Compositions and methods for treating cancer with Anti-CD38 immunotherapy

Abstract

The present application relates to compositions and methods for the treatment of cancer with anti-CD38 immunotherapy. Chimeric antigen receptors comprising a CD38 antigen binding domain are disclosed. 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] This application is a divisional application of the Chinese patent application with an application date of November 27, 2019, application number "201980090184.6", and invention name "Compositions and methods for treating cancer with anti-CD38 immunotherapy". The original application is the Chinese national phase application of international application PCT / US2019 / 063761.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0003] This invention was made in performance of a cooperative research and development agreement with the National Institutes of Health, Department of Health and Human Services. The U.S. Government has certain rights in this invention. Technical Field

[0004] The present application relates to the field of cancer, and in particular to a CD38 antigen binding domain and a chimeric antigen receptor (CAR) comprising such a CD38 antigen binding domain and methods of using the same. Background Art

[0005] 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 a 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 metastasize and grow rapidly in an uncontrolled manner, which makes treatment extremely difficult.

[0006] Multiple myeloma ("multiple myeloma, MM") is a debilitating and often incurable disease with more than 30,000 new cases diagnosed in the United States each year (Source: MM Research Foundation). In the United States, MM is the second most common blood cancer after non-Hodgkin's lymphoma ("Non-Hodgkin's Lymphoma, NHL") (Smith L, McCourt O, Henrich M et al., Multiple myeloma and physical activity: a scoping review. BMJ Open. 2015; 5: e009576). MM affects plasma cells in the bone marrow and can lead to bone marrow failure and patient death (National Cancer Institute. Asnapshot of myeloma. November 5, 2014. See cancer.gov on the World Wide Web). Complications of myeloma include bone pain, bone loss, anemia, immunosuppression, renal dysfunction, and neuropathy (Mayo Clinic staff. Diseases and conditions: multiple myeloma: treatments and drugs. December 4, 2015).

[0007] First-line treatment for MM includes proteasome inhibitors, immunomodulatory drugs, steroids, histone deacetylase (HDAC) inhibitors and chemotherapy. These approaches aim to kill MM cells, however, many of them are also associated with extensive immunosuppression and systemic toxicity.

[0008] Immunotherapy approaches for MM include the FDA-approved monoclonal antibodies Empliciti (elotuzumab, targeting SLAMF7) and Darzalex (daratumumab, targeting CD38). In addition, checkpoint inhibitors are being evaluated in clinical trials for patients with MM. Despite the abundance of treatment options, the survival rate for patients with advanced MM is only 83 months for stage II MM and 43 months for stage III (American Cancer Society, see cancer.org). Therefore, for MM and other CD38 + Malignant tumors urgently need better treatments.

[0009] Like MM, B cell chronic lymphocytic leukemia ("chronic lymphocytic leukemia, CLL") is another difficult-to-treat malignancy. CLL is a lymphoproliferative disease with different survival prognoses (ranging from several months to decades). The disease mainly affects the elderly, and the median onset time is more than 65 years old. However, despite the high age of onset, CLL diagnosis tends to shorten the patient's life expectancy (Shanafelt, Tait. "Treatment of older patients with chronic lymphocytic leukemia: key questions and current answers." ASH Education Program Book 2013.1 (2013): 158-167.). The speed of disease progression usually depends on the type of BCR mutation present (Dighiero, Guillaume. "CLL biology and prognosis." ASH Education Program Book 2005.1 (2005): 278-284.). In addition, in CLL, CD38 is used as a marker for unfavorable disease prognosis. CD38 expression in CLL is associated with increased proliferation, migration, and responsiveness to BCR-mediated signaling of leukemic cells (Malavasi, Fabio et al., "CD38 and chronic lymphocytic leukemia: a decade later." Blood (2011): blood-2011). Although patients with a good prognosis may not need to be treated for CLL, it is virtually incurable, and early treatment does not improve prognosis. The first-line treatment of CLL typically includes several therapeutic agents, i.e., a combination of chemotherapeutic agents (cyclophosphamide, fludarabine), anti-CD20 antibody rituximab (Rituximab) (Rituxan), BTK inhibitor Ibrutinib (Ibrutinib), high-dose prednisolone, or Alemtuzumab (Alemtuzumab) (Campath). Despite the presence of a large number of therapeutic agents, CLL treatment regimens still tend to be aggressive, and toxicity remains a problem.Elderly patients and those with comorbidities remain particularly susceptible to CLL treatment-related toxicities, such as the effects of immunosuppression and susceptibility to infection (Barrientos, Jacqueline C. "Management of chronic lymphocytic leukemia in the elderly." Cancer Control 22.4_Suppl (2015): 17-23; Smolej,. "How I treat elderly or comorbid patients with chronic lymphocytic leukemia." Acta Medica (Hradec Kralove) 53.4 (2010): 213-220).

[0010] Due to the suboptimal efficacy and toxicity associated with the treatment of CD38-positive malignancies, better treatment options are needed. CAR-based approaches to treat CD38-positive malignancies and CD38-positive tumor microenvironment cells may provide a much-needed treatment option for patients who cannot tolerate the toxicity associated with traditional first-line treatments. In addition, the CAR T approach is not expected to have the non-selective and often irreversible toxicity associated with chemotherapy or chemo / immunotherapy combinations. In addition, CAR T cells are "living drugs" and are therefore expected to remain in the patient's body for an extended period of time or indefinitely, thereby providing a long-term therapeutic effect. These characteristics of CAR T cells are assumed to avoid the need for repeated treatments and reduce the likelihood of disease recurrence.

[0011] The CAR approach targeting CD38 is superior to chemotherapy because it is effective in eliminating CD38. + Better efficacy can be achieved in terms of tumor cells and tumor stem cells, and because it avoids the toxicity associated with chemotherapy. Importantly, CAR T cells are expected to be more effective than chemotherapy in eliminating minimal residual disease, resulting in better long-term treatment prognosis. In addition, since CAR T cells targeting CD38 ("CAR38") can help patients with high tumor burden become eligible for BMT, they can be used as a bridge to transplantation for tumor debulking.

[0012] CAR38 represents an improvement over the prior art because a unique human ScFv (hereafter referred to as "hScFv") sequence is used in the CAR design, as opposed to the murine-derived ScFvs used in CAR designs elsewhere. Mouse-derived sequences carry the risk of immunogenicity and can induce allergic or anaphylactic reactions in patients, leading to CAR T elimination or life-threatening hypersensitivity.

[0013] Chimeric antigen receptors (CARs) are hybrid molecules containing three basic units: (1) an extracellular antigen binding motif, (2) a junction / transmembrane motif, and (3) an intracellular T cell signaling motif (Long AH, Haso WM, Orentas RJ. Lessons learned from a highly-active CD22-specific chimeric antigen receptor. Oncoimmunology. 2013; 2(4): e23621). The antigen binding motif of CAR is usually formed after the single chain variable fragment (ScFv) of the minimal binding domain of the immunoglobulin (Ig) molecule. Alternative antigen binding motifs have also been modified, such as receptor ligands (i.e., 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 (e.g., 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 important work to be done to define the most active T cell populations for transduction with CAR vectors, determine the optimal culture and expansion techniques, and define the molecular details of the CAR protein structure itself.

[0014] The connection motif of CAR can be a relatively stable structural domain, such as the constant domain of IgG, or a flexible linker 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 can 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. This 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 connected 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 beads for activation, it was found that the third-generation vectors were not superior to the second-generation vectors in in vitro assays, and they did not provide a clear benefit over the second-generation vectors in mouse models of leukemia (Haso W, Lee DW, Shah NN, Stetler-Stevenson M, Yuan CM, Pastan IH, Dimitrov DS, Morgan RA, FitzGerald DJ, Barrett DM, Wayne AS, Mackall CL, Orentas RJ. Anti-CD22-chimeric antigen receptors targeting B cell precursor acute lymphoblastic leukemia. Blood. 2013; 121 (7): 1165–74; Kochenderfer JN et al., Blood. 2012; 119 (12): 2709–20). This is confirmed by the clinical success of CD19-specific CARs in the 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).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). The culture conditions for culturing CAR T cell populations are also important.

[0015] T cell-based immunotherapy has emerged as a new frontier in synthetic biology; multiple promoters and gene products are being considered to direct these highly potent cells to the tumor microenvironment, where they can both evade negative regulatory signals and mediate effective tumor killing. Elimination of unwanted T cells by drug-induced dimerization of an inducible caspase 9 construct with AP1903 illustrates a way in which a powerful switch that can control a population of T cells can be pharmacologically activated (Di Stasi A et al., N Engl J Med. 2011; 365(18): 1673–83). Generation of effector T cell populations immune to the negative regulatory effects of transforming growth factor-β by expression of decoy receptors further illustrates the extent to which effector T cells can be engineered to achieve optimal antitumor activity (Foster AE et al., J Immunother. 2008; 31(5): 500–5). Thus, although CARs appear to be able to trigger T cell activation in a manner similar to endogenous T cell receptors, the major obstacle to the clinical application of this technology to date has been the limitation of CARs to the extent that they can be used to target T cells. + In vivo expansion of T cells, rapid disappearance of cells after infusion, and disappointing clinical activity. Therefore, there is an urgent and long-standing need in the art to discover new compositions and methods for treating MM and CLL using methods that can exhibit specific and effective anti-tumor effects without the above-mentioned disadvantages (i.e., high toxicity, insufficient efficacy).

[0016] 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, the present invention as disclosed and described herein provides such CARs: which can be used to treat diseases, disorders or conditions associated with dysregulated CD38 expression, and the CARs comprise a CD38 antigen binding domain that exhibits high surface expression on transduced T cells, exhibits a high degree of cell lysis and in vivo expansion and persistence of transduced T cells. Summary of the invention

[0017] Provided herein are new anti-CD38 antibodies or their antigen binding domains and chimeric antigen receptors (CARs) comprising such CD38 antigen binding domains, as well as host cells (e.g., T cells) expressing the receptors and nucleic acid molecules encoding the receptors.CAR may consist of a single molecule expressed on the surface of effector cells, or CAR may comprise a signal transduction module and a soluble targeting module expressed by effector cells, such as when a soluble targeting module is combined with a signal transduction module expressed by a cell, to form a complete functional CAR.CAR exhibits high surface expression on transduced T cells, with a high degree of cell lysis and transduced T cells in vivo amplification and persistence.Also provided are methods for treating cancer in a subject using disclosed CAR, host cells and nucleic acid molecules, for example.

[0018] Thus, in one aspect, an isolated polynucleotide encoding a human anti-CD38 antibody or fragment thereof is provided, comprising a nucleic acid sequence selected from SEQ ID NO: 1, 3, 5, 7, 9, 11, 15, 17, 19, 21, 23, 25, 69, 71, 73 and 75.

[0019] In one embodiment, an isolated polynucleotide encoding a fully human anti-CD38 antibody or fragment thereof is provided, wherein the antibody or fragment thereof comprises a fragment selected from the group consisting of a Fab fragment, a F(ab')2 fragment, a Fv fragment, and a single-chain Fv (ScFv).

[0020] In one embodiment, an isolated polynucleotide encoding a fully human anti-CD38 antibody or a fragment thereof is provided, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 16, 18, 20, 22, 24, 26, 70, 72, 74 and 76.

[0021] In one aspect, an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR) is provided, wherein the CAR comprises from N-terminus to C-terminus at least one CD38 antigen binding domain, at least one transmembrane domain and at least one intracellular signaling domain, wherein the at least one CD38 antigen binding domain is encoded by a nucleotide sequence comprising a nucleic acid sequence selected from SEQ ID NO: 1, 3, 5, 7, 9, 11, 15, 17, 19, 21, 23, 25, 69, 71, 73 and 75.

[0022] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded extracellular CD38 antigen binding domain comprises at least one single chain variable fragment of an antibody that binds to CD38.

[0023] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded extracellular CD38 antigen binding domain comprises at least one heavy chain variable region of an antibody that binds to CD38.

[0024] In one embodiment, the targeting domain of CAR is independently expressed in the form of a monoclonal antibody, ScFv Fab, Fab'2 and comprises an antigen targeting domain coupled to an additional binding tag or epitope, wherein the antigen targeting domain comprises a nucleic acid sequence selected from SEQ ID NO: 1, 3, 5, 7, 9, 11, 15, 17, 19, 21, 23, 25, 69, 71, 73 and 75, and the effector cell expression component of CAR comprises a binding domain, which is specifically directed to bind to a tag or epitope expressed on a soluble CAR module, such as the specific binding of the soluble component of CAR to the cell binding component of CAR to form a complete functional CAR structure.

