Compositions and methods for treating cancer with Anti-CD19 immunotherapy

Abstract

To provide use of cells for producing a pharmaceutical composition that elicits antitumor immunity in a human subject.SOLUTION: The present invention provides use of an isolated T cell or natural killer cell comprising a vector comprising a nucleic acid encoding a chimeric antigen receptor (CAR) that includes at least one extracellular antigen binding domain comprising a CD19 antigen binding domain having a specific amino acid sequence, at least one transmembrane domain, at least one intracellular signaling domain, and at least one linker or spacer domain.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 62 / 559,297, filed September 15, 2017, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy created on September 13, 2018 is named SequenceListing.txt and is 93.7 kilobytes in size.

[0003] 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.

[0004] Field of the Disclosure The present application relates to the field of cancer, in particular to CD19 antigen-binding domains and chimeric antigen receptors (CARs) comprising such CD19 antigen-binding domains, and methods of use thereof. [Background technology]

[0005] 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.

[0006] CD19 is an 85-95 kDa transmembrane cell surface glycoprotein receptor. It is a member of the immunoglobulin (Ig) superfamily of proteins and contains two extracellular Ig-like domains, a transmembrane domain, and an intracellular signaling domain (Tedder TF, Isaacs CM, 1989, J Immunol 143:712-171). CD19 modifies B cell receptor signaling, lowering the B cell receptor triggering threshold for antigen (Carter RH, and Fearon DT, 1992, Science 256:105-107), and functions in conjunction with CD81 and CD21 to regulate this essential B cell signaling complex (Bradbury LE, Kansas GS, Levy S, Evans RL, Tedder TF, 1992, J Immunol 149:2841-50). During B cell ontogeny, CD19 can signal at pro-B, pre-pre-B, pre-B, and early B cell stages independently of antigen receptors, and is associated with Src family protein tyrosine kinases, where it is tyrosine phosphorylated and induces both intracellular calcium mobilization and inositol phospholipid signaling (Uckun FM, Burkhardt AL, Jarvis L, Jun X, Stealy B, Dibirdik I, Myers DE, Tuel-Ahlgren L, Bolen JB, 1983, J Biol Chem 268:21172-84). An important point in the context of the treatment of B-cell malignancies is that CD19 is expressed in a tightly regulated manner on normal B cells, restricted to early B-cell precursors at the stage of IgH gene rearrangement, mature B cells, but not on hematopoietic stem cells or mature plasma cells (Anderson, KC, Bates, MP, Slaughenhout BL, Pinkus GS, Schlossman SF, Nadler LM, 1984, Blood 63:1424-1433).

[0007] The current standard of care for B-lineage leukemia consists of remission-inducing treatment with high doses of chemotherapy or radiation, followed by consolidation therapy, which may be characterized by stem cell transplantation and additional chemotherapy courses, if necessary (see the world wide web at cancer.gov). The high toxicity associated with these treatments, as well as the risk of complications such as relapse, secondary malignancies, or GVHD, motivates the search for better alternative treatments. Expression of CD19 in both adult and pediatric (pre-B-ALL) B-cell malignancies has led to the development of this target in both antibody- and chimeric antigen receptor (CAR)-T cell-based therapies (Kochenderfer JN, Wilson WH, Janik JE, Dudley ME, Stetler-Stevenson M, Feldman SA, Maric I, Raffeld M, Nathan DA, Lanier BJ, Morgan RA, Rosenberg SA. 2010. Blood 116:4099-102; Lee DW, Kochenderfer JN, Stetler-Stevenson M, Cui YK, Delbrook C, Feldman SA, Orentas R, Sabatino M, Shah NN, Steinberg SM, Stroncek D, Tschernia N, Yuan C, Zhang H, Zhang L, Rosenberg SA, Wayne AS, Mackall CL. 2015. Lancet 385:517-28).

[0008] Several new approaches to treating B-cell leukemia and lymphoma have been developed, including bispecific antibodies that combine anti-CD19 binding motifs with T-cell binding motifs (i.e., blinatumomab, Blincyto®, indicated for the treatment of Philadelphia chromosome-negative relapsed or refractory B-cell precursor acute lymphoblastic leukemia (ALL)). To date, most of the CD19 binding moieties used in CAR constructs utilize domains derived from murine antibodies. Many of these products, including those developed by Novartis and Kite Pharmaceuticals, are currently under approval consideration. In April 2017, Novartis announced that its CTL019 (tisagenlecleucel) received Breakthrough Therapy Designation from the FDA for the treatment of adult patients with refractory or relapsed (r / r) DLBCL (diffuse large B-cell lymphoma) who have failed two or more prior therapies, with this designation now extended to include B-cell acute lymphoblastic leukemia (ALL). These indications were based on the Phase II JULIET (NCT02445248) and ELIANA (NCT02435849) trials, respectively. The JULIET trial demonstrated a 45% overall response rate (ORR) at 3 months, with 37% overall responses (CR) and 8% partial responses (PR). In the ELIANA trial, 82% of patients infused with the product achieved CR or CR with incomplete count recovery, and the 6-month recurrence-free survival rate was 60%. Kite Pharmaceuticals' CAR-T product (KTE-C19, axicabtagene ciloleucel) was granted Breakthrough Therapy Designation for diffuse large B-cell lymphoma (DLBLC), transformed follicular lymphoma (TFL), and primary mediastinal B-cell lymphoma (PMBCL). The Kite ZUMA-3 Phase II trial of KTE-C19 in r / r ALL reported a 73% CR (≥2 months). All information is from the company's press release. Regardless of whether CAR-T therapy antibodies are utilized, there remains a significant number of patients who cannot be helped by these therapies. There is considerable room for improvement in treatment approaches.

[0009] 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 smallest binding domain of an immunoglobulin (Ig) molecule, the single-chain fragment variable (ScFv). Alternative antigen-binding motifs have also been engineered, for example, receptor ligands (i.e., IL-13 has been engineered to bind to tumor-expressed IL-13 receptors), intact immune receptors, library-derived peptides, and innate immune system effector molecules (e.g., NKG2D). Alternative cellular targets for CAR expression (e.g., NK or gamma-delta T cells) are also under development (Brown CE et al. Clin Cancer Res. 2012;18(8):2199-209; Lehner M et al. PLoS One. 2012;7(2):e31210). Considerable work still needs to be done in defining the most active T cell populations for transduction with CAR vectors, determining optimal culture and expansion techniques, and defining the molecular details of the CAR protein structure itself.

[0010] The linking motif of the CAR can be a relatively stable structural domain, such as the constant domain of IgG, or can be designed as an extended, flexible linker. Structural motifs, such as those derived from the constant domain of IgG, can be used to extend the ScFv-binding domain away from the T cell membrane surface. This may be important for some tumor targets, where the binding domain is particularly close to the tumor cell surface membrane (e.g., for disialoganglioside GD2; Orentas et al., unpublished 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 by beads linked to anti-CD3 and anti-CD28 antibodies, and the presence of the canonical "signal 2" derived from CD28, no longer needs to be encoded by the CAR itself. Using bead activation, third-generation vectors were found to be no superior to second-generation vectors in in vitro assays and offered no clear benefit over second-generation vectors in mouse models of leukemia (Haso W, Lee DW, Shah NN, Stetler-Stevenson M, Yuan CM, Pastan IH, Dimitrov DS, Morgan RA, FitzGerald DJ, Barrett DM, Wayne AS, Mackall CL, Orentas RJ. Anti-CD22-chimeric antigen receptors targeting B cell precursor acute lymphoblastic leukemia. Blood. 2013;121(7):1165-74; Kochenderfer JN et al. Blood. 2012;119(12):2709-20).This is 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 formats (Porter. This is supported by the clinical success of CD19-specific CARs (DL et al., N Engl J Med. 2011;365(8):725-33). Other tumor necrosis factor receptor superfamily members, such as OX40, can also provide important sustained signals in CAR-transduced T cells (Yvon E et al. Clin Cancer Res. 2009;15(18):5852-60). Equally important are the culture conditions under which the CAR T cell population is cultured, such as the inclusion of cytokines IL-2, IL-7, and / or IL-15 (Kaiser AD et al., Cancer Gene Ther. 2015;22(2):72-78).

[0011] A current challenge in broader and more effective application of CAR therapy for cancer relates to the lack of compelling targets. While creating binders to cell surface antigens is now readily achievable, discovering cell surface antigens specific for tumors while sparing normal tissues remains a formidable challenge. One potential way to confer greater target cell specificity to CAR-expressing T cells is to use a combinatorial CAR approach. In one system, the CD3-ζ and CD28 signaling units are split between two different CAR constructs expressed in the same cell; in another system, two CARs are expressed in the same T cell, but one has a lower affinity, thus requiring the alternative CAR to be bound first for full activity of the second CAR (Lanitis). E et al. Cancer Immunol Res. 2013;1(1):43-53; Kloss CC et al. Nat Biotechnol. 2013;31(1):71-5). A second challenge for generating a single ScFv-based CAR as an immunotherapeutic agent is tumor cell heterogeneity. At least one group has developed a CAR strategy for glioblastoma in which an effector cell population simultaneously targets multiple antigens (HER2, IL-13Ra, EphA2) in the hopes of avoiding the growth of target antigen-negative populations (Hegde M et al. Mol Ther. 2013;21(11):2087-101).

[0012] T cell-based immunotherapy is an emerging field in synthetic biology; multiple promoters and gene products are envisioned to direct these highly potent cells to the tumor microenvironment, where they can escape negative regulatory signals and mediate effective tumor killing. Elimination of unwanted T cells via drug-induced dimerization of an inducible caspase-9 construct with a chemical-based dimerizer, such as AP1903, demonstrates one way in which a powerful switch capable of controlling T cell populations can be pharmacologically initiated (Di Stasi A et al., N Engl J Med. 2011;365(18):1673-83). Creation of effector T cell populations 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, CARs appear to be able to induce T cell activation in a manner similar to endogenous T cell receptors; however, the major obstacles to the clinical application of this technology to date have been the limited in vivo expansion of CAR T cells, the rapid loss of cells after infusion, and disappointing clinical activity. This may be due, in part, to the murine origin of some of the CAR sequences used.

[0013] The use of blinotumomab (a bispecific anti-CD19 and anti-CD3 antibody) has shown excellent results in severely ill patients treated with this therapy. Nevertheless, long-term remission rates are less than 40%, and only 50% of responders can be rescued with hematopoietic stem cell transplantation (HSCT) (Gore et al., 2014, NCT01471782, and Von Stackelberg et al., 2014, NCT01471782; for abstract see Benjamin, JE, Stein AS, 2016, Therapeutic Advances in Hematology 7:142-156). ) The requirement for patients to undergo HSCT after bispecific antibody or CAR-T therapy to maintain a durable response remains an area of ​​active debate. High responses, some exceeding 90%, have been reported in CD19 CAR-T trials, but if the trials were rerun as "intent-to-treat" trials, that number could be closer to 70% (Davis KL, Mackall CL, 2016, Blood Advances 1:265-268). The best reported 12-month results after CAR19 treatment at the University of Pennsylvania showed a 55% RFS and a 79% OS among patients who were able to receive T-cell products (Maude SL, Teachey DT, Rheingold SR, Shaw PA, Aplenc R, Barrett DM, Barker CS, Callahan C, Frey NV, Farzana N, Lacey SF, Zheng A, Levine B, Melenhorst JJ, Motley L, Prter DL, June CH, Grupp SA. 2016. J Clin Oncol 34, No. 15, Suppl 2016, May, pp. 3011-3011). Summary of the Invention [Problem to be solved by the invention]

[0014] Thus, there is an urgent and long-felt need in the art to discover new compositions and methods for treating B-ALL and other CD19-expressing B-cell malignancies using approaches that can exhibit specific and effective anti-tumor effects without the aforementioned drawbacks. [Means for solving the problem]

[0015] 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 inventions disclosed and described herein provide CARs that can be used to treat diseases, disorders, or conditions associated with dysregulated expression of CD19, wherein the CARs contain a CD19 antigen-binding domain that exhibits high surface expression on transduced T cells, exhibits high cytolysis of CD19-expressing cells, and exhibits in vivo proliferation and persistence of the transduced T cells.

[0016] overview The present invention provides a novel anti-CD19 antibody or its antigen-binding domain, a chimeric antigen receptor (CAR) comprising such a CD19 antigen-binding domain, a host cell (e.g., T cell) expressing the receptor, and a nucleic acid molecule encoding the receptor.The CAR exhibits high surface expression on transduced T cells, high cytolysis, and in vivo proliferation and persistence of transduced T cells.Methods of using the disclosed CAR, host cell, and nucleic acid molecule are also provided, for example, for treating cancer in a subject.

[0017] Thus, in one aspect, an isolated polynucleotide encoding a human anti-CD19 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, 13 and 15.

[0018] In one embodiment, an isolated polynucleotide encoding a fully human anti-CD19 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).

[0019] In one embodiment, an isolated polynucleotide encoding a fully human anti-CD19 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, 14 and 16.

[0020] In one embodiment, an isolated nucleic acid molecule is provided encoding a chimeric antigen receptor (CAR) comprising, from N-terminus to C-terminus, at least one CD19 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, 13, and 15.

[0021] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded extracellular CD19 antigen-binding domain comprises at least one single-chain variable fragment of an antibody that binds to CD19.

[0022] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded extracellular CD19 antigen-binding domain comprises at least one heavy chain variable region of an antibody that binds to CD19.

[0023] In yet another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded CAR extracellular CD19 antigen-binding domain further comprises at least one lipocalin-based antigen-binding domain (anticalin) that binds to CD19.

