or peptide to be expressed in the cell. The vector as a whole is not naturally occurring.

[0251] However, portions of the vector may be naturally occurring. The recombinant expression vector may contain any type of nucleotides, including but not limited to DNA and RNA, which may be single-stranded or double-stranded, synthetic or partially obtained from a natural source, and which may contain natural, non-natural or altered nucleotides. The recombinant expression vector may contain naturally occurring or non-naturally occurring internucleotide linkages or both types of linkages. Preferably, the non-naturally occurring or altered nucleotides or internucleotide linkages do not hinder transcription or replication of the vector.

[0252] In one embodiment, the recombinant expression vector can be any suitable recombinant expression vector, and can be used for transformation or transfection of any suitable host cell. Suitable vectors include vectors designed for breeding and amplification or for expression or for both, such as plasmids and viruses. Vectors can be selected from pUC series (Fermentas Life Sciences, GlenBurnie, MD), pBluescript series (Stratagene, LaJolla, CA), pET series (Novagen, Madison, WI), pGEX series (Pharmacia Biotech, Uppsala, Sweden) and pEX series (Clontech, Palo Alto, CA).

[0253] Phage vectors can also be used, e.g. λZapII (Stratagene), EMBL4 and λNMI149. Some examples of plant expression vectors include pBIOl, pBI101.2, pBHOl.3, pBI121 and pBIN19 (Clontech). Some 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 vector or a lentiviral vector. A lentiviral vector is a vector derived from at least a portion of a lentiviral genome, particularly including a self-inactivating lentiviral vector, such as provided in Milone et al., Mol. Ther. 17 (8): 1453-1464 (2009). Other examples of lentiviral vectors that can be used clinically include, for example, but not limited to, LENTIVECTOR.RTM. gene delivery technology from Oxford BioMedica plc, LENTIMAX.TM. vector system from Lentigen, etc. Non-clinical types of lentiviral vectors are also available and are known to those skilled in the art.

[0254] Many 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).

[0255] 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 nucleic acid delivery using high-speed microprojectiles (see, e.g., Klein et al., Nature, 327:70-73 (1987)).

[0256] In one embodiment, recombinant expression vectors can be prepared using standard recombinant DNA techniques as described in, for example, 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 a prokaryotic or eukaryotic host cell. The replication system can be derived from, for example, ColE1, 2μ plasmid, lambda, SV40, bovine papilloma virus, and the like.

[0257] Recombinant expression vectors may include regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host cell (e.g., bacteria, fungi, plants, or animals) into which the vector is to be introduced, as appropriate, and taking into account whether the vector is DNA or RNA based. Recombinant expression vectors may include restriction sites to facilitate cloning.

[0258] The recombinant expression vector may include one or more marker genes that allow selection of transformed or transfected host cells. Marker genes include biocide resistance, such as resistance to antibiotics, heavy metals, etc., providing prototrophic complementation in auxotrophic hosts, etc. Suitable marker genes for expression vectors of the present invention include, for example, neomycin / G418 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.

[0259] The recombinant expression vector may include a natural or non-natural promoter, which is operably connected to a nucleotide sequence encoding CAR (including its functional part and its functional variant) or to a nucleotide sequence complementary to or hybridized with the nucleotide sequence encoding CAR. The selection of promoters (e.g., strong, weak, inducible, tissue-specific and developmentally specific) is within the ordinary technical scope of the technician. Similarly, the combination of nucleotide sequence and promoter is also within the technical scope of the technician. Promoter can be a non-viral promoter or a viral promoter, such as a cytomegalovirus (CMV) promoter, SV40 promoter, RSV promoter or a promoter found in the long terminal repeat of a mouse stem cell virus.

[0260] The recombinant expression vector can be designed to be used for transient expression, for stable expression or for both. In addition, the recombinant expression vector can be used for constitutive expression or for inducible expression.

[0261] In addition, the recombinant expression vector can be prepared to include suicide genes. The term "suicide gene" used herein refers to a gene that causes cell death of the suicide gene. The suicide gene can be a gene that gives the cell expressing the gene a sensitivity to an agent (e.g., a drug), and causes cell death when the cell is contacted with an agent or exposed to an agent. Suicide genes are known in the art (see, e.g., 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.

[0262] One embodiment also provides a host cell comprising any recombinant expression vector described herein. The term "host cell" used herein refers to any type of cell that may contain the recombinant expression vector of the present invention. The host cell may be a eukaryotic cell, such as a plant, an animal, a fungus or an algae, or may be a prokaryotic cell, such as a bacterium or a protozoan. The host cell may be a cultured cell or a primary cell (i.e., directly isolated from an organism (e.g., a human)). The host cell may be an adherent cell or a suspended cell (i.e., a cell grown in suspension). Suitable host cells are known in the art, and include, for example, DH5a Escherichia coli (E.coli) cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, etc. For the purpose of amplifying or replicating the recombinant expression vector, the host cell may be a prokaryotic cell, such as a DH5a cell. For the purpose of producing a recombinant CAR, the host cell may be a mammalian cell. The host cell may be a human cell. Although the host cell may be any cell type, may be derived from any type of tissue, and may be at any developmental stage, the host cell may be a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC). The host cell may be a T cell.

[0263] For purposes herein, T cells can be any T cells, such as cultured T cells, such as primary T cells, or T cells from cultured T cell lines (such as Jurkat, SupT1, etc.), or T cells obtained from mammals. If obtained from mammals, T cells can be obtained from a variety 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 separated from people. T cells can be any type of T cells, and can be in any developmental stage, including but not limited to CD4+ / CD8+ double positive T cells, CD4+ helper T cells (such as Th1 and Th2 cells), CD8+T cells (for example, cytotoxic T cells), tumor infiltrating cells, memory T cells, memory stem cells (i.e., Tscm), immature T cells, etc. T cells can be CD8+T cells or CD4+T cells.

[0264] In one embodiment, the CAR described herein can be used for suitable non-T cells. Such cells are those with immune effector functions, such as NK cells and T-like cells produced by pluripotent stem cells.

[0265] One embodiment also provides a cell group comprising at least one host cell described herein. The cell group can be a heterogeneous group, which includes a host cell containing any recombinant expression vector described, and in addition includes at least one other cell (e.g., host cell (e.g., T cell)) that does not contain any recombinant expression vector, or cells other than T cells, such as B cells, macrophages, neutrophils, erythrocytes, hepatocytes, endothelial cells, epithelial cells, muscle cells, brain cells, etc. Alternatively, the cell group can be a substantially homogeneous group, wherein the group mainly includes (e.g., substantially consisting of)) host cells containing a recombinant expression vector. The group can also be a clonal group of cells, wherein all cells of the group are clones of a single host cell containing a recombinant expression vector, so that all cells of the group include the recombinant expression vector. In one embodiment of the invention, the cell group is a clonal group comprising a host cell containing a recombinant expression vector as described herein.