[0025] In another embodiment, the targeting domain of CAR is independently expressed in the form of a monoclonal antibody, ScFv Fab, Fab'2 and comprises an antigen targeting domain and an additional ScFv, wherein the antigen targeting domain comprises a nucleic acid sequence selected from SEQ ID NO: 1, 3, 5, 7, 9, 11, 15, 17, 19, 21, 23, 25, 69, 71, 73 and 75, and the effector cell expression component of CAR comprises a tag or epitope that specifically reacts with the additional ScFv expressed on the soluble CAR module, such as the specific binding of the soluble component of CAR to the cell binding component of CAR to form a complete functional CAR structure.

[0026] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded CAR extracellular CD38 antigen binding domain further comprises at least one lipocalin-based antigen binding antigen (anticalin) that binds to CD38.

[0027] In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD38 antigen binding domain is connected to the transmembrane domain via a linker domain.

[0028] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded CD38 extracellular antigen binding domain is preceded by a sequence encoding a leader or signal peptide.

[0029] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the CAR comprises at least one CD38 antigen binding domain, wherein the at least one CD38 antigen binding domain is encoded by a nucleotide sequence comprising a nucleic acid sequence selected from SEQ ID NO: 1, 3, 5, 7, 9, 11, 15, 17, 19, 21, 23, 25, 69, 71, 73 and 75, and wherein the CAR further encodes an extracellular antigen binding domain targeting an antigen, including but not limited to CD19, CD20, CD22, ROR1, mesothelin, CD33, CD38, CD138, BCMA (CD269), GPC2, GPC3, FGFR4, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, NY-ESO-1 TCR, MAGE A3 TCR, or any combination thereof.

[0030] In certain embodiments, an isolated nucleic acid molecule encoding a CAR is provided, wherein the additionally encoded extracellular antigen binding domain comprises an anti-CD19 ScFv antigen binding domain, an anti-CD20 ScFv antigen binding domain, 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-c-Met ScFv antigen binding domain, an anti-PMSA ScFv antigen binding domain, an anti-glycolipid F77 ScFv antigen binding domain. ScFv antigen binding domain, anti-EGFRvIII ScFv antigen binding domain, anti-GD-2 ScFv antigen binding domain, anti-NY-ESo-1 TCR 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.

[0031] In one aspect, a CAR provided herein further comprises a linker or spacer domain.

[0032] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the extracellular CD38 antigen binding domain, the intracellular signaling domain, or both are connected to the transmembrane domain via a linker or spacer domain.

[0033] 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.

[0034] 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, CD86, CD134, CD137 and CD154, or a combination thereof.

[0035] 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.

[0036] 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 CD3 zeta intracellular domain.

[0037] 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.

[0038] 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.

[0039] 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 a nucleotide sequence of SEQ ID NO: 13, SEQ ID NO: 39, SEQ ID NO: 41 or SEQ ID NO: 43.

[0040] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded leader sequence comprises the amino acid sequence of SEQ ID NO: 14, SEQ ID NO: 40, SEQ ID NO: 42 or SEQ ID NO: 44.

[0041] In one aspect, provided herein is a chimeric antigen receptor (CAR) comprising, from N-terminus to C-terminus, at least one CD38 antigen binding domain, at least one transmembrane domain, and at least one intracellular signaling domain.

[0042] In one embodiment, a CAR is provided, wherein the extracellular CD38 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.

[0043] 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 and CD154, or a combination thereof.

[0044] In some embodiments, a CAR is provided, wherein the CAR further encodes an extracellular antigen binding domain comprising CD19, CD20, CD22, ROR1, mesothelin, CD33, CD38, CD123 (IL3RA), CD138, BCMA (CD269), GPC2, GPC3, FGFR4, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, 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.

[0045] In one embodiment, a CAR is provided, wherein the extracellular antigen binding domain comprises an anti-CD19 ScFv antigen binding domain, an anti-CD20 ScFv antigen binding domain, 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-c-Met ScFv antigen binding domain, an anti-PMSA ScFv antigen binding domain, an anti-glycolipid F77 ScFv antigen binding domain, an anti-EGFRvIII ScFv antigen binding domain, anti-GD-2ScFv antigen binding domain, anti-NY-ESo-1 TCR 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.

[0046] In another embodiment, a CAR is provided, wherein the extracellular antigen binding domain comprises only an immunoglobulin variable heavy chain (VH) anti-CD19 antigen binding domain, an anti-CD20 VH antigen binding domain, an anti-CD22 VH antigen binding domain, an anti-ROR1 VH antigen binding domain, an anti-mesothelin VH antigen binding domain, an anti-CD33 VH antigen binding domain, an anti-CD38 VH antigen binding domain, an anti-CD123 (IL3RA) VH antigen binding domain, an anti-CD138 VH antigen binding domain, an anti-BCMA (CD269) VH antigen binding domain, an anti-GPC2 VH antigen binding domain, an anti-GPC3 VH antigen binding domain, an anti-FGFR4 VH antigen binding domain, an anti-c-Met VH antigen binding domain, an anti-PMSA VH antigen binding domain, an anti-glycolipid F77 VH antigen binding domain, an anti-EGFRvIII VH antigen binding domain, anti-GD-2 VH antigen binding domain, anti-NY-ESO-1 TCR VH antigen binding domain, anti-MAGE A3 TCR VH antigen binding domain, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or any combination thereof.

[0047] In another embodiment, a CAR is provided, wherein the extracellular antigen binding domain comprises a protein or peptide (P) sequence capable of specifically binding to a target antigen, which may be derived from a natural or synthetic sequence comprising: an anti-CD19 P antigen binding domain, an anti-CD20 P antigen binding domain, an anti-CD22 P antigen binding domain, an anti-ROR1 P antigen binding domain, an anti-mesothelin P antigen binding domain, an anti-CD33 P antigen binding domain, an anti-CD38 P antigen binding domain, an anti-CD123 (IL3RA) P antigen binding domain, an anti-CD138 P antigen binding domain, an anti-BCMA (CD269) P antigen binding domain, an anti-GPC2 P antigen binding domain, an anti-GPC3 P antigen binding domain, an anti-FGFR4 P antigen binding domain, an anti-c-Met P antigen binding domain, an anti-PMSA P antigen binding domain, an anti-glycolipid F77 P antigen binding domain, an anti-EGFRvIII P antigen binding domain, an anti-GD-2 P antigen binding domain, an anti-NY-ESO-1 TCR P antigen binding domain, anti-MAGE A3 TCR P antigen binding domain, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or any combination thereof. In another embodiment, a CAR is provided, wherein the at least one intracellular signaling domain comprises a co-stimulatory domain and a primary signaling domain.

[0048] 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.

[0049] In one embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 77. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 78.

[0050] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 87. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 88.

[0051] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 89. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 90.

[0052] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 91. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 92.

[0053] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 93. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 94.

[0054] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 95. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 96.

[0055] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 97. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 98.

[0056] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 99. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 100.

[0057] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 101. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 102.

[0058] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 103. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 104.

[0059] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 105. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 106.

[0060] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 107. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 108.

[0061] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 109. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 110.

[0062] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 111. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 112.

[0063] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 113. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 114.

[0064] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 115. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 116.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] In another embodiment, the vector expressing CAR may also be modified to include one or more control elements for CAR T cell expression or for eliminating 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).

[0069] 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.

[0070] In another aspect, a pharmaceutical composition comprising an anti-tumor effective amount of a population of human T cells is provided, 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 CD38 antigen binding domain comprising an amino acid sequence selected from SEQ ID NO: 2, 4, 6, 8, 10, 12, 16, 18, 20, 22, 24, 26, 70, 72, 74 and 76, wherein the T cells are T cells 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 (MM), or a combination thereof.

[0071] 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, CD86, CD134, CD137 and CD154, or a combination thereof.

[0072] 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), pediatric 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.

[0073] 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 lymphoma (NHL)), minimal residual disease (MRD) in pediatric B-cell malignancies (including B-lineage ALL (acute lymphoblastic leukemia)), multiple myeloma (MM), lung cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, melanoma or other hematological cancers and solid tumors, or any combination thereof.

[0074] 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 CD38, thereby preparing a CAR-T cell.

[0075] In another aspect, a method for producing an RNA-engineered 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.

[0076] In another aspect, a method for diagnosing a disease, disorder or condition associated with CD38 expression on a cell is provided, comprising: a) contacting the cell with a human anti-CD38 antibody or fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 16, 18, 20, 22, 24, 26, 70, 72, 74 and 76; and b) detecting the presence of CD38, wherein the presence of CD38 is diagnostic of a disease, disorder or condition associated with CD38 expression.

[0077] In one embodiment, the disease, disorder or condition associated with CD38 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 pediatric B-cell malignancies including B-lineage ALL (acute lymphoblastic leukemia), multiple myeloma (MM), lung cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, melanoma or other hematological cancers and solid tumors, or any combination thereof.

[0078] In another embodiment, a method for diagnosing, prognosing or determining the risk of a CD38-related disease in a mammal is provided, comprising detecting the expression of CD38 in a sample derived from the mammal, comprising: a) contacting the sample with a human anti-CD38 antibody or a fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 16, 18, 20, 22, 24, 26, 70, 72, 74 and 76; and b) detecting the presence of CD38, wherein the presence of CD38 is diagnostic of a CD38-related disease in the mammal.

[0079] In another embodiment, a method of inhibiting CD38-dependent T cell suppression is provided, comprising contacting a cell with a human anti-CD38 antibody or fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 16, 18, 20, 22, 24, 26, 70, 72, 74, and 76. In one embodiment, the cell is selected from a tumor cell expressing CD38, a tumor-associated macrophage, and any combination thereof.

[0080] In another embodiment, a method of blocking T cell suppression mediated by cells expressing CD38 and altering the tumor microenvironment to inhibit tumor growth in a mammal is provided, comprising administering to the mammal an effective amount of a composition comprising an isolated anti-CD38 antibody or a fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 16, 18, 20, 22, 24, 26, 70, 72, 74 and 76. In one embodiment, the cell is selected from a tumor cell expressing CD38, a tumor associated macrophage, and any combination thereof.

[0081] 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 an isolated anti-CD38 antibody or fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from SEQ ID NO: 2, 4, 6, 8, 10, 12, 16, 18, 20, 22, 24, 26, 70, 72, 74 or 76. In one embodiment, the antibody or fragment thereof inhibits the interaction between a first cell and a T cell, wherein the first cell is selected from a tumor cell expressing CD38, a tumor associated macrophage, and any combination thereof.

[0082] 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.

[0083] 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 CD38 and / or one or more of the aforementioned antigens, under the following conditions, the conditions being sufficient to form an immune complex of the antigen binding domain on the CAR with the extracellular domain of CD38 and / or one or more of the aforementioned antigens in the subject.

[0084] In another embodiment, a method for treating a mammal suffering from a disease, disorder or condition associated with elevated tumor antigen expression is provided, the method comprising administering to a 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 CD38 antigen binding domain, at least one linker or spacer domain, at least one transmembrane domain, at least one intracellular signaling domain, the at least one extracellular CD38 antigen binding domain comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 16, 18, 20, 22, 24, 26, 70, 72, 74 or 76, or any combination thereof, and wherein the T cells are T cells of a subject suffering from cancer.

[0085] 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 at least one CD38 antigen binding domain, at least one linker or spacer domain, at least one transmembrane domain, at least one intracellular signaling domain, and the at least one CD38 antigen binding domain comprises an amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 16, 18, 20, 22, 24, 26, 70, 72, 74 or 76, or any combination thereof, wherein the T cell is a T cell of a subject suffering from cancer. In some embodiments of the foregoing methods, the at least one transmembrane domain comprises a transmembrane 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, CD86, CD134, CD137 and CD154, or a combination thereof.

[0086] 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 CAR, wherein the CAR comprises at least one CD38 antigen binding domain, at least one transmembrane domain, and at least one intracellular signaling domain, and the at least one CD38 antigen binding domain comprises SEQ ID NO: 2, 4, 6, 8, 10, 12, 16, 18, 20, 22, 24, 26, 70, 72, 74 or 76 amino acid sequence or any combination thereof, 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 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years or 3 years.

[0087] In one embodiment, the progeny T cells in the human comprise memory T cells. In another embodiment, the T cells are autologous T cells.

[0088] In all aspects and embodiments of the methods described herein, any of the aforementioned cancers, diseases, disorders or conditions associated with elevated tumor antigen expression can be treated or prevented or ameliorated using one or more CARs disclosed herein.

[0089] In another aspect, a kit is provided 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 elevated tumor antigen expression in a subject as described above, the kit comprising a container containing any one or any combination of the nucleic acid molecules, vectors, host cells or compositions disclosed above, and instructions for using the kit.

[0090] 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 detailed description below with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0091] Figure 1 The general structure of a CAR targeting CD38 is depicted. The anti-CD38 ScFv targeting domain is linked in frame to the CD8 hinge and transmembrane domains, the 4-1BB (CD137) co-stimulatory domain, and the CD3ζ signaling domain.

