Compositions and methods for treating cancer with Anti-CD38 immunotherapy

Abstract

To provide, in the field of cancer, specifically CD38 antigen binding domains and chimeric antigen receptors (CARs) containing such CD38 antigen binding domains and methods of use thereof.SOLUTION: Disclosed is an isolated nucleic acid molecule, encoding a chimeric antigen receptor, comprising a specific sequence. Also disclosed are a chimeric antigen receptor encoded by the nucleic acid molecule, a vector comprising the nucleic acid molecule, a cell containing the vector, and a pharmaceutical composition. Further disclosed are a method for treating or preventing cancer in a subject and a method for producing a chimeric antigen receptor T cell.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made in the implementation of a Cooperative Research and Development Agreement with the National Institutes of Health, an agency of the U.S. Department of Health and Human Services. The U.S. Government has certain rights in this invention.

[0002] Field of the Disclosure The present application relates to the field of cancer, in particular to CD38 antigen-binding domains and chimeric antigen receptors (CARs) comprising such CD38 antigen-binding domains, and methods of use thereof. [Background technology]

[0003] background Cancer is one of the most deadly threats to human health. In the United States alone, cancer affects nearly 1.3 million new patients each year, making it the second leading cause of death after cardiovascular disease, accounting for approximately one-quarter of all deaths. Solid tumors are responsible for most of these deaths. Although significant advances have been made in the medical treatment of certain cancers, the overall 5-year survival rate for all cancers has improved by only about 10% over the past 20 years. Cancer, or malignant tumors, metastasize and grow rapidly and uncontrolled, making treatment extremely difficult.

[0004] Multiple myeloma ("MM") is a debilitating and often incurable disease, with over 30,000 new cases diagnosed each year in the United States (Source: MM Research Foundation). MM is the second most prevalent blood cancer in the United States, after non-Hodgkin's lymphoma ("NHL") (Smith L, McCourt O, Henrich M, et al., Multiple myeloma and physical activity: a scoping review. BMJ Open. 2015; vol. 5: e009576). MM affects the plasma cells in the bone marrow and can lead to bone marrow failure and patient death (National (Mayo Clinic staff. Diseases and conditions: multiple myeloma: treatments and drugs. December 4, 2015). Complications of myeloma include bone pain, bone loss, anemia, immunosuppression, kidney dysfunction, and neuropathy. (Mayo Clinic staff. Diseases and conditions: multiple myeloma: treatments and drugs. December 4, 2015).

[0005] First-line treatments for MM include proteasome inhibitors, immunomodulatory drugs, steroids, histone deacetylase ("HDAC") inhibitors, and chemotherapy. These approaches aim to kill MM cells, but many of them are associated with extensive immunosuppression and systemic toxicity.

[0006] Immunotherapeutic 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 MM patients. Despite abundant treatment options, the survival rate for patients with advanced MM is only 83 months for stage II MM and only 43 months for stage III MM (American Cancer Society, see cancer.org World Wide Web). Therefore, the potential for treatment of MM and other CD38 + for malignant tumors Better treatment modalities are urgently needed.

[0007] Similar to MM, B-cell chronic lymphocytic leukemia (CLL) is another malignancy that is difficult to treat. CLL is a lymphoproliferative disorder with variable survival prognosis, ranging from months to decades. The disease primarily affects older adults, with a median age of onset exceeding 65 years. However, even at later ages, a CLL diagnosis tends to shorten a 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 rate of disease progression typically depends on the type of BCR mutation present (Dighiero, Guillaume. "CLL biology and prognosis." ASH Education Program Book 2005.1(2005):278-284). Furthermore, in CLL, CD38 serves as a marker of unfavorable disease prognosis. Expression of CD38 in CLL is associated with enhanced leukemic cell proliferation, migration, and increased responsiveness to BCR-mediated signaling (Malavasi, Fabio, et al., "CD38 and chronic lymphocytic leukemia: a decade later." Blood (2011):blood-2011). While patients with a favorable prognosis may not require treatment for CLL, the disease is virtually incurable, and early treatment does not improve prognosis. First-line treatment for CLL typically involves a combination of several therapeutic agents: chemotherapy drugs (cyclophosphamide, fludarabine), the anti-CD20 antibody rituximab (Rituxan), the BTK inhibitor ibrutinib, high-dose prednisolone, or alemtuzumab (Campath). Despite the existence of numerous treatments, CLL treatment regimens tend to be aggressive, and toxicity remains a problem.Elderly patients and those with comorbidities remain particularly vulnerable to CLL treatment-related toxicities, such as immunosuppression and susceptibility to infections (Barrientos, Jacqueline C. "Management of chronic lymphocytic leukemia in the elderly.”Cancer Control 22.4_suppl (2015): pp. 17-23; Smolej, Lukas. "How I treat elderly or comorbid patients with chronic lymphocytic leukemia." Acta Medica (Hradec Kralove) 53.4 (2010): pp. 213-220).

[0008] Due to suboptimal efficacy and toxicity associated with the treatment of CD38-positive malignancies, better treatment options are needed. CAR-based approaches to the treatment of CD38-positive malignancies and CD38-positive tumor microenvironment cells may provide a much-needed treatment option for patients who cannot tolerate the toxicities associated with conventional first-line therapy. Furthermore, CAR T approaches are expected to lack the nonselective and often irreversible toxicities associated with chemotherapy or chemo / immunotherapy combinations. Furthermore, because CAR T cells are "living drugs," they are expected to remain in patients' bodies for extended periods or indefinitely, providing long-term therapeutic benefit. These characteristics of CAR T cells are expected to eliminate the need for repeated treatments and reduce the likelihood of disease recurrence.

[0009] The CD38-targeting CAR approach + They are superior to chemotherapy because they may achieve higher efficacy in eliminating tumor cells and tumor stem cells and avoid the toxicity associated with chemotherapy. Importantly, CAR T cells are expected to be more efficient at eliminating minimal residual disease than chemotherapy and result in better long-term treatment outcomes. Furthermore, CD38-targeting CAR T cells ("CAR38") may be useful in patients with high tumor burden who are suitable for BMT. It may be used for tumor debulking as a bridge to transplantation because it may help patients achieve optimal survival.

[0010] CAR38 offers an improvement over the prior art because it uses a unique human ScFv (hScFv) sequence in the CAR design, rather than the mouse-derived ScFv used in other CAR designs, which carry the risk of immunogenicity and can induce allergic or anaphylactic reactions in patients, potentially leading to CAR T removal or life-threatening anaphylaxis.

[0011] Chimeric antigen receptors (CARs) are hybrid molecules containing three essential units: (1) an extracellular antigen-binding motif, (2) a linking / transmembrane motif, and (3) an intracellular T cell signaling motif (Long AH, Haso WM, Orentas RJ. Lessons learned from a highly active CD2-specific chimeric antigen receptor. Oncoimmunology. 2013;2(4):e23621). The antigen-binding motif of CARs is generally based on the single-chain fragment variable (scFv), the smallest binding domain of an immunoglobulin (Ig) molecule. Alternative antigen-binding motifs have also been engineered, for example, receptor ligands (i.e., IL-13 has been engineered to bind to tumor-expressed IL-13 receptors), intact immune receptors, library-derived peptides, and innate immune system effector molecules (e.g., NKG2D). Alternative cellular targets for CAR expression (e.g., NK or gamma-delta T cells) are also under development (Brown CE et al. Clin Cancer Res. 2012;18(8):2199-209; Lehner M et al. PLoS One. 2012;7(2):e31210). Considerable effort is still required to define the most active T cell population for transduction with CAR vectors, to determine optimal culture and expansion techniques, and to define the molecular details of the CAR protein structure itself.

[0012] The linking motif of a CAR can be a relatively stable structural domain, such as the constant domain of IgG, or can be designed to be an extended, flexible linker. Structural motifs, such as those derived from the constant domain of IgG, can be used to extend the scFv binding domain away from the T cell plasma membrane surface. This may be important for some tumor targets where the binding domain is particularly close to the tumor cell surface membrane (e.g., for disialoganglioside GD2; Orentas et al., unpublished observations). To date, the signaling motif used in CARs has always included the CD3-ζ chain, because this core motif is an important signal for T cell activation. The first reported second-generation CARs featured the CD28 signaling domain and CD28 transmembrane sequence. This motif was also used in third-generation CARs containing the CD137 (4-1BB) signaling motif (Zhao Y et al., J Immunol. 2009; 183(9):5563-74). With the advancement of new technologies, activation of T cells with beads linked to anti-CD3 and anti-CD28 antibodies, and the presence of the canonical "signal 2" from CD28, encoded by the CAR itself, is no longer necessary. Using bead activation, third-generation vectors were found to be no superior to second-generation vectors in in vitro assays and offered no clear benefit over second-generation vectors in mouse models of leukemia (Haso W, Lee DW, Shah NN, Stetler-Stevenson M, Yuan CM, Pastan IH, Dimitrov DS, Morgan RA, FitzGerald DJ, Barrett DM, Wayne AS, Mackall CL, Orentas RJ. Anti-CD22-chimeric antigen receptors targeting B cell precursor acute lymphoblastic leukemia. Blood. 2013). 121(7):1165-74; Kochenderfer JN et al. Blood. 2012; 119(12):2709-20). This is due to 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 modes (Porter This is supported by the clinical success of CD19-specific CARs (DL et al. N Engl J Med. 2011;365(8):725-33). In addition to CD137, other tumor necrosis factor receptor superfamily members, such as OX40, can also provide important sustained signals in CAR-transduced T cells (Yvon E et al. Clin Cancer Res. 2009;15(18):5852-60). The culture conditions under which the CAR T cell population is cultured are equally important.

[0013] T cell-based immunotherapy is an emerging field in synthetic biology; multiple promoters and gene products are envisioned to direct these highly potent cells to the tumor microenvironment, where they can escape negative regulatory signals and mediate effective tumor killing. Elimination of unwanted T cells via drug-induced dimerization of an inducible caspase-9 construct with AP1903 demonstrates one way in which a powerful switch can be pharmacologically initiated to control T cell populations (Di Stasi A et al. N Engl J Med. 2011;365(18):1673-83). Creation of an effector T cell population immune to the negative regulatory effects of transforming growth factor-β by expression of a decoy receptor further demonstrates the extent to which effector T cells can be engineered for optimal antitumor activity (Foster AE et al. J Immunother. 2008;31(5):500-5). Thus, although CARs appear to be able to induce T cell activation in a manner similar to endogenous T cell receptors, a major obstacle to the clinical application of this technology to date is the lack of a reliable mechanism for CAR activation. +Limited in vivo expansion of T cells, rapid loss of cells after infusion, and disappointing clinical activity. Thus, there is an urgent and long-felt need in the art to discover new compositions and methods for the treatment of MM and CLL using approaches that can exhibit specific and effective anti-tumor effects without the drawbacks mentioned above (i.e., high toxicity, poor efficacy). Summary of the Invention [Problem to be solved by the invention]

[0014] The present invention addresses these needs by providing CAR compositions and therapeutic methods that can be used to treat cancer and other diseases and / or conditions. In particular, the invention disclosed and described herein provides CARs that can be used to treat diseases, disorders, or conditions associated with dysregulation of CD38 expression, wherein the CAR contains a CD38 antigen-binding domain that exhibits high surface expression on transduced T cells and exhibits high levels of cytolysis and in vivo proliferation and persistence of the transduced T cells. [Means for solving the problem]

[0015] overview Provided herein are novel anti-CD38 antibodies or their antigen-binding domains, chimeric antigen receptors (CARs) comprising such CD38 antigen-binding domains, host cells (e.g., T cells) expressing the receptors, and nucleic acid molecules encoding the receptors. CARs may consist of a single molecule expressed on the surface of effector cells, or may consist of an effector cell-expressed signaling module and a soluble targeting module; for example, the soluble targeting module binds to the cell-expressed signaling module to form a fully functional CAR. CARs exhibit high surface expression on transduced T cells, high levels of cell lysis, and in vivo proliferation and persistence of transduced T cells. Methods using the disclosed CARs, host cells, and nucleic acid molecules, for example, to treat cancer in a subject, are also provided. Provided.

[0016] 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 the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 15, 17, 19, 23, 25, 69, 71, 73, and 75.

[0017] 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, an Fv fragment, and a single-chain Fv (scFv).

[0018] 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 an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 16, 18, 20, 22, 24, 26, 70, 72, 74, and 76.

[0019] In one aspect, an isolated nucleic acid molecule is provided encoding 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 encoded by a nucleotide sequence comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 15, 17, 19, 21, 23, 25, 69, 71, 73, and 75.

[0020] 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.

[0021] 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.

[0022] In one embodiment, the targeting domain of the CAR is expressed separately in the form of a monoclonal antibody, ScFv Fab, Fab'2 and comprises an antigen targeting domain comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 15, 17, 19, 21, 23, 25, 69, 71, 73, and 75 linked to an additional binding tag or epitope, while the effector cell-expressed component of the CAR comprises a binding domain specifically directed to bind to a tag or epitope expressed on a soluble CAR module, such as upon specific binding to the cell-binding component of the CAR on the soluble component of the CAR to form a complete functional CAR structure.

[0023] In another embodiment, the targeting domain of the CAR is expressed separately in the form of a monoclonal antibody, ScFv Fab, Fab'2 and comprises an antigen targeting domain comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 15, 17, 19, 21, 23, 25, 69, 71, 73, and 75, and an additional ScFv, while the effector cell expression component of the CAR comprises a tag or epitope that specifically reacts with an additional ScFv expressed on the soluble CAR module, such as upon specific binding to the cell-binding component of the CAR on the soluble component of the CAR to form a complete functional CAR structure.

[0024] In yet another embodiment, the encoded CAR extracellular CD38 antigen-binding domain binds at least one lipocalin-based antigen-binding antigen (anticalin) that binds to CD38. ) is provided an isolated nucleic acid molecule encoding a CAR, further comprising:

[0025] In one embodiment, an isolated nucleic acid molecule is provided in which the encoded extracellular CD38 antigen-binding domain is connected to the transmembrane domain by a linker domain.

[0026] 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.

[0027] In yet another embodiment, an isolated nucleic acid molecule is provided encoding a CAR comprising at least one CD38 antigen-binding domain encoded by a nucleotide sequence comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 15, 17, 19, 21, 23, 25, 69, 71, 73, and 75, wherein the CAR further encodes an extracellular antigen-binding domain that targets 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.

[0028] In certain embodiments, the further encoded extracellular antigen-binding domain is 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, an anti-GD-2 Provided is an isolated nucleic acid molecule encoding a CAR comprising an scFV antigen-binding domain, an anti-NY-ESo-1 TCR scFV antigen-binding domain, an anti-MAGE A3 TCR scFV antigen-binding domain, or an amino acid sequence thereof having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, or any combination thereof.

[0029] In one embodiment, the CAR provided herein further comprises a linker or spacer domain.

[0030] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, in which an extracellular CD38 antigen-binding domain, an intracellular signaling domain, or both, is connected to a transmembrane domain by a linker or spacer domain.

[0031] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded linker domain is derived from the extracellular domain of CD8 or CD28 and is linked to a transmembrane domain.

[0032] In another embodiment, the encoded CAR encodes a protein selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154, or a combination thereof. An isolated nucleic acid molecule encoding a CAR is provided, further comprising a transmembrane domain comprising a transmembrane domain of a protein.

[0033] In yet another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded intracellular signaling domain further comprises a CD3 zeta intracellular domain.

[0034] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded intracellular signaling domain is positioned C-terminal to the CD3 zeta intracellular domain.

[0035] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded at least one intracellular signaling domain comprises a costimulatory domain, a primary signaling domain, or a combination thereof.

[0036] In a further embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded at least one costimulatory domain comprises a functional signaling domain of OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP12, and 4-1BB (CD137), or a combination thereof.

[0037] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, further comprising a leader sequence or signal peptide, wherein the nucleotide sequence of the leader or signal peptide comprises the nucleotide sequence of SEQ ID NO: 13, SEQ ID NO: 39, SEQ ID NO: 41, or SEQ ID NO: 43.

[0038] In yet another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded leader sequence comprises the amino acid sequence of SEQ ID NO:14, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:44.

[0039] 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.

[0040] 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.

[0041] In another embodiment, a CAR is provided, wherein at least one transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154, or a combination thereof.

[0042] In some embodiments, CARs are 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 thereof having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity, or any combination thereof.

[0043] In one embodiment, the extracellular antigen-binding domain is 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, an anti-GD-2 CARs are provided that include an scFV antigen-binding domain, an anti-NY-ESo-1 TCR scFV antigen-binding domain, an anti-MAGE A3 TCR scFV antigen-binding domain, or an amino acid sequence thereof having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, or any combination thereof.

[0044] In another embodiment, the extracellular antigen-binding domain is an immunoglobulin variable heavy chain only (VH) anti-CD19 antigen-binding domain, anti-CD20 VH antigen-binding domain, anti-CD22 VH antigen-binding domain, anti-ROR1 VH antigen-binding domain, anti-mesothelin VH antigen-binding domain, anti-CD33 VH antigen-binding domain, anti-CD38 VH antigen-binding domain, anti-CD123 (IL3RA) VH antigen-binding domain, anti-CD138 VH antigen-binding domain, anti-BCMA (CD269) VH antigen-binding domain, anti-GPC2 VH antigen-binding domain, anti-GPC3 VH antigen-binding domain, anti-FGFR4 VH antigen-binding domain, anti-c-Met VH antigen-binding domain, anti-PMSA VH antigen-binding domain, anti-glycolipid F77 VH antigen-binding domain, anti-EGFRvIII VH antigen-binding domain, anti-GD-2 VH antigen-binding domain, anti-NY-ESO-1 TCR CARs are provided that comprise a VH antigen-binding domain, an anti-MAGE A3 TCR VH antigen-binding domain, or an amino acid sequence thereof having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, or any combination thereof.

[0045] In another embodiment, the extracellular antigen-binding domain is 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, anti-mesothelin P antigen-binding domain, anti-CD33 P antigen-binding domain, anti-CD38 P antigen-binding domain, anti-CD123 (IL3RA) P antigen-binding domain, anti-CD138 P antigen-binding domain, anti-BCMA (CD269) P antigen-binding domain, anti-GPC2 P antigen-binding domain, anti-GPC3 P antigen-binding domain, anti-FGFR4 P antigen-binding domain, anti-c-Met P antigen-binding domain, anti-PMSA P antigen-binding domain, anti-glycolipid F77 P antigen-binding domain, anti-EGFRvIII P antigen-binding domain, anti-GD-2 P antigen-binding domain, anti-NY-ESO-1 TCR P antigen-binding domain, anti-MAGE A3 TCR

[0010] CARs are provided, comprising 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 a P antigen-binding domain, or an amino acid sequence thereof having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity, or any combination thereof. In another embodiment, a CAR is provided, wherein at least one intracellular signaling domain comprises a costimulatory domain and a primary signaling domain.

[0046] In yet another embodiment, at least one intracellular signaling domain is a functional signal 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. CARs are provided that include a costimulatory domain that includes a transduction domain.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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. do.

[0061] In another embodiment, the nucleic acid sequence encoding the 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.

[0062] 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.

[0063] In one embodiment, the CARs disclosed herein are modified to express or contain a detectable marker for use in diagnosis, monitoring and / or prediction of treatment outcome, such as progression-free survival in cancer patients, or to monitor the progress of such treatment.

[0064] In one embodiment, the nucleic acid molecule encoding the disclosed CAR can be contained in a vector, such as a viral vector, which can be a DNA vector, an RNA vector, a plasmid vector, a cosmid vector, a herpes virus vector, a measles virus vector, a lentivirus vector, an adenovirus vector, or a retrovirus vector, or a combination thereof.

[0065] In certain embodiments, the vector further comprises a promoter that is an inducible promoter, a tissue-specific promoter, a constitutive promoter, a suicide promoter, or any combination thereof.

[0066] In yet another embodiment, the CAR-expressing vector can be further modified to include one or more operable elements to control the expression of CAR T cells or to eliminate CAR-T cells by a suicide switch. The suicide switch can include, for example, an apoptosis-inducing signaling cascade or a drug that induces cell death. In a preferred embodiment, the CAR-expressing vector can be further modified to express an enzyme such as thymidine kinase (TK) or cytosine deaminase (CD).

[0067] In another aspect, a host cell comprising a nucleic acid molecule encoding a CAR is also provided. In some embodiments, the host cell is a T cell, for example, a primary T cell obtained from a subject. In one embodiment, the host cell is a CD8 + T cells.

[0068] In yet another aspect, a pharmaceutical composition is provided comprising an antitumor effective amount of a population of human T cells, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), the CAR comprising at least one extracellular antigen-binding domain, at least one linker domain, at least one transmembrane domain, and at least one intracellular signaling domain, the extracellular antigen-binding domain comprising a CD38 antigen-binding domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 16, 18, 20, 22, 24, 26, 70, 72, 74, and 76, and the T cells are from a human with cancer. Cancers include, inter alia, hematological cancers such as leukemia (e.g., chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), or chronic myelogenous leukemia (CML)), lymphoma (e.g., mantle cell lymphoma, non-Hodgkin's lymphoma, or Hodgkin's lymphoma), or multiple myeloma (MM), or a combination thereof.

[0069] In one embodiment, at least one transmembrane domain of the CAR is selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, C Pharmaceutical compositions are provided that include a transmembrane domain of a protein selected from the group consisting of D5, CD8, CD9, CD16, CD22, mesothelin, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, or a combination thereof.

[0070] In another embodiment, pharmaceutical compositions are provided wherein the human cancer comprises oral and pharyngeal cancer (tongue, mouth, pharynx, head and neck), digestive system cancer (esophagus, stomach, small intestine, colon, rectum, anus, liver, intrahepatic bile duct, gallbladder, pancreas), respiratory system cancer (larynx, lung and bronchus), bone and joint cancer, soft tissue cancer, skin cancer (melanoma, basal cell carcinoma and squamous cell carcinoma), childhood tumors (neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma), tumors of the central nervous system (brain tumor, astrocytoma, glioblastoma, glioma), and adult cancers including cancer of the breast, reproductive system (cervix, uterus, ovary, vulva, vagina, prostate, testicle, penis, endometrium), urinary system (bladder, kidney and renal pelvis, ureter), eye and orbit, endocrine system (thyroid), and brain and other nervous system, or any combination thereof.

[0071] In yet another embodiment, a pharmaceutical composition is provided comprising an antitumor-effective amount of a population of human T cells from a human with cancer, wherein the cancer is a refractory cancer that is unresponsive to one or more chemotherapeutic agents. The cancer includes hematopoietic cancer, myelodysplastic syndrome, pancreatic cancer, head and neck cancer, skin tumor, adult B-cell malignancies including acute lymphoblastic leukemia (ALL), minimal residual disease (MRD) in acute myeloid leukemia (AML), CLL (chronic lymphocytic leukemia), CML (chronic myeloid leukemia), non-Hodgkin's lymphoma (NHL), pediatric B-cell malignancies (including B-cell lineage ALL (acute lymphocytic leukemia)), multiple myeloma (MM), lung cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, melanoma or other hematological cancers and solid tumors, or any combination thereof.

[0072] In another aspect, a method of generating CAR-containing T cells (hereinafter "CAR T cells") is provided, which method comprises transducing T cells with a vector or nucleic acid molecule encoding the disclosed CAR that specifically binds to CD38, thereby generating the CAR T cells.

[0073] In yet another aspect, a method of generating a population of RNA engineered cells is provided, comprising introducing in vitro transcribed or synthetic RNA of a nucleic acid molecule encoding a disclosed CAR into cells of a subject to generate CAR cells.

[0074] In yet another aspect, a method of diagnosing a disease, disorder, or condition associated with CD38 expression in a cell is provided, comprising the steps of: 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 NOs: 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 diagnoses the disease, disorder, or condition associated with CD38 expression.

[0075] 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 tumor, adult B-cell malignancies including acute lymphoblastic leukemia (ALL), minimal residual disease (MRD) in acute myeloid leukemia (AML), CLL (chronic lymphocytic leukemia), CML (chronic myeloid leukemia), non-Hodgkin's lymphoma (NHL), pediatric B-cell malignancies (including B-cell lineage ALL (acute lymphocytic leukemia)), multiple myeloma (MM), lung cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, melanoma or other hematological cancers and solid tumors, or any combination thereof.

[0076] In another embodiment, a method is provided for diagnosing, prognosing, or determining the risk of a disease associated with CD38 in a mammal, comprising detecting CD38 expression in a sample from a mammal, comprising the steps of: a) contacting the sample 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 NOs: 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 the disease associated with CD38 in the mammal.

[0077] In another embodiment, a method of inhibiting CD38-dependent T cell inhibition is provided, comprising the step of 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 NOs: 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 the group consisting of a CD38-expressing tumor cell, a tumor-associated macrophage, and any combination thereof.

[0078] In another embodiment, a method is provided for altering the tumor microenvironment to block T cell inhibition mediated by cells expressing CD38 and inhibit tumor growth in a mammal, the method comprising the step of 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 the group consisting of SEQ ID NOs: 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 the group consisting of a CD38-expressing tumor cell, a tumor-associated macrophage, and any combination thereof.

[0079] In another embodiment, a method is provided for inhibiting, suppressing, or preventing immunosuppression of an anti-tumor or anti-cancer immune response in a mammal, comprising the step of 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 the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 16, 18, 20, 22, 24, 26, 70, 72, 74, and 76. In one embodiment, the antibody or fragment thereof inhibits an interaction between a first cell and a T cell, wherein the first cell is selected from the group consisting of a CD38-expressing tumor cell, a tumor-associated macrophage, and any combination thereof.

[0080] In another aspect, provided is a method of inducing anti-tumor immunity in a mammal, the method comprising administering to the mammal a therapeutically effective amount of T cells transduced with a vector or nucleic acid molecule encoding a disclosed CAR.

[0081] In another embodiment, a method of treating or preventing cancer in a mammal is provided, comprising administering to the mammal one or more of the disclosed CARs in an amount effective to treat or prevent cancer in the mammal. The method comprises administering to the subject a therapeutically effective amount of host cells expressing the disclosed CARs that specifically bind to CD38 and / or one or more of the above-mentioned antigens under conditions sufficient to form an immune complex between the antigen-binding domain of the CAR and the extracellular domain of CD38 and / or one or more of the above-mentioned antigens in the subject.