[0024] In one embodiment, an isolated nucleic acid molecule is provided in which the encoded extracellular CD19 antigen-binding domain is connected to the transmembrane domain by a linker domain.

[0025] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded CD19 extracellular antigen-binding domain is preceded by a sequence encoding a leader or signal peptide.

[0026] In yet another embodiment, an isolated nucleic acid molecule is provided encoding a CAR comprising at least one CD19 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, 13, and 15, wherein the CAR further encodes an extracellular antigen-binding domain that targets an antigen including, but not limited to, CD20, CD22, ROR1, mesothelin, CD33, CD38, CD123 (IL3RA), CD138, BCMA (CD269), GPC2, GPC3, FGFR4, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, TSLPR, NY-ESO-1 TCR, MAGE A3 TCR, or any combination thereof.

[0027] In certain embodiments, the further encoded extracellular antigen-binding domain is an anti-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-TSLPR ScFv antigen-binding domain, an anti-c-Met ScFv antigen-binding domain, an anti-PMSA ScFv antigen-binding domain, or an anti-glycolipid F77 Provided is an isolated nucleic acid molecule encoding a CAR comprising an ScFv antigen-binding domain, an anti-EGFRvIII ScFv antigen-binding domain, an anti-GD-2 ScFv antigen-binding domain, an anti-NY-ESO-1 TCR ScFv antigen-binding domain, an anti-MAGE A3 TCR ScFv antigen-binding domain, or an amino acid sequence thereof having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, or any combination thereof.

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

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

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

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

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

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

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

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

[0036] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, further comprising a leader sequence or signal peptide, wherein the nucleotide sequence of the leader or signal peptide comprises the nucleotide sequence of SEQ ID NO:17.

[0037] In yet another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded leader sequence comprises the amino acid sequence of SEQ ID NO:18.

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

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

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

[0041] In some embodiments, the CAR is selected from the group consisting of CD20, CD22, ROR1, mesothelin, CD33, CD38, CD123 (IL3RA), CD138, BCMA (CD269), GPC2, GPC3, FGFR4, TSLPR, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, TSLPR, NY-ESO-1 TCR, MAGE A3 CARs are provided that further encode an extracellular antigen-binding domain comprising a TCR, or an amino acid sequence thereof having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, or any combination thereof.

[0042] In one embodiment, the extracellular antigen-binding domain is an anti-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-TSLPR ScFv antigen-binding domain, an anti-c-Met ScFv antigen-binding domain, an anti-PMSA ScFv antigen-binding domain, an anti-glycolipid F77 ScFv antigen-binding domain, or an anti-EGFRvIII ScFv. ScFv antigen-binding domain, anti-GD-2 ScFv antigen-binding domain, anti-NY-ESO-1 TCR ScFv antigen-binding domain, anti-MAGE CARs are provided that comprise an A3 TCR ScFv antigen-binding domain, or an amino acid sequence thereof having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, or any combination thereof.

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

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

[0045] In one embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 19 (LTG 2050 LP-M19217-CD8 TM-41BB-CD3 Zeta CAR nucleic acid sequence (Figure 2A)). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 20 (LTG 2050 LP-M19217-CD8 TM-41BB-CD3 Zeta CAR amino acid sequence (Figure 2A)).

[0046] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 21 (LTG 2065 LP-M19217-1-CD8 TM-41BB-CD3 zeta CAR nucleic acid sequence (Figure 2B)). In one embodiment, the nucleic acid sequence comprises SEQ ID NO: 22 (LTG 2065 LP-M19217-1-CD8 encodes a CAR containing the amino acid sequence of the TM-41BB-CD3 zeta CAR amino acid sequence (Figure 2B).

[0047] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 23 (LTG2066 LP-M19217-2-CD8 TM-41BB-CD3 Zeta CAR nucleotide sequence (Figure 2C)). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 24 (LTG 2066 LP-M19217-2-CD8 TM-41BB-CD3 Zeta CAR amino acid sequence (Figure 2C)).

[0048] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 25 (LTG 2067 LP-M19217-7-CD8 TM-41BB-CD3 Zeta CAR nucleic acid sequence (Figure 2D)). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 26 (LTG 2067 LP-M19217-7-CD8 TM-41BB-CD3 Zeta CAR amino acid sequence (Figure 2D)).

[0049] In another embodiment, the nucleic acid sequence encoding the CAR is the nucleic acid sequence of SEQ ID NO: 27 (LTG 2068 LP-M19217-23-CD8 TM-41BB-CD3 Zeta CAR nucleic acid sequence (Figure 2E)). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 28 (LTG2068 LP-M19217-23-CD8 TM-41BB-CD3 Zeta CAR amino acid sequence (Figure 2E)).

[0050] In another embodiment, the nucleic acid sequence encoding the CAR is the nucleic acid sequence of SEQ ID NO: 29 (LTG 2069 LP-M19217-29-CD8 TM-41BB-CD3 Zeta CAR nucleic acid sequence (Figure 2F)). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 30 (LTG 2069 LP-M19217-29-CD8 TM-41BB-CD3 Zeta CAR amino acid sequence (Figure 2F)).

[0051] In another embodiment, the nucleic acid sequence encoding the CAR is the nucleic acid sequence of SEQ ID NO: 31 (LTG 2070 LP-M19217-38-CD8 TM-41BB-CD3 Zeta CAR nucleic acid sequence (Figure 2G)). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 32 (LTG 2070 LP-M19217-38-CD8 TM-41BB-CD3 Zeta CAR amino acid sequence (Figure 2G)).

[0052] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 33 (LTG 2071 LP-M19217-40-CD8 TM-41BB-CD3 Zeta CAR nucleic acid sequence (Figure 2H)). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 34 (LTG 2071 LP-M19217-40-CD8 TM-41BB-CD3 Zeta CAR amino acid sequence (Figure 2H)).

[0053] 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.

[0054] 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.

[0055] In certain embodiments, the vector contains a promoter that is an inducible promoter, a tissue-specific promoter, a constitutive promoter, a suicide promoter, or any combination thereof. Further includes.

[0056] 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).

[0057] In another aspect, a host cell is also provided that comprises a nucleic acid molecule encoding a CAR. 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 cell.

[0058] In yet another aspect, a pharmaceutical composition is provided comprising an antitumor effective amount of a human T cell population, 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 human CD19 antigen-binding domain comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, or 16, and the T cells are human T cells 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, or a combination thereof.

[0059] In one embodiment, a pharmaceutical composition is provided, wherein at least one transmembrane domain of the CAR comprises a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, mesothelin, CD33, CD37, CD64, CD80, CD83, CD86, CD134, CD137, CD154, TNFRSF19, or a combination thereof.

[0060] 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.

[0061] In yet another embodiment, a pharmaceutical composition is provided comprising an antitumor-effective amount of a population of human T cells from a human having cancer, wherein the cancer is a refractory cancer that is unresponsive to one or more chemotherapeutic agents. The cancer includes hematopoietic cancer, myelodysplastic syndrome, pancreatic cancer, head and neck cancer, skin tumor, adult B-cell malignancies including acute lymphoblastic leukemia (ALL), minimal residual disease (MRD) in acute myeloid leukemia (AML), CLL (chronic lymphocytic leukemia), CML (chronic myeloid leukemia), non-Hodgkin's lymphoma (NHL), pediatric B-cell malignancies (including B-cell lineage ALL (acute lymphocytic leukemia)), multiple myeloma, lung cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, melanoma or other hematological cancers and solid tumors, or any combination thereof.

[0062] 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 CD19, thereby generating the CAR T cells.

[0063] 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-expressing cells.

[0064] In yet another aspect, a method is provided for diagnosing a disease, disorder, or condition associated with CD19 expression in a cell, the method comprising the steps of: (a) contacting the cell with a human anti-CD19 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, 14, 16; and (b) detecting the presence of CD19, wherein the presence of CD19 diagnoses the disease, disorder, or condition associated with CD19 expression.

[0065] In one embodiment, the disease, disorder or condition associated with CD19 expression is cancer, including hematopoietic cancer, myelodysplastic syndrome, pancreatic cancer, head and neck cancer, skin tumor, adult B-cell malignancies including acute lymphoblastic leukemia (ALL), minimal residual disease (MRD) in acute myeloid leukemia (AML), CLL (chronic lymphocytic leukemia), CML (chronic myeloid leukemia), non-Hodgkin's lymphoma (NHL), pediatric B-cell malignancies (including B-cell lineage ALL (acute lymphocytic leukemia)), multiple myeloma, lung cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, melanoma or other hematological cancers and solid tumors, or any combination thereof.

[0066] In another embodiment, a method is provided for diagnosing, prognosing, or determining the risk of a disease associated with CD19 in a mammal, comprising detecting CD19 expression in a sample from a mammal, comprising the steps of: a) contacting the sample with a human anti-CD19 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, 14, or 16; and b) detecting the presence of CD19, wherein the presence of CD19 is diagnostic of the disease associated with CD19 in the mammal.

[0067] In another embodiment, a method of inhibiting CD19-dependent T cell inhibition is provided, comprising contacting a cell with a human anti-CD19 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, 14, or 16. In one embodiment, the cell is selected from the group consisting of a CD19-expressing tumor cell, a tumor-associated macrophage, and any combination thereof.

[0068] In another embodiment, a method is provided for altering a tumor microenvironment to block T cell inhibition mediated by cells expressing CD19 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-CD19 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, 14 and 16. In one embodiment, the cell is selected from the group consisting of a CD19-expressing tumor cell, a tumor-associated macrophage, and any combination thereof.

[0069] In another embodiment, a method for inhibiting, suppressing, or preventing immunosuppression of an anti-tumor or anti-cancer immune response in a mammal is provided, comprising administering to a mammal an isolated anti-CD19 antibody or fragment thereof. Methods are provided that include administering an effective amount of a composition to a mammal, 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, 14, and 16. In one embodiment, the antibody or fragment thereof inhibits an interaction between a first cell and a T cell, and the first cell is selected from the group consisting of a tumor cell expressing CD19, a tumor-associated macrophage, and any combination thereof.

[0070] 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.

[0071] In another embodiment, a method of treating or preventing cancer in a mammal is provided, comprising administering to the mammal one or more of the disclosed CARs in an amount effective to treat or prevent cancer in the mammal. The method comprises administering to the subject a therapeutically effective amount of host cells expressing the disclosed CARs that specifically bind to CD19 and / or one or more of the above-mentioned antigens under conditions sufficient to form an immune complex between the antigen-binding domain of the CAR and the extracellular domain of CD19 and / or one or more of the above-mentioned antigens in the subject.

[0072] In yet another embodiment, a method is provided for treating a mammal having a disease, disorder, or condition associated with elevated expression of a tumor antigen, comprising administering to the subject an anti-tumor effective amount of a pharmaceutical composition comprising a population of T cells, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises at least one extracellular CD19 antigen-binding domain comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, or 16, 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.

[0073] 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 CD19 antigen-binding domain comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, or 16, 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, CD19, CD22, mesothelin, CD33, CD37, CD64, CD80, CD83, CD86, CD134, CD137, CD154, TNFRSF16, TNFRSF19, or a combination thereof.

[0074] 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 CD19 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, 14, or 16, 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.

[0075] In one embodiment, the progeny T cells in the human comprise memory T cells. In another embodiment, the T cells are autologous T cells.

[0076] 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.

[0077] 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.

[0078] 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]

[0079] [Figure 1] Schematic diagram showing the general domain structure of a CAR with a novel extracellular CD19 antigen-binding domain sequence. The chimeric antigen receptor is composed of an extracellular CD19-binding ScFv domain, a CD8 spacer and transmembrane domain, an intracellular signaling CD137 costimulatory domain, and a CD3 z signaling domain. [Figure 2A]

[0023] Figure 2A shows the nucleic acid and amino acid sequences of several chimeric antigen receptors (CARs) containing novel human extracellular CD19 antigen-binding domain sequences. The general scheme for a CAR includes, from N- to C-terminus, a signal peptide, a human anti-CD19 binder single-chain fragment variable (ScFv), an extracellular linker, a transmembrane domain, a 4-1BB (CD137) signaling domain, and a CD3 zeta signaling domain. Figure 2A shows a lentiviral vector expressing the CAR LTG2050 (19217 ScFv-CD8 TM-41BB-CD3 zeta) nucleic acid sequence (SEQ ID NO: 19) and the encoded amino acid sequence (SEQ ID NO: 20). [Figure 2B]

[0023] Figure 2B shows the nucleic acid and amino acid sequences of several chimeric antigen receptors (CARs) containing novel human extracellular CD19 antigen-binding domain sequences. The general scheme for a CAR includes, from N- to C-terminus, a signal peptide, a human anti-CD19 binder single-chain fragment variable (ScFv), an extracellular linker, a transmembrane domain, a 4-1BB (CD137) signaling domain, and a CD3 zeta signaling domain. Figure 2B shows the lentiviral vector expressing the CAR LTG2065 (M19217-1 ScFv-CD8 TM-41BB-CD3 zeta) nucleic acid sequence (SEQ ID NO: 21) and the encoded amino acid sequence (SEQ ID NO: 22). [Figure 2C]

[0023] Figure 2C shows the nucleic acid and amino acid sequences of several chimeric antigen receptors (CARs) containing novel human extracellular CD19 antigen-binding domain sequences. The general schematic for a CAR includes, from N- to C-terminus, a signal peptide, a human anti-CD19 binder single-chain fragment variable (ScFv), an extracellular linker, a transmembrane domain, a 4-1BB (CD137) signaling domain, and a CD3 zeta signaling domain. Figure 2C shows the lentiviral vector expressing the CAR LTG2066 (M19217-2 ScFv CD8 TM-41BB-CD3 zeta) nucleic acid sequence (SEQ ID NO: 23) and the encoded amino acid sequence (SEQ ID NO: 24). [Figure 2D]