[0092] Figure 2Surface expression of CD38-targeted CAR T constructs on human primary T cells is depicted. CAR T expression was determined by flow cytometry. T cells were cultured with Miltenyi Biotec TransAct in the presence of IL-2 as described in Materials and Methods. TM CD3 CD28 reagent activation, and transduction with LV.On the 8th day of culture, CD38-His reagent was followed by anti-His-PE to determine the CAR surface expression of surviving transduced T cells (7-AAD negative).The CAR construct LV used in transduction is listed below each bar.The bar represents the percentage of the CAR T positive group relative to the untransduced T cell control (UTD).The data represent three independent experiments performed with CAR T cells from three independent donors.

[0093] Figures 3A to 3C In vitro CAR T cell cytotoxicity was depicted. Using the CD38-positive tumor line RPMI-8226 ( Figure 3A ) and MM1.S( Figure 3B ) or the CD38-negative cell line 293T ( Figure 3C ) was used for luciferase-based cytotoxicity assay. The bars represent the mean + SD values ​​from three technical replicates. The data represent three independent experiments performed with CAR T cells from three independent donors.

[0094] Figures 4A to 4B CAR T cell factor release in response to MM cell lines is depicted. Figure 4A Shown are the IFNg cytokines produced by the CAR-Ts listed on the x-axis measured using ELISA after overnight co-culture with RPMI-8226 and MM1.S tumor lines at an E:T ratio of 10:1. Figure 4B The TNFα cytokines listed on the x-axis are shown after co-culture with RPMI-8226 and MM1.S tumor lines at an E:T ratio of 10:1 overnight using ELISA to measure the production of CAR-T. The bars represent the mean + SD of three replicates. The data represent three independent experiments performed with CAR T cells from three independent donors. DETAILED DESCRIPTION

[0095] definition

[0096] 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 a conflict, this specification (including the explanation of the term) 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:

[0097] 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.

[0098] 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. (eds.), 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.

[0099] The present disclosure provides CD38 antibodies or fragments thereof and chimeric antigen receptors (CARs) with such CD38 antigen binding domains. The enhancement of the functional activity of CAR is directly related to the enhancement of the functional activity of T cells expressing CAR. As a result of one or more of these modifications, CAR exhibits both high cytokine-induced cell lysis and cell surface expression on transduced T cells, as well as the persistence of the T cell amplification level in vivo and the transduced CAR-expressing T cells.

[0100] 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.

[0101] 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 they 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.

[0102] Surprisingly and unexpectedly, it has now been discovered that the use of fully human antigen-binding domains in CARs rather than mouse-derived antigen-binding fragments that are prone to inducing anti-mouse immune responses and CAR T elimination in the host (see, the UPenn-sponsored clinical trial using mouse derived SS1 ScFv sequence, NCT02159716) can also determine the functional activity of T cells expressing CARs.

[0103] CAR disclosed herein is expressed at high levels 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 CD38 antigens bound to CAR on their surface. The use of human extracellular CD38 antigen binding domains leads to the production of CARs that work better in vivo, while avoiding the induction of anti-CAR immunity and killing of CAR T cell populations in the host immune response. CARs expressing fully human extracellular CD38ScFv antigen binding domains exhibit excellent activity / characteristics, including i) preventing poor CAR T persistence and function as seen in the case of mouse-derived binding sequences; ii) making the lack of regional delivery of CAR effective; and iii) being able to produce CAR T cell designs based on both high-affinity and low-affinity binders to CD38. The latter property allows researchers to better tune the potency versus toxicity and / or tissue specificity of CAR T products, since lower affinity binders may have higher specificity for tumors compared to normal tissues due to higher expression of CD38 on tumors compared to normal tissues, which may prevent on-target off tumor toxicity and bystander cell killing.

[0104] The following is a detailed description of the CAR of the present invention, including a description of its extracellular CD38 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.

[0105] A. Chimeric Antigen Receptor (CAR)

[0106] The CAR disclosed herein comprises at least one CD38 antigen binding domain capable of binding to CD38, at least one transmembrane domain and at least one intracellular domain.

[0107] 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 receptors (T cell receptor, TCR) α and β chains.

[0108] 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 that includes an intracellular domain of a T cell receptor (e.g., but not limited to, an intracellular portion of a CD3ζ protein). A co-stimulatory signaling domain refers to a portion of a CAR that includes 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.

[0109] 1. Extracellular domain

[0110] 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.

[0111] 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, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, 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 tumor antigens disclosed herein are TNF-α, ...

[0112] 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 antigens, such as carcinoembryonic antigen (CEA). In B-cell lymphomas, tumor-specific idiotype immunoglobulins constitute the unique real tumor-specific immunoglobulin antigens of individual tumors. B cell differentiation antigens (such as CD19, CD20 and CD37) are other candidates for target antigens in B-cell lymphomas. Some of these antigens (CEA, HER-2, CD19, CD20, idiotype) have been used as targets for passive immunotherapy with monoclonal antibodies, but with limited success.

[0113] In a preferred embodiment, the tumor antigen is CD38, and tumors associated with CD38 expression include lung mesothelioma, ovarian cancer, and pancreatic cancer, which express high levels of the extracellular protein CD38, or any combination thereof.

[0114] 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.

[0115] Some non-limiting examples of TSAs or TAAs include the following: differentiation antigens, such as MART-1 / MelanA (MART-1), 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, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, α-fetoprotein, β-HCG, BCA225, BTAA, CA125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associated protein, TAAL6, TAG72, TLP and TPS.

[0116] In one embodiment, the antigen binding domain portion of CAR targets antigens including but not limited to the following: CD19, CD20, CD22, ROR1, CD123, CD33, CD38, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, MY-ESO-1TCR, MAGE A3 TCR, etc.

[0117] In a preferred embodiment, the antigen binding domain portion of the CAR targets the extracellular CD38 antigen.

[0118] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD38 hScFv M3801 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 CD38 hScFv M3801 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.

[0119] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD38 hScFv M3802 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 CD38 hScFv M3802 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.

[0120] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD38 hScFv M3803 antigen binding domain comprises the nucleotide sequence of SEQ ID NO: 5, 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 CD38 hScFv M3803 antigen binding domain comprises the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0121] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD38 hScFv M3804 antigen binding domain comprises the nucleotide sequence of SEQ ID NO: 7, 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 CD38 hScFv M3804 antigen binding domain comprises the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0122] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD38 hScFv M3806 antigen binding domain comprises the nucleotide sequence of SEQ ID NO: 9, 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 CD38 hScFv M3806 antigen binding domain comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0123] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD38 hScFv M3808 antigen binding domain comprises the nucleotide sequence of SEQ ID NO: 11, 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 CD38 hScFv M3808 antigen binding domain comprises the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0124] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD38 hScFv M3809 antigen binding domain comprises the nucleotide sequence of SEQ ID NO: 15, 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 CD38 hScFv M3809 antigen binding domain comprises the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0125] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD38 hScFv M3810 antigen binding domain comprises the nucleotide sequence of SEQ ID NO: 17, 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 CD38 hScFv M3810 antigen binding domain comprises the amino acid sequence of SEQ ID NO: 18, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0126] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD38 hScFv M3811 antigen binding domain comprises the nucleotide sequence of SEQ ID NO: 19, 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 CD38 hScFv M3811 antigen binding domain comprises the amino acid sequence of SEQ ID NO: 20, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0127] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD38 hScFv M3812 antigen binding domain comprises the nucleotide sequence of SEQ ID NO: 21, 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 CD38 hScFv M3812 antigen binding domain comprises the amino acid sequence of SEQ ID NO: 22, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0128] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD38 hScFv M3815 antigen binding domain comprises the nucleotide sequence of SEQ ID NO: 23, 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 CD38 hScFv M3815 antigen binding domain comprises the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0129] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD38 hScFv M3815_P antigen binding domain comprises the nucleotide sequence of SEQ ID NO: 25, 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 CD38 hScFv M3815_P antigen binding domain comprises the amino acid sequence of SEQ ID NO: 26, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0130] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD38 hScFv M3816 antigen binding domain comprises the nucleotide sequence of SEQ ID NO: 69, 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 CD38 hScFv M3816 antigen binding domain comprises the amino acid sequence of SEQ ID NO: 70, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0131] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD38 hScFv M3817 antigen binding domain comprises the nucleotide sequence of SEQ ID NO: 71, 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 CD38 hScFv M3817 antigen binding domain comprises the amino acid sequence of SEQ ID NO: 72, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0132] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD38 hScFv M8DR H_L antigen binding domain comprises the nucleotide sequence of SEQ ID NO: 73, 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 CD38 hScFv M8DR H_L antigen binding domain comprises the amino acid sequence of SEQ ID NO: 74, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0133] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD38 hScFv M8DR L_H antigen binding domain comprises the nucleotide sequence of SEQ ID NO: 75, 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 CD38 hScFv M8DR L_H antigen binding domain comprises the amino acid sequence of SEQ ID NO: 76, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto with the amino acid sequence of SEQ ID NO: 74.

[0134] The generation and binding properties of specific CD38 variable heavy chain only and ScFv antigen binding fragments or antigen binders described herein are shown in Example 1.

[0135] In various embodiments of the CD38-specific CAR disclosed herein, the general scheme is Figure 1 , and comprises, from N-terminus to C-terminus, a signal or leader peptide, anti-CD38 hScFv, an extracellular linker, CD8 transmembrane, 4-1BB, CD3ζ, wherein bold text indicates a cloning site for linking domains.

[0136] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO:77, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO:78.

[0137] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO:77, or a sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO:78, or a sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0138] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO:87, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO:88.

[0139] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO:87, or a sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO:88, or a sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0140] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO:89, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO:90.

[0141] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO:89, or a sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO:90, or a sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0142] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO:91, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO:92.

[0143] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO:91, or a sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO:92, or a sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0144] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO:93, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO:94.

[0145] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO:93, or a sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO:94, or a sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0146] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO:95, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO:96.

[0147] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO:95, or a sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO:96, or a sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0148] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO:97, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO:98.

[0149] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO:97, or a sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO:98, or a sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0150] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO:99, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO:100.

[0151] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO:99, or a sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO:100, or a sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0152] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO:101, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO:102.

[0153] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 101, or a sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO: 102, or a sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0154] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO:103, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO:104.

[0155] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 103, or a sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO: 104, or a sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0156] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO:105, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO:106.

[0157] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 105, or a sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO: 106, or a sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0158] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO:107, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO:108.

[0159] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 107, or a sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO: 108, or a sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0160] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO:109, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO:110.

[0161] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 109, or a sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO: 110, or a sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0162] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO:111, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO:112.

[0163] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 111, or a sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO: 112, or a sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0164] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO:113, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO:114.

[0165] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 113, or a sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO: 114, or a sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0166] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO:115, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO:116.

[0167] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 115, or a sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO: 116, or a sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0168] The surface expression of anti-CD38 CARs incorporating immunoglobulin heavy chain variable domains (VH) and single-chain variable fragments (ScFv) sequences that react with CD38 antigens is shown in Example 2 below. CAR T expression is determined by flow cytometry. On the 8th day of culture, CD38-His reagent was followed by anti-His-PE to determine the CAR surface expression of surviving transduced T cells (7-AAD negative). The CAR construct LV used in transduction is listed below each bar. The bar represents the percentage of the CAR T positive population relative to the untransduced T cell control (UTD). The data represent three independent experiments performed with CAR T cells from three independent donors.

[0169] The cytotoxic potential of CAR38 candidates was evaluated in an overnight luciferase-based killing assay using CD38-positive tumor lines RPMI-8226 and MM1.S or CD38-negative cell lines 293T stably transduced with luciferase. The bars represent the mean + SD values ​​from three technical replicates. The data represent three independent experiments performed with CAR T cells from three independent donors ( Figures 3A to 3C CAR T cells (effector) and tumor cells (target) were co-incubated overnight at an effector to target ratio (E:T) of 5, 10, or 20. + In the RPMI-8226 tumor line, most CAR constructs showed very potent tumor lytic activity ( Figure 3A ), which were comparable to the positive control constructs LTG2525 and LTG2526 containing comparative ScFv sequences in the VL-VH or VH-VL orientation, respectively. The exceptions were constructs LTG2098, LTG2099, which were less cytotoxic than the positive controls LTG2525 and 2526 ( Figure 3A In contrast, no significant lysis was observed for the negative control UTD (untransduced T cells), indicating that tumor lysis was CAR-dependent ( Figure 3A ). + In the killing assay of MM1.S cells, the potency differences between the CAR constructs were more prominent ( Figure 3B ), the CD38 +MM1.S cells are less susceptible to CAR T killing than RPMI-8226. The CAR test constructs with the strongest anti-MM1.S lytic activity were LTG2091 and LTG2092, which achieved greater lytic capacity than the control constructs LTG2525 and LTG2526 ( Figure 3B Most other constructs achieved MM1.S lysis in the high-moderate range, and constructs LTG2099, 2093, 2098 had low lytic activity in this cell line ( Figure 3B ).