[0082] In yet another embodiment, a method of treating a mammal having a disease, disorder, or condition associated with elevated expression of a tumor antigen comprises administering to the subject an anti-tumor effective amount of a pharmaceutical composition comprising a population of T cells, wherein the T cells express a nucleic acid 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 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 the T cell is a T cell of a subject with cancer.

[0083] In yet another embodiment, a method of treating cancer in a subject in need thereof is provided, comprising: administering to the subject an anti-tumor effective amount of a pharmaceutical composition comprising a population of T cells, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises at least one 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, at least one linker or spacer domain, at least one transmembrane domain, at least one intracellular signaling domain, and wherein the T cells are from a subject with cancer. In some embodiments of the above-described methods, at least one transmembrane domain comprises the transmembrane alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, mesothelin, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154, or a combination thereof.

[0084] In yet another embodiment, a method is provided for generating a persistent population of engineered T cells in a human diagnosed with cancer. In one embodiment, the method comprises administering to the human T cells engineered to express a CAR, wherein the CAR comprises at least one CD38 antigen-binding domain, at least one transmembrane domain, and at least one intracellular signaling 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 persistent population of engineered T cells, or a population of progeny of the T cells, persists in the human for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, or 3 years after administration.

[0085] In one embodiment, the progeny T cells in the human comprise memory T cells. In another embodiment, the T cells are autologous T cells.

[0086] In all of the aspects and embodiments of the methods described herein, any of the above-mentioned cancers, diseases, disorders, or conditions associated with elevated expression of tumor antigens can be treated or prevented or ameliorated using one or more of the CARs disclosed herein.

[0087] In yet another aspect, there is provided a kit for generating the chimeric antigen receptor T cells as described above, or for preventing, treating, or ameliorating any of the cancers, diseases, disorders, or conditions associated with elevated expression of a tumor antigen in the subject as described above, the kit comprising a container containing any one of the nucleic acid molecules, vectors, host cells, or compositions disclosed above, or any combination thereof, and instructions for using the kit.

[0088] It is understood that the CARs, host cells, nucleic acids, and methods are useful beyond the specific aspects and embodiments described in detail herein. The foregoing features and advantages of the present disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying drawings. [Brief explanation of the drawings]

[0089]

Figure 1

[0023] Figure 1 shows the general structure of a CD38-targeting CAR: An anti-CD38 ScFv targeting domain was linked in-frame to a CD8 hinge and transmembrane domain, a 4-1BB (CD137) costimulatory domain, and a CD3 zeta signaling domain.

Figure 2

Figure 3A

Figure 3B

Figure 3C

Figure 4A

[0033] Figure 4A shows CAR T cytokine release in response to MM cell lines. Figure 4A shows IFNg cytokine production by CAR-Ts listed on the x-axis when co-cultured overnight with RPMI-8226 and MM1.S tumor lines at an E:T ratio of 10:1, as measured using ELISA. Figure 4B shows TNFa cytokine production by CAR-Ts listed on the x-axis when co-cultured overnight with RPMI-8226 and MM1.S tumor lines at an E:T ratio of 10:1, as measured using ELISA. Bars represent the mean + SD of three replicate samples. Data are representative of three independent experiments performed with CAR T cells from three separate donors.

Figure 4B

[0033] Figure 4A shows CAR T cytokine release in response to MM cell lines. Figure 4A shows IFNg cytokine production by CAR-Ts listed on the x-axis when co-cultured overnight with RPMI-8226 and MM1.S tumor lines at an E:T ratio of 10:1, as measured using ELISA. Figure 4B shows TNFa cytokine production by CAR-Ts listed on the x-axis when co-cultured overnight with RPMI-8226 and MM1.S tumor lines at an E:T ratio of 10:1, as measured using ELISA. Bars represent the mean + SD of three replicate samples. Data are representative of three independent experiments performed with CAR T cells from three separate donors. DETAILED DESCRIPTION OF THE INVENTION

[0090] Detailed Description definition As used herein, the singular forms "a," "an," and "the" refer to both the singular and the plural unless the context clearly indicates otherwise. For example, the term "an antigen" includes single or multiple antigens and can be considered equivalent to the phrase "at least one antigen." As used herein, the term "comprises" means "includes." Thus, "comprising an antigen" means "including one antigen" without excluding other elements. The phrase "and / or" means "and" or "or." It should be further understood that any and all base or amino acid sizes and all molecular weight or molecular mass values ​​given for nucleic acids or polypeptides are approximate and provided for convenience unless otherwise specified. While many methods and materials similar or equivalent to those described herein can be used, particularly suitable methods and materials are described below. In case of conflict, the present specification, including explanations of terms, controls. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. In order to facilitate review of the various embodiments, the following explanations of terms are provided.

[0091] The term "about," when referring to a measurable value, e.g., amount, temporal duration, etc., is meant to encompass variations of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% from the specified value, where such variations are appropriate for practicing the disclosed methods.

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

[0093] The present disclosure provides a CD38 antibody or a fragment thereof, and a chimeric antigen receptor (CAR) having such a CD38 antigen-binding domain. The enhanced functional activity of the CAR is directly related to the enhanced functional activity of T cells expressing the CAR. As a result of one or more of these modifications, the CAR exhibits both high cytokine-induced cytolysis and cell surface expression on transduced T cells, along with increased in vivo T cell proliferation and persistence levels of transduced CAR-expressing T cells.

[0094] The unique ability to combine functional moieties from different protein domains is a key and innovative feature of chimeric antigen receptors (CARs). The selection of each of these protein domains, as well as the specific combinations they employ, is a key design feature. Each design domain is an essential component that can be used to manipulate lymphocyte function in various CAR platforms. For example, the selection of an extracellular binding domain can enable an otherwise ineffective CAR.

[0095] The non-variable framework components of the immunoglobulin-derived protein sequences used to create the extracellular antigen-binding domain of a CAR can be completely neutral or self-associate, rendering the T cell metabolically exhausted and therefore the therapeutic T cells expressing this CAR highly ineffective. This occurs independently of the antigen-binding function of this CAR domain. Furthermore, the choice of intracellular signaling domain(s) also influences the efficacy of the CAR for immunotherapy. The activity and durability of the resulting therapeutic lymphocyte population can be determined. While the ability of these extracellular and intracellular domains to bind to target antigens and transmit activation signals to T cells, respectively, are important aspects of CAR design, it has also become clear that the choice of source of the extracellular antigen-binding fragment can have a significant effect on the efficacy of the CAR and therefore play a crucial role in the function and clinical utility of the CAR.

[0096] Surprisingly and unexpectedly, it has now been discovered that the functional activity of CAR-expressing T cells can also be determined by using a fully human antigen-binding domain in the CAR, rather than using a mouse-derived antigen-binding fragment (see UPenn-funded clinical trial, NCT02159716, using a mouse-derived SS1 ScFv sequence), which tends to induce anti-mouse immune responses and CAR T elimination in the host.

[0097] The CARs disclosed herein are expressed at high levels in cells. Cells expressing CARs have a high proliferation rate in vivo, produce large amounts of cytokines, and have high cytotoxicity against cells bearing the CD38 antigen to which the CAR binds. The use of a human extracellular CD38 antigen-binding domain results in the generation of CARs that function better in vivo, while avoiding the induction of anti-CAR immunity in the host immune response and the death of the CAR T cell population. CARs expressing a fully human extracellular CD38 ScFv antigen-binding domain exhibit excellent activity / properties, including: i) prevention of the lack of persistence and function of CAR T cells seen with mouse-derived binding sequences; ii) no need for local delivery of CARs to be effective; and iii) the ability to generate CAR T cell designs based on both high-affinity and low-affinity binders for CD38. This last property allows researchers to better tune the efficacy and / or tissue specificity of CAR T products to toxicity, as tumors express more CD38 than normal tissues, allowing lower affinity binders to have greater specificity for tumors over normal tissues, thereby preventing non-on-target tumor toxicity and bystander cell killing.

[0098] Below is a detailed description of the CARs of the present invention, including a description of their extracellular CD38 antigen-binding domain, transmembrane domain, and intracellular domain, along with further description of CARs, antibodies and antigen-binding fragments thereof, conjugates, nucleotides, expression, vectors, and host cells, methods of treatment, compositions, and kits using the disclosed CARs.

[0099] A. Chimeric Antigen Receptor (CAR) The CARs disclosed herein comprise at least one CD38 antigen-binding domain capable of binding to CD38, at least one transmembrane domain, and at least one intracellular domain.

[0100] Chimeric antigen receptors (CARs) are artificially constructed hybrid proteins or polypeptides containing an antibody antigen-binding domain (e.g., a single-chain variable fragment (scFv)) linked to a T cell signaling domain via a transmembrane domain. Characteristics of CARs include their ability to redirect T cell specificity and reactivity toward selected targets, leveraging the antigen-binding properties of monoclonal antibodies in a non-MHC-restricted manner. Non-MHC-restricted antigen recognition confers on CAR-expressing T cells the ability to recognize antigens independently of antigen processing, thus bypassing a major mechanism of tumor escape. Furthermore, when expressed in T cells, CARs advantageously do not dimerize with the alpha and beta chains of endogenous T cell receptors (TCRs).

[0101] As disclosed herein, the intracellular T cell signaling domain of a CAR can include, for example, a T cell receptor signaling domain, a T cell costimulatory signaling domain, or any combination thereof. Both may be included. A T cell receptor signaling domain refers to a portion of a CAR that includes the intracellular domain of a T cell receptor, such as, but not limited to, the intracellular portion of a CD3 zeta protein. A costimulatory signaling domain refers to a portion of a CAR that includes the intracellular domain of a costimulatory molecule, which is a cell surface molecule other than an antigen receptor or its ligand, that is required for an efficient response of lymphocytes to antigens.

[0102] 1. Extracellular domain In one embodiment, CAR comprises target-specific binding element, otherwise called antigen-binding domain or part.The selection of domain depends on the type and number of ligands that define the surface of target cell.For example, antigen-binding domain can be selected to recognize the ligand that acts as a cell surface marker on target cells that is associated with specific disease state.Therefore, the examples of cell surface markers that can act as ligands for the antigen-binding domain in CAR include those associated with virus, bacteria and parasite infection, autoimmune disease and cancer cell.

[0103] In one embodiment, CAR can be engineered to target the tumor antigen of interest by engineering the desired antigen binding domain that specifically binds to the antigen on tumor cell.Tumor antigen is the protein produced by tumor cell that induces immune response, especially T cell-mediated immune response.The choice of antigen binding domain depends on the specific type of cancer to be treated. Tumor antigens include, for example, glioma-associated antigen, carcinoembryonic antigen (CEA), beta-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, Her2 / neu, survivin, and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, and CD38. The tumor antigens disclosed herein are included by way of example only; this list is not intended to be exhaustive, and further examples will be readily apparent to those of skill in the art.

[0104] In one embodiment, the tumor antigen comprises one or more antigenic cancer epitopes associated with malignant tumors. Malignant tumors express several proteins that can serve as target antigens for immune attack. These molecules include, but are not limited to, tissue-specific antigens, such as MART-1, tyrosinase, and GP100 in melanoma, and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation-related molecules, such as the oncogene HER-2 / Neu / ErbB-2. Yet another group of target antigens is carcinoembryonic antigens, such as carcinoembryonic antigen (CEA). In B-cell lymphomas, tumor-specific idiotypic immunoglobulins constitute truly tumor-specific immunoglobulin antigens unique to each individual tumor. B-cell differentiation antigens, such as CD19, CD20, and CD37, are other candidate target antigens in B-cell lymphomas. Some of these antigens (CEA, HER-2, CD19, CD20, idiotype) have been used as targets for passive immunotherapy with monoclonal antibodies with limited success.

[0105] In one preferred embodiment, the tumor antigen is CD38, and tumors associated with CD38 expression include lung mesothelioma, ovarian and pancreatic cancer, or any combination thereof, which express high levels of the extracellular protein CD38.

[0106] The type of tumor antigen can also be a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). TSAs are unique to tumor cells and do not exist on other cells in the body. TAAs are not unique to tumor cells, but are instead expressed on normal cells under conditions that cannot induce a state of immunological tolerance to the antigen. The expression of an antigen on a tumor can occur under conditions that allow the immune system to respond to the antigen. A TAA can be an antigen that is expressed on normal cells during fetal development, when the immune system is immature and unable to respond, or it can be an antigen that is normally present at very low levels on normal cells but is expressed at a significantly higher level on tumor cells.

[0107] Non-limiting examples of TSAs or TAAs include differentiation antigens such as MART-1 / MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, HER-2 / neu; unique tumor antigens resulting from chromosomal translocations such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3 / CA 27.29 / BCAA, CA 195, CA These include 242, CA-50, CAM43, CD68 / P1, CO-029, FGF-5, G250, Ga733 / EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 / Mac-2 binding protein / cyclophilin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0108] In one embodiment, the antigen binding domain portion of the CAR targets an antigen including, but not limited to, CD19, CD20, CD22, ROR1, CD123, CD33, CD38, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, MY-ESO-1 TCR, MAGE A3 TCR, etc.

[0109] In a preferred embodiment, the antigen-binding domain portion of the CAR targets the extracellular CD38 antigen.

[0110] In one preferred embodiment, an 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 thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD38 hScFv M3801 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 2.

[0111] 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 thereof with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity. In one embodiment, the encoded extracellular CD38 hScFv M3802 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 4, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 4. Isolated nucleic acid molecules are provided that contain an amino acid sequence that has 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.

[0112] In one preferred embodiment, an 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 thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD38 hScFv M3803 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 6.

[0113] In one preferred embodiment, an 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 thereof with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD38 hScFv M3804 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 8.

[0114] In one preferred embodiment, an 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 thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD38 hScFv M3806 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 10.

[0115] In one preferred embodiment, an 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 thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD38 hScFv M3808 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 12.

[0116] In one preferred embodiment, an 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 thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD38 hScFv M3809 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 16.

[0117] In one preferred embodiment, an 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 thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD38 hScFv M3810 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 18, or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 18.

[0118] In one preferred embodiment, an 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 thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD38 hScFv M3811 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 20, or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 20.

[0119] In one preferred embodiment, an 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 thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD38 hScFv M3812 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 22, or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 22.

[0120] In one preferred embodiment, an 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 thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD38 hScFv M3815 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 24.

[0121] In one preferred embodiment, an 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 thereof with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD38 hScFv M3815 P antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 26, or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 26.

[0122] In one preferred embodiment, an 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 thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD38 hScFv M3816 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 70, or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 70.

[0123] In one preferred embodiment, an 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 thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD38 hScFv M3817 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 72, or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 72.

[0124] In one preferred embodiment, extracellular CD38 hScFv M8DR H_L antigen binding An 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 thereof with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity. In one embodiment, an isolated nucleic acid molecule is provided wherein the encoded extracellular CD38 hScFv M8DR H_L antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 74, or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 74.

[0125] In one preferred embodiment, an 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 thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD38 hScFv M8DR L_H antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 76, or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 74.

[0126] The generation and binding characteristics of the specific CD38 variable heavy chain only and ScFv antigen-binding fragments or antigen binders described herein are shown in Example 1.

[0127] In various embodiments of the CD38-specific CAR disclosed herein, a general schematic is shown in FIG. 1, which includes, from N-terminus to C-terminus, a signal or leader peptide, anti-CD38 Contains hScFv, extracellular linker, CD8 transmembrane domain, 4-1BB, CD3 zeta, bold text indicates cloning site of linking domain.

[0128] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 77, and encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 78.

[0129] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 77, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, and encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 78, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.

[0130] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 87, and encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 88.

[0131] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 87, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, and encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 88, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.

[0132] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 89, which encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 90.

[0133] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 89, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, and encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 90, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.

[0134] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 91, and encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 92.

[0135] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 91, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, and encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 92, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.

[0136] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 93, and encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 94.

[0137] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 93, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, and encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 94, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.

[0138] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 95, and encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 96.

[0139] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 95, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, and encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 96, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.

[0140] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 97, and encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 98.

[0141] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 97, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, and encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 98, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.

[0142] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 99, which encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 100.

[0143] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 99, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, and encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 100, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.

[0144] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 101, and encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 102.

[0145] In yet another embodiment, the nucleic acid sequence encoding the CAR is the nucleic acid sequence of SEQ ID NO: 101, or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity. and encoding a CAR comprising the amino acid sequence set forth in SEQ ID NO: 102, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.

[0146] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 103, and encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 104.

[0147] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 103, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, and encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 104, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.

[0148] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 105, and encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 106.

[0149] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 105, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, and encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 106, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.

[0150] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 107, and encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 108.

[0151] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 107, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, and encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 108, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.

[0152] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 109, and encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO:110.

[0153] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 109, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, and encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 110, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.

[0154] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO:111, and encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO:112.

[0155] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 111, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, and encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 112, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.

[0156] In yet another embodiment, the nucleic acid sequence encoding the CAR is the nucleic acid of SEQ ID NO: 113. and encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 114.

[0157] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 113, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, and encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 114, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.

[0158] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 115, and encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO:116.

[0159] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 115, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, and encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 116, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.

[0160] The surface expression of anti-CD38 CARs incorporating an immunoglobulin heavy chain variable domain (VH) and single-chain fragment variable (ScFv) sequence reactive with the CD38 antigen is shown in Example 2 below. CAR expression was determined by flow cytometry. On day 8 of culture, viable transduced T cells (7-AAD negative) were assayed for CAR surface expression using CD38-His reagent followed by anti-His-PE. The CAR construct LV used for transduction is listed below each bar. The bars represent the percentage of the CAR T-positive population relative to the untransduced T cell control (UTD). Data are from CAR T-positive T cells from three separate donors. Representative of three independent experiments performed with T cells.

[0161] The cytotoxic potential of CAR38 candidates was assessed in a luciferase-based overnight killing assay using the CD38-positive tumor line RPMI-8226, and MM1.S, stably transduced with luciferase, or the CD38-negative cell line 293T. Bars represent the mean + SD values ​​from three technical replicates. Data are representative of three independent experiments performed with CAR T cells from three separate donors (Figure 3A-C). 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. Most CAR constructs exhibited CD38 + The constructs showed highly potent oncolytic activity against the tumor line RPMI-8226 (Figure 3A), comparable to the positive control constructs LTG2525 and LTG2526, which consist of comparator ScFv sequences in VL-VH or VH-VL orientation, respectively. The exceptions were constructs LTG2098 and LTG2099, which were less cytotoxic than the positive controls LTG 2525 and 2526 (Figure 3A). On the other hand, no significant lysis was observed with the negative control UTD (untransduced T cells), demonstrating that tumor lysis was CAR-dependent (Figure 3A). CD38 T cells were less susceptible to CAR T killing than RPMI-8226. +In the killing assay against MM1.S cells, the differences in potency among the CAR constructs were more pronounced (Figure 3B). 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-to-moderate range, with constructs LTG2099, 2093, and 2098 exhibiting low lytic activity in this cell line (Figure 3B).

[0162] All CAR candidates and controls were then tested in a killing assay against the CD38 cell line 293T (Figure 3C). In this case, most CAR constructs did not exhibit lytic activity above background levels compared to the positive control CAR LTG 2526. However, this indicates that these CAR constructs require CD38 expression for activation, a safety feature of CARs. The exception to this observation was constructs 2501 and 2503, which exhibited low levels of CD38-independent killing activity at high E:T ratios (Figure 3C). The nonspecific killing activity of these two constructs cannot be predicted based on binder data alone and is therefore not evident. Overall, the cytolytic activity of most CD38 CARs in this set observed against CD38-expressing MM1.S and RPMI-8226 tumor cells is target-specific and CAR-dependent.

[0163] Next, we evaluated the ability of anti-CD38 CAR T cells to secrete the proinflammatory cytokines IFNg and TNFa (Figure 4). Cytokine production by the CAR Ts listed on the x-axis was measured using ELISA when cocultured overnight with RPMI-8226 and MM1.S tumor lines at an E:T ratio of 10:1. Notably, CAR T-expressing cells LTG2091, LTG2092, and LTG2095 produced high levels of IFNgamma and TNFalpha comparable to the positive controls LTG2525 and LTG2526, whereas most other CAR constructs, as well as the negative control UTD group, produced low or no appreciable cytokine induction (Figure 4A-B). This result is consistent with the most potent in vitro cytolytic function of LTG2091, LTG2092, and LTG2095 (see Figure 3A-C). A second subset of constructs that did not produce high levels of cytokines but still had high to moderate antitumor activity includes LTG2089, 2090, 2094, 2096, and 2097. This finding also suggests that it may be possible to select CAR38 constructs that efficiently kill tumors, have a lower risk of inducing cytokine release syndrome, and therefore a better safety profile.

[0164] Without intending to be limited to any particular mechanism of action, possible reasons for the enhanced therapeutic function associated with exemplary CARs of the present invention are believed to include, for example, but not by way of limitation, a) improved lateral movement within the plasma membrane, allowing for more efficient signaling; b) superior location within plasma membrane microdomains, such as lipid rafts, and a greater ability to interact with transmembrane signaling cascades associated with T cell activation; c) superior location within the plasma membrane due to preferential movement away from inhibitory or down-regulatory interactions, e.g., less proximity to or fewer interactions with phosphatases such as CD45; and d) superior assembly into the T cell receptor signaling complex (i.e., the immune synapse), or any combination thereof.

[0165] Although the present disclosure is 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 may be used to obtain CD38 antigen-binding domains for use in the CARs described herein.

[0166] Depending on the desired antigen to be targeted, the CAR can be further engineered to contain an appropriate antigen-binding domain specific for the desired antigen target. For example, if CD19 is the desired antigen to be targeted, an antibody to CD19 can be used as the antigen-binding domain incorporated into the CAR.

[0167] In one exemplary embodiment, the antigen-binding domain portion of the CAR further targets CD19. Preferably, the antigen-binding domain in the CAR is an anti-CD19 scFV, wherein the nucleic acid sequence of the anti-CD19 scFV comprises the sequence set forth 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 the CAR comprises the amino acid sequence set forth in SEQ ID NO: 38.

[0168] In one embodiment of the present invention, a CAR is provided that is capable of binding to a non-TSA or non-TAA, including, for example, but not limited to, an antigen derived from Retroviridae (e.g., human immunodeficiency viruses, e.g., HIV-1 and HIV-LP), Picornaviridae (e.g., poliovirus, hepatitis A virus, enterovirus, human coxsackievirus, rhinovirus, and echovirus), rubella virus, coronavirus, vesicular stomatitis virus, rabies virus, Ebola virus, parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus, influenza virus, hepatitis B virus, parvovirus, Adenoviridae, Herpesviridae (e.g., herpes simplex virus types 1 and 2 (HSV), varicella-zoster virus, cytomegalovirus (CMV), and herpes viruses), Poxviridae (e.g., smallpox virus, vaccinia virus, and poxvirus), or hepatitis C virus, or any combination thereof.

[0169] In another aspect of the present invention, a CAR capable of binding to an antigen derived from a bacterial strain of Staphylococci, Streptococcus, Escherichia coli, Pseudomonas, or Salmonella is provided. In particular, a CAR capable of binding to an antigen derived from an infectious bacterium, such as Helicobacter pyloris, Legionella pneumophilia, a bacterial strain of Mycobacteria sp. (e.g., M. tuberculosis, M. avium, M. intracellulare, M. kansaii, or M. gordonea), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitides, Listeria monocytogenes, Streptococcus pyogenes, Group A Streptococcus, Group B Streptococcus (Streptococcus agalactiae), Streptococcus pneumoniae, or Clostridium tetani, or a combination thereof, is provided.

[0170] 2. Transmembrane domain With respect to the transmembrane domain, the CAR comprises one or more transmembrane domains fused to the extracellular CD33 antigen-binding domain of the CAR.

[0171] Transmembrane domains can be derived from either natural or synthetic sources. If the source is natural, the domain can be derived from any membrane-bound or transmembrane protein.

[0172] The transmembrane region particularly used in the CAR described herein can be derived from (i.e., can include at least one of) the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, mesothelin, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154. Alternatively, the transmembrane domain can be synthetic, in which case it predominantly contains hydrophobic residues such as leucine and valine. Preferably, a phenylalanine, tryptophan, and valine triplet is found at each end of the synthetic transmembrane domain. Optionally, a short oligopeptide or polypeptide linker, preferably between 2 and 10 amino acids in length, can form the link between the transmembrane domain and the cytoplasmic signaling domain of the CAR. A glycine-serine duo provides a particularly suitable linker.

[0173] In one embodiment, a transmembrane domain naturally associated with one of the domains in the CAR is used in addition to the transmembrane domain.

[0174] 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.

[0175] In one embodiment, the transmembrane domain in the CAR of the present invention is a CD8 transmembrane domain. In one embodiment, the CD8 transmembrane domain comprises the nucleic acid sequence of SEQ ID NO: 27. In one embodiment, the CD8 transmembrane domain comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 28. In another embodiment, the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 28.

[0176] In one embodiment, the encoded transmembrane domain comprises an amino acid sequence having at least one, two, or three alterations (e.g., substitutions) but not more than 20, 10, or 5 alterations (e.g., substitutions) of the amino acid sequence of SEQ ID NO:28, or a sequence having 95-99% identity to the amino acid sequence of SEQ ID NO:28.

[0177] In some instances, the transmembrane domain of the CAR comprises a CD8 alpha hinge domain. In one embodiment, the CD8 hinge domain comprises the nucleic acid sequence of SEQ ID NO: 29. In one embodiment, the CD8 hinge domain comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 30. In another embodiment, the CD8 hinge domain comprises the amino acid sequence of SEQ ID NO: 30, or a sequence thereof with 95-99% identity.

[0178] 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.

[0179] 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 the nucleic acid sequence of SEQ ID NO: 51. In one embodiment, the TNFRSF19 transmembrane domain comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 52. In another embodiment, the TNFRSF19 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 52.

[0180] In one embodiment, the encoded transmembrane domain comprises an amino acid sequence having at least one, two, or three alterations (e.g., substitutions) but not more than 20, 10, or 5 alterations (e.g., substitutions) of the amino acid sequence of SEQ ID NO: 52, or a sequence having 95-99% identity to the amino acid sequence of SEQ ID NO: 52.

[0181] 3. Spacer domain In CARs, a spacer domain, also called a hinge domain, can be located between the extracellular domain and the transmembrane domain or between the intracellular domain and the transmembrane domain. A spacer domain refers to any oligopeptide or polypeptide that functions to link the transmembrane domain to the extracellular domain and / or the transmembrane domain to the intracellular domain. A spacer domain contains up to 300 amino acids, preferably 10-100 amino acids, and most preferably 25-50 amino acids.