[0266] CAR (including its functional parts and variants), nucleic acids, recombinant expression vectors, host cells (including its groups) and antibodies (including its antigen binding portions) can be separated and / or purified. For example, a purified (or separated) host cell preparation is a preparation in which the host cell is purer than cells in the natural environment in vivo. Such host cells can be produced, for example, by standard purification techniques. In some embodiments, the preparation of the host cell is purified so that the host cell accounts for at least about 50%, for example, at least about 70% of the total cell content of the preparation. For example, the purity can be at least about 50%, can be greater than about 60%, about 70% or about 80%, or can be about 100%.

[0267] E. Treatment methods

[0268] It is expected that the CAR disclosed herein can be used in a method for treating or preventing a disease in a mammal. In this regard, one embodiment provides a method for treating or preventing cancer in a mammal, comprising administering a CAR, a nucleic acid, a recombinant expression vector, a host cell, a cell population, an antibody and / or an antigen binding portion thereof and / or a pharmaceutical composition to the mammal in an amount effective to treat or prevent cancer in the mammal.

[0269] One embodiment also includes lymphodepleting the mammal prior to administering the CAR disclosed herein. Some examples of lymphodepleting include, but are not limited to, non-myeloablative lymphodepleting chemotherapy, myeloablative lymphodepleting chemotherapy, whole body irradiation, etc.

[0270] For the purpose of the method wherein administering host cells or cell groups, cells can be mammalian allogeneic or autologous cells. Preferably, cells are mammalian autologous. Allogeneic as used herein means any material derived from different animals of the same species as the individual of the introduced material. When the genes on one or more loci are different, two or more individuals are considered to be allogeneic to each other. In some aspects, allogeneic materials from individuals of the same species can be genetically different enough to interact antigenically. "Autologous" as used herein means any material derived from the same individual of the individual of the material subsequently reintroduced.

[0271] Mammals mentioned herein can be any mammals. The term "mammal" as used herein refers to any mammal, including but not limited to mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits. Mammals can be from the order Carnivora, including felines (cats) and canines (dogs). Mammals can be from the order Artiodactyla, including Bovidae (cows) and Suidae (pigs), or the order Perssodactyla, including Equine (horses). Mammals can be Primates, Ceboid or Simoid (monkeys), or the order Anthropoids (humans and apes). Preferably, mammals are humans.

[0272] With respect to the methods, the cancer can be any cancer, including any of the following: acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer (e.g., bladder cancer), bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, anal cancer, anal canal cancer, or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervical cancer, gallbladder cancer, or pleural cancer, nasal cancer, nasal cavity cancer, or middle ear cancer, oral cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer. carcinoma, fibrosarcoma, gastrointestinal carcinoid tumors, 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 cancer, omental cancer and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumors, synovial sarcoma, stomach cancer, testicular cancer, thyroid cancer and ureteral cancer.

[0273] As used herein, the terms "treat" and "prevent" and words derived therefrom do not necessarily mean 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention that one of ordinary skill in the art would consider to have a potential benefit or therapeutic effect. In this regard, the method can provide any amount or any level of treatment or prevention of cancer in a mammal.

[0274] In addition, the treatment or prevention provided by the method may include treating or preventing one or more conditions or symptoms of the disease (e.g., cancer) being treated or prevented. Moreover, for purposes herein, "prevention" may encompass delaying the onset of a disease or its symptoms or conditions.

[0275] Another embodiment provides a method for detecting the presence of cancer in a mammal, comprising: (a) contacting a sample comprising one or more cells from a mammal with a CAR, a nucleic acid, a recombinant expression vector, a host cell, a cell population, an antibody and / or an antigen binding portion thereof, or a pharmaceutical composition, thereby forming a complex, (b) and detecting the complex, wherein detection of the complex indicates the presence of cancer in the mammal.

[0276] The sample can be obtained by any suitable method (e.g., biopsy or autopsy). A 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. The experiment may include an experiment to determine whether the individual suffers from and / or is suffering from a certain condition or disease state. The condition or disease can be, for example, cancer.

[0277] In one embodiment of a method for detecting the presence of a proliferative disorder (e.g., cancer) in a mammal, the sample comprising mammalian cells can be a sample comprising 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). If the sample comprises whole cells, the cell can be any cell of a mammal, such as a cell of any organ or tissue, including a blood cell or an endothelial cell.

[0278] For mammals, contacting can occur in vitro or in vivo. Preferably, contacting is in vitro.

[0279] In addition, the detection of the complex can be carried out by any number of methods known in the art. For example, a detectable label (e.g., a radioisotope, a fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)) disclosed above), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase) and element particles (e.g., gold particles)) can be used to label the CAR disclosed herein, the polypeptide described herein, protein, nucleic acid, recombinant expression vector, host cell, cell group or antibody or its antigen binding portion thereof.

[0280] The method for testing the ability of CAR to recognize target cells and antigen specificity is known in the art. For example, Clay et al., J.Immunol, 163: 507-513 (1999) teaches measuring cytokines (e.g., interferon-γ, granulocyte / monocyte colony stimulating factor (granulocyte / monocyte colony stimulating factor, GM-CSF), tumor necrosis factor α (tumor necrosis factor α, TNF-α) or interleukin 2 (interleukin 2, IL-2)) release method. In addition, CAR function can be evaluated by measuring the cytotoxicity of cells, such as Zhao et al., J.Immunol. 174: 4415-4423 (2005) described in.

[0281] Another embodiment provides the use of CAR, nucleic acid, recombinant expression vector, host cell, cell group, antibody or antigen binding portion thereof and / or pharmaceutical composition of the present invention for treating or preventing proliferative disorders (e.g., cancer) in mammals. Cancer can be any cancer described herein.

[0282] Any method of administration can be used for the disclosed therapeutic agent, including local administration and systemic administration. For example, surface, oral, intravascular (e.g., intravenous), intramuscular, intraperitoneal, intranasal, intradermal, intrathecal, and subcutaneous administration can be used. The specific mode of administration and dosage regimen will be selected by the attending clinician considering the specific circumstances of the case (e.g., the subject, the disease, the disease state involved, and whether the treatment is preventive). In the case where more than one agent or composition is administered, one or more routes of administration can be used; for example, a chemotherapeutic agent can be administered orally, and an antibody or antigen binding fragment or conjugate or composition can be administered intravenously. The method of administration includes injection, in which case CAR, CAR T cells, conjugates, antibodies, antigen binding fragments, or compositions are provided in, for example, the following non-toxic pharmaceutically acceptable carriers: water, saline, Ringer's solution, dextrose solution, 5% human serum albumin, fixed oil, ethyl oleate, or liposomes. In some embodiments, local administration of the disclosed compound can be used, for example, by applying an antibody or antigen binding fragment to a tissue area where a tumor is removed or an area suspected of being prone to tumors. In some embodiments, sustained intratumoral (or peritumoral) release of a pharmaceutical formulation comprising a therapeutically effective amount of an antibody or antigen-binding fragment may be beneficial. In other examples, the conjugate is applied topically to the cornea as eye drops, or intravitreally to the eye.