[0023] Figure 2D shows the nucleic acid and amino acid sequences of several chimeric antigen receptors (CARs) containing novel human extracellular CD19 antigen-binding domain sequences. The general scheme for a CAR includes, from N- to C-terminus, a signal peptide, a human anti-CD19 binder single-chain fragment variable (ScFv), an extracellular linker, a transmembrane domain, a 4-1BB (CD137) signaling domain, and a CD3 zeta signaling domain. Figure 2D shows a lentiviral vector expressing the CAR LTG2067 (M19217-7 ScFv CD8 TM-41BB-CD3 zeta) nucleic acid sequence (SEQ ID NO: 25) and the encoded amino acid sequence (SEQ ID NO: 26). [Figure 2E]

[0023] Figure 2B shows the nucleic acid and amino acid sequences of several chimeric antigen receptors (CARs) containing novel human extracellular CD19 antigen-binding domain sequences. The general schematic for a CAR includes, from N- to C-terminus, a signal peptide, a human anti-CD19 binder single-chain fragment variable (ScFv), an extracellular linker, a transmembrane domain, a 4-1BB (CD137) signaling domain, and a CD3 zeta signaling domain. Figure 2E shows a lentiviral vector expressing the CAR LTG2068 (M19217-23 ScFv CD8 TM-41BB-CD3 zeta) nucleic acid sequence (SEQ ID NO: 27) and the encoded amino acid sequence (SEQ ID NO: 28). [Figure 2F]

[0023] Figure 2B shows the nucleic acid and amino acid sequences of several chimeric antigen receptors (CARs) containing novel human extracellular CD19 antigen-binding domain sequences. The general scheme for a CAR includes, from N- to C-terminus, a signal peptide, a human anti-CD19 binder single-chain fragment variable (ScFv), an extracellular linker, a transmembrane domain, a 4-1BB (CD137) signaling domain, and a CD3 zeta signaling domain. Figure 2F shows a lentiviral vector expressing the CAR LTG2069 (M19217-29 ScFv CD8 TM-41BB-CD3 zeta) nucleic acid sequence (SEQ ID NO: 29) and the encoded amino acid sequence (SEQ ID NO: 30). [Figure 2G]

[0023] Figure 2G shows the nucleic acid and amino acid sequences of several chimeric antigen receptors (CARs) containing novel human extracellular CD19 antigen-binding domain sequences. The general schematic for a CAR includes, from N- to C-terminus, a signal peptide, a human anti-CD19 binder single-chain fragment variable (ScFv), an extracellular linker, a transmembrane domain, a 4-1BB (CD137) signaling domain, and a CD3 zeta signaling domain. Figure 2G shows the lentiviral vector expressing the CAR LTG2070 (M19217-38 ScFv CD8 TM-41BB-CD3 zeta) nucleic acid sequence (SEQ ID NO: 31) and the encoded amino acid sequence (SEQ ID NO: 32). [Figure 2H]

[0023] Figure 2H shows the nucleic acid and amino acid sequences of several chimeric antigen receptors (CARs) containing novel human extracellular CD19 antigen-binding domain sequences. The general scheme for a CAR includes, from N- to C-terminus, a signal peptide, a human anti-CD19 binder single-chain fragment variable (ScFv), an extracellular linker, a transmembrane domain, a 4-1BB (CD137) signaling domain, and a CD3 zeta signaling domain. Figure 2H shows a lentiviral vector expressing the CAR LTG2071 (M19217-40 ScFv CD8 TM-41BB-CD3 zeta) nucleic acid sequence (SEQ ID NO: 33) and the encoded amino acid sequence (SEQ ID NO: 34). [Figure 3-1]Figure 3 shows anti-CD19 CAR surface expression on primary human T cells. CAR T cells (as indicated in each panel) redirected to the CD19 tumor antigen using the ScFv domain were generated by lentiviral transduction with the CAR expression construct. CAR detection was performed by flow cytometry. T cells were washed twice with chilled PBS-EDTA buffer and stained with CD19-Fc peptide, followed by anti-Fc-AF647 reagent. At least 20,000 cells were obtained for each analysis. Cells were gated based on forward and side scatter, singlet discrimination, and 7AAD negativity to ensure that only viable cells were analyzed. Data were acquired on a MACSQuant 10 flow cytometer in the APC channel. UTD refers to untransduced negative control cells. Markers in each panel identify the CAR-expressing population and are expressed as a percentage of the total transduced T cells analyzed. [Figure 3-2] Figure 3 shows anti-CD19 CAR surface expression on primary human T cells. CAR T cells (as indicated in each panel) redirected to the CD19 tumor antigen using the ScFv domain were generated by lentiviral transduction with the CAR expression construct. CAR detection was performed by flow cytometry. T cells were washed twice with chilled PBS-EDTA buffer and stained with CD19-Fc peptide, followed by anti-Fc-AF647 reagent. At least 20,000 cells were obtained for each analysis. Cells were gated based on forward and side scatter, singlet discrimination, and 7AAD negativity to ensure that only viable cells were analyzed. Data were acquired on a MACSQuant 10 flow cytometer in the APC channel. UTD refers to untransduced negative control cells. Markers in each panel identify the CAR-expressing population and are expressed as a percentage of the total transduced T cells analyzed. [Figure 4-1]Figure 4 shows anti-CD19 CAR T cells incorporating ScFv binders (LTG2050, 2065-2071) mediating cytolysis of CD19-positive tumors in vitro. CAR T cells expressing the anti-CD19 constructs were incubated overnight with CD19-positive cell lines (Raji and Reh) or CD19-negative lines stably transduced with firefly luciferase (K562 and 293T) at effector-to-target ratios of 5, 10, 20, and 40 (x-axis). CAR T cytotoxicity activity was then assessed by luciferase activity measurement, as described in Materials and Methods. Each bar is the mean of three technical replicates, and error bars indicate SD. UTD is the untransduced T cell negative control, and 1538 is the LTG1538 FMC63 mouse anti-CD19 CAR positive control. [Figure 4-2] Figure 4 shows anti-CD19 CAR T cells incorporating ScFv binders (LTG2050, 2065-2071) mediating cytolysis of CD19-positive tumors in vitro. CAR T cells expressing the anti-CD19 constructs were incubated overnight with CD19-positive cell lines (Raji and Reh) or CD19-negative lines stably transduced with firefly luciferase (K562 and 293T) at effector-to-target ratios of 5, 10, 20, and 40 (x-axis). CAR T cytotoxicity activity was then assessed by luciferase activity measurement, as described in Materials and Methods. Each bar is the mean of three technical replicates, and error bars indicate SD. UTD is the untransduced T cell negative control, and 1538 is the LTG1538 FMC63 mouse anti-CD19 CAR positive control. [Figure 5] Figure 1 shows CD19-specific CART cell production of high levels of cytokines when co-cultured with a CD19-positive leukemia line (dark gray) or when T cells were cultured alone (gray). Assays were performed overnight at an E:T ratio of 10:1, after which supernatants were analyzed by ELISA for cytokine concentrations. N=2 technical replicates + / - SD. Negative controls: UT for untransduced T cells, 1538 for T cells transduced with the FMC63 murine CD19 positive control. The LTG number of each LV used to transduce human T cells is indicated on the x-axis. DETAILED DESCRIPTION OF THE INVENTION

[0080] 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 singular 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 further be 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. Although many methods and materials similar or equivalent to those described in the specification can be used, particularly suitable methods and materials are described below. In case of conflict, the present specification, including explanations of terms, will control. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting. In order to facilitate review of the various embodiments, the following explanations of terms are provided.

[0081] 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.

[0082] 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.

[0083] The present disclosure provides a CD19 antibody or fragment thereof, and a chimeric antigen receptor (CAR) having such a CD19 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.

[0084] 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.

[0085] The nonvariable framework components of immunoglobulin-derived protein sequences used to create the extracellular antigen-binding domain of CARs can be completely neutral or self-associate, rendering T cells metabolically exhausted and rendering therapeutic T cells expressing the CAR highly ineffective. This occurs regardless of the antigen-binding function of the CAR domain. Furthermore, the choice of intracellular signaling domain(s) can also determine the activity and durability of therapeutic lymphocyte populations used in immunotherapy. While the ability of these extracellular and intracellular domains to bind target antigens and deliver activation signals to T cells, respectively, are important aspects of CAR design, it has also become clear that the choice of source of the extracellular antigen-binding fragment can have a significant effect on CAR efficacy and therefore play a crucial role in CAR function and clinical utility.

[0086] 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.

[0087] In light of this discovery, we developed a series of CD19 binders from a human scFv expression library. These fully human CD19 CARs are no longer of murine origin and are therefore less likely to induce allergic or rejection reactions in patients (see Maus MV, Haas AR, Beatty GL, Albeda SM, Levine BL, Liu X, Zhao Y, Kalos M, June CH, 2013, Cancer Immunology Research, 1:26-31). Therefore, these "fully human" CARs are likely to be more effective when expressed in T cells and then infused into patients. These human sequence-derived CAR binders can be used to treat human cancers, leukemias, and lymphomas that express the CD19 antigen, including, but not limited to, B-ALL, DLBCL, and FL.

[0088] 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 CD19 antigen to which the CAR binds. The use of a human extracellular CD19 antigen-binding domain results in the generation of CARs that function better in vivo, while avoiding the induction of anti-CAR immunity in the host immune response and the death of CAR T cell populations. CARs expressing a fully human extracellular CD19ScFv antigen-binding domain exhibit excellent activity / characteristics, including: i) prevention of the lack of persistence and function of CAR T cells observed with mouse-derived binding sequences; ii) lack of effective local (i.e., intrapleural) delivery of CARs; and iii) the ability to generate CAR T cell designs based on both high-affinity and low-affinity binders for CD19. This last property allows researchers to better tune the efficacy and / or tissue specificity of CAR T products to toxicity, as tumors express more CD19 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.

[0089] Below is a detailed description of the CARs of the present invention, including a description of their extracellular CD19 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.

[0090] A. Chimeric Antigen Receptor (CAR) The CARs disclosed herein comprise at least one CD19 antigen-binding domain capable of binding to CD19, at least one transmembrane domain, and at least one intracellular domain.

[0091] 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).

[0092] 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.

[0093] 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.

[0094] 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 CD19. The tumor antigens disclosed herein are included by way of example only; this list is not intended to be exhaustive, and further examples will be readily apparent to those of skill in the art.

[0095] In one embodiment, the tumor antigen comprises one or more antigenic cancer epitopes associated with malignant tumors. Malignant tumors express several proteins that can serve as target antigens for immune attack. These molecules include, but are not limited to, tissue-specific antigens, such as MART-1, tyrosinase, and GP100 in melanoma, and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation-related molecules, such as the oncogene HER-2 / Neu / ErbB-2. Yet another group of target antigens is carcinoembryonic antigens, such as carcinoembryonic antigen (CEA). In B-cell lymphomas, tumor-specific idiotypic immunoglobulins constitute truly tumor-specific immunoglobulin antigens unique to individual tumors. B-cell differentiation antigens, such as CD19, CD20, CD22, BCMA, ROR1, and CD37, are other candidate target antigens in B-cell lymphomas. Some of these antigens (CEA, HER-2, CD19, CD20, idiotype) have been used as targets for passive immunotherapy with monoclonal antibodies with limited success.

[0096] In one preferred embodiment, the tumor antigen is CD19, and tumors associated with CD19 expression include lung mesothelioma, ovarian and pancreatic cancer, or any combination thereof, which express high levels of the extracellular protein CD19.

[0097] 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.

[0098] 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.

[0099] In one embodiment, the antigen binding domain portion of the CAR targets an antigen including, but not limited to, CD19, CD20, CD22, ROR1, CD33, CD38, CD123, CD138, BCMA, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, FGFR4, TSLPR, NY-ESO-1 TCR, MAGE A3 TCR, etc.

[0100] In a preferred embodiment, the antigen binding domain portion of the CAR targets the extracellular CD19 antigen.

[0101] In a preferred embodiment, an isolated nucleic acid molecule encoding the extracellular CD19 VH-2 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 CD19 VH-2 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.

[0102] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD19 VH-4 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 CD19 VH-4 antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 4. or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:4.

[0103] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD19 ScFv 9 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 CD19 ScFv 9 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.

[0104] In one preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD19 ScFv 10 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 CD19 ScFv 10 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.

[0105] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD19 ScFv 12 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 CD19 ScFv 12 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.

[0106] In one preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD19 ScFv 15 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 CD19 ScFv 15 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.

[0107] In one preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD19 ScFv 15 antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 13, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD19 ScFv 15 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 14.

[0108] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD19 ScFv 15 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, the encoded extracellular CD19 ScFv 15 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. An isolated nucleic acid molecule is provided, comprising:

[0109] In various embodiments of the CD19-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-CD19 Contains ScFv, extracellular linker, CD8 transmembrane domain, 4-1BB, CD3 zeta, bold text indicates cloning site of linking domain.

[0110] In one embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 19, which encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 20 [LTG2050 LP-M19217-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in Figure 2A)].

[0111] In one embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 19, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, and encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 20, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity [LTG2050 LP-M19217-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in Figure 2A)].

[0112] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 21, which encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 22 [LTG2065 LP-M19217-1-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in Figure 2B)].

[0113] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 21, 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: 22, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity [LTG2065 LP-M19217-1-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in Figure 2B)].

[0114] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 23, which encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 24 [LTG2066 LP-M19217-2-CD8 TM-41BB-CD3 Zeta CAR amino acid sequence (shown in Figure 2C)].