[0182] In some embodiments, the linker may include a spacer element, which, if present, increases the size of the linker, thereby increasing the distance between the effector molecule or detectable marker and the antibody or antigen-binding fragment. Exemplary spacers are known to those of skill in the art, including those described in U.S. Patent Nos. 7,964,566, 7,498,298, 6,884,869, 6,323,315, 6,239,104, 6,034,065, and 5,788,912. 0,588, U.S. Patent No. 5,665,860, U.S. Patent No. 5,663,149, U.S. Patent No. 5,635,483, U.S. Patent No. 5,599,902, U.S. Patent No. 5,554,725, U.S. Patent No. 5,530,097, U.S. Patent No. 5,521,284, U.S. Patent No. 5,504,191, U.S. Patent No. 5,410,024, U.S. Patent No. 5,138,036, U.S. Patent No. 5,076, No. 973, U.S. Pat. No. 4,986,988, U.S. Pat. No. 4,978,744, U.S. Pat. No. 4,879,278, U.S. Pat. No. 4,816,444 and U.S. Pat. No. 4,486,414, as well as those listed in U.S. Patent Application Publication No. 20110212088 and U.S. Patent Application Publication No. 20110070248, each of which is incorporated herein by reference in its entirety.

[0183] The spacer domain preferably has a sequence that promotes the binding of CAR to the antigen and enhances signal transduction into the cell. Examples of amino acids that are predicted to promote binding include cysteine, charged amino acids, and serine and threonine in potential glycosylation sites, and these amino acids can be used as amino acids that constitute the spacer domain.

[0184] The spacer domain may be the entire or a portion of the hinge region of CD8 alpha (NCBI RefSeq: NP_001759.3), amino acids 118-178 (SEQ ID NO: 31), CD8 beta (GenBank: AAA35664.1), amino acids 315-396 of CD4 (NCBI RefSeq: NP_000607.1), or amino acids 137-152 of CD28 (NCBI RefSeq: NP_006130.1). Alternatively, the spacer domain may be a portion of the constant region of an antibody heavy or light chain (e.g., the CH1 region or CL region, e.g., a peptide having the amino acid sequence set forth in SEQ ID NO: 32). Furthermore, the spacer domain may be an artificially synthesized sequence.

[0185] Furthermore, all or part of the amino acids comprising the constant region of human IgG4 (UniProt ID: P01861), including CH1 (amino acid numbers 1-98), hinge, SEQ ID NO: 80, and corresponding nucleotide SEQ ID NO: 79, (amino acid numbers 99-110), CH2, amino acid number 81 and corresponding nucleotide SEQ ID NO: 80, (amino acid numbers 111-220), and CH3, SEQ ID NO: 84 and corresponding nucleotide SEQ ID NO: 83, (amino acid numbers 221-327), or combinations thereof, can be used, for example, the IgG4 hinge CH2 CH3 domain, SEQ ID NO: 86, and corresponding nucleotide SEQ ID NO: 85.

[0186] In one embodiment, the spacer domain of the CAR comprises a TNFRSF19 hinge domain comprising the nucleic acid sequence of SEQ ID NO: 53. In one embodiment, the TNFRSF19 hinge domain comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 54. In another embodiment, the TNFRSF19 hinge domain comprises the amino acid sequence of SEQ ID NO: 54, or a sequence thereof with 95-99% identity.

[0187] In one embodiment, the spacer domain of the CAR comprises a TNFRSF19 truncated hinge domain comprising the nucleic acid sequence of SEQ ID NO: 55. In one embodiment, the TNFRSF19 truncated hinge domain comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 56. In another embodiment, the TNFRSF19 truncated hinge domain comprises the amino acid sequence of SEQ ID NO: 56, or a sequence thereof with 95-99% identity.

[0188] 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. The SF19 hinge and transmembrane domain comprises the amino acid sequence of SEQ ID NO: 50, or a sequence thereof with 95-99% identity.

[0189] In one embodiment, the CD8a hinge domain is fused to a TNFRSF19 transmembrane domain comprising the nucleic acid sequence of SEQ ID NO: 57. In one embodiment, the CD8a hinge domain is fused to a TNFRSF19 transmembrane domain and comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 58. In another embodiment, the CD8a hinge domain is fused to a TNFRSF19 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 58, or a sequence thereof with 95-99% identity.

[0190] Furthermore, a signal peptide sequence, also called a leader peptide, may be linked to the N-terminus of a CAR. Signal peptide sequences are present at the N-terminus of many secretory proteins and membrane proteins and have a length of 15 to 30 amino acids. Many of the protein molecules referred to above as intracellular domains have signal peptide sequences, and these signal peptides can be used as signal peptides for a CAR. In one embodiment, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 14.

[0191] In one embodiment, the CD8 alpha leader peptide comprises the nucleic acid sequence of SEQ ID NO: 43. In one embodiment, the CD8 alpha 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 thereof with 95-99% identity.

[0192] 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 thereof with 95-99% identity.

[0193] 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 CD8 alpha 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 a TNFRSF19 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 42, or a sequence thereof with 95-99% identity.

[0194] In one embodiment, the tag sequence encoding a truncated sequence of epidermal growth factor receptor (tEGFR) comprises the nucleic acid sequence of SEQ ID NO: 67. In one embodiment, the 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 thereof having 95-99% identity.

[0195] In one embodiment, the furin recognition site and downstream T2A self-cleaving peptide sequence designed for simultaneous bicistronic expression of the tag sequence and CAR sequence comprise the nucleic acid sequence of SEQ ID NO: 65. In one embodiment, the furin and T2A sequences comprise a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 66. In another embodiment, the tEGFR tag comprises the amino acid sequence of SEQ ID NO: 66, or a sequence thereof having 95-99% identity.

[0196] In one embodiment, the upstream furin recognition site and T2A self-cleaving peptide sequence and the downstream furin recognition site designed for 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, In this case, the tEGFR tag comprises the amino acid sequence of SEQ ID NO: 68, or a sequence thereof having 95 to 99% identity.

[0197] In one embodiment, the targeting domain of the 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-expressed component of the CAR contains a binding domain specifically directed to bind to a tag or epitope expressed on a soluble CAR module, such that upon specific binding to a cell-binding component on the soluble component of the CAR, a complete functional CAR structure is formed.

[0198] 4. Intracellular domain The cytoplasmic domain or other intracellular signaling domain of a CAR is responsible for activating at least one of the normal effector functions of the immune cell in which the CAR is placed. The term "effector function" refers to a specialized function of a cell. For example, the effector function of a T cell can be cytolytic activity or helper activity, including cytokine secretion. Thus, the term "intracellular signaling domain" refers to the portion of a protein that transmits an effector function signal and instructs the cell to perform a specialized function. While the entire intracellular signaling domain can usually be used, it is often not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the intact chain, as long as it transmits the effector function signal. Thus, the term intracellular signaling domain is meant to include any truncated portion of the intracellular signaling domain sufficient to transmit the effector function signal.

[0199] Preferred examples of intracellular signaling domains for use in CARs include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivatives or variants of these sequences, and any synthetic sequences with the same functional capability.

[0200] It is known that signals generated through the TCR alone are insufficient for full activation of T cells, and that secondary or costimulatory signals are also required. Thus, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences) and those that act antigen-independently to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences).

[0201] Primary cytoplasmic signaling sequences regulate the primary activation of the TCR complex in either a stimulatory or inhibitory manner. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs, or ITAMs.

[0202] Examples of ITAMs containing primary cytoplasmic signaling sequences of particular use in the CARs disclosed herein include those derived from TCR zeta (CD3 zeta), FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. Specific, non-limiting examples of ITAMs include amino acids 51-164 of CD3 zeta (NCBI RefSeq: NP_932170.1), amino acids 45-86 of Fc epsilon RI gamma (NCBI RefSeq: NP_004097.1), amino acids 201-244 of Fc epsilon RI beta (NCBI RefSeq: NP_000130.1), amino acids 139-182 of CD3 gamma (NCBI RefSeq: NP_000064.1), and amino acids 128-17 of CD3 delta (NCBI RefSeq: NP_000723.1). 1, amino acids 153 to 207 of CD3 epsilon (NCBI RefSeq: NP_000724.1), amino acids 402 to 495 of CD5 (NCBI RefSeq: NP_055022.2), amino acids 707 to 847 of CD5 (NCBI RefSeq: NP_001762.2), amino acids 166 to 226 of CD79a (NCBI RefSeq: NP_001774.1), amino acids 182 to 229 of CD79b (NCBI RefSeq: NP_000617.1), and CD66d (NCBI RefSeq: NP_000617.1). Included herein are peptides having a sequence of amino acid numbers 177 to 252 of NCBI RefSeq:NP_001806.2), as well as variants thereof having the same functions as these peptides. The amino acid numbers based on the amino acid sequence information in NCBI RefSeq ID or GenBank described herein are numbered based on the full length of the precursor of each protein (including signal peptide sequences, etc.). In one embodiment, the cytoplasmic signaling molecule in the CAR comprises a cytoplasmic signaling sequence derived from CD3 zeta.

[0203] In a preferred embodiment, the intracellular domain of the CAR can be designed to include a CD3-zeta signaling domain, either alone or in combination with any other desired cytoplasmic domain(s) useful in the context of the CAR. For example, the intracellular domain of the CAR can include a CD3 zeta chain portion and a costimulatory signaling region. The costimulatory signaling region refers to a portion of the CAR that includes the intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for efficient lymphocyte response to antigens. Examples of such costimulatory molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83. Specific, non-limiting examples of such costimulatory molecules include amino acids 236-351 of CD2 (NCBI RefSeq: NP_001758.2), amino acids 421-458 of CD4 (NCBI RefSeq: NP_000607.1), amino acids 402-495 of CD5 (NCBI RefSeq: NP_055022.2), amino acids 207-235 of CD8 alpha (NCBI RefSeq: NP_001759.3), amino acids 196-210 of CD83 (GenBank: AAA35664.1), amino acids 181-220 of CD28 (NCBI RefSeq: NP_006130.1), and CD137 (4-1BB, NCBI The present disclosure primarily exemplifies 4-1BB as a costimulatory signaling element, but other costimulatory elements are within the scope of the present disclosure.

[0204] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR can be linked to each other randomly or in a specific order. Optionally, a short oligopeptide or polypeptide linker, preferably between 2 and 10 amino acids in length, can form the linkage. Glycine-serine duplexes provide particularly suitable linkers.

[0205] In one embodiment, the intracellular domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of CD28. In another embodiment, the intracellular domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of 4-1BB. In yet another embodiment, the intracellular domain is designed to comprise the signaling domain of CD3-zeta and the signaling domains of CD28 and 4-1BB.

[0206] In one embodiment, the intracellular domain in the CAR is designed to comprise the signaling domain of 4-1BB and the signaling domain of CD3-zeta, wherein the signaling domain of 4-1BB comprises the nucleic acid sequence set forth in SEQ ID NO:33, SEQ ID NO:45, or SEQ ID NO:59, respectively, and the signaling domain of CD3-zeta comprises the nucleic acid sequence set forth in SEQ ID NO:35, SEQ ID NO:47, or SEQ ID NO:61, respectively.

[0207] In one embodiment, the intracellular domain in the CAR is designed to comprise the signaling domain of 4-1BB and the signaling domain of CD3-zeta, wherein the signaling domain of 4-1BB comprises 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-zeta comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:36, SEQ ID NO:48, or SEQ ID NO:62.

[0208] In one embodiment, the intracellular domain in the CAR is designed to comprise the signaling domain of 4-1BB and the signaling domain of CD3-zeta, wherein the signaling domain of 4-1BB comprises the amino acid sequence set forth in SEQ ID NO:34, SEQ ID NO:46, or SEQ ID NO:60, respectively, and the signaling domain of CD3-zeta comprises the amino acid sequence set forth in SEQ ID NO:36, SEQ ID NO:48, or SEQ ID NO:62, respectively.

[0209] In one embodiment, the intracellular domain in the CAR is designed to comprise the signaling domain of CD28 and the signaling domain of CD3-zeta, wherein the signaling domain of CD28 comprises the nucleic acid sequence set forth in SEQ ID NO:45 or SEQ ID NO:59, respectively, and the signaling domain of CD3-zeta comprises the nucleic acid sequence set forth in SEQ ID NO:35, SEQ ID NO:47, or SEQ ID NO:61, respectively.

[0210] In one embodiment, the intracellular domain in the CAR is designed to comprise the signaling domain of CD28 and the signaling domain of CD3-zeta, 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-zeta 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.

[0211] In one embodiment, the intracellular domain in the CAR is designed to comprise the signaling domain of CD28 and the signaling domain of CD3-zeta, wherein the signaling domain of CD28 comprises the amino acid sequence set forth in SEQ ID NO:46 or SEQ ID NO:60, respectively, and the signaling domain of CD3-zeta comprises the amino acid sequence set forth in SEQ ID NO:36, SEQ ID NO:48, or SEQ ID NO:62, respectively.

[0212] 5. Further description of CAR The functional portion of the CAR disclosed herein is also expressly included within the scope of the present invention. The term "functional portion", when used in reference to a CAR, refers to any one or more parts or fragments of the CAR disclosed herein, which part or fragment retains the biological activity of the CAR (parent CAR) of which it is a part. A functional portion includes, for example, a part of a CAR that retains the ability to recognize target cells or detect, treat or prevent disease to a similar degree, the same degree, or a higher degree than the parent CAR. With respect to a parent CAR, a functional portion can, for example, comprise about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95% or more of the parent CAR.

[0213] A functional portion can include additional amino acids at the amino or carboxy terminus of the portion, or at both termini, that are not found in the amino acid sequence of the parent CAR. Desirably, the additional amino acids are involved in, for example, target cell recognition. and do not interfere with the biological function of the functional moiety, such as detecting, treating, or preventing cancer. More desirably, the additional amino acids enhance the biological activity compared to the biological activity of the parent CAR.

[0214] The functional variants of the CAR disclosed herein are included within the scope of this disclosure.The term "functional variant" as used herein refers to a CAR, polypeptide or protein that has substantial or significant sequence identity or similarity with the parent CAR, and this functional variant retains the biological activity of the CAR it is a variant of.Functional variants include, for example, variants of the CAR (parent CAR) described herein that retain the ability to recognize target cells to a similar degree, the same degree, or a higher degree than the parent CAR.With respect to the parent CAR, functional variants can be, for example, at least about 30%, 50%, 75%, 80%, 90%, 98% or more identical in amino acid sequence to the parent CAR.

[0215] A functional variant can, for example, comprise the amino acid sequence of a parent CAR with at least one conservative amino acid substitution. Alternatively, or in addition, a functional variant can comprise the amino acid sequence of a parent CAR with at least one non-conservative amino acid substitution. In this case, it is preferred that the non-conservative amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. The non-conservative amino acid substitution can enhance the biological activity of the functional variant, so that the biological activity of the functional variant is increased compared to the parent CAR.

[0216] The amino acid substitutions in the CAR are preferably conservative amino acid substitutions. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid with certain physical and / or chemical properties is replaced with another amino acid with the same or similar chemical or physical properties. For example, a conservative amino acid substitution can be an acidic / negatively charged polar amino acid substituting another acidic / negatively charged polar amino acid (e.g., Asp or Glu), an amino acid having a nonpolar side chain substituting another amino acid having a nonpolar side chain (e.g., Ala, Gly, Val, He, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), a basic / positively charged polar amino acid substituting another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.), an uncharged amino acid with a polar side chain substituting another uncharged amino acid with a polar side chain (e.g., Asn, Gin, Ser, Thr, Tyr, etc.), an amino acid with a beta-branched side chain substituting another amino acid with a beta-branched side chain (e.g., He, Thr, and Val), an amino acid with an aromatic side chain substituting another amino acid with an aromatic side chain (e.g., His, Phe, Trp, and Tyr), etc.

[0217] A CAR can consist essentially of the specified amino acid sequence(s) described herein, such that other components, e.g., other amino acids, do not significantly alter the biological activity of the functional variant.

[0218] CARs (including functional portions and functional variants) can be of any length, i.e., comprise any number of amino acids, provided that the CAR (or functional portion or variant thereof) retains its biological activity, e.g., the ability to specifically bind to an antigen, detect diseased cells in a mammal, or treat or prevent a disease in a mammal, etc. For example, a CAR can be about 50 to about 5,000 amino acids in length, e.g., 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1,000 or more amino acids in length.

[0219] CARs (including functional portions and functional variants of the invention) can contain synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids can be used to These include, for example, aminocyclohexanecarboxylic acid, norleucine, -amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3-hydroxyproline and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydrobenzoylalanine, α-isopropyl ... Examples of suitable hydroxybenzoates include 2-amino-2-methyl-2-propanol, ...

[0220] CARs (including functional portions and functional variants) can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized, e.g., via disulfide bridges, or converted into acid addition salts and / or optionally dimerized or polymerized, or conjugated.

[0221] CAR (including its functional part and functional variant) can be obtained by methods known in the art.CAR can be produced by any suitable method of producing polypeptide or protein.Suitable methods for de novo synthesis of polypeptide and protein are described in references such as Chan et al., Fmoc Solid Phase Peptide Synthesis, Oxford University Press, Oxford, United Kingdom, 2000; Peptide and Protein Drug Analysis, Reid, R. (ed.), Marcel Dekker, Inc., 2000; Epitope Mapping, Westwood et al. (ed.), Oxford University Press, Oxford, United Kingdom, 2001; and U.S. Patent No. 5,449,752.In addition, polypeptide and protein can be produced recombinantly using the nucleic acid described herein by using standard recombinant methods. For example, see Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. Furthermore, some CARs (including functional parts and functional variants thereof) can be isolated and / or purified from sources such as plants, bacteria, insects, mammals, for example, rats, humans, etc. Isolation and purification methods are well known in the art. Alternatively, the CARs described herein (including functional parts and functional variants thereof) can be commercially synthesized by companies. In this regard, CARs can be synthetic, recombinant, isolated, and / or purified.

[0222] B. Antibodies and Antigen-Binding Fragments One embodiment further provides a CAR, a T cell expressing a CAR, an antibody that specifically binds to one or more of the antigens disclosed herein, or an antigen-binding domain or portion thereof. As used herein, "a T cell expressing a CAR" or "CAR T cell" refers to a T cell that expresses a CAR, e.g., has antigen specificity determined by the antibody-derived targeting domain of the CAR.

[0223] As used herein, an "antigen-binding domain" can include antibodies and antigen-binding fragments thereof. The term "antibody" is used herein in the broadest sense and encompasses various antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antigen-binding fragments thereof, so long as they exhibit the desired antigen-binding activity. Non-limiting examples of antibodies include, for example, intact immunoglobulins known in the art, as well as variants and fragments thereof that retain binding affinity to antigens.

[0224] A "monoclonal antibody" is an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and directed against a single antigenic epitope. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. In some examples, a monoclonal antibody is an antibody produced by a single clone of B lymphocytes, or an antibody produced by a cell transfected with nucleic acid encoding the antibody light and heavy chain variable regions of a single antibody (or antigen-binding fragment thereof), or its progeny. In some examples, a monoclonal antibody is isolated from a subject. A monoclonal antibody may have conservative amino acid substitutions that have substantially no effect on antigen binding or other immunoglobulin functions. Exemplary methods for producing monoclonal antibodies are known, see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual, 2nd ed. Cold Spring Harbor Publications, New York (2013).

[0225] Typically, immunoglobulins have heavy (H) chains and light (L) chains interconnected by disulfide bonds. Immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable domain genes. There are two types of light chains, lambda (λ) and kappa (κ). There are five major heavy chain classes (or isotypes): IgM, IgD, IgG, IgA, and IgE, which determine the functional activity of antibody molecules.

[0226] Each heavy and light chain contains a constant region (or constant domain) and a variable region (or variable domain; see, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p. 91 (2007)). In some embodiments, the variable regions of the heavy and light chains combine to specifically bind to an antigen. In further embodiments, only the heavy chain variable region is required. For example, naturally occurring camelid antibodies consisting only of heavy chains are functional and stable in the absence of light chains. (See, e.g., Hamers-Casterman et al., Nature, 363:446-448, 1993; Sheriff et al., Nat. Struct. Biol., 3:733-736, 1996). References to "VH" or "VH" refer to the variable region of an antibody heavy chain, including antigen-binding fragments such as Fv, scFv, dsFv, or Fab. References to "VL" or "VL" refer to the variable domain of an antibody light chain, including those of Fv, scFv, dsFv, or Fab.

[0227] The variable regions of the light and heavy chains contain a "framework" region interrupted by three hypervariable regions, also called "complementarity-determining regions" or "CDRs" (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, USDapartment of Health and Human Services, 1991). The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, which is the combined framework regions of the constituent light and heavy chains, is arranged in three-dimensional space. It functions to position and align the CDRs.

[0228] CDRs are primarily responsible for binding to an epitope of an antigen. The amino acid sequence boundaries of a given CDR can be determined by the methods described by Kabat et al. ("Sequences of Proteins of Immunological Interest", 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991; "Kabat" numbering scheme), Al-Lazikani et al. (JMB 273, 927-948, 1997; "Chothia" numbering scheme), and Lefranc et al. ("IMGT unique numbering scheme"). for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev. Comp. Immunol., 27:55-77, 2003; "IMGT" numbering scheme). The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3 (N- to C-terminus) and are also typically identified by the chain in which a particular CDR is located. Thus, a VH CDR3 is the CDR3 from the variable domain of the heavy chain of the antibody in which it is found, while a VL CDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found. Light chain CDRs are sometimes referred to as LCDR1, LCDR2, and LCDR3. Heavy chain CDRs are sometimes referred to as HCDR1, HCDR2, and HCDR3.

[0229] "Antigen-binding fragments" are portions of full-length antibodies that retain the ability to specifically recognize their cognate antigen, as well as various combinations of such portions. Non-limiting examples of antigen-binding fragments include Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. Antibody fragments include antigen-binding fragments produced by engineering whole antibodies or those synthesized de novo using recombinant DNA methodologies (see, e.g., Kontermann and Dubel (eds.), Antibody Engineering, Vols. 1-2, 2nd Edition, Springer Press, 2010).

[0230] Single-chain antibodies (scFv) are genetically engineered molecules containing the VH and VL domains of one or more antibodies (or antibodies) linked by a suitable polypeptide linker as a genetically fused single-chain molecule (see, e.g., Bird et al., Science, 242:423-426, 1988; Huston et al., Proc. Natl. Acad. Sci., 85:5879-5883, 1988; Ahmad et al., Clin. Dev. Immunol., 2012, doi:10.1155 / 2012 / 980250; Marbry, IDrugs, 13:543-549, 2010). The intramolecular orientation of the VH and VL domains in scFvs is typically not critical for scFvs. Thus, scFvs with both possible configurations (VH domain-linker domain-VL domain; VL domain-linker domain-VH domain) can be used.

[0231] In dsFv, the heavy and light variable chains are mutated to introduce disulfide bonds to stabilize the association of the chains. Also included are diabodies, which are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain but use a linker that is too short to allow pairing between the two domains on the same chain, thereby pairing the domains with complementary domains on another chain and creating two antigen-binding sites (e.g., Holliger et al., Proc. Natl. Acad. Sci., 90:6444-6448, 1993; Poljak et al., Structure and Biology, 1999). , Vol. 2: 1121-1123, 1994).

[0232] Antibodies also include genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies) and heteroconjugate antibodies (e.g., bispecific antibodies). See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rd ed., W.H. Freeman & Co., New York, 1997.

[0233] Non-naturally occurring antibodies can be constructed using solid-phase peptide synthesis, can be produced recombinantly, or can be obtained by screening combinatorial libraries consisting of variable heavy and variable light chains, as described, for example, in Huse et al., Science 246:1275-1281 (1989), which is incorporated herein by reference. These and other methods of generating, for example, chimeric, humanized, CDR-grafted, single-chain, and bifunctional antibodies are well known to those of skill in the art (Winter and Harris, Immunol. Today 14:243-246 (1993); Ward et al., Nature 14:243-246 (1993)). 341:544-546 (1989); Harlow and Lane, supra, 1988; Hilyard et al., Protein Engineering: A practical approach (IRL Press 1992); Borrabeck, Antibody Engineering, 2nd Edition (Oxford University Press 1995); each of which is incorporated herein by reference.

[0234] An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competition assay; conversely, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competition assay. Antibody competition assays are known, and exemplary competition assays are provided herein.

[0235] A "humanized" antibody or antigen-binding fragment comprises a human framework region and one or more CDRs derived from a non-human (e.g., mouse, rat, or synthetic) antibody or antigen-binding fragment. The non-human antibody or antigen-binding fragment providing the CDRs is referred to as the "donor," and the human antibody or antigen-binding fragment providing the framework is referred to as the "acceptor." In one embodiment, all CDRs are derived from the donor immunoglobulin in the humanized immunoglobulin. Constant regions need not be present, but if present, can be substantially identical to human immunoglobulin constant regions, e.g., at least about 85-90%, e.g., about 95% or more identical. Thus, all parts of a humanized antibody or antigen-binding fragment, except possibly for the CDRs, are substantially identical to the corresponding parts of a natural human antibody sequence.

[0236] A "chimeric antibody" is an antibody that contains sequences from two different antibodies, typically from different species. In some instances, a chimeric antibody contains one or more CDRs and / or framework regions from one human antibody and CDRs and / or framework regions from another human antibody.

[0237] A "fully human antibody" or "human antibody" is an antibody that contains sequences derived from the human genome (or derived from the human genome) but does not contain sequences derived from another species. In some embodiments, a human antibody contains CDRs, framework regions, and (if present) an Fc region derived from the human genome (or derived from the human genome). Human antibodies can be identified and isolated using technology to create sequences based on sequences derived from the human genome, for example, by phage display, or using transgenic animals (see, e.g., Barbas et al., Phage display: A Laboratory Manual). uel. 1st ed. New York: Cold Spring Harbor Laboratory Press, 2004. Print.; Lonberg, Nat. Biotech., 23:1117-1125, 2005; Lonenberg, Curr. Opin. Immunol., 20:450-459, 2008).

[0238] An antibody can have one or more binding sites. If more than one binding site is present, these binding sites may be identical to one another or different. For example, naturally occurring immunoglobulins have two identical binding sites, single-chain antibodies or Fab fragments have one binding site, while bispecific or bifunctional antibodies have two different binding sites.