[0283] The disclosed therapeutic agents can be formulated into unit dosage forms suitable for individual administration of precise dosages. In addition, the disclosed therapeutic agents can be administered in single doses or multiple dose regimens. A multiple dose regimen is a regimen in which the primary course of treatment may have more than one single dose (e.g., 1 to 10 doses), and then other doses are 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 a compound over a period of days to months or even years. Therefore, the dosage regimen will also be determined at least in part based on the specific needs of the subject to be treated and will depend on the judgment of the administering operator.

[0284] A typical dosage of the antibody or conjugate may be about 0.01 to about 30 mg / kg, such as about 0.1 to about 10 mg / kg.

[0285] In some specific examples, a therapeutic composition comprising one or more of a conjugate, an antibody, a composition, a CAR, a CAR T cell, or another agent is administered to a subject with multiple daily dosing regimens (e.g., at least two consecutive days, 10 consecutive days, etc., for example, for a period of weeks, months, or years). In one example, a conjugate, an antibody, a composition, or another agent is administered to a subject for at least 30 days, for example, 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.

[0286] In some embodiments, the disclosed method includes providing surgery, radiotherapy and / or chemotherapeutic agents to the subject in combination with a disclosed antibody, antigen binding fragment, conjugate, CAR or T cell expressing CAR (e.g., sequentially, substantially simultaneously or simultaneously). Such medicaments and treatment methods and therapeutic doses are known to those skilled in the art and can be determined by skilled clinicians. The preparation and dosing regimen for other medicaments can be used according to the manufacturer's instructions or determined empirically by skilled practitioners. Preparations and dosing regimens for such chemotherapy are also described in Chemotherapy Service, (1992) Ed., MCPerry, Williams & Wilkins, Baltimore, Md.

[0287] In some embodiments, the combination therapy may include administering to the subject a therapeutically effective amount of an additional cancer inhibitor. Some non-limiting examples of additional therapeutic agents that can be used with the combination therapy include microtubule binders, DNA intercalators or crosslinkers, DNA synthesis inhibitors, DNA and RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, gene regulators, and angiogenesis inhibitors. These agents (which are administered in a therapeutically effective amount) and treatments can be used alone or in combination. For example, any suitable anticancer agent or anti-angiogenic agent can be administered in combination with CAR, CAR-T cells, antibodies, antigen binding fragments, or conjugates disclosed herein. The methods and therapeutic doses of such agents are known to those skilled in the art and can be determined by skilled clinicians.

[0288] Additional chemotherapeutic agents include, but are not limited to, alkylating agents such as nitrogen mustards (e.g., chlorambucil, chlormethine, cyclophosphamide, ifosfamide, and melphalan), nitrosoureas (e.g., carmustine, fotemustine, lomustine, and streptozotocin), platinum compounds (e.g., carboplatin, cisplatin, oxaliplatin, and BBR3464), busulfan, dacarbazine, mechlorethamine, and chloramphenicol. amine), 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 podophyllotoxins (e.g., etoposide and teniposide), taxanes (e.g., docetaxel and paclitaxel), vinca alkaloids (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); n)), 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;Photosensitizers such as aminolevulinic acid, methyl aminolevulinate, porfimersodium, and verteporfin; and other agents such as alitretinoin, altretamine, amsacrine, anagrelide, arsenic trioxide, asparaginase, axitinib, bexarotene, bevacizumab, bortezomib, celecoxib, denileukin diftitox), erlotinib, estramustine, gefitinib, hydroxyurea, imatinib, lapatinib, pazopanib, pentostatin, masoprocol, mitotane, pegaspargase, tamoxifen, sorafenib, sunitinib, vemurafinib, vandetanib, and tretinoin. The selection and therapeutic dosage of such agents are known to those skilled in the art and can be determined by a skilled clinician. ;

[0289] Combination therapy can provide synergy and demonstrate synergy, i.e., the effect achieved when the active ingredients are used together is greater than the sum of the effects produced by the compounds used alone. Synergy can be achieved when the active ingredients are: (1) co-formulated in a combined unit dosage formulation and administered or delivered simultaneously; (2) delivered as separate formulations in alternation or in parallel; or (3) by some other regimen. When delivered in alternation, synergy can be achieved when the compounds are administered or delivered sequentially, such as by separate injections in separate syringes. Generally, during alternation, the effective dose of each active ingredient is administered sequentially (i.e., one after the other), whereas in combination therapy, the effective doses of two or more active ingredients are administered together.

[0290] In one embodiment, an effective amount of an antibody or antigen-binding fragment or its conjugate that specifically binds to one or more antigens disclosed herein is administered to a subject with a tumor after anti-cancer treatment. After a sufficient amount of time has passed to allow the administered antibody or antigen-binding fragment or conjugate to form immune complexes with the antigens expressed on each cancer cell, immune complexes are detected. The presence (or absence) of immune complexes indicates the effectiveness of treatment. For example, an increase in immune complexes compared to a control obtained before treatment indicates that treatment is ineffective, while a decrease in immune complexes compared to a control obtained before treatment indicates that treatment is effective.

[0291] F. Biopharmaceutical Compositions

[0292] Provided herein are biopharmaceuticals or biopharmaceutical compositions (hereinafter referred to as "compositions") for gene therapy, immunotherapy and / or cell therapy, the compositions being included in a carrier (e.g., a pharmaceutically acceptable carrier) and one or more of the disclosed CARs, or T cells expressing CARs, antibodies, antigen binding fragments, conjugates, CARs, or T cells expressing CARs that specifically bind to one or more antigens disclosed herein. The composition can be prepared in a unit dose form for administration to a subject. The amount and time of administration are determined by the treating clinician to achieve the desired outcome. The composition can be formulated for systemic (e.g., intravenous) or local (e.g., intratumoral) administration. In one example, the disclosed CARs, or T cells expressing CARs, antibodies, antigen binding fragments, and conjugates are formulated for parenteral administration, such as intravenous administration. Compositions comprising CARs disclosed herein, or T cells expressing CARs, conjugates, antibodies, or antigen binding fragments are used, for example, to treat and detect tumors, such as, but not limited to, neuroblastoma. In some examples, the composition can be used to treat or detect cancer. Compositions comprising a CAR, or a T cell expressing a CAR, a conjugate, an antibody, or an antigen-binding fragment disclosed herein are also used, for example, to detect pathological angiogenesis.