[0170] Next, relative to CD38 - All CAR candidates and controls were tested in a killing assay on the cell line 293T ( Figure 3C ). In this case, no lytic activity above background levels was observed for most CAR constructs compared to the positive control CAR LTG 2526, indicating that these CAR constructs require CD38 expression for activation, which is a feature of CAR safety. The exception to this observation is constructs 2501, 2503, which exhibit low levels of CD38-independent killing activity at high E:T ratios ( Figure 3C ). The non-specific killing activity of these two constructs cannot be predicted based on the binder data alone and is therefore not obvious. Overall, the cell lytic activity of most CD38CARs in this group observed for MM1.S and RPMI-8226 tumor cells expressing CD38 is both target-specific and CAR T-dependent.

[0171] The ability of anti-CD38 CAR T cells to secrete proinflammatory cytokines IFNg and TNFa was then evaluated (Figure 4). The cytokines produced by the CAR-T listed on the x-axis were measured using ELISA after co-culture with RPMI-8226 and MM1.S tumor lines at an E:T ratio of 10:1 overnight. It is noteworthy that CAR T-expressing cells LTG2091, LTG2092, and LTG2095 produced high levels of IFNγ and TNFα comparable to the positive controls LTG2525 and LTG2526, while most other CAR constructs as well as the negative control UTD group produced low or no significant cytokine induction ( Figures 4A to 4B This result is consistent with the strongest in vitro cell lysis function of LTG2091, LTG2092 and LTG2095 (see Figures 3A to 3C). The second subset of constructs that do not produce high levels of cytokines but still have high-moderate anti-tumor activity includes LTG2089, 2090, 2094, 2096, 2097. This finding also suggests that it may be possible to select CAR38 constructs that are effective in killing tumors but have a low risk of inducing cytokine release syndrome, thereby having a better safety profile.

[0172] 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 the T cell receptor signaling complex (i.e., immunological synapse), or any combination thereof.

[0173] Although the present disclosure has been exemplified using exemplary extracellular CD38 variable heavy chain only and ScFv antigen binding domains, other nucleotide and / or amino acid variants within the CD38 variable heavy chain only and ScFv antigen binding domains can be used to derive the CD38 antigen binding domains used in the CARs described herein.

[0174] Depending on the desired antigen to be targeted, the CAR can be additionally engineered to include a suitable antigen binding domain specific for the desired antigen target. For example, if CD19 is the desired antigen to be targeted, an antibody against CD19 can be used as an antigen binding domain incorporated into the CAR.

[0175] In an exemplary embodiment, the antigen binding domain portion of CAR also targets CD19. Preferably, the antigen binding domain in CAR is an anti-CD19 ScFv, wherein the nucleic acid sequence of the anti-CD19ScFv comprises the sequence shown in SEQ ID NO:37. In one embodiment, the anti-CD19 ScFv comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:30. In another embodiment, the anti-CD19 ScFv portion of CAR comprises the amino acid sequence shown in SEQ ID NO:38.

[0176] 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.

[0177] In another aspect of the invention, a CAR is provided that is capable of binding to an antigen from the following bacterial strains: Staphylococci, Streptococcus, Escherichia coli, Pseudomonas, or Salmonella. In particular, CARs are provided that are capable of binding to antigens from infectious bacteria, such as Helicobacter pyloris, Legionella pneumophilia, bacterial strains of the genus 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), and Streptococcus pyogenes. agalactiae), Streptococcus pneumoniae, or Clostridium tetani, or a combination thereof.

[0178] 2. Transmembrane domain

[0179] With respect to transmembrane domains, the CAR comprises one or more transmembrane domains fused to the extracellular CD33 antigen binding domain of the CAR.

[0180] 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.

[0181] The transmembrane region particularly used for CAR described herein may be derived from the following (ie, at least comprising its transmembrane region): α, β or ζ chain of T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, mesothelin, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. 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.

[0182] 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.

[0183] In some cases, transmembrane domains may be selected by amino acid substitution to avoid binding of such domains to transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

[0184] 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: 27. In one embodiment, the CD8 transmembrane domain comprises a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO: 28. In another embodiment, the CD8 transmembrane domain comprises an amino acid sequence of SEQ ID NO: 28.

[0185] 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:28, or a sequence with 95% to 99% identity to an amino acid sequence of SEQ ID NO:28.

[0186] 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:29. In one embodiment, the CD8 hinge domain comprises a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO:30. In another embodiment, the CD8 hinge domain comprises an amino acid sequence of SEQ ID NO:30, or a sequence having 95% to 99% identity thereto.

[0187] 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.

[0188] In one embodiment, the transmembrane domain in the CAR of the present invention is a TNFRSF19 transmembrane domain. In one embodiment, the TNFRSF19 transmembrane domain comprises a nucleic acid sequence of SEQ ID NO:51. In one embodiment, the TNFRSF19 transmembrane domain comprises a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO:52. In another embodiment, the TNFRSF19 transmembrane domain comprises an amino acid sequence of SEQ ID NO:52.

[0189] 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:52, or a sequence with 95% to 99% identity to an amino acid sequence of SEQ ID NO:52.

[0190] 3. Spacer domain

[0191] In CAR, a spacer domain, also referred to as a hinge 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.

[0192] 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 described in U.S. Pat. Nos. 7,964,5667, 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.

[0193] 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.

[0194] As the spacer domain, all or part of the following can be used: amino acids 118 to 178 (which is the hinge region) of CD8.α. (NCBI RefSeq: NP.sub.--001759.3) (SEQ ID NO: 31), 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 (CH1 region or CL region, for example, a peptide having the amino acid sequence shown in SEQ ID NO: 32) can be used. In addition, the spacer domain may be an artificially synthesized sequence.

[0195] In addition, all or part of the amino acid comprising the constant region of human IgG4 (UniProt ID: P01861) may be used, which comprises CH1 (amino acids 1 to 98), hinge (SEQ ID NO: 80, and corresponding nucleotides SEQ ID NO: 79) (amino acids 99 to 110), CH2 (amino acids SEQ ID NO: 81 and corresponding nucleotides SEQ ID NO: 80) (amino acids 111 to 220) and CH3 (SEQ ID NO: 84 and corresponding nucleotides SEQ ID NO: 83) (amino acids 221 to 327) or a combination thereof, such as the IgG4 hinge CH2 CH3 domain (SEQ ID NO: 86 and corresponding nucleotides SEQ ID NO: 85).

[0196] In one embodiment, the spacer domain of the CAR comprises a TNFRSF19 hinge domain comprising a nucleic acid sequence of SEQ ID NO: 53. In one embodiment, the TNFRSF19 hinge domain comprises a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO: 54. In another embodiment, the TNFRSF19 hinge domain comprises an amino acid sequence of SEQ ID NO: 54, or a sequence having 95% to 99% identity thereto.

[0197] In one embodiment, the spacer domain of the CAR comprises a truncated hinge domain of TNFRSF19 comprising a nucleic acid sequence of SEQ ID NO: 55. In one embodiment, the truncated hinge domain of TNFRSF19 comprises a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO: 56. In another embodiment, the truncated hinge domain of TNFRSF19 comprises an amino acid sequence of SEQ ID NO: 56, or a sequence having 95% to 99% identity thereto.

[0198] In one embodiment, the TNFRSF19 hinge and transmembrane domain comprises the nucleic acid sequence of SEQ ID NO: 49. In one embodiment, the TNFRSF19 hinge and transmembrane domain comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 50. In another embodiment, the TNFRSF19 hinge and transmembrane domain comprises the amino acid sequence of SEQ ID NO: 50, or a sequence with 95% to 99% identity thereof.

[0199] In one embodiment, the CD8a hinge domain is fused to a TNFRSF19 transmembrane domain comprising a nucleic acid sequence of SEQ ID NO: 57. In one embodiment, the CD8a hinge domain is fused to a TNFRSF19 transmembrane domain comprising a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO: 58. In another embodiment, the CD8a hinge domain is fused to a TNFRSF19 transmembrane domain comprising an amino acid sequence of SEQ ID NO: 58 or a sequence having 95% to 99% identity thereto.

[0200] In addition, in CAR, a signal peptide sequence (also referred to as a leader peptide) may 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 for CAR. In one embodiment, the signal peptide comprises the amino acid sequence shown in SEQ ID NO: 14).

[0201] In one embodiment, the CD8α leader peptide comprises the nucleic acid sequence of SEQ ID NO: 43. In one embodiment, the CD8α leader peptide comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 44. In another embodiment, the CD8a hinge domain is fused to a TNFRSF19 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 44 or a sequence having 95% to 99% identity thereto.

[0202] In another embodiment, the GMCSF leader peptide comprises the nucleic acid sequence of SEQ ID NO: 39. In one embodiment, the GMCSF leader peptide comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 40. In another embodiment, the CD8a hinge domain is fused to a TNFRSF19 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 40, or a sequence having 95% to 99% identity thereof.

[0203] In another embodiment, the TNFRSF19 leader peptide comprises the nucleic acid sequence of SEQ ID NO: 41. In one embodiment, the TNFRSF19 leader peptide and the CD8α leader peptide comprise a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 42. In another embodiment, the CD8a hinge domain is fused to the TNFRSF19 transmembrane domain, which comprises the amino acid sequence of SEQ ID NO: 42 or a sequence having 95% to 99% identity thereto.

[0204] In one embodiment, the tag sequence encoding the truncated sequence of epidermal growth factor receptor (tEGFR) comprises the nucleic acid sequence of SEQ ID NO: 67. In one embodiment, tEGFR comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 68. In another embodiment, the tEGFR tag comprises the amino acid sequence of SEQ ID NO: 68, or a sequence having 95% to 99% identity thereto.

[0205] In one embodiment, the furin recognition site and downstream T2A self-cleaving peptide sequence designed for simultaneous bicistronic expression of the tag sequence and the CAR sequence comprise a nucleic acid sequence of SEQ ID NO: 65. In one embodiment, the furin and T2A sequences comprise a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO: 66. In another embodiment, the tEGFR tag comprises an amino acid sequence of SEQ ID NO: 66 or a sequence having 95% to 99% identity thereto.

[0206] In one embodiment, the upstream furin recognition site and T2A self-cleaving peptide sequence and the furin recognition downstream site designed for the simultaneous bicistronic expression of the tag sequence and the CAR sequence comprise the nucleic acid sequence of SEQ ID NO: 67. In one embodiment, the furin and T2A sequences comprise a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 68. In another embodiment, the tEGFR tag comprises the amino acid sequence of SEQ ID NO: 68 or a sequence having 95% to 99% identity thereto.

[0207] In one embodiment, the targeting domain of CAR is expressed separately in the form of a monoclonal antibody, ScFv Fab, Fab'2, and is contained in a binding tag or epitope, while the effector cell expression component of CAR comprises a binding domain that is specifically directed to bind to a tag or epitope expressed on a soluble CAR module, such as the specific binding of the soluble component of CAR to the cell binding component to form a complete functional CAR structure.

[0208] 4. Intracellular domain

[0209] 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.

[0210] 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.

[0211] 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).

[0212] 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 contain a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM.

[0213] 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.

[0214] In a preferred embodiment, the intracellular domain of CAR can be designed to include 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 CD3ζ chain portion and costimulatory signaling region. Costimulatory signaling region refers to a part of CAR containing the intracellular domain of costimulatory molecules. 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 specifically binding 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.

[0215] 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.

[0216] 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.

[0217] 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: 33, SEQ ID NO: 45 or SEQ ID NO: 59, respectively, and the signaling domain of CD3-ζ includes the nucleic acid sequence shown in SEQ ID NO: 35, SEQ ID NO: 47 or SEQ ID NO: 61, respectively.

[0218] 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 comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 34, SEQ ID NO: 46 or SEQ ID NO: 60, respectively, and the signaling domain of CD3-ζ comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 36 or SEQ ID NO: 48 or SEQ ID NO: 62.

[0219] 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 comprises the amino acid sequence shown in SEQ ID NO: 34, SEQ ID NO: 46, or SEQ ID NO: 60, respectively, and the signaling domain of CD3-ζ comprises the amino acid sequence shown in SEQ ID NO: 36, SEQ ID NO: 48, or SEQ ID NO: 62, respectively.

[0220] In one embodiment, the intracellular domain in the CAR is designed to include 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:45 or SEQ ID NO:59, respectively, and the signaling domain of CD3-ζ comprises the nucleic acid sequence shown in SEQ ID NO:35, SEQ ID NO:47 or SEQ ID NO:61, respectively.

[0221] In one embodiment, the intracellular domain in the CAR is designed to comprise the signaling domain of CD28 and the signaling domain of CD3-ζ, wherein the signaling domain of CD28 comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 46 or SEQ ID NO: 60, respectively, and the signaling domain of CD3-ζ comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 36, or SEQ ID NO: 48, or SEQ ID NO: 62.

[0222] In one embodiment, the intracellular domain in the 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 amino acid sequence shown in SEQ ID NO:46 or SEQ ID NO:60, respectively, and the signaling domain of CD3-ζ comprises the amino acid sequence shown in SEQ ID NO:36, SEQ ID NO:48, or SEQ ID NO:62, respectively.