[0239] Methods for testing antibodies for the ability to bind to any functional portion of a CAR are known in the art and include any antibody-antigen binding assay, such as radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation, and competitive inhibition assays (see, e.g., Janeway et al., infra, U.S. Patent Application Publication No. 2002 / 0197266 A1, and U.S. Patent No. 7,338,929).

[0240] CARs, T cells expressing CARs, antibodies or antigen-binding portions thereof can also be modified to include detectable labels, such as radioisotopes, fluorophores (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), enzymes (e.g., alkaline phosphatase, horseradish peroxidase), and elemental particles (e.g., gold particles).

[0241] C. Conjugate CARs, T cells expressing CARs, or monoclonal antibodies or antigen-binding fragments thereof specific for one or more of the antigens disclosed herein can be conjugated to agents such as effector molecules or detectable markers using several means known to those skilled in the art. Both covalent and non-covalent binding means can be used. Conjugates include, but are not limited to, molecules in which an effector molecule or detectable marker is covalently linked to an antibody or antigen-binding fragment that specifically binds to one or more of the antigens disclosed herein. Those skilled in the art will recognize that the conjugation of an effector molecule or detectable marker to an antibody or antigen-binding fragment that specifically binds to one or more of the antigens disclosed herein can be used to conjugate an effector molecule or detectable marker to an antibody or antigen-binding fragment that specifically binds to one or more of the antigens disclosed herein. 125 I, 32 P, 14 C. 3 H and 35 It will be understood that a variety of effector molecules and detectable markers may be used, including, but not limited to, S, as well as other labels, targeting moieties, ligands, and the like.

[0242] The choice of a particular effector molecule or detectable marker will depend on the particular target molecule or cell and the desired biological effect. Thus, for example, the effector molecule can be a cytotoxin used to bring about the death of a particular target cell (e.g., a tumor cell).

[0243] The procedure for attaching an effector molecule or detectable marker to an antibody or antigen-binding fragment varies according to the chemical structure of the effector. Polypeptides typically contain various functional groups, such as carboxylic acid (COOH), free amine (-NH2), or sulfhydryl (-SH) groups, which are available for reaction with suitable functional groups on an antibody to result in attachment of an effector molecule or detectable marker. Alternatively, the antibody or antigen-binding fragment is derivatized to expose or attach additional reactive functional groups. Derivatization can include attachment of any of several known linker molecules, such as those available from Pierce Chemical Company, Rockford, IL. Linkers connect the antibody or antigen-binding fragment to the effector molecule or detectable marker. The linker may be any molecule used to bind to a detectable marker. The linker is capable of forming a covalent bond to both the antibody or antigen-binding fragment and the effector molecule or detectable marker. Suitable linkers are well known to those skilled in the art, and include, but are not limited to, straight-chain or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. When the antibody or antigen-binding fragment and the effector molecule or detectable marker are polypeptides, the linkers may be attached to the constituent amino acids via their side groups (for example, via a disulfide linkage to cysteine) or to the amino and carboxyl groups of the alpha carbon of the terminal amino acid.

[0244] In some embodiments, the linker may include a spacer element, which, if present, increases the size of the linker, thereby increasing the distance between the effector molecule or detectable marker and the antibody or antigen-binding fragment. Exemplary spacers are known to those of skill in the art, including those described in U.S. Patent Nos. 7,964,566, 7,498,298, 6,884,869, 6,323,315, 6,239,104, 6,034,065, 5,780,588, 5,665,860, 5,663,149, 5,635,483, 5,599,902, 5,554,725, 5,530,097, 5,521 Nos. 5,284, 5,504,191, 5,410,024, 5,138,036, 5,076,973, 4,986,988, 4,978,744, 4,879,278, 4,816,444 and 4,486,414, as well as those listed in U.S. Patent Application Publication Nos. 20110212088 and 20110070248, each of which is incorporated herein by reference in its entirety.

[0245] In some embodiments, the linker is cleavable under intracellular conditions, such that cleavage of the linker releases the effector molecule or detectable marker from the antibody or antigen-binding fragment in the intracellular environment. In yet other embodiments, the linker is non-cleavable, and the effector molecule or detectable marker is released, for example, by antibody degradation. In some embodiments, the linker is cleavable by a cleaving agent present in the intracellular environment (e.g., within a lysosome, endosome, or caveolae). The linker can be, for example, a peptide linker that is cleaved by an intracellular peptidase or protease enzyme, including, but not limited to, a lysosomal or endosomal protease. In some embodiments, the peptide linker is at least two amino acids long or at least three amino acids long. However, the linker can also be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids long, e.g., 1-2, 1-3, 2-5, 3-10, 3-15, 1-5, 1-10, or 1-15 amino acids long. Proteases can include cathepsins B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives, resulting in the release of active drugs inside target cells (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). For example, a peptide linker cleavable by the thiol-dependent protease cathepsin B can be used (e.g., a phenylalanine-leucine or glycine-phenylalanine-leucine-glycine linker). Other examples of such linkers are described, for example, in U.S. Pat. No. 6,214,345, which is incorporated herein by reference. In a specific embodiment, the peptide linker cleavable by intracellular proteases is a valine-citrulline linker or a phenylalanine-lysine linker (see, e.g., U.S. Pat. No. 6,214,345, which describes the synthesis of doxorubicin using a valine-citrulline linker).

[0246] In other embodiments, the cleavable linker is pH sensitive, i.e., it reacts with the cleavable linker at certain pH values. The pH-sensitive linker is sensitive to hydrolysis under acidic conditions. Typically, the pH-sensitive linker is hydrolyzable under acidic conditions. For example, an acid-labile linker (e.g., hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal, etc.) that is hydrolyzable in the lysosome can be used (see, e.g., U.S. Pat. No. 5,122,368; U.S. Pat. No. 5,824,805; U.S. Pat. No. 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661). Such linkers are relatively stable under neutral pH conditions, such as in blood, but are unstable at pHs below 5.5 or 5.0, which is the approximate pH of the lysosome. In certain embodiments, the hydrolyzable linker is a thioether linker (such as a thioether attached to a therapeutic agent via an acylhydrazone bond) (see, eg, US Pat. No. 5,622,929).

[0247] In other embodiments, the linker is cleavable under reducing conditions (eg, a disulfide linker). A variety of disulfide linkers are known in the art, including, for example, those that can be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene)-, SPDB, and SMPT (see, e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (C.W. Vogel, ed., Oxford University Press, 1987); Phillips et al., Cancer Res. 68:9280-9290, 2008. See also U.S. Patent No. 4,880,935.

[0248] In yet other specific embodiments, the linker is a malonate linker (Johnson et al., 1995, Anticancer Res. 15:1387-93), a maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1299-1304), or a 3'-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1305-12).

[0249] In still other embodiments, the linker is non-cleavable and the effector molecule or detectable marker is released by antibody degradation (see U.S. Patent Application Publication No. 2005 / 0238649, the entire contents of which are incorporated herein by reference).

[0250] In some embodiments, the linker is resistant to cleavage in an extracellular environment. For example, when the conjugate is present in an extracellular environment (e.g., in plasma), about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 3% or less, or about 1% or less of the linkers in a sample of the conjugate are cleaved. Whether a linker is resistant to cleavage in an extracellular environment can be determined, for example, by incubating the conjugate containing the desired linker with plasma for a predetermined period (e.g., 2, 4, 8, 16, or 24 hours), and then quantifying the amount of free effector molecule or detectable marker present in the plasma. Various exemplary linkers that can be used in the conjugates are described in WO2004-010957, U.S. Patent Application Publication No. 2006 / 0074008, U.S. Patent Application Publication No. 20050238649, and U.S. Patent Application Publication No. 2006 / 0024317, each of which is incorporated herein by reference in its entirety. It can be enjoyed.

[0251] In some embodiments, conjugates of a CAR, a T cell expressing a CAR, an antibody or antigen-binding portion thereof, and one or more small molecule toxins, such as calicheamicin, maytansinoids, dolastatins, auristatins, trichothecines, and CC1065, and derivatives of these toxins that have toxin activity, are provided.

[0252] Maytansine compounds suitable for use as maytansinoid toxin moieties are well known in the art and can be isolated from natural sources according to known methods, produced using genetic engineering techniques (see Yu et al. (2002) PNAS 99:7968-7973), or synthetically prepared maytansinol and maytansinol analogs according to known methods. Maytansinoids are mitotic inhibitors that act by inhibiting tubulin polymerization. Maytansine was first isolated from the East African shrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it was discovered that certain microorganisms also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042). Synthetic maytansinol and its derivatives and analogs are described, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428; and 4,311,140. Nos. 3,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533, each of which is incorporated herein by reference. Maytansinoid-containing conjugates, methods for making same, and their therapeutic uses are disclosed, for example, in U.S. Patent Nos. 5,208,020; 5,416,064; 6,441,163 and European Patent EP 0 425 235 B1, the disclosures of which are expressly incorporated herein by reference.

[0253] Additional toxins can be used with CARs, CAR-expressing T cells, antibodies, or antigen-binding portions thereof. Exemplary toxins include Pseudomonas exotoxin (PE), ricinus toxin, abrin, diphtheria toxin and its subunits, ribotoxin, ribonuclease, saporin and calicheamicin, and botulinum toxins A-F. These toxins are well known in the art, and many are readily available from commercial sources (e.g., Sigma Chemical Company, St. Louis, MO). Contemplated toxins also include variants of these toxins (see, e.g., U.S. Patent Nos. 5,079,163 and 4,689,401).

[0254] Saporin is a toxin derived from Saponaria officinalis that disrupts protein synthesis by inactivating the 60S portion of the ribosomal complex (Stirpe et al., Bio / Technology, 10:405-412, 1992). However, this toxin does not have a mechanism for specific entry into cells and therefore requires conjugation to an antibody or antigen-binding fragment that recognizes an internalized cell surface protein in order to be efficiently taken up by cells.

[0255] Diphtheria toxin is isolated from Corynebacterium diphtheriae. Typically, diphtheria toxin for use in immunotoxins is mutated to reduce or eliminate nonspecific toxicity. A mutant known as CRM107, which has full enzymatic activity but significantly reduced nonspecific toxicity, has been known since the 1970s (Laird and Groman, J. Virol. 19:220, 1976) and has been used in human clinical trials. See U.S. Patent Nos. 5,792,458 and 5,208,021.

[0256] Castor toxin is the lectin RCA60 from Ricinus communis (castor bean). For examples of castor toxins, see U.S. Patent Nos. 5,079,163 and 4,689,401. Ricinus communis agglutinin (RCA) is a lectin derived from the plant Ricinus communis (castor bean), according to its molecular weight of approximately 65 kD and 120 kD, respectively. 60 and RCA 120 The toxin exists in two forms, termed the A chain and the B chain (Nicholson and Blaustein, J. Biochim. Biophys. Acta 266:543, 1972). The A chain is responsible for inactivating protein synthesis and killing the cell. The B chain binds the toxin to cell surface galactose residues and facilitates transport of the A chain into the cytosol (Olsnes et al., Nature 249:627-631, 1974 and U.S. Pat. No. 3,060,165).

[0257] Ribonucleases have also been conjugated to targeting molecules for use as immunotoxins (see Suzuki et al., Nat. Biotech. 17:265-70, 1999). Exemplary ribotoxins, such as α-sarcin and restrictocin, are discussed, for example, in Rathore et al., Gene 190:31-5, 1997; and Goyal and Batra, Biochem. 345, Part 2:247-54, 2000. Calicheamicin, originally isolated from Micromonospora echinospora, is a member of the enediyne antitumor antibiotic family, generating double-strand breaks in DNA that lead to apoptosis (see, for example, Lee et al., J. Antibiot. 42:1070-87, 1989). This drug is the toxic moiety of an immunotoxin in clinical trials (see, eg, Gillespie et al., Ann. Oncol. 11:735-41, 2000).

[0258] Abrin includes toxic lectins from Abrus precatorius. The toxicants, abrins a, b, c, and d, have molecular weights of approximately 63 kD and 67 kD and are composed of two disulfide-linked polypeptide chains, A and B. The A chain inhibits protein synthesis; the B chain (abrin-b) binds to D-galactose residues (see Funatsu et al., Agr. Biol. Chem. 52:1095, 1988; and Olsnes, Methods Enzymol. 50:330-335, 1978).

[0259] CARs, CAR-expressing T cells, monoclonal antibodies specific for one or more of the antigens disclosed herein, and antigen-binding fragments thereof can also be conjugated to detectable markers; for example, detectable by ELISA, spectrophotometry, flow cytometry, microscopy, or imaging techniques (e.g., computed tomography (CT), computed axial tomography (CAT) scan, magnetic resonance imaging (MRI), nuclear magnetic resonance imaging (NMRI), magnetic resonance tomography (MTR), ultrasound, fiber optic testing, and laparoscopic testing). Specific non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzymatic linkages, radioactive isotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI). For example, useful detectable markers include: Fluorescent compounds include fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, and the like. Bioluminescent markers, such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP), are also used. CARs, CAR-expressing T cells, antibodies, or antigen-binding portions thereof, can also be conjugated to enzymes useful for detection, such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, glucose oxidase, and the like. When a CAR, CAR-expressing T cells, antibodies, or antigen-binding portions thereof, is conjugated to a detectable enzyme, it can be detected by adding an additional reagent that the enzyme uses to produce a discernible reaction product. For example, when the agent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine results in a visually detectable colored reaction product. CARs, CAR-expressing T cells, antibodies, or antigen-binding portions thereof, can also be conjugated with biotin and detected via indirect measurement of avidin or streptavidin binding. Note that avidin itself can be conjugated with an enzyme or fluorescent label.

[0260] CAR, CAR-expressing T cells, antibodies, or their antigen-binding portions can be conjugated with paramagnetic agents such as gadolinium. Paramagnetic agents such as superparamagnetic iron oxide can also be used as labels. Antibodies can also be conjugated with lanthanides (e.g., europium and dysprosium) and manganese. Antibodies or antigen-binding fragments can also be labeled with a predetermined polypeptide epitope recognized by a secondary reporter (e.g., leucine zipper pair sequence, binding site for secondary antibody, metal binding domain, epitope tag).

[0261] CAR, CAR-expressing T cells, antibodies, or antigen-binding portions thereof can also be conjugated with radiolabeled amino acids.Radiolabels can be used for both diagnostic and therapeutic purposes.For example, radiolabels can be used to detect one or more of the antigens and antigen-expressing cells disclosed herein by x-ray, emission spectroscopy, or other diagnostic techniques.In addition, radiolabels can be used therapeutically as toxins for treating tumors in subjects, for example, for treating neuroblastoma.Examples of labels for polypeptides include, but are not limited to, the following radioisotopes or radionucleotides: 3 H, 14 C. 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I.

[0262] Means for detecting such detectable markers are well known to those skilled in the art. Thus, for example, radiolabels can be detected using photographic film or scintillation counters, fluorescent markers can be detected using a photodetector to detect emitted illumination, enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and chromogenic labels are detected by simply visualizing the colored label.

[0263] D. Nucleotides, Expression, Vectors and Host Cells Further provided by one embodiment of the present invention is a nucleic acid comprising a nucleotide sequence encoding any of the CARs, antibodies, or antigen-binding portions thereof described herein (including functional portions and functional variants thereof). The nucleic acids of the present invention may comprise a nucleotide sequence encoding any of the leader sequences, antigen-binding domains, transmembrane domains, and / or intracellular T cell signaling domains described herein.

[0264] In some embodiments, the nucleotide sequence may be codon-modified. Without being bound by theory, codon optimization of the nucleotide sequence increases the translation efficiency of the mRNA transcript. It is believed that codon optimization of a nucleotide sequence can involve replacing a native codon with another codon that encodes the same amino acid but can be translated by a more readily available tRNA in the cell, thereby increasing translation efficiency. Optimization of a nucleotide sequence can also reduce secondary mRNA structures that interfere with translation, thereby increasing translation efficiency.

[0265] In embodiments of the invention, a nucleic acid can comprise a codon-modified nucleotide sequence encoding the antigen-binding domain of a CAR of the invention. In another embodiment of the invention, a nucleic acid can comprise a codon-modified nucleotide sequence encoding any of the CARs described herein (including functional portions and functional variants thereof).

[0266] "Nucleic acid," as used herein, includes "polynucleotides," "oligonucleotides," and "nucleic acid molecules," and generally refers to a polymer of DNA or RNA that can be single- or double-stranded, synthetic or obtained from natural sources (e.g., isolated and / or purified), and can contain natural, non-natural, or modified nucleotides, and can contain natural, non-natural, or modified internucleotide linkages, e.g., phosphoramidate or phosphorothioate linkages, in place of the phosphodiesters found between nucleotides in unmodified oligonucleotides. In some embodiments, a nucleic acid does not contain any insertions, deletions, inversions, and / or substitutions. However, in some cases, as discussed herein, it may be appropriate for a nucleic acid to contain one or more insertions, deletions, inversions, and / or substitutions.

[0267] Recombinant nucleic acids can have sequences that do not occur in nature or that are created by the artificial combination of two otherwise separate segments of sequence. This artificial combination is often achieved by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acid, for example, by genetic engineering techniques such as those described in Sambrook et al., supra. Nucleic acids can be constructed based on chemical synthesis and / or enzymatic ligation reactions using procedures known in the art. See, for example, Sambrook et al., supra and Ausubel et al., supra. For example, nucleic acids can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides (e.g., phosphorothioate derivatives and acridine-substituted nucleotides) designed to increase the biological stability of the molecule or to increase the physical stability of the duplex formed upon hybridization.Examples of modified nucleotides that can be used to generate nucleic acids include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-substituted adenines, 7-methylguanine, and the like. Examples of nucleic acids include, but are not limited to, uracil-5-oxyacetic acid (v), ... It can be purchased from companies such as Technologies (Coralville, IA, USA).

[0268] The nucleic acid can comprise any isolated or purified nucleotide sequence encoding a CAR or any of its functional portions or variants. Alternatively, the nucleotide sequence can comprise a nucleotide sequence that is degenerate to any of the sequences, or a combination of degenerate sequences.

[0269] One embodiment also provides an isolated or purified nucleic acid comprising a nucleotide sequence that is complementary to or that hybridizes under stringent conditions to the nucleotide sequence of any of the nucleic acids described herein.

[0270] Nucleotide sequences that hybridize under stringent conditions may also hybridize under high stringency conditions. "High stringency conditions" means that a nucleotide sequence specifically hybridizes to a target sequence (any nucleotide sequence of a nucleic acid described herein) in an amount detectably stronger than nonspecific hybridization. High stringency conditions include conditions that distinguish polynucleotides with exact complementary sequences, or polynucleotides containing only a few scattered mismatches, from random sequences that happen to have several small regions (e.g., 3-10 bases) that match the nucleotide sequence. Such small regions of complementarity are more easily melted than full-length complements of 14-17 or more bases, and high stringency hybridization makes them readily distinguishable. Relatively high stringency conditions include low salt and / or high temperature conditions, such as those provided by about 0.02-0.1 M NaCl or equivalent at temperatures of about 50-70°C. Such highly stringent conditions tolerate little, if any, mismatch between the nucleotide sequence and the template or target strand and are particularly suitable for detecting expression of any of the CARs of the present invention. It is generally understood that conditions can be made more stringent by the addition of increasing amounts of formamide.

[0271] 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.

[0272] In one embodiment, the nucleic acid may be incorporated into a recombinant expression vector. In this regard, one embodiment provides a recombinant expression vector comprising any of the nucleic acids. For purposes of this specification, the term "recombinant expression vector" refers to a genetically engineered oligonucleotide or polynucleotide construct that allows a host cell to express an mRNA, protein, polypeptide, or peptide when the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide, and when the vector is contacted with a cell under conditions sufficient for the mRNA, protein, polypeptide, or peptide to be expressed in the cell. The vector as a whole does not exist in nature.

[0273] However, portions of the vector may be naturally occurring. Recombinant expression vectors may be single-stranded or double-stranded, synthetic or derived from partially natural sources, and may contain any type of nucleotide, including, but not limited to, DNA and RNA, which may contain natural, non-natural, or altered nucleotides. Recombinant expression vectors may contain naturally occurring or non-naturally occurring internucleotide linkages, or both types of linkages. Preferably, non-naturally occurring or altered nucleotides or internucleotide linkages are not present in the vector. It does not interfere with transcription or replication.

[0274] In one embodiment, the recombinant expression vector may be any suitable recombinant expression vector and may be used to transform or transfect any suitable host cell. Suitable vectors include those designed for propagation and propagation or for expression, or both, such as plasmids and viruses. The vector may be selected from the group consisting of the pUC series (Fermentas Life Sciences, Glen Burnie, MD), the pBluescript series (Stratagene, LaJolla, CA), the pET series (Novagen, Madison, WI), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, CA).

[0275] Bacteriophage vectors, such as λυTΙO, λυTΙ 1, λZapII (Stratagene), EMBL4, and λΝΜΙ 149, can also be used. Examples of plant expression vectors include pBI01, pBI101.2, pBHO1.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). The recombinant expression vector can be a viral vector, such as a retroviral or lentiviral vector. Lentiviral vectors are vectors derived from at least a portion of the lentiviral genome, including, in particular, self-inactivating lentiviral vectors such as those provided in Milone et al., Mol. Ther. 17(8):1453-1464 (2009). Other examples of lentiviral vectors that may be used in the clinic include, for example, but not limited to, Oxford BioMedica plc's LENTIVECTOR® gene delivery technology, Lentigen's LENTIMAX™ vector system, etc. Non-clinical forms of lentiviral vectors are also available and known to those skilled in the art.

[0276] Several transfection techniques are generally known in the art (see, e.g., Graham et al., Virology, 52:456-467 (1973); Sambrook et al., supra; Davis et al., Basic Methods in Molecular Biology, Elsevier (1986); and Chu et al., Gene, 13:97 (1981)).

[0277] Transfection methods include calcium phosphate coprecipitation (see, e.g., Graham et al., supra), direct microinjection into cultured cells (see, e.g., Capecchi, Cell 22:479-488 (1980)), electroporation (see, e.g., Shigekawa et al., BioTechniques 6:742-751 (1988)), liposome-mediated gene transfer (see, e.g., Mannino et al., BioTechniques 6:682-690 (1988)), lipid-mediated transduction (see, e.g., Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987)), and high velocity micropropellants. Microprojectile delivery (see, eg, Klein et al., Nature, 327:70-73 (1987)) is also included.

[0278] In one embodiment, recombinant expression vectors can be prepared using standard recombinant DNA techniques, for example, as described in Sambrook et al., supra, and Ausubel et al., supra. Circular or linear expression vector constructs can be prepared to contain a replication system functional in prokaryotic or eukaryotic host cells. Replication systems can be derived from, for example, ColEl, 2μ plasmid, λ, SV40, bovine papilloma virus, etc.

[0279] Recombinant expression vectors, taking into account whether the vector is DNA- or RNA-based, can optionally include regulatory sequences, e.g., transcriptional and translational initiation and termination codons, specific to the type of host cell (e.g., bacterial, fungal, plant, or animal) into which the vector will be introduced. Recombinant expression vectors can include restriction sites to facilitate cloning.

[0280] The recombinant expression vector may contain one or more marker genes to allow for the selection of transformed or transfected host cells. Marker genes include biocide resistance, resistance to, e.g., antibiotics, heavy metals, etc., complementation in auxotrophic hosts to provide prototrophy, etc. Suitable marker genes for the expression vectors of the present invention include, for example, the neomycin / G418 resistance gene, the hygromycin resistance gene, the histidinol resistance gene, the tetracycline resistance gene, and the ampicillin resistance gene.

[0281] The recombinant expression vector may comprise a native or non-native promoter operably linked to the nucleotide sequence encoding the CAR (including its functional portion and functional variant), or to a nucleotide sequence complementary to or hybridizing with the nucleotide sequence encoding the CAR. The selection of a promoter, for example, strong, weak, inducible, tissue-specific, and developmentally specific, is within the skill of those skilled in the art. Similarly, combining a nucleotide sequence with a promoter is also within the skill of those skilled in the art. The promoter may be a non-viral promoter or a viral promoter, such as a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, or a promoter found in the long terminal repeat of murine stem cell virus.

[0282] Recombinant expression vectors can be designed for transient expression, stable expression, or both, and can be made for constitutive or inducible expression.

[0283] Furthermore, recombinant expression vectors can be made to contain suicide genes. As used herein, the term "suicide gene" refers to a gene that causes cells that express the suicide gene to die. A suicide gene can be a gene that confers sensitivity to a drug or other agent on the cell in which the gene is expressed, or a gene that causes the cell to die when contacted with or exposed to a drug. Suicide genes are known in the art (see, for example, Suicide Gene Therapy: Methods and Reviews, Springer, Caroline J. (Cancer Research UK Centre for Cancer Therapeutics at the Institute of Cancer Research, Sutton, Surrey, UK), Humana Press, 2004), and include, for example, herpes simplex virus (HSV) thymidine kinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase, and nitroreductase.

[0284] One embodiment further provides a host cell comprising any of the recombinant expression vectors described herein. As used herein, the term "host cell" refers to any type of cell that can contain a recombinant expression vector of the present invention. Host cells can be eukaryotic cells, such as plants, animals, fungi, or algae, or prokaryotic cells, such as bacteria or protists. Host cells can be cultured or primary cells, i.e., directly isolated from an organism, such as a human. Host cells can be adherent or suspension cells, i.e., cells that grow in suspension. Suitable host cells are known in the art and include, for example, DH5a E. coli cells, Chinese hamster ovary cells, monkey VERO cells, CO2 cells, and the like. The host cell may be a prokaryotic cell, such as a DH5a cell, for example, a S cell, a HEK293 cell, or the like. For the purpose of amplifying or replicating a 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. The host cell may be of any cell type, originate from any type of tissue, or be at any developmental stage, but 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.

[0285] For purposes herein, T cells can be any T cells, e.g., cultured T cells, e.g., primary T cells, or T cells from cultured T cell lines, e.g., Jurkat, SupTl, etc., or T cells obtained from a mammal. If obtained from a mammal, T cells can be obtained from a number of sources, including, but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. T cells can also be enriched or purified. T cells can be human T cells. T cells can be T cells isolated from a human. T cells can be CD4 + / CD8 + Double positive T cells, CD4 + Helper T cells, e.g., Th1 and Th2 cells, CD8 + The T cells can be of any type and at any stage of development, including, but not limited to, 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 + It may be a T cell.