[0293] The composition for administration may include a solution of CAR, or T cells expressing CAR, conjugates, antibodies or antigen binding fragments dissolved in a pharmaceutically acceptable carrier (e.g., an aqueous carrier). A variety of aqueous carriers may be used, such as buffered saline, etc. These solutions are sterile and generally do not contain undesirable substances. These compositions can be sterilized by conventional, well-known sterilization techniques. The composition may contain pharmaceutically acceptable auxiliary substances as needed to approach physiological conditions, such as pH regulators and buffers, toxicity regulators, adjuvants, etc., such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. In these preparations, the concentration of CAR, or T cells expressing CAR, antibodies or antigen binding fragments or conjugates may vary widely, and will be selected mainly based on fluid volume, viscosity, body weight, etc. according to the specific mode of administration selected and the needs of the subject. The actual method of preparing such dosage forms for gene therapy, immunotherapy and / or cell therapy is known or will be obvious to those skilled in the art.

[0294] Typical compositions for intravenous administration include about 0.01 mg / kg to about 30 mg / kg of antibody or antigen binding fragment or conjugate (or a corresponding dose of CAR, or T cells expressing CAR, or a conjugate comprising an antibody or antigen binding fragment) per subject per day. Actual methods for preparing administrable compositions will be known or apparent to those skilled in the art, and are described in more detail in publications such as Remington's Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, PA (1995).

[0295] CAR, or T cells expressing CAR, antibodies, antigen binding fragments, or conjugates can be provided in a lyophilized form and rehydrated with sterile water before administration, but they can also be provided as a sterile solution of known concentration. The solution of CAR, or T cells expressing CAR, antibodies, antigen binding fragments, or conjugates is then added to an infusion bag containing 0.9% sodium chloride (USP) and in some cases administered at a dose of 0.5 mg / kg to 15 mg / kg body weight. There is considerable experience in the art in administering antibody or antigen binding fragment or conjugate drugs; for example, since 1997, Since its approval, antibody drugs have been listed in the United States. CAR, or T cells expressing CAR, antibodies, antigen binding fragments thereof, or conjugates can be administered by slow infusion rather than by intravenous push or rapid bolus injection (bolus). In one example, a higher loading dose is administered, and the administered dose is subsequently maintained at a lower level. For example, an initial loading dose of 4 mg / kg antibody or antigen binding fragment (or a conjugate comprising an antibody or antigen binding fragment of a corresponding dose) can be infused within about 90 minutes, and then if the previous dose is well tolerated, a maintenance dose of 2 mg / kg per week is infused within 30 minutes for 4 to 8 weeks.

[0296] Controlled release parenteral formulations can be prepared as implants, oily injections or as particle systems. For a broad overview of protein delivery systems, see Banga, AJ, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Technomic Publishing Company, Inc., Lancaster, PA, (1995). Particle systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres and nanoparticles. Microcapsules contain therapeutic proteins (e.g., cytotoxins or drugs) as cores. In microspheres, therapeutic agents are dispersed throughout the particles. Particles, microspheres and microcapsules less than about 1 μm are generally referred to as nanoparticles, nanospheres and nanocapsules, respectively. The diameter of the capillary is about 5 μm, so that only nanoparticles are administered intravenously. The diameter of the microparticle is generally about 100 μm, and is 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 & Tabibi, Treatise on Controlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, NY, pp. 315-339, (1992).

[0297] Polymers can be used for CAR disclosed herein or T cells expressing CAR, antibodies or AFs or conjugate compositions for controlled release of ions. A variety of degradable and non-degradable polymer matrices for controlled drug delivery are known in the art (Langer, Accounts Chem. Res. 26: 537-542, 1993). For example, block copolymer poloxamer 407 exists as a viscous but flowing liquid at low temperatures, but forms a semisolid gel at body temperature. It has been shown to be an effective carrier 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 controlled release of proteins (Ijntema et al., Int. J. Pharm. 112: 215-224, 1994). In another aspect, liposomes are used for controlled release of lipid-encapsulated drugs and drug targeting (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA (1993)). Many additional systems for controlled delivery of therapeutic proteins are known (see U.S. Pat. No. 5,055,303, U.S. Pat. No. 5,188,837, U.S. Pat. No. 4,235,871, U.S. Pat. No. 4,501,728, U.S. Pat. No. 4,837,028, U.S. Pat. No. 4,957,735, U.S. Pat. No. 5,019,369, U.S. Pat. No. 5,055,303, U.S. Pat. No. 5,514,670, U.S. Pat. No. 5,413,797, U.S. Pat. No. 5,268,164, U.S. Pat. No. 5,004,697, U.S. Pat. No. 4,902,505, U.S. Pat. No. 5,506,206, U.S. Pat. No. 5,271,961, U.S. Pat. No. 5,254,342, and U.S. Pat. No. 5,534,496).

[0298] G.Medicine box

[0299] In one aspect, a kit using the CAR disclosed herein is also provided. For example, a kit for treating a tumor in a subject or preparing a CAR T cell expressing one or more CARs disclosed herein. The kit will generally contain a disclosed antibody, antigen binding fragment, conjugate, nucleic acid molecule, CAR or T cell expressing CAR as disclosed herein. More than one disclosed antibody, antigen binding fragment, conjugate, nucleic acid molecule, CAR or T cell expressing CAR may be included in the kit.

[0300] The medicine box may include a container and a label or package insert on or attached to the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container may be formed of a variety of materials (e.g., glass or plastic). The container typically contains one or more compositions comprising the disclosed antibodies, antigen binding fragments, conjugates, nucleic acid molecules, CARs, or T cells expressing CARs. In several embodiments, the container may have a sterile inlet (e.g., the container may be an intravenous solution bag or a vial having a plug that can be pierced by a hypodermic needle). The label or package insert indicates that the composition is used to treat a specific condition.

[0301] The label or package insert will generally also include instructions for using disclosed antibodies, antigen binding fragments, conjugates, nucleic acid molecules, CARs or T cells expressing CARs, for example, in methods for treating or preventing tumors or preparing CAR T cells. The package insert generally includes instructions customarily included in the commercial packaging of therapeutic products, which include information about indications, usage, dosage, administration, contraindications and / or warnings related to the use of such therapeutic products. The illustrative material may be written in electronic form (e.g., a computer floppy disk or CD), or may be visual (e.g., a video file). The kit may also include additional components that are conducive to the specific application for which the kit is designed. Therefore, for example, the kit may additionally include tools for detecting labels (e.g., enzyme substrates for enzyme labeling, filter devices for detecting fluorescent labels, suitable secondary labels (e.g., secondary antibodies), etc.). The kit may additionally include buffers and other reagents conventionally used to practice a particular method. Such kits and suitable contents are well known to those skilled in the art.