[0115] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 23, 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: 24, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity [LTG2066 LP-M19217-2-CD8 TM-41BB-CD3 Zeta CAR amino acid sequence (shown in Figure 2C)].

[0116] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 25, which encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 26 [LTG2067 LP-M19217-7-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in Figure 2D)].

[0117] In yet another embodiment, the nucleic acid sequence encoding the CAR is the nucleic acid sequence of SEQ ID NO: 25, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity. 26, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity [LTG2067 LP-M19217-7-CD8 encodes a CAR containing the TM-41BB-CD3 amino acid sequence (shown in Figure 2D )].

[0118] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 27, which encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 28 [LTG2068 LP-M19217- 23-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in Figure 2E)].

[0119] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 27, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, and the amino acid sequence set forth in SEQ ID NO: 28, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity [LTG2068 LP-M19217- 23-CD8 encodes a CAR containing the TM-41BB-CD3 zeta amino acid sequence (shown in Figure 2E)].

[0120] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 29, which encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 30 [(LTG2069 LP-M19217-29 -CD8 TM-41BB-CD3 zeta amino acid sequence (shown in Figure 2F)].

[0121] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 29, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, and the amino acid sequence set forth in SEQ ID NO: 30, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity [LTG2069 LP-M19217-29-CD8 encodes a CAR containing the TM-41BB-CD3 zeta amino acid sequence (shown in Figure 2F)].

[0122] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 31, which encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 32 [(LTG2070 LP-M19217-38-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in Figure 2G)].

[0123] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 31, 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: 32, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity [(LTG2070 LP-M19217-38-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in Figure 2G)].

[0124] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 33, and encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 34 [(LTG2071 LP-M19217-40-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in Figure 2H)].

[0125] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 33, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, and the amino acid sequence set forth in SEQ ID NO: 34, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity [(LTG207 1 LP-M19217-40-CD8 encodes a CAR containing the TM-41BB-CD3 zeta amino acid sequence (shown in Figure 2H)].

[0126] Surface expression of anti-CD19 CARs incorporating single-chain fragment variable (ScFv) sequences reactive with the CD19 antigen is shown in Example 2 below and summarized in Table 2. Expression levels for each CAR containing ScFv or VH were determined by flow cytometry analysis of LV-transduced T cells from two healthy donors using recombinant CD19-Fc peptide followed by anti-human Fc F(ab')2 fragments conjugated to AF647 and detected in the APC channel (see Figure 3). The ScFv-based anti-CD19 CAR constructs LTG2050, LTG2065, and LTG2071 were highly expressed in human primary T cells (indicated by gated populations) compared with non-transduced T cell controls (ungated cell populations). Representative results from one donor are shown.

[0127] As shown in Example 2 and Figure 4, lentiviral vectors (LVs) expressing the following CARs were created and tested for anti-leukemia activity, demonstrating the high cytolytic activity of the CD19 CARs. Each experimental CAR contains a 4-1BB / CD3-zeta chain signaling motif and a specific anti-CD19 binding motif / domain as specified therein. Leukemia target lines with various CD19 surface expression were used: Raji and Reh; and CD19-negative K562 and 293T. The ScFv-based anti-CD19 CAR constructs LTG2050 and LTG2065-2071 were able to efficiently lyse the CD19-high tumor lines Raji and Reh, while lacking specific lytic activity against K562 or 293T (see Figure 4). These results demonstrate the efficiency and specificity of the generated CAR constructs.

[0128] We then evaluated the ability of anti-CD19 CAR T cells to secrete cytokines. Tumor cells were co-incubated with CAR T cells or control T cells overnight at an effector-to-target ratio of 10:1, and culture supernatants were analyzed by ELISA for IFN-gamma, TNF-alpha, and IL-2 (see Figure 5). It is noteworthy that CAR T-expressing cells LTG2065, LTG2066, LTG2067, LTG2068, LTG2069, LTG2070, and LTG2071 produced high levels of IFN-gamma, TNF-alpha, and IL-2, whereas the negative control (untransduced, UN) produced no obvious cytokine induction. Surprisingly, CD19 CAR LTG2050 produced significantly lower levels of induced cytokines against the tumor lines tested. The CAR tested as a positive control, LTG1538, which expressed the murine FMC63 CD19 binder (SEQ ID NO: 47), was less active than all binders tested except for LTG2050. The high in vitro cytolytic function of LTG2050, combined with its low cytokine production potential, suggests that multiple CAR T functional endpoints need to be tested on a construct-by-construct basis. Furthermore, the superiority of the LTG2065-2071 human CD19 binder over the murine FMC63 scFv was clearly demonstrated in cytokine production assays.

[0129] 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.

[0130] Although the present disclosure is exemplified using an exemplary extracellular human CD19 ScFv antigen-binding domain, other nucleotide and / or amino acid variants within the CD19 variable ScFv antigen-binding domain may be used to obtain a heavy chain single binding domain or subset thereof, and thus include a CD19 antigen-binding domain for use in a CAR as described herein. In one embodiment, other nucleotide and / or amino acid variants within the CD19 variable ScFv antigen-binding domain include, for example, the CD19 variable ScFv antigen-binding domain-containing variant labeled as FMC63 (SEQ ID NO: 46 and SEQ ID NO: 47 (nucleotide and amino acid sequences, respectively (see Example 1))).

[0131] 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.

[0132] In one exemplary embodiment, the antigen-binding domain portion of the CAR further targets CD33. Preferably, the antigen-binding domain in the CAR is an anti-CD33 ScFv, wherein the nucleic acid sequence of the anti-CD33 ScFv comprises the sequence set forth in SEQ ID NO: 48. In one embodiment, the anti-CD33 ScFv comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 49. In another embodiment, the anti-CD33 ScFv portion of the CAR comprises the amino acid sequence set forth in SEQ ID NO: 49.

[0133] In one exemplary embodiment, the antigen-binding domain portion of the CAR further targets mesothelin. Preferably, the antigen-binding domain in the CAR is an anti-mesothelin ScFv, wherein the nucleic acid sequence of the anti-mesothelin ScFv comprises the nucleotide sequence set forth in SEQ ID NO: 50. In one embodiment, the anti-mesothelin ScFv comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 51. In another embodiment, the anti-mesothelin ScFv portion of the CAR comprises the amino acid sequence set forth in SEQ ID NO: 51.

[0134] 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.

[0135] In another aspect of the present invention, a CAR is provided that is capable of binding to antigens derived from bacterial strains of Staphylococci, Streptococcus, Escherichia coli, Pseudomonas, or Salmonella, particularly infectious bacteria such as Helicobacter pyloris, Legionella pneumophilia, bacterial strains of Mycobacteria sp. (e.g., M. tuberculosis, M. avium, M. intracellulare, M. kansaii, or M. gordonea), Staphylococcus aureus, and the like. us, 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.

[0136] 2. Transmembrane domain With respect to the transmembrane domain, the CAR comprises one or more transmembrane domains fused to the extracellular CD19 antigen-binding domain of the CAR.

[0137] 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.

[0138] 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, CD83, CD86, CD134, CD137, CD154, TNFRSF16, or TNFRSF19. Alternatively, the transmembrane domain can be synthetic, in which case it predominantly contains hydrophobic residues such as leucine and valine. Preferably, a triad of phenylalanine, tryptophan, and valine is found at each end of the synthetic transmembrane domain. Optionally, a short oligopeptide or polypeptide linker, preferably between 2 and 10 amino acids in length, can form the link between the transmembrane domain and the cytoplasmic signaling domain of the CAR. A glycine-serine doublet provides a particularly suitable linker.

[0139] In one embodiment, a transmembrane domain naturally associated with one of the domains in the CAR is used in addition to the transmembrane domain.

[0140] In some cases, the transmembrane domain may be selected or by amino acid substitution to avoid binding of such domain to transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

[0141] In one embodiment, the transmembrane domain in the CAR of the present invention is a CD8 transmembrane domain. In one embodiment, the CD8 transmembrane domain comprises the nucleic acid sequence of SEQ ID NO: 35. In one embodiment, the CD8 transmembrane domain comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 20. In another embodiment, the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 36.

[0142] 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: 36, or a sequence having 95-99% identity to the amino acid sequence of SEQ ID NO: 36.

[0143] 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: 37. In one embodiment, the CD8 hinge domain comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 38. In another embodiment, the CD8 hinge domain comprises the amino acid sequence of SEQ ID NO: 38, or a sequence thereof with 95-99% identity.

[0144] 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.

[0145] 3. Spacer domain In CARs, a spacer domain can be located between the extracellular domain and the transmembrane domain, or between the intracellular domain and the transmembrane domain. A spacer domain refers to any oligopeptide or polypeptide that functions to link the transmembrane domain to the extracellular domain and / or the transmembrane domain to the intracellular domain. A spacer domain contains up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids.

[0146] 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.

[0147] 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.

[0148] The spacer domain may be the entire or a portion of the hinge region of CD8 alpha (NCBI RefSeq: NP_001759.3), amino acids 137-206 (SEQ ID NO: 39), CD8 beta (GenBank: AAA35664.1), amino acids 315-396 of CD4 (NCBI RefSeq: NP_000607.1), or amino acids 137-152 of CD28 (NCBI RefSeq: NP_006130.1). Alternatively, a portion of the constant region of an antibody heavy or light chain may be used. Furthermore, the spacer domain may be an artificially synthesized sequence.

[0149] Furthermore, a signal peptide sequence may be linked to the N-terminus of a CAR. Signal peptide sequences are present at the N-terminus of many secretory proteins and membrane proteins and are 15 to 30 amino acids in length. Many of the protein molecules referred to above as intracellular domains have signal peptide sequences, and these signal peptides may be used as signal peptides for a CAR. In one embodiment, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 18.

[0150] 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.

[0151] 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.

[0152] It is known that signals generated through the TCR alone are insufficient for full activation of T cells, and that secondary or costimulatory signals are also required. Thus, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences) and those that act antigen-dependently to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences).

[0153] 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.

[0154] Examples of ITAMs containing primary cytoplasmic signaling sequences of particular use in the CARs disclosed herein include those derived from TCR zeta (CD3 zeta), FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. Specific, non-limiting examples of ITAMs include amino acids 51-164 of CD3 zeta (NCBI RefSeq: NP_932170.1), amino acids 45-86 of Fc epsilon RI gamma (NCBI RefSeq: NP_004097.1), amino acids 201-244 of Fc epsilon RI beta (NCBI RefSeq: NP_000130.1), amino acids 139-182 of CD3 gamma (NCBI RefSeq: NP_000064.1), amino acids 128-171 of CD3 delta (NCBI RefSeq: NP_000723.1), amino acids 153-207 of CD3 epsilon (NCBI RefSeq: NP_000724.1), and CD5 (NCBI RefSeq: NP_000724.1). The amino acids 402 to 495 of CD79a (NCBI RefSeq:NP_055022.2), 707 to 847 of CD79a (NCBI RefSeq:NP_001762.2), 166 to 226 of CD79a (NCBI RefSeq:NP_001774.1), 182 to 229 of CD79b (NCBI RefSeq:NP_000617.1), and CD66d (NCBI RefSeq:NP_000617.1) were identified. Peptides having the sequence of amino acid numbers 177 to 252 of RefSeq:NP_001806.2, and variants thereof having the same functions as these peptides. The amino acid numbers based on the amino acid sequence information of 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.

[0155] 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.

[0156] The cytoplasmic signaling sequences in the cytoplasmic signaling portion of 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 doublets provide a particularly suitable linker.

[0157] 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.

[0158] 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: 40, and the signaling domain of CD3-zeta comprises the nucleic acid sequence set forth in SEQ ID NO: 42 and the variant nucleic acid sequence set forth in SEQ ID NO: 44. 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: 40, and the signaling domain of CD3-zeta comprises the nucleic acid sequence set forth in SEQ ID NO: 42. or in another embodiment, the CD3-zeta variant nucleic acid sequence set forth in SEQ ID NO:44.

[0159] 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: 41, and the signaling domain of CD3-zeta comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 43, or in another embodiment, a CD3-zeta variant nucleic acid encoding the amino acid sequence of SEQ ID NO: 45.

[0160] 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: 41 and the signaling domain of CD3-zeta comprises the amino acid sequence set forth in SEQ ID NO: 43 and the variant amino acid sequence set forth in SEQ ID NO: 45.

[0161] 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, constitute about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95% or more of the parent CAR.

[0162] The functional portion can comprise additional amino acids at the amino or carboxy end of the portion, or at both ends, which additional amino acids are not found in the amino acid sequence of the parent CAR. Preferably, the additional amino acids do not interfere with the biological function of the functional portion, such as, for example, recognizing target cells, detecting cancer, treating or preventing cancer, etc. More preferably, the additional amino acids enhance the biological activity of the functional portion compared to the biological activity of the parent CAR.

[0163] 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.

[0164] 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.

[0165] 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.

[0166] 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.

[0167] 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.

[0168] CARs (including functional portions and functional variants of the invention) can contain synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, -amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3-hydroxyproline and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydrobenzoic ... Examples of suitable hydroxybenzoates include 2-amino-2-methyl-2-propanol, ...

[0169] 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.

[0170] CARs (including functional portions and functional variants thereof) can be obtained by methods known in the art. CARs can be prepared by any suitable method for producing polypeptides or proteins. Polypeptides and proteins can be produced by methods.Suitable methods for de novo synthesis of polypeptides and proteins 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.Polypeptides and proteins can also be produced recombinantly using standard recombinant methods and the nucleic acids described herein.See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. Furthermore, some CARs (including functional portions and functional variants thereof) can be isolated and / or purified from sources such as plants, bacteria, insects, mammals, such as rats, humans, etc. Isolation and purification methods are well known in the art. Alternatively, the CARs described herein (including functional portions and functional variants thereof) can be commercially synthesized by companies. In this regard, CARs can be synthetic, recombinant, isolated, and / or purified.