[0286] In one embodiment, the CARs described herein can be used in suitable non-T cells, such as cells with immune effector function, such as NK cells and T-like cells generated from pluripotent stem cells.

[0287] Also provided by one embodiment is a population of cells comprising at least one host cell described herein. The population of cells can be a heterogeneous population comprising host cells comprising any of the described recombinant expression vectors in addition to at least one other cell, e.g., a host cell (e.g., a T cell), that does not comprise any recombinant expression vector, or a cell other than a T cell, e.g., a B cell, macrophage, neutrophil, erythrocyte, hepatocyte, endothelial cell, epithelial cell, muscle cell, brain cell, etc. Alternatively, the population of cells can be a substantially homogeneous population, wherein the population primarily comprises host cells comprising (e.g., consisting essentially of) the recombinant expression vector. The population can also be a clonal population of cells, wherein all cells in the population are clones of a single host cell comprising the recombinant expression vector, such that all cells in the population comprise that recombinant expression vector. In one embodiment of the present invention, the population of cells is a clonal population comprising host cells comprising the recombinant expression vector described herein.

[0288] CARs (including functional portions and variants thereof), nucleic acids, recombinant expression vectors, host cells (including populations thereof), and antibodies (including antigen-binding portions thereof) can be isolated and / or purified. For example, a purified (or isolated) host cell preparation is one in which the host cells are more pure than the cells in their natural environment in the body. Such host cells can be produced, for example, by standard purification techniques. In some embodiments, a host cell preparation is purified such that the host cells represent at least about 50%, e.g., at least about 70%, of the total cell content of the preparation. For example, the purity can be at least about 50%, greater than about 60%, about 70%, or about 80%, or can be about 100%.

[0289] E. Treatment Method It is contemplated that the CARs disclosed herein can be used in methods for treating or preventing disease in a mammal. In this regard, one embodiment relates to a method for treating or preventing cancer in a mammal, comprising administering to the mammal a CAR, a nucleic acid, a recombinant expression vector, a host cell, a population of cells, an antibody and / or an antigen-binding portion thereof, and / or a pharmaceutical composition in an amount effective to treat or prevent cancer in the mammal. to provide.

[0290] One embodiment further comprises lymphodepleting the mammal prior to administering a CAR disclosed herein. Examples of lymphodepletion can include, but are not limited to, non-myeloablative lymphodepleting chemotherapy, myeloablative lymphodepleting chemotherapy, total body irradiation, etc.

[0291] For the purpose of the method of administering host cells or a group of cells, the cells can be allogeneic or autologous to the mammal.Preferably, the cells are autologous to the mammal.As used herein, allogeneic refers to any material derived from a different animal of the same species as the individual into which the material is introduced.Two or more individuals are said to be allogeneic to each other if the genes are not identical at one or more loci.In some embodiments, allogeneic materials from individuals of the same species can be genetically sufficiently different to interact antigenically.As used herein, "autologous" refers to any material derived from the same individual that is subsequently reintroduced into the individual.

[0292] The mammal referred to herein may be any mammal. As used herein, the term "mammal" refers to any mammal, including, but not limited to, mammals of the order Rodentia, e.g., mice and hamsters, and mammals of the order Lagomorpha, e.g., rabbits. The mammal may be from the order Carnivora, including Feline (cat) and Canine (dog). The mammal may be from the order Artiodactyla, including Bovine (cow) and Swine (pig), or from the order Perissodactyla, including Equine (horse). The mammal may be from the order Primates, Ceboids, or Simoids (monkeys), or Anthropoids (humans and apes). Preferably, the mammal is a human.

[0293] For these methods, the cancer may be acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer (e.g., bladder carcinoma), bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, cancer of the anus, anal canal, or anorectum, eye cancer, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid carcinoma The cancer may be any cancer, including any of the following: colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, head and neck cancer (e.g., head and neck squamous cell carcinoma), Hodgkin's lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, leukemia, liquid tumors, liver cancer, lung cancer (e.g., non-small cell lung cancer and lung adenocarcinoma), lymphoma, mesothelioma, mast cell tumor, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin's lymphoma, B-chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia (ALL) and Burkitt's lymphoma, ovarian cancer, pancreatic cancer, peritoneal, omental and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumor, synovial sarcoma, gastric cancer, testicular cancer, thyroid cancer, and ureteral cancer.

[0294] The terms "treat" and "prevent," and words derived therefrom, as used herein, do not necessarily mean 100% or complete treatment or prevention. Rather, there are various degrees of treatment or prevention that one of skill in the art would recognize as having a potential beneficial or therapeutic effect. In this regard, the method may provide any amount or level of treatment or prevention of cancer in a mammal.

[0295] Furthermore, the treatment or prevention provided by this method can include treatment or prevention of one or more conditions or symptoms of the disease, such as cancer, being treated or prevented. For purposes of this specification, "prevention" can include delaying the onset of the disease, or its symptoms or conditions.

[0296] Another embodiment provides a method of detecting the presence of cancer in a mammal, the method comprising: (a) contacting a sample comprising one or more cells from the mammal with a CAR, a nucleic acid, a recombinant expression vector, a host cell, a population of cells, an antibody and / or antigen-binding portion thereof, or a pharmaceutical composition, thereby forming a complex; and (b) detecting the complex, wherein detection of the complex indicates the presence of cancer in the mammal.

[0297] The sample can be obtained by any suitable method, for example, biopsy or autopsy.Biopsy is the removal of tissue and / or cells from an individual.Such removal can be the collection of tissue and / or cells from an individual in order to perform experiments on the removed tissue and / or cells.This experiment can include experiments to determine whether the individual has a certain condition or disease state and / or whether they are suffering from a certain condition or disease state.The condition or disease can be, for example, cancer.

[0298] For embodiments of methods for detecting the presence of a proliferation disorder, e.g., cancer, in a mammal, the sample containing mammalian cells can be a sample containing whole cells, a lysate thereof, or a fraction of a whole cell lysate, e.g., a nuclear or cytoplasmic fraction, a whole protein fraction, or a nucleic acid fraction. When the sample contains whole cells, these cells can be any cells of a mammal, e.g., cells of any organ or tissue, including blood cells or endothelial cells.

[0299] The contacting step can occur in vitro or in vivo with respect to a mammal. Preferably, the contacting step is in vitro.

[0300] Also, detection of complexes can be carried out by many methods known in the art.For example, the CAR disclosed herein, polypeptide, protein, nucleic acid, recombinant expression vector, host cell, cell population, or antibody or its antigen-binding portion can be labeled with detectable label, such as the radioisotope disclosed above, fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), enzyme (e.g., alkaline phosphatase, horseradish peroxidase) and element particle (e.g., gold particle), etc.

[0301] The method of testing CAR for its ability to recognize target cells and antigen specificity is known in the art.For example, Clay et al., J.Immunol, vol. 163: 507-513 (1999) teaches a method for measuring the release of cytokines (e.g., interferon-γ, granulocyte / monocyte colony stimulating factor (GM-CSF), tumor necrosis factor α (TNF-α) or interleukin 2 (IL-2)).In addition, CAR function can be evaluated by measuring cytotoxicity, as described in Zhao et al., J.Immunol. vol. 174: 4415-4423 (2005).

[0302] Another embodiment provides the use of the CARs, nucleic acids, recombinant expression vectors, host cells, populations of cells, antibodies or antigen-binding portions thereof, and / or pharmaceutical compositions of the invention to treat or prevent a proliferative disorder, such as cancer, in a mammal. The cancer can be any of the cancers described herein.

[0303] Any method of administration can be used for the disclosed therapeutic agents, including local and systemic administration, for example, topical, oral, intravascular such as intravenous, intramuscular, intraperitoneal, intranasal, intradermal, intrathecal. Intracutaneous and subcutaneous administration may be used. The specific mode of administration and dosing regimen will be selected by the attending clinician, taking into account the characteristics of the case (e.g., the subject, the disease, the disease state involved, and whether the treatment is prophylactic). When more than one agent or composition is being administered, one or more administration routes may be used; for example, a chemotherapeutic agent may be administered orally, and an antibody or antigen-binding fragment or conjugate or composition may be administered intravenously. Administration methods include injection, in which the CAR, CAR T cell, conjugate, antibody, antigen-binding fragment, or composition is provided in a non-toxic pharmaceutically acceptable carrier, such as water, saline, Ringer's solution, dextrose solution, 5% human serum albumin, solid oil, ethyl oleate, or liposomes. In some embodiments, local administration of the disclosed compounds may be used, for example, by applying the antibody or antigen-binding fragment to an area of ​​tissue from which a tumor has been removed or to an area suspected of being prone to tumor development. In some embodiments, sustained intratumoral (or near-tumoral) release of a pharmaceutical preparation containing a therapeutically effective amount of an antibody or antigen-binding fragment may be beneficial. In other examples, the conjugate is applied topically as eye drops to the cornea or intravitreally to the eye.

[0304] The disclosed therapeutic agents can be formulated in unit dosage forms suitable for individual administration of precise dosage amounts. Furthermore, the disclosed therapeutic agents can be administered in a single dose or in a multiple-dose schedule. A multiple-dose schedule is one in which the main course of treatment may involve more than one discrete dose, e.g., 1 to 10 doses, followed by other doses given at subsequent time intervals as needed to maintain or enhance the effect of the composition. Treatment may involve a daily dose or multiple daily doses of the compound(s) over a period of several days to several months or even years. Thus, the dosing regime will also be determined, at least in part, based on the specific needs of the subject being treated and will be dependent on the judgment of the administering practitioner.

[0305] Typical dosages of antibodies or conjugates can range from about 0.01 to about 30 mg / kg, for example, from about 0.1 to about 10 mg / kg.

[0306] In particular examples, the subject is administered a therapeutic composition comprising one or more of the conjugate, antibody, composition, CAR, CAR T cell, or additional agent in a multiple daily dosing schedule, e.g., at least 2 consecutive days, 10 consecutive days, etc., for a period of, e.g., weeks, months, or years. In one example, the subject is administered the conjugate, antibody, composition, or additional agent for a period of at least 30 days, e.g., at least 2 months, at least 4 months, at least 6 months, at least 12 months, at least 24 months, or at least 36 months.

[0307] In some embodiments, the disclosed methods include providing a subject with surgery, radiation therapy, and / or chemotherapy in combination with the disclosed antibodies, antigen-binding fragments, conjugates, CARs, or CAR-expressing T cells (e.g., sequentially, substantially simultaneously, or simultaneously). Such agents and treatment methods and therapeutic dosages are known to those skilled in the art and can be determined by a skilled clinician. Preparation and dosing schedules for additional agents can be used according to manufacturer's instructions or can be as empirically determined by one skilled in the art. Preparation and dosing schedules for such chemotherapy are also described in Chemotherapy Service, (1992) MC Perry (ed.), Williams & Wilkins, Baltimore, Md.

[0308] In some embodiments, the combination therapy may include administering to the subject a therapeutically effective amount of an additional cancer inhibitor. Non-limiting examples of additional therapeutic agents that can be used in the combination therapy include microtubule binding agents, DNA intercalators or crosslinkers, DNA synthesis inhibitors, DNA and RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, gene regulators, and angiogenesis inhibitors. These agents (administered in therapeutically effective amounts) and treatments may be administered alone or in combination. For example, any suitable anti-cancer or anti-angiogenic agent can be administered in combination with the CAR, CAR-T cell, antibody, antigen-binding fragment, or conjugate disclosed herein. Methods and therapeutic dosages of such agents are known to those skilled in the art and can be determined by a skilled clinician.

[0309] Additional chemotherapeutic agents include alkylating agents such as nitrogen mustards (e.g., chlorambucil, chlormethine, cyclophosphamide, ifosfamide, and melphalan), nitrosoureas (e.g., carmustine, fotemustine, lomustine, and streptozocin), platinum compounds (e.g., carboplatin, cisplatin, oxaliplatin, and BBR3464), busulfan, dacarbazine, mechlorethamine, procarbazine, temozolomide, thiotepa, and uramustine; antimetabolites such as folates (e.g., methotrexate, pemetrexed, and raltitrexed), purines (e.g., cladribine, clofarabine, fludarabine, mercaptopurine, and thioguanine), pyrimidines (e.g., capecitabine), cytarabine, fluorouracil, and gemcitabine; plant alkaloids, such as For example, podophyllum (e.g., etoposide and teniposide), taxanes (e.g., docetaxel and paclitaxel), vincas (e.g., vinblastine, vincristine, vindesine, and vinorelbine); cytotoxic / antitumor antibiotics, such as members of the anthracycline family (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, and valrubicin), bleomycin, rifampicin, hydroxyurea, and mitomycin; topoisomerase inhibitors, such as topotecan and irinotecan; monoclonal antibodies, such as alemtuzumab, bevacizumab, cetuximab, gemtuzumab, rituximab, panitumumab, pertuzumab, and trastuzumab; tumor-affinity photosensitizers, such as aminolevulinic acid, methyl aminolevulinate, porfimer sodium, and verteporfin;and other drugs, such as alitretinoin, altretamine, amsacrine, anagrelide, arsenic trioxide, asparaginase, axitinib, bexarotene, bevacizumab, bortezomib, celecoxib, denileukin diftitox, erlotinib, estramustine, gefitinib, hydroxycarbamide, imatinib, lapatinib, pazopanib, pentostatin, masoprocol, mitotane, pegaspargase, tamoxifen, sorafenib, sunitinib, vemurafenib, vandetanib, and tretinoin. The selection and therapeutic dosage of such drugs are known to those skilled in the art and can be determined by a skilled clinician.

[0310] Combination therapy can provide synergistic effects and can be proven to be synergistic, that is, the effect achieved when active ingredients are used together is greater than the sum of the effects that can be obtained from using these compounds separately.Synergistic effects can be achieved when active ingredients are (1) co-formulated and administered or delivered simultaneously as a combined unit dosage formulation; (2) delivered alternately or in parallel as separate formulations; or (3) by some other regimen.When delivered alternately, synergistic effects can be achieved when these compounds are administered or delivered sequentially, for example, by different injections in separate syringes.Generally, during alternation, each active ingredient in an effective dosage is administered continuously, that is, sequentially, whereas in combination therapy, two or more active ingredients in an effective dosage are administered together.

[0311] In one embodiment, an effective amount of an antibody or antigen-binding fragment or conjugate thereof that specifically binds to one or more of the antigens disclosed herein is administered to a tumor-bearing subject after anti-cancer treatment. After a sufficient amount of time has passed for the administered antibody or antigen-binding fragment or conjugate to form an immune complex with the antigen expressed on each cancer cell, the immune complex is detected. The presence (or absence) of the immune complex indicates the effectiveness of the treatment. For example, an increase in immune complexes compared to a control taken before treatment indicates that the treatment is ineffective, and a decrease in immune complexes compared to a control taken before treatment indicates that the treatment is ineffective. indicates that the treatment is effective.

[0312] F. Biopharmaceutical Compositions Provided herein are biopharmaceutical or biologic compositions (hereinafter "compositions") for use in gene therapy, immunotherapy, and / or cell therapy, comprising one or more of the disclosed CARs, or CAR-expressing T cells, antibodies, antigen-binding fragments, conjugates, CARs, or T cells expressing CARs that specifically bind to one or more antigens disclosed herein, in a carrier (e.g., a pharmaceutically acceptable carrier). These compositions can be prepared in unit dosage forms for administration to a subject. The amount and timing of administration to achieve a desired outcome is at the discretion of the treating clinician. These compositions can be formulated for systemic (e.g., intravenous) or local (e.g., intratumoral) administration. In one example, the disclosed CARs, or CAR-expressing T cells, antibodies, antigen-binding fragments, conjugates, are formulated for parenteral administration, such as intravenous administration. Compositions comprising the disclosed CARs, or CAR-expressing T cells, conjugates, antibodies, or antigen-binding fragments, are used for, e.g., treatment and detection of tumors, such as, but not limited to, neuroblastoma. In some examples, these compositions are useful for treating or detecting cancer. Compositions comprising the CARs disclosed herein, or T cells, conjugates, antibodies, or antigen-binding fragments expressing the CARs, are also used, for example, to detect pathological angiogenesis.

[0313] Compositions for administration can include a solution of CAR, or CAR-expressing T cells, conjugates, antibodies, or antigen-binding fragments, dissolved in a pharmaceutically acceptable carrier, such as an aqueous carrier. A variety of aqueous carriers, such as buffered saline, can be used. These solutions are sterile and generally free of undesirable substances. The compositions can be sterilized by conventional, well-known sterilization techniques. The compositions can contain pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, adjuvant drugs, etc., such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of CAR, or CAR-expressing T cells, antibodies, or antigen-binding fragments, or conjugates in these formulations can vary widely and is selected primarily based on fluid volume, viscosity, body weight, etc., according to the particular mode of administration selected and the needs of the subject. Actual methods for preparing such dosage forms for use in gene therapy, immunotherapy, and / or cell therapy are known or will become apparent to those skilled in the art.

[0314] A typical composition for intravenous administration contains about 0.01 to about 30 mg / kg of antibody or antigen-binding fragment or conjugate (or a corresponding dose of CAR, or T cells expressing a CAR, or a conjugate comprising the antibody or antigen-binding fragment) per subject per day. Actual methods for preparing administrable compositions will be known or apparent to those of skill in the art and are described in more detail in publications such as Remington's Pharmaceutical Science, 19th Edition, Mack Publishing Company, Easton, PA (1995).

[0315] CARs, or CAR-expressing T cells, antibodies, antigen-binding fragments, or conjugates, can be provided in lyophilized form and rehydrated with sterile water before administration, but they can also be provided in sterile solutions of known concentrations. The CAR, or CAR-expressing T cells, antibodies, or antigen-binding fragments, or conjugates, solution is then added to an infusion bag containing 0.9% sodium chloride, USP, and in some cases administered at a dosage of 0.5 to 15 mg / kg body weight. Considerable experience is available in the art for administering antibody or antigen-binding fragment and conjugate drugs; for example, antibody drugs have been commercially available in the United States since the approval of RITUXAN® in 1997. CARs, or CAR-expressing T cells, antibodies, antigen-binding fragments, and their conjugates, can be administered intravenously or intravenously. It may be administered by slow infusion rather than as a bolus. In one example, a higher loading dose is administered with subsequent maintenance doses administered at lower levels. For example, an initial loading dose of 4 mg / kg of the antibody or antigen-binding fragment (or a corresponding dose of a conjugate comprising the antibody or antigen-binding fragment) may be infused over a period of approximately 90 minutes, followed by weekly maintenance doses of 2 mg / kg for 4 to 8 weeks infused over a period of 30 minutes if the previous dose was well tolerated.

[0316] Controlled-release parenteral formulations can be prepared as implants, oily injections, or granular systems. For a broad review of protein delivery systems, see Banga, AJ, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Technomic Publishing Company, Inc., Lancaster, PA (1995). Granular systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain a therapeutic protein, such as a cytotoxin or drug, as a central core. In microspheres, the therapeutic agent is dispersed throughout the particle. Particles smaller than about 1 μm, microspheres, and microcapsules, are commonly referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Capillaries have a diameter of approximately 5 μm, so only nanoparticles can be administered intravenously. Microparticles are typically approximately 100 μm in diameter and are administered subcutaneously or intramuscularly. See, e.g., Kreuter, J., Colloidal Drug Delivery Systems, J. Kreuter (ed.), Marcel Dekker, Inc., New York, NY, pp. 219-342 (1994); and Tice and Tabibi, Treatise on Controlled Drug Delivery, A. Kydonieus (ed.), Marcel Dekker, Inc., New York, NY, pp. 315-339 (1992).

[0317] The polymers can be used for ion-controlled release of the CARs disclosed herein, or T cells, antibodies or antigen-binding fragments, or conjugate compositions expressing the CARs. Various degradable and non-degradable polymer matrices used for controlled drug delivery are known in the art (Langer, Accounts Chem. Res. 26:537-542, 1993). For example, the block copolymer polaxamer 407 exists as a viscous but still mobile liquid at low temperatures, but forms a semi-fluid gel at body temperature. It has been shown to be an effective vehicle for the formulation and sustained delivery of recombinant interleukin-2 and urease (Johnston et al., Pharm. Res. 9:425-434, 1992; and Pec et al., J. Parent. Sci. Tech. 44(2):58-65, 1990). Alternatively, hydroxyapatite has been used as a microcarrier for the controlled release of proteins (Ijntema et al., Int. J. Pharm. 112:215-224, 1994). In yet another embodiment, liposomes are used for the controlled release and drug targeting of lipid-encapsulated drugs (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA (1993)). Numerous additional systems for controlled delivery of therapeutic proteins are known (see U.S. Patent Nos. 5,055,303; 5,188,837; 4,235,871; 4,501,728; 4,837,028; 4,957,735; 5,019,369; 5,055,303; 5,514,670; 5,413,797; 5,268,164; 5,004,697; 4,902,505; 5,506,206; 5,271,961; 5,254,342 and 5,534,496).

[0318] G.Kit In one embodiment, also provided is a kit that uses the CAR disclosed herein.For example, a kit for treating tumor in a subject or a kit for producing CAR T cells that express one or more of the CARs disclosed herein.The kit typically includes the disclosed antibody, antigen-binding fragment, conjugate, nucleic acid molecule, CAR or CAR-expressing T cells disclosed herein.More than one of the disclosed antibody, antigen-binding fragment, conjugate, nucleic acid molecule, CAR or CAR-expressing T cells can be included in the kit.

[0319] The kit may include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container may be formed from a variety of materials, such as glass or plastic. The container typically holds a composition comprising one or more of the disclosed antibodies, antigen-binding fragments, conjugates, nucleic acid molecules, CARs, or CAR-expressing T cells. In some embodiments, the container may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle). The label or package insert indicates that the composition is used to treat a specific condition.

[0320] The label or package insert typically further includes instructions for using the disclosed antibody, antigen-binding fragment, conjugate, nucleic acid molecule, CAR, or CAR-expressing T cell, for example, in a method for treating or preventing tumors or in a method for generating CAR T cells. The package insert typically includes instructions customarily included in the commercial packaging of a therapeutic product, including information about indications, usage, dosage, administration, contraindications, and / or warnings regarding the use of the therapeutic product. The instructional material can be written in electronic form (e.g., a computer diskette or compact disc) or visual (e.g., a video file). The kit can also include additional components to facilitate the specific application for which the kit is designed. Thus, for example, the kit can further include a means for detecting the label (e.g., an enzyme substrate for an enzymatic label, a filter set for detecting a fluorescent label, an appropriate secondary label such as a secondary antibody, etc.). The kit can further include buffers and other reagents routinely used for the implementation of a particular method. Such kits and their appropriate contents are well known to those skilled in the art.

[0321] Example The present invention is further illustrated by the following examples, which should not be construed as imposing limitations on the scope of the present invention in any way. On the contrary, it is readily understood that recourse must be had to various other embodiments, modifications, and equivalents thereof, which may occur to those skilled in the art after reading the description herein without departing from the spirit of the present invention and / or the scope of the appended claims. [Example]

[0322] Isolation of human CD38-specific antibodies from a fully human yeast-displayed ScFv library material and method: A large yeast-displayed human naive 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 over 60 individuals. Three rounds of magnetic-activated cell sorting (MACS) were performed to enrich for human ScFv binders to the recombinant human CD38 extracellular domain fused to human IgG1 Fc, designated CD38-Fc. In the first round of yeast library panning, yeast display Lei ScFv library (5 × 10 10 The yeast cells were incubated with 5 μg / mL CD38-Fc in 15 ml PBSA (0.1% bovine serum albumin (BSA) in Dulbecco's phosphate-buffered saline (PBS) buffer) for 1.5 hours on a rotator at room temperature. After two washes with 25 ml PBSA, the yeast library mix was incubated with 100 μL Protein G microbeads (Miltenyi Biotec) for 30 minutes on a rotator at room temperature. After one wash, the library mix was resuspended in 50 ml PBSA and loaded onto a MACS cell separation column (LS column). After three washes with 10 ml PBSA, yeast cells displaying ScFv binders were subsequently eluted twice with 2 ml PBSA. These eluted yeast cells were combined and resuspended in 50 ml of SDCAA medium (1 L water, 20 g D-glucose, 6.7 g BD Difco™ Yeast Nitrogen Base without amino acids, 5 g Bacto™ Casamino Acids, 5.4 g Na2.HPO4, 8.56 g NaH2PO4.H2O) and amplified for 20 hours with shaking at 225 rpm and 30°C. The amplified pool was then induced in SGCAA medium (same composition as SDCAA medium, but with galactose instead of glucose), shaken at 225 rpm and 30°C for an additional 16 hours, and used for the next round of panning. The same process was repeated two more times to enrich for CD38-Fc-specific binders.

[0323] 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 derived pool was incubated with 1 μg / ml CD38-Fc at room temperature for 1 hour, then stained with Anti-c-Myc-Alexa 488 and goat anti-Hu-Fc PE conjugate. The top 1% of pools with the highest PE-to-FITC signal were gated and sorted. The sorted pool was amplified in SDCAA medium, yeast plasmid DNA was extracted, and single clones were transformed into bacteria for DNA sequencing. 40 random clones were sequenced, identifying 36 unique sequences. Fourteen CD38 ScFv clones, designated M3801, M3802, M3803, M3804, M3806, M3808, M3809, M3810, M3811, M3812, M3815, M3816, and M3817, respectively, were cloned into CAR constructs for CAR-T functional screening, as shown in Example 2, Table 1. M3815_P, 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. Additionally, M8DR H_L and M8DR L_H, derived from the monoclonal antibody daratumumab, which were used as positive controls for the novel human anti-CD38 ScFv-based CAR constructs, were cloned into CAR constructs for CAR-T functional screening, as shown in Example 2, Table 1. [Example]

[0324] Generation of CD38-targeting CAR T constructs incorporating fully human binder ScFv sequences from a yeast display library Few treatment options are available for AML, and treatment-related toxicity and post-treatment disease relapse are common. Furthermore, immunotherapies using non-human sequences, such as mouse-derived antibodies, can lead to treatment rejection or side effects in patients. To develop a new CAR T treatment for AML, we designed 15 CD38-targeting CAR T constructs incorporating a fully human ScFv targeting domain and evaluated their antitumor activity.

[0325] material and method: (a) Cell line MM cell lines MM1.S and RPMI-8226, and the human embryonic kidney line 293T The cells 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 encoding firefly luciferase (Lentigen). 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+ and CD8+ human T cells were purified from the buffy coats by positive selection using a 1:1 mixture of CD4- and CD8-MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's protocol.