[0223] 5. Additional description of CAR

[0224] The functional part of CAR disclosed herein is also clearly included within 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 CAR parts that retain the ability to identify 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.

[0225] 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.

[0226] 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.

[0227] 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.

[0228] 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.

[0229] The CAR may consist essentially of the specific amino acid sequence described herein, such that other components (e.g., other amino acids) do not substantially alter the biological activity of the functional variant.

[0230] 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.

[0231] 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, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, γ-diaminobutyric acid, β-diaminopropionic acid, homophenylalanine and α-tert-butylglycine.

[0232] 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.

[0233] 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, editor, Reid, R., Marcel Dekker, Inc., 2000; Epitope Mapping, editor, 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, for example, 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.

[0234] B. Antibodies and Antigen-binding Fragments

[0235] 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.

[0236] 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.

[0237] "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 a subject. 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).

[0238] 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.

[0239] 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., p. 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. Reference to "VL" or "VL" refers to the variable domain of an antibody light chain, including the variable domain of an Fv, ScFv, dsFv or Fab.

[0240] 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, US Department 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.

[0241] 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 Tcell 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 found, and VL CDR1 is the CDR1 of the variable domain of the light chain of the antibody in which it is found. Light chain CDRs are sometimes referred to as LCDR1, LCDR2, and LCDR3. Heavy chain CDRs are sometimes referred to as HCDR1, HCDR2, and HCDR3.

[0242] "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).

[0243] 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.

[0244] 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 with a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and create two antigen binding sites (see, e.g., Holliger et al., Proc. Natl. Acad. Sci., 90:64446448, 1993; Poljak et al., Structure, 2:1121 1123, 1994).

[0245] 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.

[0246] 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).

[0247] An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks 50% or more of the binding of the reference antibody to its antigen in a competition assay, and conversely, the reference antibody blocks 50% or more of the binding of the antibody to its antigen in a competition assay. Antibody competition assays are known, and exemplary competition assays are provided herein.

[0248] "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., having 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.

[0249] A "chimeric antibody" is an antibody comprising sequences derived from two different antibodies (which are usually from different species). In some examples, a chimeric antibody comprises one or more CDRs and / or framework regions from one human antibody and CDRs and / or framework regions from other human antibodies.

[0250] "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. Phagedisplay: 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).

[0251] 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.

[0252] 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).

[0253] 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).

[0254] C. Conjugates

[0255] 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 covalent attachments of effector molecules or detectable markers 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.

[0256] 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).

[0257] 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 an effector molecule or a detectable marker. 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 may 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, linear 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 attached via its side groups to the constituent amino acids (e.g., to cysteine ​​via a disulfide bond) or to the alpha carbon amino and carboxyl groups of the terminal amino acids.

[0258] In several embodiments, the linker may include a spacer element that, when present, increases the size of the linker so 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 the following: U.S. Patent Nos. 7,964,5667, 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, 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.

[0259] 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 within 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, e.g., in U.S. Pat. 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. Pat. No. 6,214,345, which describes the synthesis of doxorubicin with a valine-citrulline linker).

[0260] In other embodiments, the cleavable linker is pH sensitive, i.e., sensitive to hydrolysis at certain pH values. Generally speaking, 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. Patent 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., such as a thioether attached to the therapeutic agent via an acylhydrazone bond (see, e.g., U.S. Pat. No. 5,622,929).

[0261] 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).

[0262] 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).

[0263] 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).

[0264] 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 in a sample of the conjugate is cleaved. Whether the linker is resistant to cleavage in the extracellular environment 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. 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.

[0265] 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.

[0266] 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 (US Pat. No. 3,896,111). Subsequently, it was found that some microorganisms also produced maytansine compounds, such as maytansinol and C-3 maytansinol esters (US Pat. 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 425235 Bl, the disclosures of which are expressly incorporated herein by reference.

[0267] 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, see U.S. Patent Nos. 5,079,163 and 4,689,401).

[0268] Saponin is a toxin 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 for efficient uptake by the cell.

[0269] Diphtheria toxin is isolated from Corynebacterium diphtheriae. Generally speaking, diphtheria toxins used for immunotoxins are mutated to reduce or eliminate non-specific toxicity. A mutant called CRM107 with full enzyme activity but significantly reduced non-specific 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.

[0270] Ricin is a lectin RCA60 from Ricinus communis (castor bean). For examples of ricin, see U.S. Pat. No. 5,079,163 and U.S. Pat. No. 4,689,401. Based on their molecular weights, Ricinus communis agglutinin (RCA) is referred to as RCA in the form of approximately 65 and 120 kD, respectively. 60 and RCA 120 There are two forms of ricin (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).

[0271] 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).

[0272] Abrins comprise toxic lectins from Abrus precatorius. Toxin mechanism, abrin a, b, c and d have a molecular weight of about 63 to 67 kD and are composed of two disulfide-linked polypeptide chains, A and B. Chain A inhibits protein synthesis; chain B (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).

[0273] CAR, T cells expressing CAR, monoclonal antibodies specific for one or more antigens disclosed herein, and 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, fiberoscopy, and laparoscopy). Some specific non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzyme linkages, radioisotopes, 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 are also useful, such as luciferase, green fluorescent protein (GFP), yellow fluorescent protein (YFP). 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.

[0274] 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.

[0275] 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 polypeptide labels 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.

[0276] Means 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 labels can be detected using photodetectors to detect emitted radiation. 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.

[0277] D. Nucleotides, expression, vectors and host cells

[0278] 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.

[0279] In some embodiments, the nucleotide sequence can be codon-modified. Without being bound by a particular theory, it is believed that codon optimization of nucleotide sequences improves the translation efficiency of mRNA transcripts. Codon optimization of nucleotide sequences can involve replacing natural codons 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 nucleotide sequences can also reduce secondary mRNA structures that interfere with translation, thereby improving translation efficiency.

[0280] 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).

[0281] "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, the nucleic acid does not include any insertion, deletion, inversion and / or replacement. However, as discussed herein, in some cases, it may be appropriate for the nucleic acid to include one or more insertions, deletions, inversions and / or replacements.

[0282] Recombinant nucleic acid can be a nucleic acid having a non-naturally occurring sequence or having a sequence prepared by an artificial combination of two otherwise separated sections of a sequence. This artificial combination is usually by chemical synthesis, or more commonly by the artificial manipulation of separated nucleic acid sections, 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, and the modified nucleotides 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 by 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-galactosylquenoside, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-substituted adenine In some embodiments, the nucleic acid of the present invention can be purine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosyl quercetin, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyl adenine, uracil-5-oxyacetic 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).

[0283] 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.

[0284] 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.

[0285] 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 for distinguishing 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 complementary sequences 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, for example, 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.

[0286] 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.

[0287] 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.

[0288] 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.

[0289] 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).

[0290] 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 Milone et al., Mol. Ther. 17 (8): 1453-1464 (2009) provided. Other examples of lentiviral vectors that can be used for clinical use 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.

[0291] 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).

[0292] 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)).

[0293] 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.

[0294] 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.

[0295] 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., complementation in auxotrophic hosts to provide prototrophy, 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.

[0296] 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 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.

[0297] 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.

[0298] 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. Suicide genes can be genes that give cells expressing genes sensitivity to reagents (e.g., drugs), and cause cell death when cells are contacted or exposed to reagents. 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, Institute of Cancer Research, Sutton, Surrey, UK), Humana Press, 2004), and include, for example, herpes simplex virus (HSV) thymidine kinase (TK) genes, cytosine deaminase, purine nucleoside phosphorylase, and nitroreductase.

[0299] 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 suspension 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.

[0300] 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 (e.g., 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 isolated from humans. T cells can be any type of T cell and can be at any stage of development, including but not limited to CD4 T cells. + / CD8 + Double positive T cells, CD4 + Helper T cells (such as Th1 and Th2 cells), CD8 + T cells (e.g., cytotoxic T cells), tumor infiltrating cells, memory T cells, memory stem cells (i.e., Tscm), naive T cells, etc. T cells can be CD8 + T cells or CD4 + T cells.

[0301] 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.

[0302] 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, myocytes, brain cells, etc. Alternatively, the cell group can be a substantially homogeneous group, wherein the group mainly includes (e.g., is substantially composed of) host cells containing recombinant expression vectors. 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 described herein.

[0303] 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 has a higher purity 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%.

[0304] E. Treatment methods

[0305] 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 to the mammal an amount of CAR, nucleic acid, recombinant expression vector, host cell, cell population, antibody and / or antigen binding portion thereof and / or pharmaceutical composition effective for treating or preventing cancer in the mammal.

[0306] 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.

[0307] For methods in which host cells or cell populations are administered, 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 of 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 from the same individual of an individual of which material is subsequently reintroduced.

[0308] 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 Lagomorpha, 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 Perissodactyla, including Equine (horses). Mammals can be Primates, Ceboid or Simoid (monkeys), or the order Anthropoids (humans and apes). Preferably, mammals are humans.

[0309] For the method, the cancer can be any cancer, including any of the following: acute lymphocytic carcinoma, 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 carcinoma, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumors, head and neck cancer (e.g., head and neck squamous cell carcinoma), Hodgkin 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 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.

[0310] 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.

[0311] In addition, the treatment or prevention provided by the method may include treating or preventing one or more disorders 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 disorders.

[0312] 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 to form a complex, (b) and detecting the complex, wherein detection of the complex indicates the presence of cancer in the mammal.

[0313] 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 an individual has and / or suffers from a certain condition or disease state. The condition or disease can be, for example, cancer.

[0314] 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.

[0315] The contacting can occur in vitro or in vivo with respect to the mammal. Preferably, the contacting is in vitro.

[0316] In addition, the detection of the complex can be performed 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)) as disclosed above), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase) and an element particle (e.g., gold particles)) can be used to label the CAR disclosed herein, the polypeptide described herein, the protein, the nucleic acid, the recombinant expression vector, the host cell, the cell group or the antibody or its antigen binding portion thereof.

[0317] 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) teach 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.

[0318] 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.

[0319] Any method of administration can be used for the disclosed therapeutic agent, including local administration and systemic administration. For example, topical, 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 details of the case (e.g., the subject, the disease, the disease state involved, and whether the treatment is preventive). In the case of administering more than one agent or composition, 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, wherein CAR, CAR T cells, conjugates, antibodies, antigen binding fragments, or compositions are provided in a non-toxic pharmaceutically acceptable carrier (e.g., water, saline, Ringer's solution, glucose 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 topically applied to the cornea as eye drops, or intravitreally administered into the eye.

[0320] The disclosed therapeutic agents can be formulated into unit dosage forms suitable for separate administration of precise doses. 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 main course of treatment may have more than one separate 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 the 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.

[0321] 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.

[0322] 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, e.g., at least 2 months, at least 4 months, at least 6 months, at least 12 months, at least 24 months, or at least 36 months.

[0323] In some embodiments, the disclosed method includes combining with disclosed antibodies, antigen binding fragments, conjugates, CARs or T cells expressing CARs, providing surgery, radiotherapy and / or chemotherapeutic agents (e.g., sequentially, substantially simultaneously or simultaneously) to the subject. Such agents 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 of additional agents can be used according to the manufacturer's instructions or determined empirically by skilled practitioners. Such chemotherapeutic preparation and dosing regimens are also described in Chemotherapy Service, (1992) editor, MCPerry, Williams & Wilkins, Baltimore, MD.

[0324] 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 other 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 (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 a skilled clinician.

[0325] 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, 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), 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, porfimer sodium, and trastuzumab. sodium) and verteporfin; and other agents, such as alitretinoin, hexamethylmelamine, amridine, 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.

[0326] Combination therapy can provide and prove synergistic, 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 form and administered or delivered simultaneously; (2) delivered in alternation or in parallel as separate formulations; or (3) by some other regimen. Synergy can be achieved when the compounds are administered or delivered sequentially, e.g., by different injections in separate syringes, when delivered in alternation. Generally, during alternation, an effective dose of each active ingredient is administered sequentially (i.e., sequentially), whereas in combination therapy, effective doses of two or more active ingredients are administered together.

[0327] In one embodiment, an effective amount of an antibody or antigen-binding fragment or conjugate thereof 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 taken before treatment indicates that treatment is ineffective, while a decrease in immune complexes compared to a control taken before treatment indicates that treatment is effective.

[0328] F. Biopharmaceutical Compositions

[0329] 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) disclosed in CAR, or T cells expressing CAR, antibodies, antigen binding fragments, conjugates, CAR, or T cells expressing CAR specifically bound to one or more antigens disclosed herein. The composition can be prepared in unit dosage 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 CAR, or T cells expressing CAR, antibodies, antigen binding fragments, and conjugates are formulated for parenteral administration, such as intravenous administration. Compositions comprising CAR, or T cells expressing CAR, conjugates, antibodies, or antigen binding fragments as disclosed herein 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 as disclosed herein are also used, for example, to detect pathological angiogenesis.