[0171] 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.

[0172] 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.

[0173] 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, being 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, for example, Harlow & Lane, Antibodies, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, 1999. publications, New York (2013).

[0174] 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.

[0175] 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.

[0176] 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, functions to position and align the CDRs in three-dimensional space.

[0177] 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 in Kabat et al. ("Sequences of Proteins of Immunological Interest", 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991; "Kabat" numbering scheme), Al-Lazikani et al. (JMB 273, 927-948, 1997; "Chothia" numbering scheme), and Lefranc et al. ("IMGT unique numbering for immunoglobulin and T cell receptor 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 LCDR1, LCDR2, and LCDR3.

[0178] "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).

[0179] Single-chain antibodies (ScFvs) are genetically engineered molecules containing the VH and VL domains of one or more antibodies (or antibodies) linked by a suitable polypeptide linker as a genetically fused single-chain molecule (see, e.g., Bird et al., Science, 242:423-426, 1988; Huston et al., Proc. Natl. Acad. Sci., 85:5879-5883, 1988; Ahmad et al., Clin. Dev. Immunol., 2012, doi:10.1155 / 2012 / 980250; Marbry, IDrugs, 13:543-549, 2010). The intramolecular orientation of the VH and VL domains in ScFvs is typically not critical for ScFvs. Thus, ScFvs with both possible configurations (VH domain-linker domain-VL domain; VL domain-linker domain-VH domain) can be used.

[0180] In dsFv, the heavy and light variable chains are mutated to introduce disulfide bonds to stabilize the association of the chains. Also included are diabodies, which are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain but use a linker that is too short to allow pairing between the two domains on the same chain, thereby pairing the domains with complementary domains on another chain to create two antigen-binding sites (see, for example, Holliger et al., Proc. Natl. Acad. Sci., 90:6444-6448, 1993; Poljak et al., Structure, 2:1121-1123, 1994).

[0181] 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.

[0182] 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 ed. (Oxford University Press 1995); each of which is incorporated herein by reference. ).

[0183] 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.

[0184] 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.

[0185] 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.

[0186] A "fully human antibody" or "human antibody" is an antibody that contains sequences derived from the human genome but does not contain sequences derived from another species. In some embodiments, a human antibody contains CDRs, framework regions, and (if present) an Fc region derived from the human genome. Human antibodies can be identified and isolated using technology to create sequences based on sequences derived from the human genome, for example, by phage display, or using transgenic animals (see, e.g., Barbas et al., Phage display: A Laboratory Manuel. 1st ed. New York: Cold Spring Harbor Laboratory Press, 2004 Print.; Lonberg, Nat. Biotech., 23:1117-1125, 2005; Lonenberg, Curr. Opin. Immunol., 20:450-459, 2008).

[0187] 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.

[0188] 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).

[0189] Additionally, the CAR, CAR-expressing T cell, antibody, or antigen-binding portion thereof, may be labeled with a detectable label, such as a radioisotope, a fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), an enzyme (e.g., alkaline phosphatase), or a fluorophore (e.g., fluorophore (FITC) or phycoerythrin (PE)). The antibody may be modified to include other enzymes such as enzymes like hydroxylase, horseradish peroxidase, and elemental particles (e.g., gold particles).

[0190] 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.

[0191] 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).

[0192] The procedure for attaching an effector molecule or detectable marker to an antibody or antigen-binding fragment varies depending on the chemical structure of the effector. Polypeptides typically contain various functional groups, such as carboxylic acid (COOH), free amine (-NH), or sulfhydryl (-SH) groups, which are available for reaction with appropriate functional groups on an antibody to attach an effector molecule or detectable marker. Alternatively, the antibody or antigen-binding fragment can be derivatized to expose or attach additional reactive functional groups. Derivatization can include attachment of any of several known linker molecules, such as those available from Pierce Chemical Company, Rockford, IL. The linker can be any molecule used to attach an antibody or antigen-binding fragment to an effector molecule or detectable marker. The linker can form covalent bonds to both the antibody or antigen-binding fragment and the effector molecule or detectable marker. Suitable linkers are well known to those skilled in the art and include, but are not limited to, straight-chain or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. When the antibody or antigen-binding fragment and the effector molecule or detectable marker are polypeptides, the linkers can be attached to the constituent amino acids through their side groups (e.g., via a disulfide linkage to cysteine) or to the amino and carboxyl groups of the alpha carbon of the terminal amino acid.

[0193] 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 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, and 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. 5,076,973, U.S. Patent No. 4,986,988, U.S. Patent No. 4,978,744, U.S. Patent No. 4,879,278, U.S. Patent No. 4,811,149. Nos. 6,444 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.

[0194] 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).

[0195] In other embodiments, the cleavable linker is pH-sensitive, i.e., sensitive to hydrolysis at a certain pH value. Typically, pH-sensitive linker is hydrolyzable under acidic conditions. For example, acid-labile linkers (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amides, orthoesters, acetals, ketals, etc.) that are hydrolyzable in lysosomes can be used (see, for example, U.S. Patent No. 5,122,368; U.S. Patent No. 5,824,805; U.S. Patent No. 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661). Such linkers are relatively stable under neutral pH conditions, such as in blood, but are unstable at pHs below 5.5 or 5.0, which is approximately the pH of the lysosome. In certain embodiments, the hydrolyzable linker is a thioether linker (e.g., a thioether attached to a therapeutic agent via an acylhydrazone bond) (see, e.g., U.S. Pat. No. 5,622,929).

[0196] In other embodiments, the linker is cleavable under reducing conditions (e.g., a disulfide linker). A variety of disulfide linkers are known in the art, including, for example, those that can be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene)-, SPDB, and SMPT (e.g., T Horpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., Immunoconjugates: Antibody C onjugates in Radioimagery and Therapy of Cancer (CW Vogel, ed., Oxford U. Press, 1987; Phillips et al., Cancer Res. 68:9280-9290, 2008). See also U.S. Patent No. 4,880,935.

[0197] 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).

[0198] 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).

[0199] In some embodiments, the linker is resistant to cleavage in an extracellular environment. For example, when the conjugate is present in an extracellular environment (e.g., in plasma), about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 3% or less, or about 1% or less of the linkers in the sample of the conjugate are cleaved. Whether a linker is resistant to cleavage in an extracellular environment can be determined, for example, by incubating the conjugate containing the desired linker with plasma for a predetermined period (e.g., 2, 4, 8, 16, or 24 hours), and then quantifying the amount of free effector molecule or detectable marker present in the plasma. Various exemplary linkers that can be used in the conjugates are described in WO2004-010957, U.S. Patent Application Publication No. 2006 / 0074008, U.S. Patent Application Publication No. 20050238649, and U.S. Patent Application Publication No. 2006 / 0024317, each of which is incorporated herein by reference in its entirety.

[0200] 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.

[0201] 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; and 4,3 Nos. 4,308,268; 4,308,269; 4,309,428; 4,313,946; 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. Conjugates containing maytansinoids, methods for making them, and their therapeutic uses are disclosed, for example, in U.S. Pat. Nos. 5,208,020; 5,416,064; 6,441,163 and European Patent EP 0 425 235 B1, the disclosures of which are expressly incorporated herein by reference.

[0202] 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).

[0203] 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.

[0204] 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.

[0205] 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).

[0206] Ribonucleases have also been conjugated to targeting molecules for use as immunotoxins (Suzuki et al., Nat. Biotech. 17:265-70, 1999). (See, e.g., Lee et al., J. Antibiot. 42:1070-87, 1989). Exemplary ribotoxins, such as α-sarcin and restrictocin, are discussed, for example, in Rathore et al., Gene 190:31-5, 1997; and Goyal and Batra, Biochem. 345(2):247-54, 2000. Calicheamicin, originally isolated from Micromonospora echinospora, is a member of the enediyne antitumor antibiotic family, producing double-strand breaks in DNA that lead to apoptosis (see, e.g., Lee et al., J. Antibiot. 42:1070-87, 1989). This drug is the toxic moiety of an immunotoxin in clinical trials (see, e.g., Gillespie et al., Ann. Oncol. 11:735-41, 2000).

[0207] 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).

[0208] CARs, CAR-expressing T cells, monoclonal antibodies specific for one or more of the antigens disclosed herein, and antigen-binding fragments thereof can also be conjugated to a detectable marker; for example, a detectable marker detectable by ELISA, spectrophotometry, flow cytometry, microscopy, or imaging techniques (e.g., computed tomography (CT), computed axial tomography (CAT) scan, magnetic resonance imaging (MRI), nuclear magnetic resonance imaging (NMRI), magnetic resonance tomography (MTR), ultrasound, fiber optic testing, and laparoscopic testing). Specific non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzymatic linkages, radioactive isotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI). For example, useful detectable markers include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, and the like. Bioluminescent markers, such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP), are also used. CARs, CAR-expressing T cells, antibodies, or antigen-binding portions thereof, can also be conjugated to enzymes useful for detection, such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, glucose oxidase, and the like. When a CAR, CAR-expressing T cells, antibodies, or antigen-binding portions thereof, is conjugated to a detectable enzyme, it can be detected by adding an additional reagent that the enzyme uses to produce a discernible reaction product. For example, when the agent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine results in a visually detectable colored reaction product. CARs, CAR-expressing T cells, antibodies, or antigen-binding portions thereof, can also be conjugated with biotin and detected via indirect measurement of avidin or streptavidin binding. Note that avidin itself can be conjugated with an enzyme or fluorescent label.

[0209] CARs, CAR-expressing T cells, antibodies, or antigen-binding portions thereof, can be conjugated with paramagnetic agents such as gadolinium. Paramagnetic agents such as superparamagnetic iron oxide are also 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 conjugated to a predetermined polypeptide epitope (e.g., leucine zipper) recognized by a secondary reporter. The antibody may be labeled with a specific antibody sequence, a binding site for a secondary antibody, a metal binding domain, or an epitope tag.

[0210] 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.

[0211] 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.

[0212] 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.

[0213] In some embodiments, nucleotide sequence can be codon modified.Without being bound by any particular theory, it is believed that codon optimization of nucleotide sequence can increase the translation efficiency of mRNA transcript.The codon optimization of nucleotide sequence can include replacing native codon with another codon that encodes the same amino acid but can be translated by tRNA that is more readily available in cells, thus increasing translation efficiency.The optimization of nucleotide sequence can also reduce the secondary mRNA structure that interferes with translation, thus increasing translation efficiency.

[0214] 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).

[0215] "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.

[0216] Recombinant nucleic acids can have sequences that do not occur in nature or that are created by the artificial combination of two otherwise separate segments of sequence. This artificial combination is often achieved by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acid, for example, by genetic engineering techniques such as those described in Sambrook et al., supra. Nucleic acids can be constructed based on chemical synthesis and / or enzymatic ligation reactions using procedures known in the art. See, for example, Sambrook et al., supra and Ausubel et al., supra. For example, nucleic acids can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides (e.g., phosphorothioate derivatives and acridine-substituted nucleotides) designed to increase the biological stability of the molecule or to increase the physical stability of the duplex formed upon hybridization.Examples of modified nucleotides that can be used to generate nucleic acids include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-substituted adenines, 7-methylguanine, and the like. Examples of nucleic acids include, but are not limited to, uracil-5-oxyacetic acid (v), ubutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleic acids of the present invention can be purchased from a company such as Integrated DNA Technologies (Coralville, IA, USA).

[0217] 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.

[0218] 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.

[0219] Nucleotide sequences that hybridize under stringent conditions can 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 exactly 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 can be 14-1. They melt more easily than full-length complements of 7 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 a temperature of about 50-70°C. Such high stringency conditions tolerate little, if any, mismatch between the nucleotide sequence and the template or target strand and are particularly suitable for detecting the 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.

[0220] 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.

[0221] 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.

[0222] However, portions of the vector may be naturally occurring. Recombinant expression vectors may be single-stranded or double-stranded, synthetic or derived from partially natural sources, and may contain any type of nucleotide, including, but not limited to, DNA and RNA, which may contain natural, non-natural, or modified nucleotides. Recombinant expression vectors may contain naturally occurring or non-naturally occurring internucleotide linkages, or both types of linkages. Preferably, the non-naturally occurring or modified nucleotides or internucleotide linkages do not interfere with the transcription or replication of the vector.

[0223] 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).

[0224] 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 particularly those derived from at least a portion of the lentiviral genome, including 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, but are 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.

[0225] 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)).

[0226] 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.

[0227] 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.

[0228] 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.

[0229] 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.

[0230] The recombinant expression vector may comprise a native or non-native promoter operably linked to a nucleotide sequence encoding a CAR (including functional portions and functional variants thereof) or to a nucleotide sequence complementary to or hybridizing to the nucleotide sequence encoding a 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, for example, a cytomegalovirus (CMV) promoter, an SV40 promoter, a R promoter, or a ribosomal protein (RI). It may be the SV promoter, or the promoter found in the long terminal repeat of the murine stem cell virus.

[0231] Recombinant expression vectors can be designed for transient expression, stable expression, or both, and can be made for constitutive or inducible expression.

[0232] 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.