[0326] (b) Creation of chimeric antigen receptor (CAR)-expression vector The CAR antigen-binding domain, scFv, sequence was derived from human anti-CD38 ScFv. The CAR T construct was generated by linking the binder sequence in frame to the CD8a binding and transmembrane domain (aa123-191, Ref Sequence ID NP001759.3), followed by the 4-1BB (CD137, aa214-255, UniProt Sequence ID Q07011) signaling domain and the CD3 zeta signaling domain (CD247, aa52-163, Ref Sequence ID: NP_000725.1). CAR positive control constructs LTG2525 and LTG2526 were constructed in a similar manner, incorporating a control ScFv, herein referred to as M8DR, whose heavy and light chains were derived from the monoclonal antibody daratumumab, in either a VH-VL or VL-VH orientation, respectively. The CAR construct sequence was cloned into a third-generation lentiviral plasmid backbone (Lentigen Technology Inc., Gaithersburg, MD). Supernatant containing the lentiviral vector (LV) was generated by transient transfection of HEK293T cells, and the vector was pelleted by centrifugation and stored at -80°C.

[0327] (c) Primary T cell purification and transduction Human primary T cells from healthy volunteers were transfected with CD4 T cells according to the manufacturer's protocol (Miltenyi Biotec, Bergisch Gladbach, Germany). + and CD8 + T cells were purified from whole blood or buffy coat (purchased from a commercial provider with written consent from the donor) using immunomagnetic bead selection at a density of 0.3–2 × 10 6Cells / ml were cultured in TexMACS™ medium supplemented with 30 IU / ml IL-2, activated with CD3 / CD28MACS® GMP T cell TransAct reagent (Miltenyi Biotec), transduced with a lentiviral vector encoding a CAR construct 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. Cultures were propagated in TexMACS™ medium supplemented with 30 IU / ml IL-2 until harvest on day 8–10.

[0328] (d) Immune effector assays (CTL and cytokines) To determine cell-mediated cytotoxicity (CTL assay), 5,000 target cells stably transduced with firefly luciferase were combined with CAR T cells at various 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). Target-only wells (maximum CPS) and target-only wells plus 1% Tween-20 (minimum CPS) were used to determine the assay range. Percent specific lysis was calculated as (1 - (sample CPS - minimum CPS) / (maximum CPS - minimum CPS)). Supernatants from 10:1 E:T co-cultures were removed and analyzed for IFNγ and TNFα concentrations using ELISA (eBioscience, San Diego, CA). ) was analyzed.

[0329] (e) Flow cytometry analysis of CAR surface expression For cell staining, 500,000 CAR T-transduced cells were harvested from culture and washed twice with cold AutoMACS buffer supplemented with 0.5% bovine serum albumin (Miltenyi Biotec). CAR surface expression was detected by staining with CD38-His peptide (Thermo Fisher Scientific, Waltham, Massachusetts), followed by secondary anti-His-PE detection reagent (Miltenyi Biotec, Bergisch Gladbach, Germany). Anti-CD4 antibodies conjugated with VioBlue fluorophore (Miltenyi Biotec) were used as needed, according to the vendor's protocol. Non-transduced cells served as negative controls. In all studies, dead cells were excluded by 7AAD staining (BD Biosciences, San Jose, CA). Cells were washed twice and resuspended in 200 μl of staining buffer before quantitative analysis by flow cytometry. Flow cytometry analysis was performed on a MACSQuant® 10 Analyzer (Miltenyi Biotec) and data plots were generated using FlowJo software (Ashland, OR).

[0330] result: This example demonstrates the ability of MM and other CD38 + We describe the creation of CAR T cells targeting the tumor antigen CD38 for the treatment of malignant tumors and the killing of CD38-expressing tumor suppressor cells in the tumor microenvironment. Each CAR was composed of a human ScFv binder derived from a human yeast display library, a CD8 hinge and transmembrane domain, a 4-1BB costimulatory domain, and a CD3z activation domain (Figure 1).

[0331] Table 1 below details the CAR38 constructs developed, designated by LTG number, and the corresponding designations of the ScFv sequences used in each construct. Comparative CAR sequences LTG2525 and LTG2526 incorporate ScFvs derived from the heavy and light chains of the daratumumab antibody in heavy-light and light-heavy orientations, respectively.

[0332] [Table 1]

[0333] An overview of the CD38 CAR design is shown in Figure 1. A fully human ScFv binder targeting CD38 was linked in-frame to the CD8 hinge and transmembrane domains, 4-1BB costimulatory domain, and CD3 zeta 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 anti-CD38 CARs incorporating single-chain fragment variable (ScFv) sequences is shown in Figure 2. Expression levels for each CAR containing ScFv were determined by flow cytometry analysis of LV-transduced T cells from healthy donors using a two-step staining procedure: Step 1: CD38-His tag peptide; followed by Step 2: anti-His PE reagent. A subset of ScFv-based anti-CD38 CAR constructs, as detected by CD38-His, was highly expressed in human primary T cells compared to untransduced T cell controls (UTD) (Figure 2). All transduction constructs were found to be highly CAR T-positive (40%-95%) with the exception of constructs LTG2097 and LTG2503, which had less than 20% CAR T-positive T cells. + The CAR T cells expressed 100% CAR T expression (expressed as %). Negative controls—untransduced T cells from the same donor (UTD) and an anti-His PE control (FMO PE)—did not show any obvious CAR T expression, demonstrating the specificity of CAR T staining. As shown in Figure 3A-C, high cytolytic activity of anti-CD38 CARs was demonstrated for the majority of the analyzed constructs. Untransduced T cells (UTD) were used as a negative control for CAR cytolytic function. Human primary T cells were transduced with LV encoding the CAR constructs (see Methods) and then transduced with CD38 +The tumor cells were incubated for 18 hours with the tumor lines MM1.S, RPMI-8226, or the CD38 negative control line HEK293. Firefly luciferase was stably transduced into each target line to facilitate detection of the viable tumor cell fraction in a luminescence-based in vitro killing assay. To compare and contrast the efficacy of different CAR constructs, effector CAR T cells and tumor cells were combined at effector-to-target (E:T) ratios of 5, 10, and 20 (Figure 3A-C). RPMI-8226 cells were the most sensitive cell line to CD38 CAR-mediated tumor killing, with most CAR constructs achieving greater than 80% tumor lysis at the lowest E:T ratio of 5 (Figure 3A). All tested constructs effectively lysed RPMI-8226, while the negative control UTD group did not produce significant tumor lysis (the highest cytolytic activity observed for UTD was 10). At the highest E:T ratio of 20, only 10% lysis was observed, and lower E:T ratios did not result in detectable tumor lysis. Thus, lysis of RPMI-8226 tumors by CD38-targeted T cell constructs was antigen-specific. The constructs with the lowest levels of cytolytic activity against RPMI-8226 tumors were LTG2090, LTG2098, and LTG2099, which achieved <70%, <40%, and <10% tumor killing, respectively, at an E:T ratio of 1:5 (Figure 3A). Notably, the decline in cytolytic function of these CAR constructs did not correlate with their surface expression levels, which were relatively high at >45%, >75%, and >65%, respectively, as shown in Figure 2. Therefore, the function of CAR constructs cannot be predicted based solely on CAR surface expression, and selecting the best CAR candidate is not straightforward. The second CD38 construct tested +The tumor line MM1.S showed a similar distribution of killing potency by the anti-CD38 CAR constructs tested, and because it was less sensitive to killing compared to RPMI-8228, the differences in killing potency of the CAR constructs were more pronounced, allowing 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 weakest of the set, again confirming that the cytolytic activity of CD38-targeted CARs is tumor antigen-specific and that assessment of CAR-CAR function can be generalized across CD38-positive cell lines. Furthermore, the MM1S killing assay revealed moderate cytolytic activity of the CAR construct LTG2502 compared to the other CAR constructs tested (Figure 3B). Similar to RPMI-8226, the negative assay control UTD had no discernible killing activity in the MM1.S tumor line, demonstrating that the killing was CAR-dependent (Figure 3B).

[0334] The 293T tumor line, lacking CD38 expression, was tested as a negative / specificity control for CAR function (Figure 3C). Background cell lysis levels in this assay, determined based on the UTD negative control, did not exceed 10% (Figure 3C). With the exception of the CAR construct LTG2501 at an E:T ratio of 10:1 and 20:1, none of the CD38-targeted CAR T constructs consistently demonstrated lysis of 293T cells above a background level of 10% (Figure 3C). Thus, while the CAR T constructs tested in this set are target-specific, construct LTG2501 may have low specificity and therefore mediate off-target toxicity. The discovery of this CAR feature required empirical testing and could not have been predicted, again demonstrating the nonobviousness of the present invention.

[0335] Next, we evaluated the ability of anti-CD38 CAR T cells to produce cytokines in response to antigen-expressing target cells (Figure 4A-B). IFN-gamma and TNF-alpha are proinflammatory cytokines known to be secreted at high rates in association with T cell activation and cytolytic activity. The CD38-positive tumor lines RPMI-8226 and MM1.S, or the CD38-negative tumor line 293T, were co-incubated overnight with CAR T cells incorporating the CAR38 construct at an effector-to-target ratio of 10:1 or with negative untransduced T cells (UTD). Culture supernatants were analyzed by ELISA for IFN-gamma (Figure 4A) and TNF-alpha (Figure 4B). The cytokine patterns produced by the tested CAR constructs were similar for IFN-gamma and TNF-alpha. The CAR constructs with the highest supernatant levels of IFN-gamma and TNF-alpha 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, demonstrating that the ability of the CAR constructs to secrete cytokines was consistent across the two cytokines tested (Figures 4A and 4B). The levels of cytokines produced in this subset of vectors were 2500–3500 pg / ml for IFN-gamma and 1500–2500 pg / ml for TNF-alpha when incubated with the tumor lines RPMI-H226 and MM1.S (Figures 4A and 4B). CAR construct LTG2092 produced high levels when incubated alone and with the target lines RPMI-8226 and MM1.S. tNF-alpha was produced at the bell (Figure 4B), thus demonstrating sustained signaling. Again, this data demonstrates the nonobviousness of the CAR invention and the need for empirical testing to identify the best CAR candidates. Without intending to be limited to any particular mechanism of action, possible reasons for the enhanced therapeutic function associated with the exemplary CD38 targeting CARs of the present invention may include, for example, but not by way of limitation, a) improved lateral movement within the plasma membrane, allowing for more efficient signaling; b) superior location within plasma membrane microdomains, such as lipid rafts, and a greater ability to interact with transmembrane signaling cascades associated with T cell activation; c) superior location within the plasma membrane, e.g., less proximity to or fewer interactions with phosphatases such as CD45, due to preferential movement away from inhibitory or downregulatory interactions; and d) superior assembly into the T cell receptor signaling complex (i.e., immune synapse), or e) superior ability to engage tumor antigens due to the two distinct targeting domains present in each CAR molecule, or any combination thereof.

[0336] Each application and patent cited herein, and each document or reference cited therein (including each issued patent in litigation, the "Application Citations"), and each PCT and foreign application or patent corresponding to and / or claiming priority to any of these applications and patents, and each document cited or referenced in each Application Citation, are hereby expressly incorporated herein by reference and may be used in the practice of the invention. More generally, documents or references are cited either in the text, in a reference list before the claims, or in the text itself, and each such document or reference (the "In-Herein Cited References"), and each document or reference cited in each In-Herein Cited Reference (including any manufacturer's specifications, instructions, etc.), is hereby expressly incorporated herein by reference.

[0337] The foregoing description of some specific embodiments provides sufficient information to enable others, by applying knowledge of the present invention, to easily modify or adapt the specific embodiments for various applications without departing from the general concept; therefore, such adaptations and modifications should, and are intended to, be understood as being within the meaning and range of equivalents of the disclosed embodiments. It is understood that the words or terms used herein are for purposes of description and not of limitation. In the drawings and description, exemplary embodiments are disclosed, and although specific terminology may be employed, unless otherwise noted, they are used in a generic and descriptive sense only, and not for purposes of limitation, and the claims are therefore not so limited. Furthermore, those skilled in the art will recognize that certain steps of methods discussed herein can be sequenced in a different order or steps can be combined. Accordingly, it is intended that the appended claims not be limited to the precise embodiments disclosed herein. Those skilled in the art will be able to recognize and ascertain, using no more than routine experimentation, many equivalents to the embodiments of the invention described herein. Such equivalents are encompassed by the following claims.

[0338] Reference to sequence listing This application contains a Sequence Listing that has been submitted electronically to the United States Patent and Trademark Office in a PDF file titled "Sequence Listing." The Sequence Listing is incorporated by reference.