[0302] Example

[0303] The present invention is further illustrated by the following examples, which should not be construed in any way as limiting its scope. On the contrary, it should be clearly understood that a variety of other embodiments, modifications and equivalents thereof may be adopted without departing from the spirit of the present invention and / or the scope of the appended claims, which, after reading the description herein, will themselves be suggested to those skilled in the art.

[0304] Example 1

[0305] Development of CD19 and CD20 dual-targeting CAR T constructs

[0306] Adoptive immunotherapy for cancer using genetically engineered autologous human T cells is currently being evaluated in many centers. A common approach to establishing cell populations for adoptive immunotherapy is to isolate T cells from patients by apheresis and transduce these cells ex vivo with retroviral or lentiviral vectors that integrate into the host genome and express chimeric antigen receptors (CARs), reviewed in [1]. Chimeric antigen receptors are generated by linking functional sequence domains from different subunits of immunologically active proteins. For example, the scFv domain generated from the VH and VL domains of anti-CD19 or anti-CD20 antibodies can be linked to transmembrane sequences derived from CD28 or CD8, and then to intracellular signaling domains derived from the CD3-ζ chain and CD28 or CD137 [2,3]. Thus, the CAR confers both the binding domain derived from the scFv and the linked signaling domain in a single transmembrane protein, thereby activating the vector-transduced T cells. This transduced population of T cells (CAR-T) can now functionally target cells bearing the cognate antigen for destruction by active cell lysis as well as indirect immune effector mechanisms marshaled by the production of cytokines such as interferon-γ (IFNγ), interleukin-2 (IL-2), and tumor necrosis factor-α (TNFα). Adoptive immunotherapy with chimeric antigen receptor-modified T cells specifically targeting CD19 has been shown to be effective against pediatric pre-B ALL [4,5]. In adult hematologic malignancies, the effectiveness of CAR-modified T cells is more heterogeneous.

[0307] While the experience with anti-CD19 CAR-T therapy in three patients with CLL at the University of Pennsylvania appears to indicate generally positive responses, the Surgery Branch of the NCI reported a mixture of partial responses, stable disease, and one complete response in a different set of eight patients with adult B-cell malignancies [6,7]. Thus, anti-CD19 CARs are not universally effective and may benefit from further enhancement of their antitumor targeting potential. The escape mechanisms employed by B-ALL during anti-CD19 CAR-T therapy have been well characterized by Thomas-Tikhonenko's laboratory, which include alternative splicing, frameshift mutations, and missense mutations of CD19 [8]. One approach to both broadening the target range of CAR-T products and targeting malignancies with greater efficacy is to include two binding domains in a single CAR construct. 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 villous lymphocytes (SLVL) [9]. A single CAR vector targeting both antigens will target a wider range of hematological malignancies and can target them more effectively.

[0308] In this example, we describe the development of fully human CAR T cells that simultaneously target both CD19 and CD20 tumor antigens to prevent tumor antigen escape. We also describe the selection of the optimal CAR T structure and the most effective hinge and transmembrane CAR domains.

[0309] Materials and methods

[0310] Cell lines

[0311] The Burkitt lymphoma cell line Raji was purchased from the American Tissue Culture Collection (ATCC, Manassas, VA). REH and NALM-6 leukemia lines were purchased from DSMZ (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). Wild-type tumor cell lines were stably transduced with a lentiviral vector (Lentigen Technology, Inc., Gaithersburg, MD) encoding firefly luciferase and GFP linked to a 2A peptide, followed by selection of luciferase-positive clones to generate single cell clones expressing luciferase and GFP cell lines. The human embryonic kidney cell 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 the wild-type tumor cell line with a lentiviral vector encoding firefly luciferase (Lentigen Technology, Inc., Gaithersburg, MD) followed by selection of luciferase-positive cells.

[0312] Generation of CAR constructs and lentiviral vector production

[0313] Human anti-CD19 and CD20 chimeric antigen receptor (CAR) constructs are designed as tandem CAR or bicistronic CAR. Multiple single-chain variable fragment (ScFv) sequences targeting the extracellular domain of human CD19 are identified internally, and the ScFv targeting CD20 is derived from Ofatumumab. For tandem CAR, anti-CD20 scFv is connected to anti-CD19 scFv via a G4S joint, followed by CD8 or CD28 hinge transmembrane domain, 4-1BB or CD28_4-1BB costimulatory domain and CD3-ζ activation domain sequence. Bicistronic CAR includes a single CAR targeting CD20 and a single CAR targeting CD19, connected to a P2A ribosomal skipping element. Single CAR constructs use CD8 hinge regions, CD8 or OX40 transmembrane domains, costimulatory domains and CD3-ζ activation domains derived from human 4-1BB, CD28, ICOS, OX40 proteins. Previously developed tandem CAR20_19 (LTG1497) with mouse CD19 and CD20 scFv 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 5, 42, 2017). The CAR sequence was cloned into a lentiviral vector (LV) expression cassette under the control of the human EF-lα promoter (Lentigen Technology Inc., Gaithersburg, MD). Lentiviral particles were produced by transient transfection of HER293T cells, precipitated by centrifugation and stored at -80 ° C until transduction.

[0314] Primary T cell preparation and transduction

[0315] Healthy donor primary T cells were isolated from leukapheresis collections (AllCells, Alameda, CA) or processed buffy coats (Oklahoma Blood Institute, Tulsa, OK) with written consent from the donors. CD4-positive and CD8-positive 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 stained with CD3 / CD28 GMP T Cell TransAct reagent (Miltenyi Biotec) was activated and 1×10 6 The cells were cultured at a density of 0.5 × 10 cells / ml in serum-free TexMACS medium supplemented with 30 IU / ml IL-2. In addition, activated T cells were transduced with lentiviral vector particles encoding the CAR construct at an MOI of 80 on day 1. On day 3, the transduced T cells were washed and resuspended to 0.5 × 10 6 After this, the cultures were supplemented with fresh TexMACS medium containing 30 IU / ml IL-2 every 2 to 3 days until harvest on day 8 to 10.

[0316] Flow cytometric analysis of CAR surface expression

[0317] 500,000 CAR T cells were washed in cold 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 antibodies labeled with violet-blue fluorescent dye were used to separate CD4 and CD8 populations. Untransduced cells (UTD) were used as negative controls. The cells were washed twice, resuspended in 200 μl running buffer, and acquired by flow cytometry. Flow cytometric analysis was performed on a 10 flow cytometer (Miltenyi Biotec), and data graphs were generated using FlowJo software (Ashland, OR).