[0233] One embodiment further provides a host cell containing any of the recombinant expression vectors described herein. As used herein, the term "host cell" refers to any type of cell that can contain a recombinant expression vector of the present invention. The host cell can be a eukaryotic cell, such as a plant, animal, fungus, or algae, or a prokaryotic cell, such as a bacterium or protist. The host cell can be a cultured cell or a primary cell, i.e., directly isolated from an organism such as a human. The host cell can be an adherent cell or a suspension cell, i.e., a cell that grows in suspension. Suitable host cells are known in the art and include, for example, DH5a E. coli cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, etc. For the purpose of amplifying or replicating a recombinant expression vector, the host cell can be a prokaryotic cell, such as a DH5a cell. For the purpose of producing a recombinant CAR, the host cell can be a mammalian cell. The host cell can be a human cell. The host cell can be of any cell type, originate from any type of tissue, and be at any stage of development, but the host cell can be a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC). The host cell can be a T cell.

[0234] For purposes herein, T cells can be any T cells, e.g., cultured T cells, e.g., primary T cells, or T cells from cultured T cell lines, e.g., Jurkat, SupTl, etc., or T cells obtained from a mammal. When obtained from a mammal, T cells can be obtained from a number of sources, including, but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. T cells can also be enriched or purified. T cells can be human T cells. T cells can be T cells isolated from a human. T cells can be any type of T cell and at any developmental stage, including, but not limited to, CD4+ / CD8+ double-positive T cells, CD4+ helper T cells, e.g., Th1 and Th2 cells, CD8+ T cells (e.g., cytotoxic T cells), tumor-infiltrating cells, memory T cells, memory stem cells, i.e., Tscm, naive T cells, etc. T cells can be CD8+ T cells or CD4+ T cells.

[0235] In one embodiment, the CARs described herein can be used in suitable non-T cells. Such cells can be cells with immune effector function, e.g., cells generated from pluripotent stem cells. These include NK cells and T-like cells.

[0236] 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.

[0237] 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%.

[0238] E. Treatment Method It is contemplated that the CARs disclosed herein can be used in methods for treating or preventing disease in mammals. In this regard, one embodiment provides a method for treating or preventing cancer in a mammal, comprising administering to the mammal a CAR, a nucleic acid, a recombinant expression vector, a host cell, a population of cells, an antibody and / or an antigen-binding portion thereof, and / or a pharmaceutical composition in an amount effective to treat or prevent cancer in the mammal.

[0239] 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.

[0240] 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.

[0241] 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, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits. Mammals may be from the order Carnivora, including Feline (cat) and Canine (dog). Mammals The article may be from the order Artiodactyla, which includes Bovine (cow) and Swine (pig), or Perssodactyla, which includes 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.

[0242] 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.

[0243] 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.

[0244] Furthermore, the treatment or prevention provided by this method can include treatment or prevention of one or more conditions or symptoms of the disease, such as cancer, being treated or prevented. Also, for purposes herein, "prevention" can include delaying the onset of the disease, or its symptoms or conditions.

[0245] 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.

[0246] 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.

[0247] 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., blood cells. The cells may be cells of any organ or tissue, including cytoplasmic or endothelial cells.

[0248] The contacting step can occur in vitro or in vivo with respect to a mammal. Preferably, the contacting step is in vitro.

[0249] 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.

[0250] 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).

[0251] 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.

[0252] Any administration method, including local and systemic administration, can be used for the disclosed therapeutic agents. For example, topical, oral, intravascular (e.g., intravenous), intramuscular, intraperitoneal, intranasal, intradermal, intrathecal, and subcutaneous administration can be used. The specific administration mode and dosing regimen will be selected by the attending clinician, taking into account the characteristics of the case (e.g., the subject, the disease, the disease state involved, and whether the treatment is preventive). When more than one agent or composition is administered, one or more administration routes can be used; for example, the chemotherapeutic agent can be administered orally, and the antibody or antigen-binding fragment or conjugate or composition can be administered intravenously. Administration methods include injection, in which the CAR, CAR T cell, conjugate, antibody, antigen-binding fragment, or composition is provided in a non-toxic pharmaceutically acceptable carrier, such as water, saline, Ringer's solution, dextrose solution, 5% human serum albumin, solid oil, ethyl oleate, or liposome. In some embodiments, local administration of the disclosed compounds can be used, for example, by applying an antibody or antigen-binding fragment to an area of ​​tissue from which a tumor has been removed or to an area suspected of being prone to tumor development. In some embodiments, sustained intratumoral (or near-tumoral) release of a pharmaceutical preparation containing a therapeutically effective amount of an antibody or antigen-binding fragment can be beneficial. In other examples, the conjugate is applied topically to the cornea as eye drops or intravitreally to the eye.

[0253] 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 be with more than one separate 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 be with a daily dose or multiple daily doses over a period of days to months or even years. Accordingly, the dosage regime will also be determined, at least in part, by the particular need of the subject being treated and will be dependent on the judgment of the administering practitioner.

[0254] 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.

[0255] 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.

[0256] 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.

[0257] In some embodiments, the combination therapy may include administering to the subject a therapeutically effective amount of an additional cancer inhibitor.Non-limiting examples of additional therapeutic agents that can be used in the combination therapy include microtubule binding agents, DNA intercalators or crosslinkers, DNA synthesis inhibitors, DNA and RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, gene regulators, and angiogenesis inhibitors.These agents (administered in therapeutically effective amounts) and treatments can be used alone or in combination.For example, any suitable anti-cancer or anti-angiogenic agent can be administered in combination with the CAR, CAR-T cells, antibodies, antigen-binding fragments, or conjugates disclosed herein.The methods and therapeutic dosages of such agents are known to those skilled in the art and can be determined by skilled clinicians.

[0258] 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 folic acid (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, e.g., podophyllum (e.g., etoposide and teniposide), taxanes (e.g., docetaxel and paclitaxel), vincas (e.g., vinblastine, vincristine, vindesine, and vinorelbine); cytotoxic / antitumor antibiotics, e.g., members of the anthracycline family (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, and valrubicin), bleomycin, rifampicin, hydroxyurea, and mitomycin; topoisomerase inhibitors, e.g., topotecan and irinotecan; monoclonal antibodies, e.g., cyclosporin ... monoclonal antibodies, such as alemtuzumab, bevacizumab, cetuximab, gemtuzumab, rituximab, panitumumab, pertuzumab, and trastuzumab; tumor-affinity photosensitizing dyes, such as aminolevulinic acid, methyl aminolevulinate, porfimer sodium, and verteporfin; and other drugs, such as alitretinoin, altretamine, amsacrine, anagrelide, arsenic trioxide, asparaginase, axitinib, bexilin, thiazolinone ... These include, but are not limited to, salotene, 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 agents are known to those skilled in the art and can be determined by a skilled clinician.

[0259] 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.

[0260] In one embodiment, an effective amount of an antibody or antigen-binding fragment or conjugate thereof that specifically binds to one or more of the antigens disclosed herein is administered to a subject with a tumor after anti-cancer treatment. After a sufficient amount of time has passed for the administered antibody or antigen-binding fragment or conjugate to form an immune complex with the antigen expressed on each cancer cell, the immune complex is detected. The presence (or absence) of the immune complex indicates the effectiveness of the treatment. For example, an increase in immune complexes compared to a control taken before treatment indicates that the treatment is ineffective, and a decrease in immune complexes compared to a control taken before treatment indicates that the treatment is effective.

[0261] 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.

[0262] The composition for administration may include CA dissolved in a pharmaceutically acceptable carrier, such as an aqueous carrier. The compositions may contain solutions of CAR, or CAR-expressing T cells, conjugates, antibodies, or antigen-binding fragments. Various aqueous carriers, such as buffered saline, may be used. These solutions are sterile and generally free of undesirable substances. The compositions may be sterilized by conventional, well-known sterilization techniques. The compositions may 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 may 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.

[0263] 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).

[0264] CARs, or CAR-expressing T cells, antibodies, antigen-binding fragments, or conjugates, can be provided in lyophilized form and rehydrated with sterile water before administration, but they can also be provided in sterile solutions of known concentrations. The CAR, or CAR-expressing T cells, antibodies, or antigen-binding fragments, or conjugates, solution is then added to an infusion bag containing 0.9% sodium chloride, USP, and in some cases administered at a dosage of 0.5 to 15 mg / kg body weight. Considerable experience is available in the field for administering antibody or antigen-binding fragment and conjugate drugs; for example, antibody drugs have been commercially available in the United States since the approval of Rituxan® in 1997. CARs, or CAR-expressing T cells, antibodies, antigen-binding fragments, and their conjugates, can be administered by slow infusion rather than intravenous injection or intravenous bolus. In one example, a higher loading dose is administered with subsequent maintenance doses administered at lower levels. For example, an initial loading dose of 4 mg / kg of the antibody or antigen-binding fragment (or a corresponding dose of a conjugate comprising the antibody or antigen-binding fragment) can be infused over a period of approximately 90 minutes, followed by weekly maintenance doses of 2 mg / kg for 4-8 weeks infused over a period of 30 minutes if the previous dose was well tolerated.

[0265] 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, edited by J. Kreuter, 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).

[0266] 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 poloxamer 407 exists as a viscous but still mobile liquid at low temperatures, but forms a semi-fluid gel at body temperature. This 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).

[0267] 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.

[0268] 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.

[0269] The label or package insert typically includes, for example, instructions on how to treat or prevent tumors, or The kit may further comprise instructions for using the disclosed antibodies, antigen-binding fragments, conjugates, nucleic acid molecules, CARs, or CAR-expressing T cells in methods for generating CAR T cells or CAR T cells. The package insert typically includes instructions customarily included in commercial packaging of therapeutic products, including information about indications, usage, dosage, administration, contraindications, and / or warnings regarding the use of the therapeutic product. The instructional material may be written in electronic form (e.g., a computer diskette or compact disc) or visual (e.g., a video file). The kit may also include additional components to facilitate the particular application for which the kit is designed. Thus, for example, the kit may further include 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 may 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 of skill in the art. [Example]

[0270] 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.

[0271] Example 1 Isolation of CD19-specific antibodies from phage- and yeast-displayed fully human ScFv libraries material and method: a) Production of human ScFv and CD19-specific antibodies A naive human ScFv (recombinant single-chain fragment of immunoglobulin variable) phage display library (approximate diversity, 10) constructed from peripheral blood B cells of 50 healthy donors was used. 10The phage-displayed ScFv10 (species-specific specificity) (ZYZhu and DSDimitrov, unpublished data) was used for the selection of ScFv on recombinant human CD19 protein (Miltenyi Biotec, unpublished data). 12 The amplified species library was incubated with 5, 3, or 1 μg of coated CD19 in 5 × 100 μl volumes and distributed equally among five wells of a 96-well plate for 2 hours at room temperature during the first, second, and third rounds of biopanning. After each round of incubation, wells were washed with phosphate-buffered saline containing 0.05% Tween 20 (PBST) five times in the first round and ten times in subsequent rounds to remove nonspecifically bound phages. Bound phages were mixed with TG1 competent cells at 37°C for 1 hour, and phages were amplified from the infected cells and used in the next round of biopanning. After the third round of biopanning, 380 clones were randomly selected from the infected TG1 cells and each was inoculated into 150 μl of 2YT medium containing 100 μg / ml carbenicillin and 0.2% glucose in a 96-well plate using an automated BioRobotics BioPick colony picking system (Genomic Solutions, Ann Arbor, MI). After the bacterial culture reached an optical density at 600 nm (OD600) of 0.5, helper phage M13K07 at a multiplicity of infection (MOI) of 10 and 50 μg / ml (final concentration) of kanamycin were added to the medium, and the plate was further incubated overnight at 30°C on a shaker at 250 rpm. The phage supernatant was mixed with a 3% nonfat milk solution in PBS at a volume ratio of 4:1 and used in enzyme-linked immunosorbent assay (ELISA) to identify phage clones displaying ScFv or VH with high CD19 binding affinity. The supernatant was incubated with 50 ng of coated recombinant human CD19 per well in a 96-well plate for 2 hours at room temperature, washed five times with PBST, and incubated overnight at 4°C. The wells were blocked with 3% nonfat milk in PBS and washed three times with PBS containing 0.05% Tween 20. Phage binding to CD19 was detected using a goat anti-M13 antibody conjugated with horseradish peroxidase. After incubation with the antibody, the wells were washed to remove nonspecifically bound antibody, and 3,3',5,5'-tetramethylbenzidine (TMB) substrate was added. The absorbance of the solution at 450 nm (A450) was measured. Clones that bound to CD19 and had an A450 greater than 1.0 were selected for further characterization.

[0272] b) Expression and purification of selected soluble ScFvs The VH and VL of selected clones were DNA sequenced, and ScFvs encoded by clones with unique sequences were expressed and purified as described below. Plasmids extracted from these clones were used to transform HB2151 cells. Single colonies were picked from plates containing freshly transformed cells and inoculated into 200 ml of 2YT medium containing 100 μg / ml ampicillin and 0.2% glucose, and incubated at 37°C with shaking at 250 rpm. When the culture OD at 600 nm reached 0.90, isopropyl-β-d-thiogalactopyranoside was added to a final concentration of 0.5 mM, and the culture was further incubated overnight at 30°C. After centrifugation at 8,000 × g for 20 min, the bacterial pellet was collected and resuspended in PBS buffer containing 0.5 mU polymyxin B (Sigma-Aldrich, St. Louis, MO). After incubation for 30 min at room temperature with rotation at 50 rpm, the resuspended pellet was centrifuged at 25,000 × g for 25 min at 4°C, and the supernatant was used for ScFv purification using Ni-NTA resin according to the supplier's protocol (Qiagen).