[0339] Sequences of the present disclosure The nucleic acid and amino acid sequences listed below are shown using standard abbreviations for nucleotide bases, and one-letter or three-letter codes for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the represented strand. In the accompanying sequence listing: SEQ ID NO: 1 Nucleotide sequence of CD38 hScFv binder M3801 GAAGTGCAATTGGTCCAAAGCGGTGCAGAAGTCAAGAAACCTGGTTCGAGCGTGAAAGTGTCCTGCAAGGCCTCTGGCGGCACCTTCTCATCCTACGCCATTAGCTGGGTCCGCCAAGCCCCGGGCCAGGGACTTGAGTGGATGGGCGGAATCATCCCTATCTTCGGGACCGCCAATTACGCCCAGAAGTTCCAGGGCCGCGTGACCATCACCGCGGACAAGTCCACATCAACCGCCTATATGGAGCTCTCGTCGCTGAGATCAGAGGACACTGCTGTCTACTACTGTGCGAGGGCCCCATACGACGATGCATTCGACATCTGGGGACAGGGTACCATGGTCACTGTGTCCAGCGGGGGCGGAGGCTCCGGGGGGGGCGGATCGGGGGGCGGCGGATCCAACTTCATGCTGACCCAGCCGCACTCGGTGTCAGAGAGCCCCGGAAAGACTGTGACCATTAGCTGCACCCGGTCCAGCGGAAGCATCGCCTCCAACTACGTGCAGTGGTACCAGCAGCGGCCGGGCTCCGCGCCGACCACTGTGATCTACGAAGATAACCAGCGCCCCTCCGGTGTCCCTGGACGGTTCTCCGGATCCATTGACACCTCGTCCAACTCCGCCTCGCTGACGATCTCCGGACTGGAGACTGAAGATGAAGCTGTGTACTACTGCCAATCCTACGACTCCGGATTTCCCTACGCCGTGTTCGGTGGCGGGACCCAGCTCACCGTGCTGGGA Amino acid sequence of SEQ ID NO:2 CD38 hScFv binder M3801[[ID=**3**]] EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARAPYDDAFDIWGQGTMVTVSSGGGGS GGGGSGGGGSNFMLTQPHSVSESPGKTVTISCTRSSGSIASNYVQWYQQRPGSAPTTVIYEDNQRPSGVPGRFSGSIDTSSNSASLTISGLETEDEAVYYCQSYDSGFPYAVFGGGTQLTVLG SEQ ID NO: 3 Nucleotide sequence of CD38 hScFv binder M3802 GAAGTGCAACTCGTCGAATCTGGTGGAGGATCCGTGCAGCCTGGTGGAAGCCTGAGACTGTCGTGTGCCGCCTCCGAGTTCACCTTCTCCTCGTACTGGATGCACTGGGTCAGACAGGCGCCCGGAAAGGGCCTGGAATGGGTGGCCGTGATCTCATACGACGGCGCGAAGAAGTACTACGCCGATTCCGTGAAGGGCCGCTTTACCATTTCCCGGGACAATTCAAAGAACACCCTCTACCTTCAAATGAACTCCCTGAGGGCCGAGGATACTGCAGTCTACTACTGCGCTCGGGATGACGGGAGCGGGGGCTTCGACTACTGGGGACAGGGAACCCTCGTGACTGTGTCATCCGGTGGCGGCGGCTCCGGAGGCGGCGGTTCCGGGGGGGGGGGATCCGACATCGTGATGACGCACACTCCTCTGTCGCTGTCCGTGACCCCCGGCCAGCCTGCCTCAATCTCGTGCAAGAGCTCCCAGTCGCTGCTGCATAGCGATGGAAAGACCTACCTGTATTGGTACCTTCAAAAGCCGGGACAGCCGCCGCAGTTGCTGATCTATGAAGTCAGCAACCGCTTCTCGGGAGTGCCCGACCGCTACTCCGGCTCGGGGAGCGGAACCGACTTCACCCTGAAAATTTCCCGGGTCGAGGCAGAGGACGTGGGAGTGTACTACTGCATGCAAAGCATTCAGTTGCCACTGACTTTCGGTGGAGGAACCAAGCTCGAAATCAAA Amino acid sequence of SEQ ID NO:4 CD38 hScFv binder M3802 EVQLVESGGGSVQPGGSLRLSCAASEFTFSSYWMHWVRQAPGKGLEWVAVISYDGAKKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDDGSGGFDYWGQGTLVTVSSGGGG SGGGGSGGGGSDIVMTHTPLSLSVTPGQPASISCKSSQSLLHSDGKTYLYWYLQKPGQPPQLLIYEVSNRFSGVPDRYSGSGSGTDFTLKISRVEAEEDVGVYYYCMQSIQLPLTFGGGTKLEIK SEQ ID NO: 5 Nucleotide sequence of CD38 hScFv binder M3803 GAAGTGCAATTGGTGCAGAGCGGTGGAGGACTTGTGAAACCTGGTGGATCCCTGAGACTTTCCTGTGCCGCTTCGGGCTTCACCTTCTCGGACTACTACATGTCCTGGATTCGCCAGGCCCCTGGAAAGGGACTGGAATGGGTGTCATACATCAGCTCCTCCGGTTCCACCATCTACTATGCCGATTCCGTGAAGGGCAGATTCACCATCTCGCGCGACAACGCCAAGAACACTCTCTATCTGCAAATGAACTCACTGCGGGCTGAGGACACCGCGGTCTACTACTGCGCCCGGGACCTCAGCGGAAAGTCCAGCGGATGGTCCCATTACTTCGATTACTGGGGACAGGGAACCCTGGTCACCGTGTCCAGCGGCGGGGGGGGCTCGGGTGGCGGCGGCTCCGGCGGCGGCGGGAGCAACTTCATGCTGACTCAGCCCCACTCCGTGTCCGAGAGCCCGGGAAAGACCGTGACTATTTCGTGCACACGGTCCTCCGGGAGCATTGCGAACAACTACGTGCAGTGGTACCAGCAGCGGCCCGATAGGGCCCCAACCACTGTGATCTACGAAGATGACCAGCGGCCGTCTGGAGTCCCGGACCGCTTCTCGGGGTCCATCGACTCATCATCCAATTCCGCATCGCTGACGATCAGCGGACTGAAGATCGAGGACGAAGCCGATTACTACTGCCAGTCCTACGACGGCACCAACTGGGTCTTTGGGGGTGGAACCAAGCTGACTGTGCTCGGA Amino acid sequence of SEQ ID NO: 6 CD38 hScFv binder M3803 EVQLVQSGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARDLSGKSSGWSHYFDYWGQGTLVTVSSG GGGSGGGGSGGGGSNFMLTQPHSVSESPGKTVTISCTRSSGSIANNYVQWYQQRPDRAPTTVIYEDDQRPSGVPDRFSGSIDSSSNSASLTISGLKIEDEADYYCQSYDGTNWVFGGGTKLTVLG SEQ ID NO: 7 Nucleotide sequence of CD38 hScFv binder M3804 GAAGTGCAACTTGTCGAAAGCGGTGGAGGTCTTGTCCAACCTGGTCGCTCCCTGAGGCTCTCGTGTGCCGCGAGCGGATT CACCTTCTCATCGTACGCTATGTCCTGGGTCAGACAGGCTCCTGGAAAGGGCCTGGAATGGGTGGCCGTGATCTCCTACGACGGCAGCAACAAGTATTACGCCGACTCAGTGAAGGGCCGGTTCACCATTTCCCGGGACAACAGCAAGAACACCCTGTACTTGCAAATGAACTCCCTGCGGGCCGAGGATACCGCGGTGTACTACTGCGCCCACCTCCGCTTTGGATACGGAATGGATGTCTGGGGACAGGGAACTACCGTGACCGTGTCGTCCGGGGGGGGGGGAAGCGGCGGCGGGGGATCGGGTGGCGGCGGATCCCAGACTGTGGTCACCCAAGAGCCTTCACTGACCGTGTCCCCGGGTGGCACCGTGACGCTGACTTGCGCGTCATCTACCGGGGCCGTGACCTCGGACCACTACCCCTGCTGGTTCCAGCAGAAACCCGGACATCCACCGAGAGCCCTGGTGTACTCCACTGACACCATCCACTCCTGGACTCCGGCCCGGTTCTCCGGAAGCCTCCTGGGCGGGAAGGCCGCACTGACAGTGTCCGGAGTGCAGCCCGAGGATGAAGCCGACTACTACTGTCTGCTCTACTATGGGGGAGCACGCGTGTTCGGTGGCGGCACTCAGCTGACCGTGCTGGGA Amino acid sequence of SEQ ID NO: 8 CD38 hScFv binder M3804 EVQLVESGGGLVQPGRSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAHLRFGYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCASSTGAVTSDHYPCWFQQKPGHPPRALVYSTDTIHSWTPARFSGSLLGGKAALTVSGVQPEDEADYYCLLYYGGARVFGGGTQLTVLG Nucleotide sequence of SEQ ID NO: 9 CD38 hScFv binder M3806 CAAGTGCAACTCGTCCAATCCGGAGCTGAAGTCAAGAAGCCAGGATCTTCCGTGAAAGTGTCGTGCAAGGCCTCCGGGGGAACATTCTCCTCATATGCGATCAGCTGGGTCCGCCAGGCTCCGGGACAGGGCTTGGAGTGGATGGGTATCATTAACCCTTCGGGCGGCTCAACTAGCTACGCCCAGAAGTTCCAGGGCAGAGTGACCATGACCAGGGACACCAGCACTTCGACGGTGTACATGGAACTGTCCTCACTGCGGTCCGAGGACACCGCCGTGTACTACTGCGCCCGGGAGTATTCCTCCTCCCGCGTGGACGCCTTCGATATCTGGGGACAGGGTACCATGGTCACTGTGTCGTCCGGCGGCGGCGGAAGCGGGGGTGGCGGGAGCGGTGGCGGCGGATCCAGCTACGAACTCACCCAGCCGCCGTCAGTGTCCGTGAGCCCGGGACAGACCGCAACCATTACTTGTTCCGGGGATGACCTGGGATCCAAATACGTGTGCTGGTACCAACAGAAGCCTGGTCACTCGCCCGTGCTGATCATCTACGACGACTCAGACCGGCCCAGCGGCATCCCCGAGAGATTTTCCGGATCCAACAGCGGAAACACCGCCACTCTGACTATTTCGCGCGTCGAGGCCGGCGACGAAGCGGATTACTACTGCCAAGTCTGGGACAGCTCCTCGGATCATGCCGTGTTCGGGGGCGGAACCCAGCTTACCGTGCTGGGG Amino acid sequence of SEQ ID NO: 10 CD38 hScFv binder M3806 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREYSSSRVDAFDIWGQGTMVTVSSG GGGSGGGGSGGGGSSYELTQPPSVSVSPGQTATITCSGDDLGSKYVCWYQQKPGHSPVLIIYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSSDHAVFGGGGTQLTVLG SEQ ID NO: 11 Nucleotide sequence of CD38 hScFv binder M3808 GAAGTGCAGTTGGTGGAGAGCGGTGGAGGACTTGTGCAACCTGGTGGATCCCTGAGATTGTCGTGTGCCGCCTCCGGTTTCACCTTCTCCTCGTACTGGATGAGCTGGGTCCGCCAGGCACCCGGGAAGGGACTGGAATGGGTGGCGGACATTAAGGATGACGGCTCCGAGCGGTACTACGTGGACTCCGTGAAGGGCCGGTTCACTATCTCAAGAGACAATGCCAAGAACAGCCTGTACCTCCAAATGAACTCGCTGCGGGCCGAGGATACCGCAGTGTATTACTGCGCCCGCGACGTGTGGGCTGGGATGGATGTCTGGGGCCAGGGGACCACTGTCACTGTGTCTAGCGGGGGCGGCGGAAGCGGCGGCGGCGGATCCGGTGGTGGCGGAAGCGACATTCAGCTGACCCAGTCCCCATCATTCCTGTCCGCCTCCGTGGGCGACAGGGTCACTATCACTTGCCAAGCCAGCCAGGACATCTCCAACTACCTGAACTGGTACCAGCAGAAGCCTGGAAAAGCTCCGAAGCTCCTCATCTACGACGCCTCGAACCTCGAAACCGGAGTGCCCTCACGGTTTTCCGGATCGGGATCGGGGACCGATTTCTCCTTCACCATTTCATCCCTGCAACCCGAGGACATCGCGACCTACTACTGCCAACAGACATACTCCCCGCCGATTACGTTCGGACAGGGAACCCGCCTGGAAATCAA Amino acid sequence of SEQ ID NO: 12 CD38 hScFv binder M3808 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVADIKDDGSERYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDVWAGMDVWGQGTTVTVSSGG GGSGGGGSGGGGSDIQLTQSPSFLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFSFTISSLQPEDIATYYCQQTYSPPITFGQGTRLEIK SEQ ID NO: 13 Nucleotide sequence of leader / signal peptide sequence atgctgctgctggtgaccagcctgctgctgtgcgaactgccgcatccggcgtttctgctgattccg SEQ ID NO: 14: Amino acid sequence of leader / signal peptide sequence MLLLVTSLLLCELPHPAFLLIP SEQ ID NO: 15 Nucleotide sequence of CD38 hScFv binder M3809 CAAGTCCAACTCGTCCAGTCCGGTGCCGAAGTCAAGAAGCCTGGCTCATCCGTGAAAGTGTCCTGCAAAGCATCGGGCGG AACCTTCTCCTCCTATGCCATTTCCTGGGTCCGCCAGGCACCGGGCCAGGGTCTGGAGTGGATGGGCGGGATTATCCCTATCTTCGGAACTGCCAACCACGCCCAAAAGTTCCAGGGACGCGTGACCATTACCGCCGATGAATCAACCTCAACCGCCTAC ATGGAACTGTCCAGCTTGAGGTCCGAGGACACCGCCGTGTACTACTGCGCGTTCATGATGGTGCCGGAGTACTACTTTGACTACTGGGCCAGGGAACCCTTGTGACCGTGTCGTCCGGTGGTGGCGGATCCGGGGGGGGGATCTGGGGGCGGCGGAAG CGATATCCAGATGACCCAGTCGCCATCGAGCCTGTCCGCTTCCGTGGGCGACAGAGTGACGATCACTTGCCGGGCTTCACAAGGCATCAGAAATGACCTGGGCTGGTATCAGCAGAAGCCCGGAGAAGCGCCCAAGCGGCTGATCTACGCGGCCAGCACCC TGCAAAACGGAGTGCCTTCGCGGTTCCGGGAGCGGCTCCGGAACTGACTTCACTCTGACTATTAACAGCCTCCAGCCCGAGGATTTCGCCACATACTACTGTCAGCAGTACAACAGCTACCCGTACACCTTCGGACAGGGAACTAAGCTCGAAATCAAG SEQ ID NO: 16. Amino acid sequence of CD38 hScFv binder M3809 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANHAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAFMMVPEYYFDYWGQGTLVTVSSG GGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGEAPKRLIYAASTLQNGVPSRFSGSGSGTDFTLTINSLQPEDFATYYCQQYNSYPYTFGQGTKLEIK SEQ ID NO: 17. Nucleotide sequence of CD38 hScFv binder M3810 AAAGTGCAACTCCAAGAAAGCGGCCCCGGACTTGTCAAACCGTCCCAGACTCTCTCCCTGACCTGTGCCGTCAGCGGTGGCTCAATTTCGAGCGGTGGCTACTACTGGTCCTGGATCAGGCAGCATCCGGGAAAGGGCTTGGAGTGGATTGGATACATCTACTACTCCGGGTCCACCTACTACAATCCCTCGCTGAAGTCGAGAGTCACCATCAGCGTGGACACCTCCAAGAACCAGTTTAGCCTGAAGCTGTCCTCAGTGACCGCAGCTGACACCGCCGTGTACTACTGCGCCCGCGGCGGAGTGATGGGCGGAGACTTCGACTACTGGGGACAGGGAACCCTGGTCACTGTGTCCAGCGGGGGGGGGGGATCCGGCGGCGGCGGTTCCGGTGGCGGAGGGTCCCAGTCAGTGCTGACTCAGCCACCTTCCGTGTCTGTGTCGCCCGGACAAACCGCCAGCATCACGTGCTCCGGCGACAACCTCGGTGATCACTATGTGTGCTGGTACCAACAGCGGCCGGGGCAGTCACCGGTGCTGATTATGTACGAGGATACTAAGCGCCCTTCCGGAATCCCTGACCGGTTCTCGGGAAGCAACTCCGGAAACACCGCCACCCTGACCATCTCCGGAACCCAGACAATGGATGAAGCGGACTACTATTGCGTGGCGTGGGACGATTCGCTGTCCGGCTGGGTGTTCGGCGGGGGAACTCAGCTGACTGTGCTCGGA Amino acid sequence of SEQ ID NO: 18 CD38 hScFv binder M3810 KVQLQESGPGLVKPSQTLSLTCAVSGGSISSGGYYWSWIRQHPGKGLEWIGYIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGGVMGGDFDYWGQGTLVTVSSGG GGSGGGGSGGGGSQSVLTQPPSVSVSPGQTASITCSGDNLGDHYVCWYQQRPGQSPVLIMYEDTKRPSGIPDRFSGSNSGNTATLTISGTQTMDEADYYCVAWDDSLSGWVFGGGTQLTVLG SEQ ID NO: 19 Nucleotide sequence of CD38 hScFv binder M3811 CAACTCCAACTCCAAGAATCTGGACCAGGCCTCGTGAAGCCCTCCCAAACTCTGTCCCTGACCTGTACCGTGTCGGGTGGAAGCATTTCGAGCGGTGGATACTACTGGTCCTGGATCAGGCAGCATCCTGGAAAGGGACTGGAGTGGATTGGGTACATCTACTACTCCGGCTCAACCTACTACAACCCGTCCTTGAAATCGCGCGTGACGATCTCCGTGGACACTTCAAAGAACCAGTTCAGCCTGAAGCTTTCCTCCGTGACCGCGGCCGATACAGCGGTGTACTACTGCGCTCGGGATCAGAGCGTGGCCGACCCTGGTGGCGGCTACTACTACTACGGAATGGATGTCTGGGGACAGGGAACCACCGTGACTGTGTCCAGCGGGGGCGGCGGATCCGGGGGGGGGGGATCGGGCGGCGGCGGTTCGCAGTCCGTGCTGACCCAGCCACCTAGCGTGTCAGTGGCACCGGGACAGACCGCCTCCATTTCCTGCGGGGGAAATGACTTCGGTAGCCGCTCCGTGTCATGGTATCACCAGAAGCCGGGACAGGCCCCGGTGCTGGTCATCTATGACGACAACGACAGACCCTCGGGCATCCCCGAACGGTTTTCGGGAAGCACCTCCGGAGACACTGCCACCCTGACCATCTCCCGGGTCGAGGTCGGCGATGAAGCCGATTACTACTGCCAAGTCTGGGACGACGACTCCGACCACTGGGTGTTCGGCGGCGGAACTAAGCTGACTGTGCTGGGG Amino acid sequence of SEQ ID NO:20 CD38 hScFv binder M3811 QLQLQESGPGLVKPSQTLSLTCTVSGGSISSGGYYWSWIRQHPGKGLEWIGYIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDQSVADPGGGYYYYGMDVWGQGTTVTV SSGGGGSGGGGSGGGGSQSVLTQPPSVSVAPGQTASISCGGNDFGSRSVSWYHQKPGQAPVLVIYDDNDRPSGIPERFSGSTSGDTATLTISRVEVGDEADYYCQVWDDDDSDHWVFGGGTKLTVLG SEQ ID NO: 21 Nucleotide sequence of CD38 hScFv binder M3812 CAAGTGCAACTCCAACAATCGGGCCCGGACTCGTGAAACCGTCCCAGACTCTGTCCCTTACTTGCGTGATTTCCGGGACTCCGTGAGCTCAAACTCGGCGGCCTGGAACTGGATTAGACAGTCCCCTTCCCGGGGTCTGGAATGGCTGGGCCGGACTTACTACCGGTCGAAGTGGCACAACGATTACGCAGTCAGCGTCGAGAGCCGCATCATCGTGAACCCGGACACCTCAAAGAAC CAGTTCAGCCTGCAACTGAATTCCGTGACCCCGAGGACACCGCTGTGTACTACTGCGCCCGCGACCCCGGATACTTCTATGGACTGGACGTCTGGGGACAGGGGACCATGGTCACCGTGTCGAGC GGCGGCGGCGGATCCGGTGGCGGGGGATCCGGCGGTGGCGGATCCAACTTCATGTTGACTCAGCCGCATTCGGTGTCGGAGTCCCCTGGAAAGACAGTCACCATCCCATGTACCCGGAGCAGCGGAA CTATCGCCGACTACTACGTGCAGTGGTACCAGCAGCGCCCTGATTCCTCACCGATCATTGTGATCTATGACGACAACCAGAGGCCCTCCGGGGTGCCGGATAGATTCAGCGGATCCATTGACTCCTC GTCCAACTCTGCCTCACTGACGATTCCGGGCTCAAGACCGAAGATGAAGCGGCCTACTACTGCCAGTCATACGACTCCACCAACCACTGGGTGTTTGGTGGCGGAACTAAGCTGACCGTGCTGGGA SEQ ID NO: 22 Amino acid sequence of CD38 hScFv binder M3812 QVQLQQSGPGLVKPSQTLSLTCVISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWHNDYAVSVESRIIVNPDTSKNQFSLQLNSVTPEDTAVYYCARDPGYFYGLDVWGQGTMVTVSSGG GGSGGGGSGGGGSNFMLTQPHSVSESPGKTVTIPCTRSSGTIADYYVQWYQQRPDSSPIIVIYDDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEAAYYCQSYDSTNHWVFGGGTKLTVLG SEQ ID NO: 23 Nucleotide sequence of CD38 hScFv binder M3815 CAAGTCACACTCAAAGAATCCGGACCGGTGCTCGTGAAGCCTACTGAAACCTTGACCCTGACATGCACCTTTTCCGGGTTCTCCCTGAGCACCTCGGGAGTCGGAGTGGCGTGGATTAGGCAGCCGCCAGGAAAAGACCTCGAGTGGCTCGCCCTTATCTACTGGGACGATGACAAGCGCTACTCACCCTCACTGGAGAGCAGACTGACGATCACCAAGGATACCTCGAAGAACCAAGTGGCCCTGACTATGTCCGACATGGACCCTGTGGACACCGCGACTTACTACTGCGCCCGGGGCGATTACTGGGGACGGCTGGACTACTGGGGACAGGGAACTCTGGTCACCGTGTCCAGCGGCGGCGGTGGTTCAGGGGGTGGCGGCAGCGGGGGGGGCGGATCGGATATCCAGCTTACCCAGTCGCCGTCCTCCCTCTCTGCATCGATTGGCGACCGCGTGACTATTACGTGTCAGGCCTCCGAGGACATCAACAACTACCTGAACTGGTACCAGCAGAAGCCCGGAAAGGCCCCAAAGCTGCTGATCTACGACGCTAGCAACTTGGAAACCGGAGTGCCGTCCCGGTTCTCCGGATCCGGGAGCGGTACCGACTTCACCTTCACCATCAACTCCCTGCAACCCGAGGATATTGCCACCTATTACTGCCAACAGTTCGACAATATGCCTCTGACTTTCGGGGGCGGCACTAAGCTCGAAATCAA Amino acid sequence of SEQ ID NO: 24 CD38 hScFv binder M3815 QVTLKESGPVLVKPTETLTLTCTFSGFSLSTSGVGVAWIRQPPGKDLEWLALIYWDDDKRYSPSLESRLTITKDTSKNQVALTMSDMDPVDTATYYCARGDYWGRLDYWGQGTLVTVSSG GGGSGGGGSGGGGSDIQLTQSPSSLSASIGDRVTITCQASEDINNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTINSLQPEDIATYYCQQFDNMPLTFGGGTKLEIK SEQ ID NO: 25 Nucleotide sequence of CD38 hScFv binder M3815 P CAAGTCACTCTGAAAGAATCCGGTCCCGTGCTCGTCAAACCCACCGAAACCCTGACCCTGACCTGTACTTTCTCCGGATTTTCCCTCTCAACCTCCGGCGTGGGCGTGGCCTGGATTCGGCAGCCTCCCGGAAAGGATTTGGAGTGGCTGGCCCTGATCTACTGGGATGACGATAAGCGCTACTCCCCATCCCTCGAGTCCCGGCTGACTATCACTAAGGACACCTCCAAGAATCAAGTCGCCCTTACTATGTCGGACATGGACCCTGTGGACACCGCTACGTACTACTGCGCTCGGGGAGACTATTGGGGGCGCCTGGACTACTGGGGACAGGGAACCCTCGTGACCGTGTCGTCTGGGGGCGGCGGACCGGGTGGCGGAGCGTCCGGGGGCGGTGGATCGGACATCCAGCTGACACAGAGCCCCAGCAGCCTGAGCGCCTCGATTGGCGACAGAGTGACCATTACGTGCCAGGCATCCGAGGACATCAACAACTACCTGAACTGGTACCAGCAGAAGCCTGGGAAGGCCCCAAAGCTGCTGATCTACGACGCCTCCAACCTGGAAACCGGAGTGCCGTCAAGGTTCAGCGGCTCGGGATCAGGAACCGATTTCACTTTCACCATCAACAGCTTGCAGCCGGAAGATATCGCGACCTACTACTGCCAACAGTTCGACAACATGCCGCTGACTTTCGGTGGCGGGACCAAGCTTGAGATTAAG Amino acid sequence of SEQ ID NO: 26 CD38 hScFv binder M3815P Q V T L K E S G P V L V K P T E T L T L T C T F S G F S L S T S G V G V A W I R Q P P G K D L E W L A L I Y W D D D K R Y S P S L E S R L T I T K D T S K N Q V A L TMSDMDPVDTATYYCARGDYW GRLDYW GQGTLVTVSSGGGGPGGGASG GGGSDI QLTQSPSSLSASIGDRVTITC QASEDI NNYLNWYQQKPGKAPKLLIYD ASNLET GVPSRFSGSGSGTDFTFTINS LQPEDI ATYYCQQFDNMPLTFGGGTKL EIK SEQ ID NO: 27 DNA CD8 transmembrane domain nucleotide sequence atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc accctttact gc SEQ ID NO: 28: Amino acid sequence of CD8 transmembrane domain Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys SEQ ID NO: 29 DNA CD8 hinge domain nucleotide sequence accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg gacttcgcct gtgat SEQ ID NO: 30 Amino acid sequence of CD8 hinge domain Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Amino acid sequence of the hinge region at amino acid positions 118 - 178 of SEQ ID NO:31 CD8.alpha (NCBI RefSeq:NP_001759.3) Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Amino acid sequence of SEQ ID NO:32 human IgG CL sequence Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala Pro Thr Glu Cys Ser SEQ ID NO: 33 Nucleotide sequence of the DNA signaling domain of 4-1BB aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggagaggtgt gaactg SEQ ID NO: 34: Amino acid sequence of the signaling domain of 4-1BB Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu SEQ ID NO: 35 Nucleotide sequence of the DNA signaling domain of CD3-zeta agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc tacgacgccc ttcacatgca ggccctgccc cctcgc CD3 Photo Information Photo Information Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp With Leu His Met Gln Ala Leu Pro Pro Arg Characteristics of 37 ScFv CD19 gataccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcaccatcagttgca gggc aagtca ggacattagt aaatatttaa attggtagca gcagaaacca gatgaactg ttaaactcct gatctaccat acatcagat tacactcagg agtcccatca aggttcagtg gcagtggtc tggaacagat tattctca ccattagcaa cctggagcaa gaagatattg ccacttactt ttgccaacag ggtaatacgc ttccgtacac gttcggaggg gggaccaagc tggagatcac aggtggcggt ggctcgggcg gtggtgggtc gggtggcggc ggatctgagg tgaaactgca ggagtcagga cctggcctgg tggcgccctc acagagcctg tccgtcacat gcactgtctc aggggtctca ttacccgact atggtgtaag ctggattcgc cagcctccac gaaagggtct ggagtggctg ggagtaatat ggggtagtga aaccacatac tataattcag ctctcaaatc cagactgacc atcatcaagg acaactccaa gagccaagtt ttcttaaaaa tgaacagtct gcaaactgat gacacagcca tttactactg tgccaaacat tattactacg gtggtagcta tgctatggac tactggggcc aaggaacctc agtcaccgtc tcctca Amino acid sequence of SEQ ID NO:38 ScFv CD19 Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser SEQ ID NO: 39 Nucleotide sequence of GMCSF leader peptide ATGCTGCTGCTGGTGACCAGCCTGCTGCTGTGCGAACTGCCGCATCCGGCGTTTCTGCTGATTCCG SEQ ID NO: 40 Amino acid sequence of GMCSF leader peptide MLLLVTSLLLCELPHPAFLLIP SEQ ID NO: 41 Nucleotide sequence of TNFRSF19 leader peptide GGCTCTGAAAGTGCTGTTGGAACAAGAAAAGACCTTCTTCACCTTGCTCGTGTTGCTGGGGTACCTGTCCTGCAAAGTCACCTGT SEQ ID NO: 42 Amino acid sequence of TNFRSF19 leader peptide MALKVLLEQEKTFFTLLVLLGYLSCKVTC SEQ ID NO: 43 Nucleotide sequence of CD8 alpha leader peptide atggcgctgccggtgaccgcgctgctgctgccgctggcgctgctgctgcatgcggcgcgc ccg SEQ ID NO: 44: Amino acid sequence of CD8 alpha leader peptide MALPVTALLLPLALLLHAARP SEQ ID NO: 45 Nucleotide sequence of CD28 costimulatory domain CGGTCGAAGAGGTCCAGACTCTTGCACTCCGACTACATGAACATGACTCCTAGAAGGCCCGGACCCACTAGAAAGCACTACCAGCCGTACGCCCCTCCTCGGGATTTCGCCGCATACCGG TCC SEQ ID NO: 46: Amino acid sequence of CD28 costimulatory domain RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS SEQ ID NO: 47 Nucleotide sequence of CD3 zeta activation domain AGAGTGAAGTTCAGCCGCTCAGCCGATGCACCGGCCTACCAGCAGGGACAGAACCAGCTCTACAACGAGCTCAACCTGGGTCGGCGGGAAGAATATGACGTGCTGGACAAACGGCGCGGCAGAGATCCGGAGATGGGGGGAAAGCCGAGGAGGAAGAACCCTCAAGAG GGCCTGTACAACGAACTGCAGAAGGACAAGATGGCGGAAGCCTACTCCGAGATCGGCATGAAGGGAGAACGCCGGAGAGGGAAGGGTCATGACGGACTGTACCAGGGCCTGTCAACTGCCACTAAGGACACTTACGATGCGCTCCATATGCAAGCTTTGCCCCCGCGG SEQ ID NO: 48 Amino acid sequence of CD3 zeta activation domain RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGER RRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 49 Nucleotide sequence of TNFRSF19 hinge and transmembrane domain (transmembrane domain underlined) GCGGCCGCGGTCGGATTCCAAGACATGGAATGCGTGCCCTGCGGCGACCCGCCACCTCCTTACGAGCCGCACTGCGCATCGAAGGTCAACCTCGTGAAGATCGCGAGCACCGCGTCCTCACCCCGGGATACTGCTCTG GCCGCCGTGATTTGTTCCGCCTTGGCCACCGTGCTTCTGGCCCTGCTGATCCTCTGTGTGATC SEQ ID NO: 50 Amino acid sequence of TNFRSF19 hinge and transmembrane domain (transmembrane domain underlined) AAAVGFQDMECVPCGDPPPPY EPHCASKVNLVKIASTASS PRDTA L A A V I C S A L A T V L L A L L I L C V I SEQ ID NO: 51 nucleotide sequence of TNFRSF19 transmembrane domain GCCGCCGTGATTTGTTCCGCCTTGGCCACCGTGCTTCTGGCCCTGCTGATCCTCTGTGTGATC SEQ ID NO: 52 Amino acid sequence of TNFRSF19 transmembrane domain AAVICSALATVLLALLILCVI SEQ ID NO: 53 Nucleotide sequence of TNFRSF19 hinge domain GCGGCCGCGGTCGGATTCCAAGACATGGAATGCGTGCCCTGCGGCGACCCGCCACCTCCTTACGAGCCGCACTGCGCATCGAAGGTCAACCTCGTGAAGATCGCGAGCACCGCGTCCTCACCCCGGGATACTGCTCTG SEQ ID NO: 54: Amino acid sequence of TNFRSF19 hinge domain AAAVGFQDMECVPCGDPPPPY EPHCASKVNLVKIASTASSPR DTAL SEQ ID NO: 55 Nucleotide sequence of truncated TNFRSF19 hinge domain TACGAGCCTCACTGCGCCAGCAAAGTCAACTTGGTGAAGATCGCGAGCACTGCCTCGTCCCCTCGGGACACTGCTCTGGC SEQ ID NO: 56: Amino acid sequence of truncated TNFRSF19 hinge domain YEPHCASKVNLVKIASTASSP RDTAL SEQ ID NO: 57 Nucleotide sequence of CD8a hinge domain fused to TNFRSF19 transmembrane domain (transmembrane sequence underlined) GCGGCCGCGCCCGCCCCTCGGCCCCCGACTCCTGCCCGACGATCGCTTCCCAACCTCTCTCGCTGCCCGGAAGCATGCCGGCCCGCCGCCGGTGGCGCTGTCCACACTCGCGGACTGGACTTTGATACCGCACTG GCGGCCGTGATCTGTAGCGCCCTGGCCACCGTGCTGCTGGCGCTGCTCATCCTTTGCGTGATCTACTGCAAGCGGCAGCCTAGG SEQ ID NO: 58 Amino acid sequence of the CD8a hinge domain fused to the TNFRSF19 transmembrane domain (transmembrane sequence underlined) AAAPAPRPPTPAPTIASQPLS LRPEACRPAAGGAVHTRGLDF DTAL A A V I C S A L A T V L L A L L I L C V I Y C K R Q P R SEQ ID NO: 59 Nucleotide sequence of CD28 costimulatory domain CGGTCGAAGAGGTCCAGACTCTTGCACTCCGACTACATGAACATGACTCCTAGAAGGCCCGGACCCACTAGAAAGCACTACCAGCCGTACGCCCCTCCTCGGGATTTCGCCGCATACCGGTCC SEQ ID NO: 60 Amino acid sequence of CD28 costimulatory domain RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS SEQ ID NO: 61 Nucleotide sequence of CD3 zeta version 2 cgcgtgaaatttagccgcagcgcggatgcgccggcgtatcagcagggccagaaccagctg tataacgaactgaacctgggccgccgcgaagaatatgatgtgctggataaacgccgcggc cgcgatccggaaatgggcggcaaaccgcgccgcaaaaacccgcaggaaggcctgtataac gaactgcagaaagataaaatggcggaagcgtatagcgaaattggcatgaaaggcgaacgc cgccgcggcaaaggccatgatggcctgtatcagggcctgagcaccgcgaccaaagatacc tatgatgcgctgcatatgcaggcgctgccgccgcgc SEQ ID NO: 62 Amino acid sequence of CD3 zeta version 2 RVKFSRSADAPAYQQGQNQLY NELNLG RREEYDVLDKRRGRDPEMGGK PRRKNP QEGLYNELQKDKMAEAYSEIG MKGERRRGKGHDGLYQGLSTA TKDTYDALHMQA LPPR SEQ ID NO: 63 Nucleotide sequence of furin P2A furin CGCGCGAAACGCAGCGGCAGCGGCGCGACCAACTTTAGCCTGCTGAAACAGGCGGGCGAT GTGGAAGAAAACCCGGGCCCGCGAGCAAAGAGG SEQ ID NO: 64 Amino acid sequence of furin P2A furin (furin sequence underlined) RAKR SGSGATNFSLLKQAGDVEENPGP RAKR SEQ ID NO: 65 Nucleotide sequence of furin T2A AGAGCTAAACGCTCTGGGTCTGGTGAAGGACGAGGTAGCCTTCTTACGTGCGGAGACGTGGAGGAAAACCCAGGACCC SEQ ID NO: 66 Amino acid sequence of furin T2A (furin sequence underlined) RAKR SGSGEGRGSLLTCGDVEENPGP SEQ ID NO: 67 Nucleotide sequence of truncated EGFR (tEGFR) tag SEQ ID NO: 68: Amino acid sequence of truncated EGFR (tEGFR) tag RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCK ATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM SEQ ID NO: 69 Nucleotide sequence of CD38 hScFv binder M3816 CAAGTGCAACTCCAAGAATCCGGTCCTGGCCTCGTCAAACCTTCCGAAACACTGTCCCTGACCTGTACCGTGTCCGGAGGGTCGATTAGCTCGTCGTCATACTACTGGGGATGGATCAGACAGCCGCCGGGAAAGGGACTCGAGTGGATCGGGTCCATCTACTACTCGGGTAGCACTTACTACAACCCGTCGCTGAAGTCCCGCGTGACTATTTCCGTGGACACCTCCAAGAACCAGTTCAGCCTGAAGCTGAGCTCCGTGACCGCTGCCGATACCGCAGTGTACTACTGCGCGCGCCGGGGAAGCATCCGCGCCTTTGACATCTGGGGACAGGGAACTATGGTCACGGTGTCCAGCGGCGGTGGCGGATCTGGCGGCGGCGGATCCGGGGGGGGGGGAAGCCAGTCAGTGCTGACTCAGCCGCCTTCGGTGTCCGAGGCGCCCGGCCAGAGGGTCACCATTTCCTGCTCTGGATCGTCCAGCAACATTGGCAACAACGCCGTCAACTGGTACCAGCAGCTGCCCGGGGAAGCCCCCAAGCTGTTGATCTACTACGACGAGTATCTGCCAAGCGGAGTGTCAGACAGATTCTCCGCGTCGAAGTCCGGCACCTCCGCCTCACTTGCAATCTCCGGCCTGCGGTCCGAGGACGAAGCCGATTACTATTGCGCCGCCTGGGACGATAATCTGTCCGGTTGGGTGTTCGGCGGTGGAACCCAGCTTACCGTGCTCGGA Amino acid sequence of SEQ ID NO: 70, CD38 hScFv binder M3816 QVQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARRGSIRAFDIWGQGTMVTVSSGGG GSGGGGSGGGGSQSVLTQPPSVSEAPGQRVTISCSGSSSNIGNNAVNWYQQLPGEAPKLLIYYDEYLPSGVSDRFSASKSGTSASLAISGLRSEDEADYYCAAWDDNLSGWVFGGGTQLTVLG SEQ ID NO: 71 Nucleotide sequence of CD38 hScFv binder M3817 CAAGTGCAACTCCAAGAATCCGGTCCTGGTCTGGTCAAACCCTCCGAAACTCTGTCCCTGACGTGCACCGTGTCGGGATACTCCATTTCCTCCGGATACTACTGGGGTTGGATCCGCCAGCCTCCGGGAAAAGGCCTGGAATGGGTCGGAACCATCGGGAAGGACG GATCCACTTTCTATTCCCCGTCGCTGAAGTCACGGATCACCATTAGCCAGGACACCTCCAAGAACCAGTTCAGCCTGAAGCTGAACTCCGTGAACGCCGCGGACACTGCCGTCTACTACTGTGCGAAGCACAAGTGGTCCTTCGACTCCGGGAACGATTACTTCGACCACTGGGGCCAGGGGACCCTCGTGACCGTGTCGTCCGGCGGCGGGGGATCCGGTGGCGGGGGAAGCGGCGGCGGCGGATCAGACATTCAGCTGACCCAGTCTCCCTCATCCCTGTCGGCTAGCGTGGGCGATAGAGTGACCATCACATGCCAGGCATCGCAGGACATTTCGAACTATCTGAACTGGTACCAGCAGAAGCCTGGAAAGGCCCCGAAGCTTTTGATCTACGACGCCAGCAACCTGGAGACTGGAGTGCCCAGCCGGTTCAGCGGATCGGGATCCGGTACCGATTTCACCTTTACCATCTCCTCACTGCAACCAGAGGATATCGCCACCTACTACTGCCAGCAGTACCAGAATCTCCCGCTCACTTTCGGACAAGGGACTAGGCTTGAGATCAAG Amino acid sequence of SEQ ID NO: 72 CD38 hScFv binder M3817 QVQLQESGPGLVKPSETLSLTCTVSGYSISSGYYWGWIRQPPGKGLEWVGTIGKDGSTFYSPSLKSRITISQDTSKNQFSLKLNSVNAADTAVYYCAKHKWSFDSGNDYFDHWGQGTLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYQNLPLTFGQGTRLEIK SEQ ID NO: 73 Nucleotide sequence of CD38 hScFv binder M8DR H_L SEQ ID NO: 74 Amino acid sequence of CD38 hScFv binder M8DR H_L EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSSASGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIK SEQ ID NO: 75 Nucleotide sequence of CD38 hScFv binder M8DR L_H SEQ ID NO: 76 Amino acid sequence of CD38 hScFv binder M8DR L_H EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIKGGGGSGGGGSGGGS EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSS SEQ ID NO: 77 LTG2089 (hScFv aCD38 CD8™ 4-1BB Nucleotide sequence of CD3 zeta ATGCTGCTGCTGGTGACCAGCCTGCTGCTGTGCGAACTGCCGCATCCGGCGTTTCTGCTGATTCCGGAAGTGCAATTGGTCCAAAGCGGTGCAGAAGTCAAGAAACCTGGTTCGAGCGTGAAAGTGTCCTGCAAGGCCTCTGGCGGCACCTTCTCATCCTACGCCATTAGCTGGGTCCGCCAAGCCCCGGGCCAGGGACTTGAGTGGATGGGCGGAATCATCCCTATCTTCGGGACCGCC SEQ ID NO: 78 LTG2089 (hScFv aCD38 CD8™ 4-1BB CD3 zeta amino acid sequence MLLLVTSLLLCELPHPAFLLIPEVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARAPY DDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSNFMLTQPHSVSESPGKTVTISCTRSSGSIASNYVQWYQQRPGSAPTTVIYEDNQRPSGVPGRFSGSIDTSSNSASLTISGLETEDEAVYYCQ SYDSGFPYAVFGGGTQLTVLGAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 79 Nucleotide sequence of human IgG4 hinge GAGAGCAAATACGGGCCGCCATGTCCCCCGTGTCCG SEQ ID NO: 80 Amino acid sequence of human IgG4 hinge ESKYGPPCPPCP SEQ ID NO: 81 Nucleotide sequence of human IgG4 CH2 domain GCACCACCAGTTGCTGGCCCTAGTGTCTTCTTGTTCCCTCCCAAGCCCAAAGACACCTTGATGATTTCCAGAACTCCTGAGGTTACCTGCGTTGTCGTAGATGTTTCTCAGGAGGACCCAGAGGTCCAATTTAACTGGTACGTTGATGGGGTGGAAGTTCACA ATGCGAAGACAAAGCCGCGGGAAGAACAATTTCAGTCCACTTACCGGGTTGTCAGCGTTCTGACGGTATTGCATCAAGACTGGCTTAATGGAAAGGAATATAAGTGTAAGGTGTCCAACAAAGGTTTGCCGAGCAGTATTGAGAAGACCATATCAAAGGCGAAG SEQ ID NO: 82 Amino acid sequence of human IgG4 CH2 domain APPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV DGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKA K SEQ ID NO: 83 Nucleotide sequence of human IgG4 CH3 domain GGGCAGCCGCGCGAGCCACAAGTTTACACTTTGCCGCCATCTCAAGAGGAAATGACTAAAAACCAGGTATCCTTGACATGCCTCGTAAAAGGATTTTATCCATCTGATATTGCTGTGGAATGGGAGTCTAACGGGCAGCCGGAAAATAATTACAAAACTA CACCACCTGTGCTCGATTCAGATGGAAGTTTCTTTCCTTTACAGTAGACTTACGGTGGACAAATCTAGGTGGCAGGAAGGGAATGTGTTTAGTTGTAGTGTAATGCACGAGGCACTTCATAACCACTATACACAGAAGTCACTGAGTTTGAGTCTTGGCAAA SEQ ID NO: 84 Amino acid sequence of human IgG4 CH3 domain GQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO: 85 Nucleotide sequence of human IgG4 hinge CH2 CH3 domain GAGAGCAAATACGGGCCGCCATGTCCCCCGTGTCCGGCACCACCAGTTGCTGGCCCTAGTGTCTTCTTGTTCCCTCCCCAAGCCCAAAGACACCTTGATGATTTCCAGAACTCCTGAGGTTACCTGCGTTGTCGTAGATGTTTCTCAGGAGGACCCAGAGG TCCAATTTAACTGGTACGTTGATGGGGTGGAAGTTCACAATGCGAAGACAAAGCCGCGGGAAGAACAATTTCAGTCCACTTACCGGGTTGTCAGCGTTCTGACGGTATTGCATCAAGACTGGCTTAATGGA AAGGAATATAAGTGTAAGGTGTCCAACAAAGGTTTGCCGAGCAGTATTGAGAAGACCATATCAAAGGCGAAGGGGCAGCCGCGCGAGCCACAAGTTTACACTTTGCCGCCATCTCAAGAGGAAATGACTAA AAACCAGGTATCCTTGACATGCCTCGTAAAAGGATTTTATCCATCTGATATTGCTGTGGAATGGGAGTCTAACGGGCAGCCGGAAAATAATTACAAAACTACACCACCTGTGCTCGATTCAGATGGAAGTT TCTTCCTTTACAGTAGACTTACGGTGGACAAATCTAGGTGGCAGGAAGGGAATGTGTTTAGTTGTAGTGTAATGCACGAGGCACTTCATAACCACTATACACAGAAGTCACTGAGTTTGAGTCTTGGCAAA SEQ ID NO: 86 Amino acid sequence of human IgG4 hinge CH2 CH3 domain ESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIE KTISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO: 87 LTG2090 (hScFv aCD38 CD8™ 4-1BB Nucleotide sequence of CD3 zeta SEQ ID NO: 88 LTG2090 (hScFv aCD38 CD8™ 4-1BB CD3 zeta amino acid sequence MLLLVTSLLLCELPHPAFLLIPEVQLVESGGGSVQPGGSLRLSCAASEFTFSSYWMHWVRQAPGKGLEWVAVISYDGAKKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDDG SGGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTHTPLSLSVTPGQPASISCKSSQSLLHSDGKTYLYWYLQKPGQPPQLLIYEVSNRFSGVPDRYSGSGSGTDFTLKISRVEAEEDVGVY YCMQSIQLPLTFGGGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 89 LTG2091 (hScFv aCD38 CD8™ 4-1BB Nucleotide sequence of CD3 zeta ATGCTGCTGCTGGTGACCAGCCTGCTGCTGTGCGAACTGCCGCATCCGGCGTTTCTGCTGATTCCGGAAGTGCAATTGGTGCAGAGCGGTGGAGGACTTGTGAAACCTGGTGGATCCCTGAGACTTTCCTGTGCCGCTTCGGGCTTCACCTTCTCGGACTACTACATGTCCTGGATTCGCCAGGCCCCTGGAAAGGGACTGGAATGGGTGTCATACATCAGCTCCTCCGGTTCCACCATCTACTATGCCGATTCCGTGAAGGGCAGATTCACCATCTCGCGCGACAACGCCAAGAACACTCTCTATCTGCAAATGAACTCACTGCGGGCTGAGGACACCGCGGTCTACTACTGCGCCCGGGACCTCAGCGGAAAGTCCAGCGGATGGTCCCATTACTTCGATTACTGGGGACAGGGAACCCTGGTCACCGTGTCCAGCGGCGGGGGGGGCTCGGGTGGCGGCGGCTCCGGCGGCGGCGGGAGCAACTTCATGCTGACTCAGCCCCACTCCGTGTCCGAGAGCCCGGGAAAGACCGTGACTATTTCGTGCACACGGTCCTC CGGGAGCATTGCGAACAACTACGTGCAGTGGTACCAGCAGCGGCCCGATAGGGCCCCAACCACTGTGATCTACGAAGATGACCAGCGGCCGTCTGGAGTCCCGGACCGCTTCTCGGGGTCCATCGACTCATCATCCAATTCCGCATCGCTGACGATCAGCGGACTGAAGATCGAGGACGAAGCCGATTACTACTGCCAGTCCTACGACGGCACCAACTGGGTCTTTGGGGGTGGAACCAAGCTGACTGTGCTCGGAGCGGCCGCAACTACCACCCCTGCCCCTCGGCCGCCGACTCCGGCCCCAACCATCGCAAGCCAACCCCTCTCCTTGCGCCCCGAAGCTTGCCGCCCGGCCGCGGGTGGAGCCGTGCATACCCGGGGGCTGGACTTTGCCTGCGATATCTACATTTGGGCCCCGCTGGCCGGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACCCTTTACTGCAAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAGCCGTTCATGCGGCCCGTGCAGACGACTCAGGAAGAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGAGGGGGGATGCGAACTGCGCGTCAAGTTCTCACGGTCCGCCGACGCCCCCGCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGG Sequence number 90 LTG2091 (hScFv aCD38 CD8 TM 4-1BB CD3 zeta amino acid sequence MLLLVTSLLLCELPHPAFLLIPEVQLVQSGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARDLSG KSSGWSHYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSNFMLTQPHSVSESPGKTVTISCTRSSGSIANNYVQWYQQRPDRAPTTVIYEDDQRPSGVPDRFSGSIDSSSNSASLTISGLKIEDEAD YYCQSYDGTNWVFGGGTKLTVLGAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCS CRFPEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 91 LTG2092 (hScFv aCD38 CD8™ 4-1BB Nucleotide sequence of CD3 zeta SEQ ID NO: 92 LTG2092 (hScFv aCD38 CD8™ 4-1BB CD3 zeta amino acid sequence MLLLVTSLLLCELPHPAFLLIPEVQLVESGGGLVQPGRSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAH LRFGYGMDVWGQGTTVTVSSGGGGSGGGGSGGGSQTVVTQEPSLTVSPGGTVTLTCASSTGAVTSDHYPCWFQQKPGHPPRALVYSTDTIHSWTPARFSGSLLGGKAALTVSGVQPEDE ADYYCLLYYGGARVFGGGTQLTVLGAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQ EEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTY DALHMQALPPR SEQ ID NO: 93 LTG2093 (hScFv aCD38 CD8™ 4-1BB Nucleotide sequence of CD3 zeta SEQ ID NO: 94 LTG2093 (hScFv aCD38 CD8™ 4-1BB CD3 zeta amino acid sequence MLLLVTSLLLCELPHPAFLLIPQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREYS SSRVDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSSYELTQPPSVSVSPGQTATITCSGDDLGSKYVCWYQQKPGHSPVLIIYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQ VWDSSSSDHAVFGGGGTQLTVLGAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 95 LTG2094 (hScFv aCD38 CD8™ 4-1BB Nucleotide sequence of CD3 zeta AGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGG SEQ ID NO: 96 LTG2094 (hScFv aCD38 CD8™ 4-1BB CD3 zeta amino acid sequence MLLLVTSLLLCELPHPAFLLIPEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVADIKDDGSERYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARD VWAGMDVWGQGTTVTVSSGGGGSGGGGSGGGSDIQLTQSPSFLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFSFTISSLQPEDIATYYCQ QTYSPPITFGQGTRLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRF PEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 97 LTG2095 (hScFv aCD38 CD8™ 4-1BB Nucleotide sequence of CD3 zeta SEQ ID NO: 98 LTG2095 (hScFv aCD38 CD8™ 4-1BB CD3 zeta amino acid sequence MLLLVTSLLLCELPHPAFLLIPQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANHAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAFMM VPEYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGEAPKRLIYAASTLQNGVPSRFSGSGSGTDFTLTINSLQPEDFATYYC QQYNSYPYTFGQGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 99 LTG2096 (hScFv aCD38 CD8™ 4-1BB Nucleotide sequence of CD3 zeta ATGCTGCTGCTGGTGACCAGCCTGCTGCTGTGCGAACTGCCGCATCCGGCGTTTCTGCTGATTCCGAAAGTGCAACTCCAAGAAAGCGGCCCCGGACTTGTCAAACCGTCCCAGACTCTCTCCCTGACCTGTGCCGTCAGCGGTGGCTCAATTTCGAGCGGTGGCTACTACTGGTCCTGGATCAGGCAGCATCCGGGAAAGGGCTTGGAGTGGATTGGATACATCTACTACTCCGGGTCCACCTACTACAATCCCTCGCTGAAGTCGAGAGTCACCATCAGCGTGGACACCTCCAAGAACCAGTTTAGCCTGAAGCTGTCCTCAGTGACCGCAGCTGACACCGCCGTGTACTACTGCGCCCGCGGCGGAGTGATGGGCGGAGACTTCGACTACTGGGGACAGGGAACCCTGGTCACTGTGTCCAGCGGGGGGGGGGGATCCGGCGGCGGCGGTTCCGGTGGCGGAGGGTCCCAGTCAGTGCTGACTCAGCCACCTTCCGTGTCTGTGTCGCCCGGACAAACCGCCAGCATCACGTGCTCCGGCGACAACCTCGGTGATCA CTATGTGTGCTGGTACCAACAGCGGCCGGGGCAGTCACCGGTGCTGATTATGTACGAGGATACTAAGCGCCCTTCCGGAATCCCTGACCGGTTCTCGGGAAGCAACTCCGGAAACACCGCCACCCTGACCATCTCCGGAACCCAGACAATGGATGAAGCGGACTACTATTGCGTGGCGTGGGACGATTCGCTGTCCGGCTGGGTGTTCGGCGGGGGAACTCAGCTGACTGTGCTCGGAGCGGCCGCAACTACCACCCCTGCCCCTCGGCCGCCGACTCCGGCCCCAACCATCGCAAGCCAACCCCTCTCCTTGCGCCCCGAAGCTTGCCGCCCGGCCGCGGGTGGAGCCGTGCATACCCGGGGGCTGGACTTTGCCTGCGATATCTACATTTGGGCCCCGCTGGCCGGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACCCTTTACTGCAAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAGCCGTTCATGCGGCCCGTGCAGACGACTCAGGAAGAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGAGGGGGGATGCGAACTGCGCGTCAAGTTCTCACGGTCCGCCGACGCCCCCGCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGG Amino acid sequence of SEQ ID NO: 100, LTG2096 (hScFv aCD38 CD8 TM 4-1BB CD3 zeta) MLLLVTSLLLCELPHPAFLLIPKVQLQESGPGLVKPSQTLSLTCAVSGGSISSGGYYWSWIRQHPGKGLEWIGYIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGG VMGGDFDYWGQGTLVTVSSGGGGSGGGGSGGGSQSVLTQPPSVSVSPGQTASITCSGDNLGDHYVCWYQQRPGQSPVLIMYEDTKRPSGIPDRFSGSNSGNTATLTISGTQTMDEADYYCVA WDDSLSGWVFGGGTQLTVLGAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 101 Nucleotide sequence of LTG2097 (hScFv aCD38 CD8™ 4-1BB CD3 Zeta) SEQ ID NO: 102 Amino acid sequence of LTG2097 (hScFv aCD38 CD8™ 4-1BB CD3 zeta) MLLLVTSLLLCELPHPAFLLIPQLQLQESGPGLVKPSQTLSLTCTVSGGSISSGGYYWSWIRQHPGKGLEWIGYIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCA RDQSVADPGGGYYYYGMDVWGQGTTVTVSSGGGGGSGGGGSGGGGSQSVLTQPPSVSVAPGQTASISCGGNDFGSRSVSWYHQKPGQAPVLVIYDDNDRPSGIPERFSGSTSGDTATLTIS RVEVGDEADYYCQVWDDDDSDHWVFGGGTKLTVLGAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQP FMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQG LSTATKDTYDALHMQALPPR SEQ ID NO: 103 Nucleotide sequence of LTG2098 (hScFv aCD38 CD8™ 4-1BB CD3 zeta) SEQ ID NO: 104 Amino acid sequence of LTG2098 (hScFv aCD38 CD8™ 4-1BB CD3 zeta) MLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVKPSQTLSLTCVISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWHNDYAVSVESRIIVNPDTSKNQFSLQLNSVTPEDTAVYYCARD PGYFYGLDVWGQGTMVTVSSGGGGSGGGGSGGGGSNFMLTQPHSVSESPGKTVTIPCTRSSGTIADYYVQWYQQRPDSSPIIVIYDDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEAAY YCQSYDSTNHWVFGGGTKLTVLGAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCS CRFPEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 105 Nucleotide sequence of LTG2099 (hScFv aCD38 CD8™ 4-1BB CD3 Zeta) AGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGG SEQ ID NO: 106 Amino acid sequence of LTG2099 (hScFv aCD38 CD8™ 4-1BB CD3 zeta) MLLLVTSLLLCELPHPAFLLIPQVTLKESGPVLVKPTETLTLTCTFSGFSLSTSGVGVAWIRQPPGKDLEWLALIYWDDDKRYSPSLESRLTITKDTSKNQVALTMSDMDPVDTATYYCARG DYWGRLDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASIGDRVTITCQASEDINNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTINSLQPEDIATYYC QQFDNMPLTFGGGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 107 Nucleotide sequence of LTG2501 (hScFv aCD38 CD8™ 4-1BB CD3 Zeta) SEQ ID NO: 108 Amino acid sequence of LTG2501 (hScFv aCD38 CD8™ 4-1BB CD3 zeta) MLLLVTSLLLCELPHPAFLLIPQVTLKESGPVLVKPTETLTLTCTFSGFSLSTSGVGVAWIRQPPGKDLEWLALIYWDDDKRYSPSLESRLTITKDTSKNQVALTMSDMDPVDTATYYCARG DYWGRLDYWGQGTLVTVSSGGGGPGGASGGGGSDIQLTQSPSSLSASIGDRVTITCQASEDINNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTINSLQPEDIATYYC QQFDNMPLTFGGGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 109 Nucleotide sequence of LTG2502 (hScFv aCD38 CD8™ 4-1BB CD3 Zeta) ATGCTGCTGCTGGTGACCAGCCTGCTGCTGTGCGAACTGCCGCATCCGGCGTTTCTGCTGATTCCGCAAGTGCAACTCCAAGAATCCGGTCCTGGCCTCGTCAAACCTTCCGAAACACTGTCCCTGACCTGTACCGTGTCCGGAGGGTCGATTAGCTCGTCGTCATACTACTGGGGATGGATCAGACAGCCGCCGGGAAAGGGACTCGAGTGGATCGGGTCCATCTACTACTCGGGTAGCACTTACTACAACCCGTCGCTGAAGTCCCGCGTGACTATTTCCGTGGACACCTCCAAGAACCAGTTCAGCCTGAAGCTGAGCTCCGTGACCGCTGCCGATACCGCAGTGTACTACTGCGCGCGCCGGGGAAGCATCCGCGCCTTTGACATCTGGGGACAGGGAACTATGGTCACGGTGTCCAGCGGCGGTGGCGGATCTGGCGGCGGCGGATCCGGGGGGGGGGGAAGCCAGTCAGTGCTGACTCAGCCGCCTTCGGTGTCCGAGGCGCCCGGCCAGAGGGTCACCATTTCCTGCTCTGGATCGTCCAGCAACATTGGCAA CAACGCCGTCAACTGGTACCAGCAGCTGCCCGGGGAAGCCCCCAAGCTGTTGATCTACTACGACGAGTATCTGCCAAGCGGAGTGTCAGACAGATTCTCCGCGTCGAAGTCCGGCACCTCCGCCTCACTTGCAATCTCCGGCCTGCGGTCCGAGGACGAAGCCGATTACTATTGCGCCGCCTGGGACGATAATCTGTCCGGTTGGGTGTTCGGCGGTGGAACCCAGCTTACCGTGCTCGGAGCGGCCGCAACTACCACCCCTGCCCCTCGGCCGCCGACTCCGGCCCCAACCATCGCAAGCCAACCCCTCTCCTTGCGCCCCGAAGCTTGCCGCCCGGCCGCGGGTGGAGCCGTGCATACCCGGGGGCTGGACTTTGCCTGCGATATCTACATTTGGGCCCCGCTGGCCGGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACCCTTTACTGCAAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAGCCGTTCATGCGGCCCGTGCAGACGACTCAGGAAGAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGAGGGGGGATGCGAACTGCGCGTCAAGTTCTCACGGTCCGCCGACGCCCCCGCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGG Amino acid sequence of SEQ ID NO: 110, LTG2502 (hScFv aCD38 CD8 TM 4-1BB CD3 zeta) MLLLVTSLLLCELPHPAFLLIPQVQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARRG SIRAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSQSVLTQPPSVSEAPGQRVTISSCSGSSSNIGNNAVNWYQQLPGEAPKLLIYYDEYLPSGVSDRFSASKSGTSASLAISGLRSEDEADYYCA AWDDNLSGWVFGGGTQLTVLGAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 111 Nucleotide sequence of LTG2503 (hScFv aCD38 CD8™ 4-1BB CD3 Zeta) SEQ ID NO: 112 Amino acid sequence of LTG2503 (hScFv aCD38 CD8™ 4-1BB CD3 zeta) MLLLVTSLLLCELPHPAFLLIPQVQLQESGPGLVKPSETLSLTCTVSGYSISSGYYWGWIRQPPGKGLEWVGTIGKDGSTFYSPSLKSRITISQDTSKNQFSLKLNSVNAADTAVYYCAK HKWSFDSGNDYFDHWGQGTLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQP EDIATYYCQQYQNLPLTFGQGTRLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQT TQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKD TYDALHMQALPPR SEQ ID NO: 113 Nucleotide sequence of LTG2525 (hScFv aCD38 CD8™ 4-1BB CD3 Zeta) SEQ ID NO: 114: Amino acid sequence of LTG2525 (hScFv aCD38 CD8™ 4-1BB CD3 zeta) MLLLVTSLLLCELPHPAFLLIPEVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKI LWFGEPVFDYWGQGTLVTVSSASGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVY YCQQRSNWPPTFGQGTKVEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 115 Nucleotide sequence of LTG2526 (hScFv aCD38 CD8™ 4-1BB CD3 Zeta) CAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGG SEQ ID NO: 116 Amino acid sequence of LTG2526 (hScFv aCD38 CD8™ 4-1BB CD3 zeta) MLLLVTSLLLCELPHPAFLLIPEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQG TKVEIKGGGGSGGGGSGGGSEVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILW FGEPVFDYWGQGTLVTVSSASAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