[0318] CAR T cell cytotoxicity and cytokine assays

[0319] To evaluate CAR T cell-mediated cytotoxicity, 5 × 10 3Tumor target cells were combined with CAR T cells at a specified effector to target ratio and incubated overnight at 37 ° C and 5% CO2. SteadyGlo reagent (Promega, Madison WI) was added to each well, and the resulting luminescence was quantified as counts per second (sample CPS). The wells with only the target (maximum CPS) and the wells with only the target plus 1% Tween-20 (minimum CPS) were used to determine the range of the assay. The specific lysis percentage was calculated as follows: (1-(sample CPS-minimum CPS) / (maximum CPS-minimum CPS)). For cytokine release analysis, 5×10 4 effectors and 5×10 3 The target cells were co-cultured overnight, and the supernatant was removed from the co-culture 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 different healthy donors.

[0320] Long-term CAR T and tumor cell co-incubation assay

[0321] CD19 and CD20 dual-targeting CAR T cells or untransduced control T cells (1×10 6 ) and 1×10 6 Raji lymphoma cells were co-cultured at an effector to target ratio of 0.3:1 for 7 days and replenished with fresh media when needed. The cell mixtures were analyzed by flow cytometry at the 4th and 7th day time points. CountBright TM Absolute counting beads (Thermofisher, Eugene, OR) were included in the cell mixture to obtain absolute cell counts. After exclusion of dead cells by 7-AAD staining, the number of live T cells (CD3 + ) counts and percentage of tumor cells (GFP + ). On day 7, when the Raji cell % was less than 10% in most cell mixtures, 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.

[0322] result

[0323] Example 1 describes the generation and in vitro evaluation of CAR T cells targeting CD20 and CD19 antigens for the treatment of B cell malignancies.

[0324] Schematic diagram of a bispecific CAR construct targeting both CD19 and CD20 antigens. Figure 1A. A fully human binder for CAR19 was developed internally, and the fully human scFv targeting CD20 is derived from Ofatumumab. The tandem CAR is designed to connect the CD20 scFv to the CD19 scFv via a G4S linker, and then connect to the CD8 or CD28 hinge and transmembrane domain, 4-1BB or CD28_4-1BB costimulatory domain, and CD3ζ activation domain in the frame. The dual CAR construct is configured as a single CD20 CAR separated from a single CD19 CAR by a P2A sequence. A single CAR comprises a CD19 or CD20 scFv, a CD8 hinge, a transmembrane domain from CD8 or OX40, a costimulatory domain 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 produce CD20_19CAR T cells under the control of the mammalian EF-lα promoter. The previously evaluated CAR construct LTG1497 with a mouse binder 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 of transduced T cells was measured by flow cytometry using CD19-Fc followed by staining with anti-Fc Alexa Flour 647. Due to the lack of detection reagents, the expression of CAR20 was not pursued. Different CD20_19CAR constructs showed higher surface expression than the positive control LTG1497 (n=4 donors). Flow cytometry (CD4 vs CAR19) from a representative donor is shown in Figure 1B For this donor, LTG1497 showed the lowest surface expression (49.7%), followed by D0144 (58.0%), and the other CAR20_19 constructs ranged from 60% to 80%, indicating robust transduction efficiency and CAR expression.

[0325] In order to evaluate the target-specific cytotoxicity of CD20_19CAR in vitro, CD19 + CD20 + Leukemia or lymphoma lines (Raji, Nalm-6, and Reh) and CD19 - CD20 - The non-leukemic line 293T was used as the target line. CAR-T cells were co-incubated with target tumor cell lines at effector to target ratios of 2.5:1; 5:1 and 10:1. After overnight co-incubation, the cytotoxicity of CAR was analyzed in a luminescence-based in vitro killing assay ( Figure 2A to C). In CD19 +Among the cell lines, Raji, Nalm-6 and Reh, CAR20_19 constructs showed similar or higher killing capacity 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 (background killing) was observed for the higher E:T ratios of UTD cells co-incubated with NALM-6. In addition, no cytotoxicity was observed for the CD19 and CD20 negative 293T cell lines ( Figure 2D ) or limited background killing, indicating the robust target-specific cytotoxic function of all designed CD20_19CAR constructs.

[0326] T cell homeostasis and production of proinflammatory cytokines IL-2, IFNγ, and TNFα of the fully human CD20_19CAR and control construct LTG1497 were examined by ELISA. After CAR T cells were co-incubated with the Raji target line at an E:T ratio of 10 overnight ( Figures 4A to 4C ), harvest culture supernatants for measuring specific cytokine release. When compared to UTD or CAR-only controls, the full-human CAR20_19 constructs exhibited significant cytokine responses, indicating robust target-specific CAR T cell cytokine responses. The intensity of cytokine release of each CAR20_19 construct was equivalent to or higher than the control construct LTG1497.

[0327] A long-term co-culture assay was designed to further characterize the CAR20_19 construct. CAR T cells were co-cultured with Raji cells at a low E:T ratio of 0.3:1, and UTD cells were included as a negative control. Flow cytometry was used to measure the culture mixture of CAT T and Raji cells to examine the percentage of remaining Raji cells and T cell fold expansion during co-culture ( Figure 4B, 4C). On day 4, in the co-culture product of Raji and CAR T cells, the percentage of Raji cells decreased, and CART cell expansion was observed. In contrast, in the co-culture of Raji and UTD cells, the percentage of Raji cells increased, and T cells did not show proliferation. In addition, CAR D0256 and D0266 showed the highest Raji clearance. On day 7, all Raji cells were killed by T cells except the UTD group. CD20_19CAR showed a close multiple expansion compared to the control CAR LTG1497 at the end of the first round of co-culture. Additional Raji cells were added to the co-culture to readjust the ET ratio to 0.15:1. On day 10, the killing efficacy between the groups could be distinguished. It is worth noting that CAR D0266 showed the best killing ability during the second round of co-culture, followed by D0255 and D0257>D0258>D0256 and the control LTG1497. D0144 CAR has only very low killing ability ( Figure 4B ). CAR T cells continued to proliferate during the second round of co-incubation. CAR LTG1497, D0256 and D0144 showed slightly more proliferation than other CAR 20_19. Similar trends were observed in different donors, and most CAR 20_19 maintained a higher killing efficacy than control CAR 1497 except CARD0144 (data not shown).

[0328] Among all CAR20_19 constructs, D0144 showed the lowest transduction efficiency and low cytotoxic function during long-term co-culture. Compared with LTG1497, all other CAR20_19 constructs showed high CAR transduction efficiency, similar or stronger cytotoxicity in overnight or long-term killing assays, and in vitro specific cytokine induction. In summary, all CAR20_19 constructs except D0144 were evaluated for in vivo tumor clearance ability.