[0330] 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 the specific mode of administration selected and the needs of the subject based on fluid volume, viscosity, body weight, etc. The actual method of preparing such a dosage form for gene therapy, immunotherapy and / or cell therapy is known or will be obvious to those skilled in the art.

[0331] Typical compositions for intravenous administration include about 0.01 to about 30 mg / kg 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 obvious to those skilled in the art, and are described in more detail in publications such as Remington's Pharmaceutical Science, 19th edition, Mack Publishing Company, Easton, PA (1995).

[0332] 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 to 15 mg / kg body weight. There is considerable experience in the art in administering antibodies or antigen binding fragments and conjugate drugs; for example, since the approval of the FDA in 1997, the FDA has been approved for use in the treatment of CAR-related diseases. Since 2005, antibody drugs have been listed in the United States. CAR, or T cells expressing CAR, antibodies, antigen binding fragments thereof, and 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 about 30 minutes for 4 to 8 weeks.

[0333] 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 usually referred to as nanoparticles, nanospheres and nanocapsules, respectively. Capillaries have a diameter of about 5 μm, so only nanoparticles are administered intravenously. The diameter of microparticles 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).

[0334] Polymers can be used for CAR disclosed herein or T cells expressing CAR, antibodies or antigen binding fragments 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 flowing liquid at low temperatures, but forms a semisolid gel at body temperature. It has been shown to be an effective carrier for the preparation 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 as well as 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).

[0335] G.Medicine box

[0336] 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.

[0337] The medicine box may include a container and a label or package insert associated with the container or on 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.

[0338] 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 a method for treating or preventing tumors or preparing CAR T cells. The package insert generally includes instructions customarily included in the commercial packaging of the therapeutic product, which includes information about the indications, usage, dosage, administration, contraindications and / or warnings for using such therapeutic products. The guidance 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 medicine box may also include additional components to facilitate the specific application of the medicine box designed for it. Therefore, for example, the medicine box may additionally include means for detecting the label (e.g., enzyme substrates for enzyme labeling, filtering devices for detecting fluorescent labels, suitable secondary labels (e.g., secondary antibodies), etc.). The medicine box may additionally include buffers and other reagents conventionally used to practice a particular method. Such medicine boxes and suitable contents are well known to those skilled in the art.

[0339] Example

[0340] The present invention is further illustrated by the following examples, which should not be construed in any way as limiting the scope of the invention. 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, are themselves suggested to those skilled in the art.

[0341] Example 1

[0342] Isolation of human CD38-specific antibodies from a fully human yeast-displayed ScFv library

[0343] Materials and methods:

[0344] A large yeast-displayed human primary single-chain variable fragment (ScFv) antibody library was used to isolate the anti-human CD38 antibodies described herein. The library was constructed using a collection of human antibody gene repertoires from more than 60 individuals. Three rounds of magnetic-activated cell sorting (MACS) were performed to enrich human ScFv binders to the recombinant human CD38 extracellular domain fused to human IgG1 Fc (referred to as CD38-Fc). For the first round of yeast library panning, the yeast-displayed ScFv library (5×10 10 5 μ g / mL CD38-Fc in 15ml PBSA (composed of 0.1% bovine serum albumin (Bovine Serum Albumin, BSA) in Dulbecco's phosphate buffered saline (PBS) buffer) was incubated on a rotator for 1.5 hours at room temperature. After washing twice with 25ml PBSA, the yeast library mixture was incubated on a rotator for 30 minutes at room temperature with 100 μL protein G micro beads (Miltenyi Biotec). After washing once, the library mixture was resuspended in 50ml PBSA and loaded on a MACS cell separation column (LS column). After washing 3 times with 10ml PBSA, the ScFv binding substances of the yeast display of the column were then eluted twice with 2ml PBSA. The yeast cells of these elutions were merged and subsequently resuspended in 50ml SDCAA culture medium (20g D-glucose, 6.7g BD Difco without amino acids in 1L water). TM Yeast nitrogen base, 5g Bacto TM The pool was then induced in SGCAA medium (composed of the same components of SDCAA medium, but containing galactose instead of glucose) at 30°C with shaking at 225 rpm for another 16 hours and used for the next round of panning. The same process was repeated twice more to enrich for CD38-Fc specific binders.

[0345] To further enrich for binders with higher affinity and better specificity, FACS-based sorting was used to isolate the strongest binders from the pool. The induced pool was incubated with 1 μg / ml CD38-Fc for 1 hour at room temperature and subsequently stained with anti-c-Myc-Alexa 488 and goat anti-Hu-Fc PE conjugates, and the top 1% of the pool with the highest PE relative to FITC signal was gated and sorted. The sorted pool was amplified in SDCAA medium, and yeast plasmid DNA was extracted and transformed into bacteria for monoclonal DNA sequencing. 40 random clones were sequenced and 36 unique sequences were identified. Fourteen (14) CD38 ScFv clones, designated as M3801, M3802, M3803, M3804, M3806, M3808, M3809, M3810, M3811, M3812, M3815, M3816 and M3817, were cloned into CAR constructs for CAR-T functional screening, as shown in Example 2, Table 1. M3815_P, which is a variant of the M3815 CD38 ScFv clone that differs from M3815 in the sequence of the intrachain linker connecting the heavy and light chains of the ScFv, was also cloned into CAR constructs for CAR-T functional screening, as shown in Example 2, Table 1. In addition, M8DR H_L and M8DR L_H, which were used as positive controls for the new human anti-CD38 ScFv-based CAR constructs and derived from the monoclonal antibody daratumumab, were cloned into the CAR constructs for CAR-T functional screening, as shown in Example 2, Table 1.

[0346] Example 2

[0347] Generation of CD38-targeted CAR T constructs incorporating fully human binder ScFv sequences derived from a yeast display library

[0348] There are few treatment options for AML, and treatment-related toxicity and disease relapse after treatment are common. In addition, immunotherapy using non-human sequences (e.g., mouse-derived antibodies) can lead to treatment rejection or adverse reactions in patients. In order to develop new CAR T treatments for AML, 15 CAR T constructs targeting CD38 incorporating fully human ScFv targeting domains were designed and evaluated for antitumor activity.

[0349] Materials and methods:

[0350] (a) Cell lines

[0351] MM cell lines MM1.S and RPMI-8226 and human embryonic kidney line 293T were purchased from the American Tissue Culture Collection (ATCC, Manassas, VA). Single cell clones of luciferase-expressing cell lines were generated by stably transducing wild-type tumor lines with a lentiviral vector (Lentigen) encoding firefly luciferase. Whole blood was collected from healthy volunteers at the Oklahoma Blood Institute (OBI) with written consent from the donors. Processed buffy coats were purchased from OBI (Oklahoma City, OK). CD4 - Microbeads and CD8 - CD4-positive and CD8-positive human T cells were purified from buffy coats by positive selection using a 1:1 mixture of ELISA microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's protocol.

[0352] (b) Production of chimeric antigen receptor (CAR)-expressing vectors

[0353] The CAR antigen binding domain ScFv sequence is derived from human anti-CD38 ScFv. The CAR T construct is generated by connecting the binder sequence to CD8a and the transmembrane domain (aa 123-191, reference sequence ID NP_001759.3) and then connecting in frame with 4-1BB (CD137, aa 214-255, UniProt sequence ID Q07011) signaling domain and CD3ζ signaling domain (CD247, aa 52-163, reference sequence ID: NP_000725.1). CAR positive control constructs LTG2525 and LTG2526 with control ScFvs of heavy and light chains derived from monoclonal antibody daratumumab (and referred to herein as M8DR) were incorporated in VH-VL or VL-VH orientations, respectively. The CAR construct sequence was cloned into a third-generation lentiviral plasmid backbone (Lentigen Technology Inc., Gaithersburg, MD). The supernatant containing the lentiviral vector (LV) was generated by transient transfection of HEK 293T cells, and the supernatant containing the lentiviral vector was centrifuged to precipitate the vector and stored at -80°C.

[0354] (c) Purification and transduction of primary T cells

[0355] Using CD4 + and CD8+ Human primary T cells from healthy volunteers were purified from whole blood or buffy coats (purchased from commercial suppliers with written consent from the donors) according to the manufacturer's protocol (Miltenyi Biotec, Bergisch Gladbach, Germany). T cells were cultured at 0.3 to 2 × 10 6 The cells were cultured at a density of 10 cells / ml and CD3 / CD28 GMP T cells were activated with TransAct reagent (Miltenyi Biotec) and transduced with lentiviral vector encoding CAR constructs overnight in the presence of 10 μg / ml protamine sulfate (Sigma-Aldrich, St. Louis, MO) on day 2, and the medium was changed on day 3. The cultures were propagated in TexMACS medium supplemented with 30 IU / ml IL-2 until harvested on days 8 to 10.

[0356] (d) Immune effector assays (CTL and cytokines)

[0357] To determine cell-mediated cytotoxicity (CTL assay), 5,000 target cells stably transduced with firefly luciferase were combined with CAR T cells in a variety of effector-to-target ratios and incubated overnight. SteadyGlo reagent (Promega, Madison WI) was added to each well and the resulting luminescence was quantified as counts per second (sample CPS). Only the target wells (maximum CPS) and only the target wells plus 1% Tween-20 (minimum CPS) were used to determine the range of the assay. The percentage of lysis was calculated as: (1-(sample CPS-minimum CPS) / (maximum CPS-minimum CPS)). The supernatant of the co-culture with an E:T ratio of 10:1 was removed and IFNγ and TNFα concentrations were analyzed by ELISA (eBioscience, San Diego, CA).

[0358] (e) Flow cytometric analysis of CAR surface expression

[0359] For cell staining, 500,000 CAR T-transduced cells were harvested from culture, washed twice in cold AutoMACS buffer supplemented with 0.5% bovine serum albumin (Miltenyi Biotec), and CAR surface expression was detected by staining with CD38-His peptide (Thermo Fisher Scientific, Waltham, Massachusetts) followed by a secondary anti-His-PE detection reagent (Miltenyi Biotec, Bergisch Gladbach, Germany). Anti-CD4 antibodies conjugated to VioBlue fluorophore (Miltenyi Biotec) were used where indicated, according to the supplier's protocol. Untransduced cells were used as negative controls. Dead cells were excluded from all studies by 7AAD staining (BD Biosciences, San Jose, CA). The cells were washed twice and resuspended in 200 μl of staining buffer and then quantitatively analyzed by flow cytometry. Flow cytometric analysis was performed on a 10 analyzer (Miltenyi Biotec), and data graphs were generated using FlowJo software (Ashland, OR).

[0360] result:

[0361] This example describes the generation of CAR T cells targeting the tumor antigen CD38 for the treatment of MM (if any) and other CD38 + malignant tumors and kill CD38-expressing tumor suppressor cells in the tumor microenvironment. Each CAR contains a human ScFv binder derived from a human yeast display library, a CD8 hinge and transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3ζ activation domain ( Figure 1 ).