Claims

1. 1. An isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), comprising at least one extracellular antigen-binding domain comprising a CD38 antigen-binding domain, at least one transmembrane domain, and at least one intracellular signaling domain, the at least one extracellular antigen-binding domain comprising 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 by a linker or spacer domain.

5. 5. The isolated nucleic acid molecule of claim 4, wherein the encoded linker or spacer domain is derived from the extracellular domain of CD8, TNFRSF19, or CD28 and is linked to a transmembrane domain.

6. 2. 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. 7. The isolated nucleic acid molecule of claim 6, wherein the leader nucleotide sequence comprises a nucleotide sequence comprising SEQ ID NO:13, which encodes the leader amino acid sequence of SEQ ID NO:14, or SEQ ID NO:39, which encodes the leader amino acid sequence of SEQ ID NO:40, or SEQ ID NO:41, which encodes the leader amino acid sequence of SEQ ID NO:42, or SEQ ID NO:43, which encodes the leader amino acid sequence of SEQ ID NO:

44.

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

9. 2. 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 thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.

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 located C-terminal to the CD3 zeta intracellular domain.

12. The at least one encoded intracellular signaling domain may comprise a co-stimulatory domain, 2. The isolated nucleic acid molecule of claim 1, comprising a primary signaling domain, a primary signaling domain, or any combination thereof.

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

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

15. The CAR of claim 14, comprising at least one extracellular antigen-binding domain comprising a 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, or 72, at least one transmembrane domain, and at least one intracellular signaling domain.

16. The CAR of claim 15, wherein the CD38 antigen-binding domain comprises at least a 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. 16. The CAR of claim 15, wherein the transmembrane domain comprises a transmembrane domain of a protein comprising the alpha, beta or zeta chain of the T-cell receptor, CD8, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154 and TNFRSF19, or any combination thereof.

19. 19. The CAR of claim 18, wherein the CD8 transmembrane domain comprises an amino acid sequence of SEQ ID NO:27, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity 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 comprising a 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, or 72, and the at least one intracellular signaling domain, or both, are connected to the transmembrane domain by a linker or spacer domain.

21. 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 linked to a transmembrane domain.

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

23. The at least one intracellular signaling 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. The CAR of claim 22, comprising a stimulatory domain.

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

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

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

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

28. A cell comprising the vector of claim 24.

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

30. The T cell is CD8 ＋ The cell of claim 28 which is a T cell.

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

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

33. 11. A method for generating a population of RNA engineered cells, comprising the step of introducing in vitro transcribed or synthetic RNA into cells, said RNA comprising the nucleic acid molecule of claim 1.

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

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

36. 1. A pharmaceutical composition comprising an anti-tumor effective amount of a population of human T cells, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), the CAR comprising at least one extracellular antigen-binding domain, including a 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, or 72, at least one linker domain, at least one transmembrane domain, at least one intracellular signaling domain, and wherein the T cells are from a human with cancer.

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

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

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

40. 40. The pharmaceutical composition of claim 39, wherein the leukemia is acute myeloid leukemia (AML), blastic plasmacytoid dendritic cell neoplasm (BPDCN), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), acute lymphoblastic T-cell leukemia (T-ALL), or acute lymphoblastic B-cell leukemia (B-ALL).

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

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

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

44. 1. A method of treating a mammal having a disease, disorder or condition associated with elevated expression of a tumor antigen, comprising administering to the subject an anti-tumor effective amount of a pharmaceutical composition comprising a population of T cells, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), the CAR comprising at least one extracellular antigen-binding domain, including a 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, or 72, at least one linker or spacer domain, at least one transmembrane domain, at least one intracellular signaling domain, and wherein the T cells are T cells of a subject with cancer.

45. 1. A method of treating cancer in a subject in need thereof, comprising the step of administering to the subject an anti-tumor effective amount of a pharmaceutical composition comprising a population of T cells, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises at least one extracellular antigen-binding domain, including a 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, or 72, at least one linker or spacer domain, at least one transmembrane domain, at least one intracellular signaling domain, and wherein the T cells are T cells of a subject with cancer.

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

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

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

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