[0329] References for Example 1

[0330] 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.

[0331] 2.Kochenderfer.J.N.,et al,Construction and pre-clinical evaluation ofan anti-CD19 chimeric antigen receptor.Journal of immunotherapy(Hagerstown,Md.:1997),2009.32(7):p.689.

[0332] 3.Jensen,M.,et al.,CD20 is a molecular target for scFvFc:zetareceptor redirected Tcells:implications for cellular immunotherapy of CD20+malignancy.Biology of Blood and Marrow Transplantation,1998.4(2):p.75-83.

[0333] 4.Lee,D.W.,et al.,T cells expressing CD19 chimeric antigen receptorsfor acutelymphoblastic leukaemia in children and young adults:a phase l dose-escalation trial.The Lancet,2015.385(9967):p.517-528.

[0334] 5.Grupp,S.A.,et al.,Chimeric antigen rcceptor-modified T cells foracutc lymphoid leukemia.New England Journal of Medicine,2013.368(16):p.1509-1518.

[0335] 6.Porter,D.L.,et al.,Chimeric antigen receptor-modified T cells inchronic lymphoid leukemia.New England Journal of Medicine,2011.365(8):p.725-733.

[0336] 7.Kochenderfer, JN, et al., B-cell depletion and remissions ofmalignancy along with cvtokine-associated toxicity in a clinical trial of anti-CD19 chimeric-antigen-receptor-transduced T cells. Blood, 2012.119(12): p.2709-2720.

[0337] 8.Sotillo, E., et al., Convergence of acquired mutations and alternativesplicing of CD19 enables resistance to CART-19 immunotherapy. Cancerdiscovery, 2015.5(12): p.1282-1295.

[0338] 9.Ginaldi, L., et al., Levels of expression of CD19 and CD20 in chronic B cell leukaemias. Joumal of clinical pathology, 1998.51(5): p.364-369.

[0339] Sequence of the present disclosure

[0340] The nucleic acid and amino acid sequences listed below are shown using standard letter abbreviations for nucleotide bases and the three-letter code for amino acids, as defined in 37 CFR 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included by reference to the strand shown. In the attached sequence listing:

[0341] SEQ ID NO: 1 Nucleotide sequence of CD20-reactive ScFv1 binding domain

[0342]

[0343] SEQ ID NO:2 Amino acid sequence of CD20-reactive ScFv1 binding domain

[0344]

[0345] SEQ ID NO:3 Nucleotide sequence of CD19-reactive ScFv2 binding domain

[0346]

[0347] SEQ ID NO:4 Amino acid sequence of CD19-reactive ScFv2 binding domain

[0348]

[0349] SEQ ID NO:5 Nucleotide sequence of CAR D0144 (CD20_CD19 CD8 BBz)

[0350]

[0351]

[0352] SEQ ID NO:6 Amino acid sequence of CAR D0144 (CD20_CD19 CD8 BBz)

[0353]

[0354] SEQ ID NO:7 Nucleotide sequence of CAR D0255 (CD20_CD19 CD28) CD28 BBz

[0355]

[0356]

[0357] SEQ ID NO:8 Amino acid sequence of CAR D0255 (CD20_CD19 CD28) CD28 BBz

[0358]

[0359] SEQ ID NO:9 Nucleotide sequence of CAR D0256 (CD20_CD19 CD8 CD28 BBz)

[0360]

[0361]

[0362] SEQ ID NO:10 Amino acid sequence of CAR D0256 (CD20_CD19 CD8 CD28 BBz)

[0363]

[0364] SEQ ID NO: 11 Nucleotide sequence of leader / signal peptide sequence (LP)

[0365]

[0366] SEQ ID NO:12 Amino acid sequence of leader / signal peptide sequence (LP)

[0367]

[0368] SEQ ID NO: 13 Nucleotide sequence of CAR D0257 (CD20 CD8 CD28z_CD19 CD8 BBz)

[0369]

[0370]

[0371] SEQ ID NO:14 Amino acid sequence of CAR D0257 (CD20 CD8 CD28z_CD19 CD8 BBz)

[0372]

[0373] SEQ ID NO: 15 Nucleotide sequence of CAR D0258 (CD20 CD8 BBz_CD19 CD8 CD28z)

[0374]

[0375]

[0376]

[0377] SEQ ID NO:16 Amino acid sequence of CAR D0258 (CD20 CD8 BBz_CD19 CD8 CD28z)

[0378]

[0379] SEQ ID NO: 17 Nucleotide sequence of CAR D0266 (CD20 CD8 OX40 OX40z_CD19 CD8 ICOSz)

[0380]

[0381]

[0382]

[0383] SEQ ID NO:18 Amino acid sequence of CAR D0266 (CD20 CD8 OX40 OX40z_CD19 CD8 ICOSz)

[0384]

[0385] SEQ ID NO: 19 Nucleotide sequence of CAR LTG1497 (mCD20_CD19 CD8 BBz)

[0386]

[0387]

[0388] SEQ ID NO:20 Amino acid sequence of CAR LTG1497 (mCD20_CD19 CD8 BBz)

[0389]

[0390] SEQ ID NO:21 Nucleotide sequence of CD28 costimulatory domain

[0391]

[0392] SEQ ID NO:22 Amino acid sequence of CD28 costimulatory domain

[0393]

[0394] SEQ ID NO:23 Nucleotide sequence of 4-IBB costimulatory domain

[0395]

[0396] SEQ ID NO:24 Amino acid sequence of 4-1BB costimulatory domain

[0397] SEQ ID NO:25 Nucleotide sequence of tandem CD28_4-1BB costimulatory domain

[0398]

[0399] SEQ ID NO:26 Amino acid sequence of the tandem CD28_4-1BB co-stimulatory domain

[0400]

[0401] SEQ ID NO:27 Nucleotide sequence of OX40 costimulatory domain

[0402]

[0403] SEQ ID NO:28 Amino acid sequence of OX40 costimulatory domain

[0404]

[0405] SEQ ID NO:29 Nucleotide sequence of ICOS costimulatory domain

[0406]

[0407] SEQ ID NO:30 Amino acid sequence of ICOS costimulatory domain

[0408]

[0409] SEQ ID NO:31 Nucleotide sequence of CD28 hinge domain

[0410]

[0411] SEQ ID NO:32 Amino acid sequence of CD28 hinge domain

[0412]

[0413] SEQ ID NO:33 Nucleotide sequence of OX40 transmembrane domain

[0414]

[0415] SEQ ID NO:34 Amino acid sequence of OX40 transmembrane domain

[0416]