[0362] Table 1 below details the developed CAR38 constructs (named by LTG number), and the corresponding ScFv sequence names used in each construct. Comparative control CAR sequences LTG2525 and LTG2526 incorporate ScFvs derived from the heavy and light chains of the daratumumab antibody in the heavy-light and light-heavy orientations, respectively. Table 1 - Anti-CD38 CAR T construct LTG numbers and corresponding ScFv names

[0363] CAR construct LTG# CD38 ScFv conjugate 2089 M3801 2090 M3802 2091 M3803 2092 M3804 2093 M3806 2094 M3808 2095 M3809 2096 M3810 2097 M3811 2098 M3812 2099 M3815 2501 M3815_P 2502 M3816 2503 M3817 2525 M8DR H_L 2526 M8DR L_H

[0364] CD38 CAR design diagram Figure 1The fully human ScFv binder targeting CD38 was linked in-frame to the CD8 hinge and transmembrane domains, the 4-1BB co-stimulatory domain, and the CD3ζ activation domain. The CAR sequence was incorporated into a third-generation lentiviral vector and applied to primary human T cells for transduction. The surface expression of the anti-CD38 CAR incorporating the single-chain variable fragment (ScFv) sequence was Figure 2 The expression level of each ScFv-containing CAR was determined by flow cytometric analysis of LV-transduced T cells from healthy donors in a two-step staining procedure as follows: Step 1: CD38-His-tagged peptide; followed by Step 2: anti-His PE reagent. A subset of ScFv-based anti-CD38 CAR constructs were highly expressed in human primary T cells, as detected by CD38-His and compared to untransduced T cell controls (UTD). Figure 2 All transduced constructs expressed CAR T cells at high rates (40% to 95%) of viable T cells. + % expressed CAR T cells, with the exception of constructs LTG2097, LTG2503, which had less than 20% CAR T positive T cells. Negative controls - untransduced T cells (UTD) from the same donor and anti-His PE control (FMO PE) did not show significant CAR T expression, indicating the specificity of CAR T staining. Figures 3A to 3C As shown in , high cytolytic activity of anti-CD38 CAR was demonstrated for most of the constructs analyzed. Untransduced T cells (UTD) were used as a negative control for CAR cytolytic function. Human primary T cells were transduced with LV encoding CAR constructs (see Methods) and then incubated with CD38 + Tumor lines MM1.S, RPMI-8226 or CD38 negative control line HEK293 were incubated for 18 hours. Each target line was stably transduced with firefly luciferase to facilitate detection of surviving tumor cell fractions in a luminescence-based in vitro killing assay. Effector CAR T cells and tumor cells were combined at effector to target (E:T) ratios of 5, 10, and 20 to compare and contrast the efficacy of different CAR constructs ( Figures 3A to 3C RPMI-8226 cells were the most susceptible cell line to CD38 CAR-mediated tumor killing, with most CAR constructs achieving more than 80% tumor lysis at the lowest E:T ratio of 5 ( Figure 3A). All constructs tested effectively lysed RPMI-8226, while the negative control UTD group did not cause significant tumor lysis (the highest cell lytic activity of UTD was observed at an E:T ratio of 20, with no detectable tumor lysis at lower E:T ratios). Therefore, the lysis of RPMI-8226 by CD38-targeted T cell constructs is antigen-specific. The constructs with the lowest levels of cell lytic activity compared to RPMI-8226 were LTG2090, LTG2098, and LTG2099, and they achieved <70%, <40%, and <10% tumor killing at an E:T ratio of 1:5, respectively ( Figure 3A ). It is noteworthy that the lower cytolytic function of these CAR constructs was independent of their surface expression levels, which were relatively high: >45%, >75%, and >65%, respectively. Figure 2 Therefore, the function of a CAR construct cannot be predicted based on CAR surface expression alone, and the selection of the best CAR candidate is not trivial. + The tumor line MM1.S exhibited a similar distribution of killing potency to the anti-CD38 CAR constructs tested, and due to lower susceptibility to killing compared to RPMI-8228, the differences in killing potency among the CAR constructs were more pronounced and allowed for a more accurate comparison of CAR potency ( Figure 3B ). In the MM1.S assay, as in the RPMI-8226 assay, constructs LTG2090, LTG2098, and LTG2099 were the least potent of the group, thus confirming once again that the cytolytic activity of CARs targeting CD38 is tumor antigen specific and that CAR-to-CAR functional assessment can be extended to CD38-positive cell lines. In addition, the moderate cytolytic activity of CAR construct LTG2502 was shown in the MM1S killing assay compared to the other tested CAR constructs ( Figure 3B ). Similar to RPMI-8226, the negative assay control UTD had no discernible killing activity in the MM1.S tumor line, indicating that killing was CAR-dependent ( Figure 3B ).

[0365] The 293T tumor line lacking CD38 expression was tested as a negative / specificity control for CAR function ( Figure 3C The background cell lysis level in this assay was determined based on the UTD negative control and did not exceed 10% ( Figure 3C ). Except for the CAR construct LTG2501 with E:T ratios of 10:1 and 20:1, all CD38-targeting CAR T constructs did not consistently exhibit lysis of 293T cells above 10% of background levels ( Figure 3C). Thus, the CAR T constructs tested in this group are target specific, whereas construct LTG2501 may be less specific and thus mediate target toxicity. The discovery of this CAR property required empirical testing and was unpredictable, thus again demonstrating the non-trivial nature of the present invention.

[0366] The ability of anti-CD38 CAR T cells to produce cytokines in response to target cells expressing the antigen was then evaluated ( Figures 4A to 4B ). IFNγ and TNFα are proinflammatory cytokines known to be secreted at high ratios consistent with T cell activation and cytolytic activity. Tumor lines RPMI-8226 and MM1.S, which are positive for CD38 expression, or 293T, which are negative for CD38 expression, were co-incubated overnight with CAR T cells incorporating the CAR38 construct or negative untransduced T cells (UTD) at an effector to target ratio of 10:1, and the culture supernatants were analyzed for IFNγ by ELISA ( Figure 4A ) and TNFα( Figure 4B ). For IFNγ and TNFα, the pattern of cytokine production by the tested CAR constructs was similar. The CAR constructs with the highest IFNγ and TNFα supernatant levels were LTG2091, LTG2095, LTG2097, and the positive control constructs LTG2525 and LTG2526, which incorporated a control ScFv derived from the monoclonal antibody daratumumab in the VH-VL or VL-VH orientation, respectively, which demonstrated that the ability of the CAR construct to secrete cytokines was consistent between the two tested cytokines ( Figure 4A , 4B When incubated with the tumor lines RPMI-H226 and MM1.S, the levels of cytokine production in this vector subset were 2500 to 3500 pg / ml for IFNg and 1500 to 2500 pg / ml for TNFα ( Figure 4A , 4B ). The CAR construct LTG2092 produced TNFα at high levels when incubated alone and when incubated with the target lines RPMI-8226 and MM1.S ( Figure 4B), thereby demonstrating tonic signaling. Again, the data demonstrates the non-triviality of the CAR of the present invention, and the necessity of empirical testing to determine the best CAR candidate. Without intending to limit any specific mechanism of action, it is believed that the possible reasons for the enhanced therapeutic function associated with the exemplary CAR targeting CD38 of the present invention include, for example, but not limited to: a) improved lateral movement within the plasma membrane to allow more efficient signal transduction, b) excellent position within the plasma membrane microdomain (such as lipid rafts), and greater ability to interact with transmembrane signaling cascades associated with T cell activation, c) by preferentially moving away from inhibitory or down-regulatory interactions (such as low proximity or interaction with phosphatases (such as CD45)) and excellent position within the plasma membrane, and d) excellent assembly into T cell receptor signaling complexes (i.e., immune synapses), or e) excellent ability to engage with tumor antigens (due to the presence of two different targeting domains in each CAR molecule), or any combination thereof.

[0367] Each application and patent cited herein, and each document or reference cited in each application and patent (including during the prosecution of each issued patent; "application cited document"), and each PCT and foreign application or patent corresponding to any of these applications and patents and / or claiming priority to any of these applications and patents, and each document cited or referenced in each application cited document, are hereby expressly incorporated herein by reference and may be used in the practice of the present invention. More generally, documents or references are cited herein, whether in the reference list preceding the claims or in the text itself; and each of these documents or references ("herein cited references"), and each document or reference cited in each herein cited reference (including any manufacturer's instructions, instructions, etc.), is hereby expressly incorporated herein by reference.

[0368] The foregoing description of some specific embodiments provides enough information so that others can easily modify or adapt multiple applications (such as specific embodiments) by applying current knowledge without departing from the general concept, and therefore, such adaptations and modifications should and are intended to be understood within the meaning and scope of the equivalent schemes of the disclosed embodiments. It should be understood that the words or terms used herein are for description rather than limitation purposes. In the drawings and descriptions, exemplary embodiments have been disclosed, and although specific terms may have been adopted, unless otherwise specified, they are only used in a general and descriptive sense and are not used for limiting purposes, so the scope of the claims is not limited thereto. In addition, those skilled in the art will understand that some steps of the methods discussed herein can be arranged in an alternative order, or the steps can be combined. Therefore, the attached claims are intended not to be limited to the specific embodiments disclosed herein. Those skilled in the art will recognize or be able to determine many equivalent schemes of the embodiments of the present invention described herein using experiments that do not exceed routine. Such equivalent schemes are covered in the following claims.

[0369] References to sequence listings

[0370] This application contains a Sequence Listing that has been submitted electronically to the United States Patent and Trademark Office via a PDF file entitled “Sequence Listing.” This Sequence Listing is incorporated by reference.

[0371] Sequence of the present disclosure

[0372] As defined in 37 C.FR 1.822, the nucleic acid and amino acid sequences listed below are shown using standard letter abbreviations for nucleotide bases and single-letter or three-letter codes for amino acids. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included by any reference to the strand shown. In the attached sequence listing:

[0373] SEQ ID NO: 1 Nucleotide sequence of CD38 hScFv binder M3801

[0374]

[0375] SEQ ID NO:2 Amino acid sequence of CD38 hScFv binder M3801

[0376] SEQ ID NO:3 Nucleotide sequence of CD38 hScFv binder M3802

[0377] SEQ ID NO:4 Amino acid sequence of CD38 hScFv binder M3802

[0378]

[0379] SEQ ID NO:5 Nucleotide sequence of CD38 hScFv binder M3803

[0380] SEQ ID NO:6 Amino acid sequence of CD38 hScFv binder M3803

[0381] SEQ ID NO:7 Nucleotide sequence of CD38 hScFv binder M3804

[0382]

[0383]

[0384] SEQ ID NO:8 Amino acid sequence of CD38 hScFv binder M3804

[0385] SEQ ID NO:9 Nucleotide sequence of CD38 hScFv binder M3806

[0386]

[0387]

[0388] SEQ ID NO: 10 Amino acid sequence of CD38 hScFv binder M3806

[0389] SEQ ID NO: 11 Nucleotide sequence of CD38 hScFv binder M3808

[0390]

[0391] SEQ ID NO: 12 Amino acid sequence of CD38 hScFv binder M3808

[0392]

[0393]

[0394] SEQ ID NO: 13 Nucleotide sequence of leader / signal peptide sequence

[0395] SEQ ID NO:14 Amino acid sequence of leader / signal peptide sequence

[0396]

[0397] SEQ ID NO: 15 Nucleotide sequence of CD38 hScFv binder M3809

[0398]

[0399] SEQ ID NO: 16 Amino acid sequence of CD38 hScFv binder M3809

[0400]

[0401]

[0402] SEQ ID NO: 17 Nucleotide sequence of CD38 hScFv binder M3810

[0403]

[0404] SEQ ID NO: 18 Amino acid sequence of CD38 hScFv binder M3810

[0405] SEQ ID NO: 19 Nucleotide sequence of CD38 hScFv binder M3811

[0406]

[0407] SEQ ID NO:20 Amino acid sequence of CD38 hScFv binder M3811

[0408]

[0409] SEQ ID NO:21 Nucleotide sequence of CD38 hScFv binder M3812

[0410]

[0411] SEQ ID NO:22 Amino acid sequence of CD38 hScFv binder M3812

[0412] SEQ ID NO:23 Nucleotide sequence of CD38 hScFv binder M3815

[0413]

[0414] SEQ ID NO:24 Amino acid sequence of CD38 hScFv binder M3815

[0415] SEQ ID NO:25 Nucleotide sequence of CD38 hScFv binder M3815_P

[0416]

[0417] SEQ ID NO:26 Amino acid sequence of CD38 hScFv binder M3815_P

[0418]

[0419] SEQ ID NO:27 Nucleotide sequence of DNA CD8 transmembrane domain

[0420]

[0421] SEQ ID NO:28 Amino acid sequence of CD8 transmembrane domain

[0422]

[0423] SEQ ID NO:29 Nucleotide sequence of DNA CD8 hinge domain

[0424]

[0425] SEQ ID NO:30 Amino acid sequence of CD8 hinge domain

[0426]

[0427] SEQ ID NO:31 Amino acid sequence of the hinge region from amino acids 118 to 178 of CD8.α. (NCBI RefSeq: NP.sub.--001759.3)

[0428]

[0429] SEQ ID NO:32 Sequence of human IgG CL amino acid sequence

[0430]

[0431] SEQ ID NO:33 Nucleotide sequence of the DNA signaling domain of 4-1BB

[0432]

[0433] SEQ ID NO:34 Amino acid sequence of the signaling domain of 4-1BB

[0434] SEQ ID NO:35 Nucleotide sequence of the DNA signaling domain of CD3-ζ

[0435]

[0436] SEQ ID NO:36 Amino acid sequence of CD3ζ

[0437]

[0438] SEQ ID NO:37 Nucleotide sequence of ScFv CD 19

[0439]

[0440] SEQ ID NO:38 Amino acid sequence of ScFv CD 19

[0441]

[0442] SEQ ID NO: 39 Nucleotide sequence of GMCSF leader peptide

[0443] SEQ ID NO:40 Amino acid sequence of GMCSF leader peptide

[0444] SEQ ID NO:41 Nucleotide sequence of TNFRSF19 leader peptide

[0445] SEQ ID NO:42 Amino acid sequence of TNFRSF19 leader peptide

[0446] SEQ ID NO:43 Nucleotide sequence of CD8α leader peptide

[0447]

[0448] SEQ ID NO:44 Amino acid sequence of CD8α leader peptide

[0449] SEQ ID NO:45 Nucleotide sequence of CD28 costimulatory domain

[0450]

[0451] SEQ ID NO:46 Amino acid sequence of CD28 costimulatory domain

[0452]

[0453] SEQ ID NO:47 Nucleotide sequence of CD3ζ activation domain

[0454]

[0455] SEQ ID NO:48 Amino acid sequence of CD3ζ activation domain

[0456]