[0273] c) ELISA binding assay: Recombinant human CD19 diluted to 2 μg / ml in PBS 50 μl was coated onto a 96-well plate overnight at 4°C. Serial dilutions of purified ScFvs bearing His and Flag tags were added to the target protein-coated wells. After washing, a 1:3000 dilution of HRP-conjugated anti-Flag antibody was added for 1 hour at RT. After washing, 3,3,5,5'-tetramethylbenzidine (TMB) substrate was added and incubated for 10 minutes at room temperature. The reaction was then stopped by adding 1N H2SO4, and the OD was read at 450 nm to quantify the relative ability of the ScFvs to bind to CD19.

[0274] d) Yeast display of scFv libraries. The same ScFv starting material as used for phage display was also incorporated into the yeast ScFv display system. To complement the phage-based scFv analysis, a yeast library expressing a human scFv library was also screened. To enrich for yeast expressing scFvs that bind both recombinant CD19-Fc and CD19 expressed on the cell surface of CHOK1 cells, cell panning was performed on CHOK1 transfected CD19 cells. For the first round of cell surface panning, CHOK1-CD19 cells were seeded into 6-well plates and grown in F12K medium to 50% confluency 2 days prior to panning. Then, 5 × 10 7Yeast cells were washed twice with PBSA buffer, resuspended in 3 mL of F12K medium, and gently added dropwise to the CHOK1-CD19 cells. After gentle shaking on ice for 2 hours, the CHOK1-CD19 cells were washed three times with ice-cold PBSA to remove yeast cells that did not bind to CHOK1-CD19. Then, 0.05% trypsin-EDTA (Gibco) was used to separate the CHOK1-CD19-bound yeast cells from the plate. The cell mix containing both yeast and CHOK1 cells was then inoculated into 10 mL of SDCAA medium and amplified overnight at 30°C, followed by induction in SGCAA medium at 30°C for 16 hours. For the second round of cell panning, a similar protocol was used, but with more stringent washing conditions. This panning method yielded the ml9217 binder. This binder and other binders obtained from phage display were subsequently isolated. Further characterization of the binder suggested that the biological properties of the CAR generated from this hit were still suboptimal and required affinity maturation.

[0275] To increase the affinity of ml9217, we used error-prone PCR to generate a yeast-display ml9217 mutant scFv library, generating random point mutations in the scFv gene sequence. After electroporation, the resulting mutant library was cultured overnight in SDCAA medium at 30°C for 16 hours, then switched to SGCAA medium at 30°C for another 16 hours. The mutant library was then sorted through MACS (immunomagnetic column, Miltenyi Biotec) using CD19-Fc as the capture antigen to downsize the library and increase the population of mutants capable of binding to CD19-Fc. The strongest binders were then selected by double-staining the pool with Anti-c-Myc-Alexa 488 and CD19-Fc / Anti-Hu-Fc and selecting binders with the highest binding affinity as well as c-Myc expression levels. This process was then repeated two more times until flow cytometry of yeast particles using fluorescently labeled antigen yielded an increased average binding affinity of the mutant pool compared to the starting construct. Binding affinities were estimated by flow cytometry of yeast pools using titrated labeled CD19. This process increased the affinity from an EC50 (effective concentration for 50% binding of labeled CD19 to ScFv-displaying yeast) of 0.5 μg / ml for M19217 to <0.01 μg / ml for affinity-matured binders (M19217-1, 19217-2, M19217-7, M19217-23, M19217-29, M19217-38, M19217-40).

[0276] result: Because phage display candidates did not yield biologically functional CAR constructs due to inherent challenges in the CD19 structure, ScFv identifications that yielded biologically active binders were generated by yeast display. Based on flow cytometry analysis of the yeast-displayed ScFvs, eight ScFv clones specific for recombinant human CD19 were identified and designated as the human anti-CD19 ScFv binder M19217 (LTG2050, established clone, EC50 0.5 μg / ml) and the following affinity-matured binders (EC50 < 0.01 μg / ml): M19217-1 (LTG2065), M19217-2 (LTG2066), M19217-7 (LTG2067), M19217-23 (LTG2068), M19217-29 (LTG2069), M19217-38 (LTG2070), and M19217-40 (LTG2071). The generation of chimeric antigen receptor expressing human anti-CD19 binders LTG2050, LTG2065, LTG2066, LTG2067, LTG2068, LTG2069, LTG2070, and LTG2071 is outlined in Example 2 below.

[0277] Example 2 CAR expressing anti-CD19 fully human binding sequence Homo sapiens CD19 (B4, CVID3, Leu-12) is a well-studied cell surface glycoprotein expressed in B-cell leukemias and lymphomas. At least two antibody-drug conjugates (SGN-CD19A denintuzumab mafodotin (Seattle Genetics) and SAR3419 coltuximab ravtansine) are being evaluated in Phase II clinical trials. A Phase 1 trial of SGN-19A demonstrated a CRc of 35% in the optimal treatment arm (Fathi AT, Borate U, DeAngelo DJ, O'Brien MM, Trippett T, Shah BD, Hale GA, Foran JM, Silverman LB, Tibes R, Cramer S, Pauly M, Kim S, Kostic A, Huang X, Pan Y, Chen R. 2015. Blood 126:1328). However, disappointing Phase II results for SAR3419 have put development on hold. (Coiffer B, Thieblemont C, de Guibert S, Dupuis J, Ribrag V, Bouabdallah R, Morschhauser F, Navarro R, Le Gouill S, Haioun C, Houot R, Cassasnovas O, Holte H, Lamy T, Broussais F, Payrard S, Hatteville L, Tilly H. 2016. Bt J Haematolo 173:722-30.) The use of a bispecific anti-CD3 / anti-CD19 antibody (brinotumomab) has been discussed above. Given the current progress in T cell-based therapy using CD19 CARs, the best approach is certainly cell-based immunotherapy, and the CAR construct presented here represents an innovative new approach to generate and implement a novel CD19-binding moiety derived from human sequences.

[0278] The novel anti-CD19 CAR-T constructs described herein have high levels of cell surface expression on primary human T cells and possess specific and potent cytotoxic and cytokine functions against CD19-positive tumor cells. CD19 CARs were designed using CD19-binding sequences derived from ScFv candidates identified by phage display, as in Example 1. For characterization, they were cloned into lentiviral expression vectors containing selected structural and signaling domains under the control of the EF1a promoter and tested in vitro for transduction efficiency, killing function, and cytokine production in both model cell lines and primary human T cells. Table 1 outlines the terms used. The CAR construct LTG#1538 is of interest, as this murine-derived sequence is the current binder used in commercial development (see KTE-C19, Kite Pharma, and CTL019, Novartis).

[0279] [Table 1]

[0280] material and method: (a) Cell line The Burkitt's lymphoma cell line Raji and chronic myeloid leukemia line K562 were purchased from the American Tissue Culture Collection (ATCC, Manassass, VA). Cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS, Hyclone, Logan, UT) and 2 mM L-Glutamax (Thermo Fisher Scientific, Grand Island, NY). The human embryonic kidney line 293T was purchased from ATCC and cultured in CD FortiCho medium (Gibco / Thermo Fisher Scientific). c, Grand Island, NY). 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 Technology, Inc., Gaithersburg, MD), followed by cloning and selection of luciferase-positive clones.

[0281] (b) Creation of chimeric antigen receptor (CAR)-expression vector The CAR antigen-binding domain, ScFv, sequence was derived from a human anti-CD19 ScFv fragment. The CAR T construct was generated by linking the binder sequence in frame to the CD8a binding and transmembrane domains (UniProt sequence ID P01732, aa 138-206), followed by the 4-1BB (CD137, aa 214-255, UniProt sequence ID Q07011) signaling domain and the CD3 zeta signaling domain (CD247, aa 52-163, Ref sequence ID: NP_000725.1). 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.

[0282] (c) Primary T cell purification and transduction Human primary T cells from healthy volunteers were cultured according to the manufacturer's protocol (Miltenyi Biotec, Bergisch Gladbach) using CD4 + and CD8 + T cells were purified from whole blood or buffy coat (purchased from a commercial provider with the donor's written consent) using immunomagnetic bead selection at a density of 0.3–2 × 10 6cells / ml, were cultured in TexMACS medium supplemented with 200 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 IL-2 until harvest on days 8–12.

[0283] (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 were removed from co-cultures at an E:T ratio of 10:1 and analyzed for IFNγ, TNFα, and IL-2 concentrations by ELISA (eBioscience, San Diego, CA).

[0284] (e) Flow cytometry analysis. For cell staining, 500,000 CAR T-transduced cells were harvested from culture, washed twice with cold AutoMACS buffer supplemented with 0.5% bovine serum albumin (Miltenyi Biotec), and stained with CD19-Fc peptide (R&D, Minneapolis, MN) followed by anti-Fc-AF647 conjugate (Jackson ImmunoResearch, West Grove, PA) to detect CAR surface expression. Non-transduced cells were used as a control. In all studies, dead cells were identified by 7AAD staining (BD Cells were filtered using a standard ELISA kit (Microfluidics Laboratory, San Jose, CA) and 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).

[0285] result: To evaluate the novel anti-CD19 fully human ScFv binding sequences, CAR constructs were designed incorporating one of the following ScFv sequences (Table 1): ScFv1 (M19217), ScFv2 (M19217-1), ScFv3 (M19217-2), ScFv4 (M19217-7), ScFv5 (M19217-23), ScFv6 (M19217-29), ScFv7 (M19217-38), and ScFv8 (M19217-40) as tumor antigen-binding domains. In each CAR design, the tumor-targeting domain was followed by a linker and transmembrane domain derived from the human CD8 protein, a 4-1BB costimulatory domain, and a CD3 zeta signaling domain (Table 2). A CAR construct encoding the FMC63 binder sequence derived from mouse immunoglobulin was used as a positive control.

[0286] [Table 2]

[0287] Anti-CD19 chimeric antigen receptor-transduced T cells demonstrate surface expression and cytolytic activity.

[0288] a) Surface expression of anti-CD19 CAR To evaluate the novel anti-CD19 CAR, a lentiviral vector (LV) encoding the CAR construct under the control of the human EF1a promoter was generated as described in Materials and Methods. Primary human T cells from healthy donors were then transduced with the lentiviral vector encoding the CAR. Non-transduced cells (NT) from the same donor or GFP-transduced cells from the same donor served as negative controls. Data represent the results of at least three assays from different donors.

[0289] On day 0 of culture, T cells were activated with TransAct T cell reagent (active engagement of CD3 and CD28 antigens, Miltenyi Biotec, Inc.) in the presence of IL-2, as described in Materials and Methods. On days 8–10 of culture, anti-CD19 CAR expression on the T cell surface was detected with CD19-Fc peptide followed by anti-Fc-AF647 and analyzed by flow cytometry. Each of the selected anti-CD19 CAR constructs demonstrated surface CAR expression. When non-transduced T cells were used as a negative control (0%), the following CAR expression levels were observed: LTG1538, 60%; LTG 2050, 46%;LTG2065, 64%;LTG2066, 21%;LTG2067, 81%;LTG2068, 54%;LTG2069, 68%;LTG2070, 47%;LTG2071, 21%.

[0290] b) Cytolytic and cytokine assays of anti-CD19 CAR To demonstrate the cytolytic function of the generated CAR T cells, an overnight cell killing assay was performed by combining CAR-T cells with CD19-positive Raji-luc cells, CD19-positive Reh-luc cells, CD19-negative K562-luc cells, or CD19-negative 293T-luc cells at E:T ratios of 40:1, 20:1, or 10:1, as described in Materials and Methods (Figure 4). Selected constructs (LTG2050, LTG2065-2071) demonstrated dose-dependent CD19-specific tumor killing. We then measured the concentrations of the proinflammatory cytokines IFN-γ, TNF-α, and IL-2 secreted by CAR T cells transduced with the CAR19 constructs when challenged with CD19-positive tumor cells (Figure 5). A CAR T cell-only control was included with each construct to test the basal levels of cytokine production. TNF-α, IFN-γ, and IL-2 levels were significantly higher in CD19 tumor cells than in the control group. + These constructs were strongly induced by T cells exposed to Raji cells. Furthermore, none of the constructs demonstrated cytokine production above baseline in the absence of tumor cell targets. Thus, the CAR T constructs LTG2050, LTG2065-2071 inhibited CD19 + Interestingly, construct LTG2050, despite efficiently killing CD19-positive cell lines in vitro, exhibited low levels of IL-2 as detected by ELISA. Thus, while some binders are active in soluble IgG or ScFv formats and suitable for expression on the T cell surface in a CAR T format, none of the CD19 - CAR design and binder selection are not straightforward because they are inefficient at killing or producing cytokines when cocultured with CD19-positive tumors. Many binders were identified during screening, but are not included here. These binders were not expressed on the surface of T cells in our CAR expression vector, or did not show strong lytic activity against CD19-positive cell lines.

[0291] In summary, the novel fully human anti-CD19 CAR constructs LTG2050, LTG2065, LTG2066, LTG2067, LTG2068, LTG2069, LTG2070, and LTG2071 (Table 1 below) were demonstrated to be highly functional. All CAR constructs except LTG2050 outperformed the positive control, murine FMC63 (LTG1538), and are therefore expected to have more potent therapeutic activity.

[0292] 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.

[0293] 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, and such adaptations and modifications should therefore be understood to be within the meaning and range of equivalents of the disclosed embodiments, and should be understood as such. It is intended to be understood that the terms or phrases employed herein are for purposes of description and not of limitation. In the drawings and description, exemplary embodiments are disclosed, and although specific terms may be employed, unless otherwise stated, they are used in a generic and descriptive sense only and not for purposes of limitation, and the claims are therefore not so limited. Moreover, those skilled in the art will recognize that certain steps of the 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.