[0417] SEQ ID NO:35 Nucleotide sequence of DNA CD8 transmembrane domain

[0418]

[0419] SEQ ID NO:36 Amino acid sequence of CD8 transmembrane domain

[0420]

[0421] SEQ ID NO:37 Nucleotide sequence of DNA CD8 hinge domain

[0422]

[0423] SEQ ID NO:38 Amino acid sequence of CD8 hinge domain

[0424]

[0425] SEQ ID NO:39 The amino acid sequence of the transmembrane region and hinge of amino acid numbers 137 to 206 of CD8.α (NCBI RefSeq: NP.sub.--001759.3)

[0426]

[0427] SEQ ID NO:40 Nucleotide sequence of the DNA signaling domain of 4-IBB

[0428]

[0429] SEQ ID NO:41 Amino acid sequence of the signaling domain of 4-IBB

[0430]

[0431] SEQ ID NO:42 Nucleotide sequence of the DNA signaling domain of CD3-ζ

[0432]

[0433] SEQ ID NO:43 Amino acid sequence of CD3ζ

[0434]

[0435] SEQ ID NO:44 Alternative nucleotide sequence of CD3ζ_1

[0436]

[0437] SEQ ID NO:45 Alternative nucleotide sequence of CD3ζ_2

[0438]

[0439] SEQ ID NO:46 Nucleotide sequence of CD28 transmembrane domain

[0440]

[0441] SEQ ID NO:47 Amino acid sequence of CD28 transmembrane domain

[0442]

[0443] SEQ ID NO:48 Nucleotide sequence of CD20 / CD19-reactive ScFv binding domain

[0444]

[0445] SEQ ID NO:49 Amino acid sequence of CD20 / CD19-reactive ScFv binding domain

[0446]

Claims

1. An isolated nucleic acid molecule encoding a fully human CD20 / CD19 tandem chimeric antigen receptor (CAR), wherein the fully human CD20 / CD19 tandem chimeric antigen receptor (CAR) comprises at least one extracellular antigen binding domain, at least one transmembrane domain and at least one intracellular signaling domain, wherein the extracellular antigen binding domain contains a fully human CD20 / CD19 antigen binding 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 via a linker or spacer domain.

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 connected to the transmembrane domain.

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

7. The isolated nucleic acid molecule of claim 6, wherein the leading nucleotide sequence comprises a nucleotide sequence of SEQ ID NO: 11 that contains a leading amino acid sequence encoding SEQ ID NO:

12.

8. The isolated nucleic acid molecule of claim 1, wherein the transmembrane domain comprises a transmembrane domain of a protein comprising: an α, β or ζ chain of a T cell receptor, CD8, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD83, CD86, CD134, CD137, CD154 and TNFRSF19, an IgG hinge, CD2, a CH3 domain, or any combination thereof.

9. 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 having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

10. 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. The isolated nucleic acid molecule of claim 10, wherein the encoded at least one intracellular signaling domain is arranged on the C-terminal side relative to the CD3ζ intracellular domain.

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

13. The isolated nucleic acid molecule of claim 12, wherein the encoded at least one co-stimulatory 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, at least one transmembrane domain and at least one intracellular signaling domain, wherein the extracellular antigen binding domain comprises a fully human CD20 / CD19 antigen binding domain comprising an amino acid sequence of SEQ ID NO. 2, 4 or 49.

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. The CAR of claim 15, wherein the transmembrane domain comprises a transmembrane domain of a protein comprising: the α, β or ζ chain of a T cell receptor, CD8, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154 and TNFRSF19 or any combination thereof.

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 that is 85%, 90%, 95%, 96%, 97%, 98% or 99% identical 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 and the at least one intracellular signaling domain or both are connected to the transmembrane domain via a linker or a spacer domain, and the extracellular antigen binding domain comprises a fully human CD20 / CD19 antigen binding domain comprising an amino acid sequence of SEQ ID NO. 2, 4 or 49.

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

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

23. The CAR of claim 22, wherein the at least one intracellular signaling domain comprises a co-stimulatory domain containing 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. The vector of claim 24, wherein the vector is selected from: a DNA vector, an RNA vector, a plasmid vector, a cosmid vector, a herpes virus vector, a measles virus vector, a lentiviral vector, an adenoviral vector or a retroviral vector, or a combination thereof.

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

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. The cell of claim 28, wherein the cell is a T cell.

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

31. The cell of claim 28, wherein the cell is a human cell.

32. A method for preparing cells, comprising transducing T cells with the vector of claim 24.

33. A method for producing a population of cells modified with RNA, comprising introducing in vitro transcribed RNA or synthetic RNA into cells, wherein the RNA comprises the nucleic acid molecule of claim 1.

34. A method of providing anti-tumor immunity in a mammal, comprising administering to the mammal an effective amount of the cell 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. A pharmaceutical composition comprising an anti-tumor effective amount of a human T cell population, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises at least one extracellular antigen binding domain, at least one linker domain, at least one transmembrane domain, and at least one intracellular signaling domain, and the extracellular antigen binding domain comprises a fully human CD20 / CD19 antigen binding domain comprising an amino acid sequence of SEQ ID NO.2, 4 or 49, and wherein the T cells are T cells of a person suffering from cancer.

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 a T cell receptor, CD8, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154 and TNFRSF19, or any combination thereof.

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

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

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

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

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

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

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

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

46. ​​The method of claim 44 or 45, wherein the at least one transmembrane domain comprises a transmembrane domain of a protein comprising: the α, β or ζ chain of the T cell receptor, CD8, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, or any combination thereof.

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

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

49. A pharmaceutical composition comprising an effective amount of a human T cell population for anti-allergy, anti-autoimmunity, anti-allogeneic immunity or anti-self-invasiveness, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises at least one extracellular antigen binding domain, at least one linker domain, at least one transmembrane domain, and at least one intracellular signaling domain, and the extracellular antigen binding domain comprises a fully human CD20 / CD19 antigen binding domain comprising an amino acid sequence of SEQ ID NO.2, 4 or 49, and wherein the T cells are T cells of a person suffering from a disease.

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

51. The pharmaceutical composition of claim 49, wherein the disease comprises an autoimmune disease, an alloimmune disease, and an autoinvasive disease, including rheumatoid arthritis, lupus, celiac disease, Sjögren's syndrome, multiple sclerosis, polymyalgia rheumatica, ankylosing spondylitis, type 1 diabetes, alopecia areata, vasculitis, temporal arteritis, poststreptococcal autoimmune disorder, anti-neuronal antibody-mediated neuropsychiatric disorder, immune-mediated extrapyramidal movement disorder, Sydenham's chorea, alloimmune hemolytic disease, pulmonary fibrosis, systemic scleroderma or fibrotic disease, or any combination thereof.

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