[0457] SEQ ID NO:49 Nucleotide sequence of hinge and transmembrane domain of TNFRSF19 (transmembrane domain is underlined)

[0458]

[0459] SEQ ID NO:50 Amino acid sequence of hinge and transmembrane domain of TNFRSF19 (transmembrane domain is underlined)

[0460]

[0461] SEQ ID NO:51 Nucleotide sequence of TNFRSF19 transmembrane domain

[0462]

[0463] SEQ ID NO:52 Amino acid sequence of TNFRSF19 transmembrane domain

[0464] SEQ ID NO:53 Nucleotide sequence of TNFRSF19 hinge domain

[0465]

[0466] SEQ ID NO:54 Amino acid sequence of TNFRSF19 hinge domain

[0467] SEQ ID NO:55 Nucleotide sequence of truncated TNFRSF19 hinge domain

[0468]

[0469] SEQ ID NO:56 Amino acid sequence of truncated TNFRSF19 hinge domain

[0470]

[0471] SEQ ID NO:57 Nucleotide sequence of CD8a hinge domain fused to TNFRSF19 transmembrane domain (transmembrane sequence is underlined)

[0472]

[0473] SEQ ID NO:58 Amino acid sequence of the CD8a hinge domain fused to the TNFRSF19 transmembrane domain (transmembrane sequence is underlined)

[0474]

[0475] SEQ ID NO:59 Nucleotide sequence of CD28 costimulatory domain

[0476]

[0477] SEQ ID NO:60 Amino acid sequence of CD28 costimulatory domain

[0478]

[0479] SEQ ID NO:61 Nucleotide sequence of CD3ζ form 2

[0480]

[0481] SEQ ID NO:62 Amino acid sequence of CD3 zeta form 2

[0482]

[0483] SEQ ID NO:63 Nucleotide sequence of furin P2A furin

[0484] SEQ ID NO:64 Amino acid sequence of Furin P2A Furin (Furin sequence is underlined)

[0485]

[0486] SEQ ID NO:65 Nucleotide sequence of Furin T2A

[0487]

[0488] SEQ ID NO:66 Amino acid sequence of Furin T2A (Furin sequence is underlined)

[0489]

[0490] SEQ ID NO:67 Nucleotide sequence of truncated EGFR (tEGFR) tag

[0491]

[0492] SEQ ID NO:68 Amino acid sequence of truncated EGFR (tEGFR) tag

[0493] SEQ ID NO:69 Nucleotide sequence of CD38 hScFv binder M3816

[0494]

[0495]

[0496] SEQ ID NO:70 Amino acid sequence of CD38 hScFv binder M3816

[0497]

[0498] SEQ ID NO:71 Nucleotide sequence of CD38 hScFv binder M3817

[0499]

[0500] SEQ ID NO:72 Amino acid sequence of CD38 hScFv binder M3817

[0501]

[0502] SEQ ID NO:73 Nucleotide sequence of CD38 hScFv binder M8DR H_L

[0503]

[0504] SEQ ID NO:74 Amino acid sequence of CD38 hScFv binder M8DR H_L

[0505]

[0506] SEQ ID NO:75 Nucleotide sequence of CD38 hScFv binder M8DR L_H

[0507]

[0508] SEQ ID NO:76 Amino acid sequence of CD38 hScFv binder M8DR L_H

[0509] SEQ ID NO: 77 Nucleotide sequence of LTG 2089 (hScFv aCD38 CD8 TM 4-1BB CD3ζ)

[0510]

[0511] SEQ ID NO:78 Amino acid sequence of LTG 2089 (hScFv aCD38 CD8 TM 4-1BB CD3ζ)

[0512]

[0513] SEQ ID NO: 79 Nucleotide sequence of human IgG4 hinge

[0514]

[0515] SEQ ID NO: 80 Amino acid sequence of human IgG4 hinge

[0516]

[0517] SEQ ID NO: 81 Nucleotide sequence of human IgG4 CH2 domain

[0518]

[0519] SEQ ID NO:82 Amino acid sequence of human IgG4 CH2 domain

[0520]

[0521] SEQ ID NO: 83 Nucleotide sequence of human IgG4 CH3 domain

[0522] SEQ ID NO:84 Amino acid sequence of human IgG4 CH3 domain

[0523] SEQ ID NO: 85 Nucleotide sequence of human IgG4 hinge CH2 CH3 domain

[0524]

[0525] SEQ ID NO:86 Amino acid sequence of human IgG4 hinge CH2 CH3 domain

[0526]

[0527] SEQ ID NO: 87 Nucleotide sequence of LTG 2090 (hScFv aCD38 CD8 TM 4-1BB CD3ζ)

[0528]

[0529] SEQ ID NO:88 Amino acid sequence of LTG 2090 (hScFv aCD38 CD8 TM 4-1BB CD3ζ)

[0530]

[0531]

[0532] SEQ ID NO: 89 Nucleotide sequence of LTG 2091 (hScFv aCD38 CD8 TM 4-1BB CD3ζ)

[0533]

[0534]

[0535] SEQ ID NO:90 Amino acid sequence of LTG 2091 (hScFv aCD38 CD8 TM 4-1BB CD3ζ)

[0536]

[0537] SEQ ID NO: 91 Nucleotide sequence of LTG 2092 (hScFv aCD38 CD8 TM 4-1BB CD3ζ)

[0538]

[0539]

[0540] SEQ ID NO:92 Amino acid sequence of LTG 2092 (hScFv aCD38 CD8 TM 4-1BB CD3ζ)

[0541]

[0542]

[0543] SEQ ID NO: 93 Nucleotide sequence of LTG 2093 (hScFv aCD38 CD8 TM 4-1BB CD3ζ)

[0544]

[0545] SEQ ID NO: 94 Amino acid sequence of LTG 2093 (hScFv aCD38 CD8 TM 4-1BB CD3ζ)

[0546]

[0547] SEQ ID NO: 95 Nucleotide sequence of LTG 2094 (hScFv aCD38 CD8 TM 4-1BB CD3ζ)

[0548]

[0549]

[0550] SEQ ID NO:96 Amino acid sequence of LTG 2094 (hScFv aCD38 CD8 TM 4-1BB CD3ζ)

[0551]

[0552] SEQ ID NO: 97 Nucleotide sequence of LTG 2095 (hScFv aCD38 CD8 TM 4-1BB CD3ζ)

[0553]

[0554]

[0555] SEQ ID NO:98 Amino acid sequence of LTG 2095 (hScFv aCD38 CD8 TM 4-1BB CD3ζ)

[0556]

[0557] SEQ ID NO: 99 Nucleotide sequence of LTG 2096 (hScFv aCD38 CD8 TM 4-1BB CD3ζ)

[0558]

[0559] SEQ ID NO: 100 Amino acid sequence of LTG 2096 (hScFv aCD38 CD8 TM 4-1BB CD3ζ)

[0560]

[0561] SEQ ID NO: 101 Nucleotide sequence of LTG 2097 (hScFv aCD38 CD8 TM 4-1BB CD3ζ)

[0562]

[0563]

[0564] SEQ ID NO: 102 Amino acid sequence of LTG 2097 (hScFv aCD38 CD8 TM 4-1BB CD3ζ)

[0565]

[0566] SEQ ID NO: 103 Nucleotide sequence of LTG 2098 (hScFv aCD38 CD8 TM 4-1BB CD3ζ)

[0567]

[0568]

[0569] SEQ ID NO: 104 Amino acid sequence of LTG 2098 (hScFv aCD38 CD8 TM 4-1BB CD3ζ)

[0570]

[0571] SEQ ID NO: 105 Nucleotide sequence of LTG 2099 (hScFv aCD38 CD8 TM 4-1BB CD3ζ)

[0572]

[0573] SEQ ID NO: 106 Amino acid sequence of LTG 2099 (hScFv aCD38 CD8 TM 4-1BB CD3ζ)

[0574]

[0575] SEQ ID NO: 107 Nucleotide sequence of LTG 2501 (hScFv aCD38 CD8 TM 4-1BB CD3ζ)

[0576]

[0577]

[0578] SEQ ID NO: 108 Amino acid sequence of LTG 2501 (hScFv aCD38 CD8 TM 4-1BB CD3ζ)

[0579]

[0580] SEQ ID NO: 109 Nucleotide sequence of LTG 2502 (hScFv aCD38 CD8 TM 4-1BB CD3ζ)

[0581]

[0582]

[0583] SEQ ID NO: 110 Amino acid sequence of LTG 2502 (hScFv aCD38 CD8 TM 4-1BB CD3ζ)

[0584]

[0585] SEQ ID NO: 111 Nucleotide sequence of LTG 2503 (hScFv aCD38 CD8 TM 4-1BB CD3ζ)

[0586]

[0587] SEQ ID NO:112 Amino acid sequence of LTG 2503 (hScFv aCD38 CD8 TM 4-1BB CD3ζ)

[0588]

[0589] SEQ ID NO: 113 Nucleotide sequence of LTG 2525 (hScFv aCD38 CD8 TM 4-1BB CD3ζ)

[0590]

[0591]

[0592] SEQ ID NO: 114 Amino acid sequence of LTG 2525 (hScFv aCD38 CD8 TM 4-1BB CD3ζ)

[0593]

[0594] SEQ ID NO: 115 Nucleotide sequence of LTG 2526 (hScFv aCD38 CD8 TM 4-1BB CD3ζ)

[0595]

[0596]

[0597] SEQ ID NO:116 Amino acid sequence of LTG 2526 (hScFv aCD38 CD8 TM 4-1BB CD3ζ)

[0598]

Claims

1. An isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the 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 comprises a CD38 antigen binding domain encoded by a nucleotide sequence comprising SEQ ID NO: 1, 3, 5, 7, 9, 11, 15, 17, 19, 21, 23, 25, 69 or 71.

2. The isolated nucleic acid molecule of claim 1, wherein the encoded at least one CD38 antigen binding domain comprises at least one single chain variable fragment of an antibody that binds to CD38.

3. The isolated nucleic acid molecule of claim 1, wherein the encoded at least one CD38 antigen binding domain comprises at least one heavy chain variable region of an antibody that binds to CD38.

4. The isolated nucleic acid molecule of claim 1, wherein the encoded at least one CD38 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, TNFRSF19 or CD28 and is connected to the transmembrane domain.

6. The isolated nucleic acid molecule of claim 1, wherein the encoded extracellular CD38 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 comprising: SEQ ID NO:13 encoding the leading amino acid sequence of SEQ ID NO:14, or SEQ ID NO:39 encoding the leading amino acid sequence of SEQ ID NO:40, or SEQ ID NO:41 encoding the leading amino acid sequence of SEQ ID NO:42, or SEQ ID NO:43 encoding the leading amino acid sequence of SEQ ID NO:

44.

8. The isolated nucleic acid molecule of claim 1, wherein the transmembrane domain comprises a transmembrane domain of a protein comprising: the alpha, beta or zeta chain of 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, or any combination thereof.

9. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid sequence encoding the extracellular CD38 antigen binding domain comprises a nucleic acid sequence comprising SEQ ID NO: 1, 3, 5, 7, 9, 11, 15, 17, 19, 21, 23, 25, 69 or 71, 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 CD38 antigen binding domain comprising an amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 16, 18, 20, 22, 24, 26, 70 or 72.

16. The CAR of claim 15, wherein the CD38 antigen binding domain comprises at least one single-chain variable fragment of an antibody that binds to CD38.

17. The CAR of claim 15, wherein the CD38 antigen binding domain comprises at least one heavy chain variable region of an antibody that binds to CD38.

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: 27, 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:

28.

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 CD38 antigen binding domain comprising an amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 16, 18, 20, 22, 24, 26, 70 or 72.

21. The CAR of claim 20, wherein the linker or spacer domain is derived from the extracellular domain of CD8, TNFRSF19, IgG4 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 comprising a functional signaling domain of a protein selected from the group consisting of OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP12, and 4-1BB (CD137), or a combination thereof.

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

25. The vector of claim 24, wherein the vector is selected from the group consisting of a DNA vector, an RNA vector, a plasmid vector, a cosmid vector, a herpes virus vector, a measles virus vector, a 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 cells.

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 an RNA-engineered cell population 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 population of human 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 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 CD38 antigen binding domain comprising an amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 16, 18, 20, 22, 24, 26, 70 or 72, 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 and CD154, 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 acute myeloid leukemia (AML), blastic plasmacytoid dendritic cell neoplasm (BPDCN), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), acute lymphocytic T-cell leukemia (T-ALL), or acute lymphocytic B-cell leukemia (B-ALL).

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), pediatric 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 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 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 CD38 antigen binding domain comprising an amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 16, 18, 20, 22, 24, 26, 70 or 72, 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 CD38 antigen binding domain comprising an amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 16, 18, 20, 22, 24, 26, 70 or 72, 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 into the cell the isolated nucleic acid of claim 1.

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 comprising a T cell.

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