[0294] Sequences of the present disclosure The nucleic acid and amino acid sequences listed below are shown using standard abbreviations for nucleotide bases and three-letter codes for amino acids as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the represented strand. In the accompanying sequence listing: SEQ ID NO: 1 is the nucleotide sequence of the CD19-reactive ScFv1 binding domain (LTG2050). GAGGTCCAGCTGGTACAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCAGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCTAGTGGTGGTAGCACAAGCTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGGATCGGGGAATTACCGCCACGGACGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAGGAGGTGGCGGGTCTGGTGGAGGCGGTAGCGGTGGTGGCGGATCCCAGTCTGTGCTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGGCAGACGGCCAAGATTACCTGTGGGGGAAGTGACATTGGAAATAAAAATGTCCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTCCTGGTCGTCTATGATGATTACGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGGACGCGGCCACCCTGACGATCAGCACGGTCGAAGTCGGGGATGAGGCCGACTATTTCTGTCAGGTGTGGGACAGTAGTGGTGATCCTTATTGGGTGTTCGGCGGAGGGACCCAGCTCACCGTTTTAGGT SEQ ID NO: 2 is the amino acid sequence of the CD19-reactive ScFv1 binding domain (LTG2050). EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSDRGITATDAFDIWGQGTMVTVSSG GGGSGGGGSGGGGSQSVLTQPPSVVAPGQTAKITCGGSDIGNKNVHWYQQKPGQAPVLVVYDDYDRPSGIPERFSGSNSGDAATLTISTVEVGDEADYFCQVWDSSGDPYWVFGGGTQLTVLG SEQ ID NO: 3 is the nucleotide sequence of the CD19-reactive ScFv2 binding domain (LTG2065). GAGGTCCAGCTGGTACAGTCTGGAGCTGAGGTGAAGAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCAGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATTAATCAACCCTAGTGGTGGTAGCACAAGCTACGCACA GAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGACACGGCGTGTATTACTGTGCGAGATCGGATCGGGGAATTACCGCCACGGACGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAGGCG GAGGAGGCTCCGGGGGAGGAGGTTCGGGGGCGGGGGTTCCCAGTCTGTGCTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGGCGGATGGCCAAGATTACCTGTGGGGGAAGTGACATTGGAAATAAAATGTCCACTGGTATCAGCAGAAGCCAGGCCAGGCCCCTGTCCTGGTTGTCTAT GATGATTACGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGGACGCGGCCACCCTGACGATCAGCACGGTCGAAGTCGGGGATGAGGCCGACTATTTCTGTCAGGTGTGGGACGGTAGTGGTGATCCTTATTGGATGTTCGGCGGAGGGACCCAGCTCACCGTTTTAGGT The sequence number 4 is the amino acid sequence of CD19 reaction ScFv2 binding domain(LTG2065). EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGLINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYCARSDRGITATDAFDIWGQGTMVTVSSGGGGGSGGGSGGGGSQSVLTQPPSVSVAPGRMAKITCG GSDIGNKNVHWYQQKPGQAPVLVVYDDYDRPSGIPERFSGSNSGDAATLTISTVEVGDEADYFCQVWDGSGDPYWMFGGGTQLTVLG SEQ ID NO: 5 is the nucleotide sequence of the CD19-reactive ScFv3 binding domain (LTG2066). GAGGTCCAGCTGGTACAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCCGGATACACCTTCACCAGCTACTACATGCACTGGGTGCGACAGGCCCCTGGACAAGGGTTTGAGTGGATGGGATTAATCAACCCTAGTGGTAGTAGCACAAGCTACGCACA GAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAACCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGGATCGGGGAATTACCGCCACGGACGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAGGCG GAGGAGGCTCCGGGGGAGGAGGTTCCGGGGGCGGGGGTTCCCAGTCTGTGCTGACTCAGCCACCCTCGGTGCCAGTGGCCCCAGGGCAGACGGCCAAGATTATCTGTGGGGGAAGTGACATTGGAAATAAAAATGTCCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTCCTGGTCGTCTAT GATGACTACGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGGACGCGGCCACCCTGACGATCAGCACGGTTGAAGTCGGGGATGAGGCCGACTATTTCTGTCAGGTGGGACGGTAGTGGTGATCCTTATTGGGTGTTCGGCGGAGGGACCCAGCTCACCGTCTTAGGT SEQ ID NO: 6 is the amino acid sequence of the CD19-reactive ScFv3 binding domain (LTG2066). EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGFEWMGLINPSGSSTSYAQKFQGRVTMTRDTSTSTVYMELSNLRSEDTAVYYCARSDRGITATDAFDIWGQGTMVTVSSG GGGSGGGGSGGGGSQSVLTQPPSVPVAPGQTAKIICGGSDIGNKNVHWYQQKPGQAPVLVVYDDYDRPSGIPERFSGSNSGDAATLTISTVEVGDEADYFCQVWDGSGDPYWVFGGGTQLTVLG SEQ ID NO: 7 is the nucleotide sequence of the CD19-reactive ScFv4 binding domain (LTG2067). GAGGTCCAGCTGGTACAGTCTGGAGCTGAGGTGACAAGCCTGGTGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCAGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATGATCAACCCTAGTGGTGGTAGCACAAGCTACGCACA GAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGACACGGCGTGTATTACTGTGCGAGATCGGATCGGGGAATTACCTCCACGGACGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAGGCG GAGGAGGCTCCGGGGGAGGAGGTTCGGGGGGCGGGGTTCCCAGTCTGTGCTGACTCAGCCACCTCGGTGTCATTGGCCCCAGGGCAGACGGCCAAGATTACCTGTGGGGGAAGTGACATTGGAAATAAAATGTCCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTCCTAGTCGTCTAT GATGATTACAACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGGACTCAGCCACCCTGACGATCAGCACGGTCGAAGTCGGGGATGAGGCCGACTATTTCTGTCAGGTGTGGGACGGTAGTGGTGATCCCTTATTGGGTGTTCGGCGGAGGGACCCAGCTCACCGTTTTAGGT The sequence number 8 is the amino acid sequence of CD19 reaction ScFv4 binding domain(LTG2067). EVQLVQSGAEVNKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGMINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSDRGITSTDAFDIWGQGTMVTVSSG GGGSGGGGSGGGGSQSVLTQPPSVSLAPGQTAKITCGGSDIGNKNVHWYQQKPGQAPVLVVYDDYNRPSGIPERFSGSNSGDSATLTISTVEVGDEADYFCQVWDGSGDPYWVFGGGTQLTVLG SEQ ID NO: 9 is the nucleotide sequence of the CD19-reactive ScFv5 binding domain (LTG2068). GAGGTCCAGCTGGTACAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCATCTGGATACACCTTCACCGGCTACTATATGCACTGGGTGCGGCAGGCCCCTGGACAAGGGCTTGAGTGGATAGGATTAATCAACCCTAGTGGTGGTAGCACAAGCTACGAACA GAAGTTCCAGGGCAGAGTCGCCATGACCAGGGACACGTCAACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGACACGGCGTGTATTACTGTGCGAGATCGGATCGGGGAATTACCGCCACGGACGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAGGCG GAGGAGGCTCCGGGGGAGGAGGTTCGGGGGGCGGGGTTCCCAGTCTGTGCTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGGCAGACGGCCAAGATTACCTGTGGGGGAAGTGACATTGGAGATAAAAATGTCCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTCCTGGTCGTCTAT GATGATTACGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGGACGCGGCCACCCTGACGATCAGCACGGTCGAAGTCGGGGATGAGGCCGACTATTTCTGTCAGGTGTGGGACGGTATTGGTGATCCCTATTGGGTGTTCGGCGGAGGGACCCAGCTCACCGTTTTAGGT The sequence number 10 is the amino acid sequence of CD19 reaction ScFv5 binding domain(LTG2068). EVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWIGLINPSGGSTSYEQKFQGRVAMTRDTSTSTVYMELSSLRSEDTAVYYCARSDRGITATDAFDIWGQGTMVTVSSG GGGSGGGGSGGGGSQSVLTQPPSVVAPGQTAKITCGGSDIGDKNVHWYQQKPGQAPVLVVYDDYDRPSGIPERFSGSNSGDAATLTISTVEVGDEADYFCQVWDGIGDPYWVFGGGTQLTVLG SEQ ID NO: 11 is the nucleotide sequence of the CD19-reactive ScFv6 binding domain (LTG2069). GAGGTCCAGCTAGTACAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCAGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATGATCAACCCTAGTGGTGGTAGCACAAGCTACGCACA GAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGACACGGCGTGTATTACTGTGCGAGATCGGATCGGGGAATTACCGCCACGGACGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAGGCG GAGGAGGCTCCGGGGGAGGAGGTTCGGGGGCGGGGGTTCCCAGTCTGTGCTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGGCAGACGGCCAAGATTACCTGTGGGGGAAGTGACATTGGAAATAAAATGCCCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTCCTGGTCGTCTAT GATGATTACGACCGGCCCTCAGGGATCTCTGAGCGATTCTCTGGCTCCAACTCTGGGGACGCGGCCACCCTGACGATCAGCACGGTCGAAGTCGGGGATGAGGCCGACTATTTCTGTCAGGTGTGGGACGGTAGTGGTGATCCTTTTTGGGTGTTCGGCGGAGGGACCCAGCTCACCCGTTTTAGGT The sequence number 12 is the amino acid sequence of CD19 reaction ScFv6 binding domain(LTG2069). EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGMINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSDRGITATDAFDIWGQGTMVTVSSG GGGSGGGGSGGGGSQSVLTQPPSVVAPGQTAKITCGGSDIGNKNAHWYQQKPGQAPVLVVYDDYDRPSGISERFSGSNSGDAATLTISTVEVGDEADYFCQVWDGSGDPFWVFGGGTQLTVLG SEQ ID NO: 13 is the nucleotide sequence of the CD19-reactive ScFv7 binding domain (LTG2070). GAGGTCCAGCTGGTACAGTCTGGAGCTGAGGTGAAGGGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCAGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACGAGGGCTTGAGTGGATGGGATTAATCAACCCTAGTGGTGGTAGCACAAGCTACGCACA GGAGTTCCAGGGCAGAGTCACCATGACCAGGGACATGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGACACGGCGTGTATTACTGTGCGAGATCGGATCGGGGAATTAGCGCCACGGACGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAGGCG GAGGAGGCTCCGGGGGAGGAGGTTCGGGGGGCGGGGTTCCCAGTCTGTGCTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGGCAGATGGCCAAGATTACCTGTGGGGGAAGTGACATTGGAAATAAAATGTCCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTCCTGGTCGTCTAT GATGATTACAACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGGACGCGGCCACCCTGACGATCAGCACGGTCGAAGTCGGGGATGAGGCCGACTATTTCTGTCAGGTATGGGACGGTAGTGGTGATCCTATTGGGTGTTCGGCGGAGGGACCCAGCTCACCGATTTAGGT Amino acid sequence of CD19 reaction ScFv7 binding domain(LTG2070) is sequence number 14. EVQLVQSGAEVKRPGASVKVSCKASGYTFTSYYMHWVRQAPGRGLEWMGLINPSGGSTSYAQEFQGRVTMTRDMSTSTVYMELSSLRSEDTAVYCARSDRGISATDAFDIWGQGTMVTVSSGGGGGSGGGSGGGGSQSVLTQPPSVSVAPGQMAKITCGGSDIGNNKNVHWYQQKPGQAPVLVVYDDYNRPSGIPERFSGSNSGDAATLTISTVEVGDEADYFCQVWDGSGDPYWVFGGTQLTDLG Nucleotide sequence 15 is CD19 reactive ScFv8 binding domain(LTG2071). GAGGTCCAGCTGGTACAGTCTGGAGCTGAGGTGAAGGGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCAGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGTGTGGATGGGGATTAATCAACCCTAGTGGTGGCAGCACAAGCTACGCACAGAAGTTCCAGGGCAGAGTCCACCATGACCAGGGACACGTCCACGAGCACAGTCTAC ATGGAGCTGAGCAGCCTGAGATCTGAGGACAGGGCCGTGTATTACTGTGCGAGATCGGATCGGGGAATTACCGCCACGGACGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAGGCGGAGGAGGCTCCGGGGAGGAGGTTCCGGGGGCGGGGTTCCCAGTCTGTGCTGACTCAGCCACCCTCGGTCTCAGTGGCCCCAGGGCAGACGGCCAAGACTACCTGGG GGAAGTGACATTGGAAATAAAAATGTCCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTCCTGGTCGTCTATGATGATTACGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTG GGGACGCGGCCACCCTGACGATCAGCACGGTCGAAGTCGGGGATGAGGCCGACTATGTCTGTCAGGTGTGGGACGGTAGTGGTGATCCTTATTGGGTGTTCGGCGGAGGGACCCAGCTCACCGTTTTAGGT SEQ ID NO: 16 is the amino acid sequence of the CD19-reactive ScFv8 binding domain (LTC2071). EVQLVQSGAEVKRPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLVWMGLINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSDRGITATDAFDIWGQGTMVTVSSG GGGSGGGGSGGGGSQSVLTQPPSVVAPGQTAKTTCGGSDIGNKNVHWYQQKPGQAPVLVVYDDYDRPSGIPERFSGSNSGDAATLTISTVEVGDEADYVCQVWDGSGDPYWVFGGGTQLTVLG SEQ ID NO: 17 is the nucleotide sequence of the leader / signal peptide sequence (LP).

[0295] atgctgctgctggtgaccagcctgctgctgtgcgaactgccgcatccggcgtttctgctgattccg SEQ ID NO: 18 is the amino acid sequence of the leader / signal peptide sequence (LP).

[0296] MLLLVTSLLLCELPHPAFLLIP SEQ ID NO: 19 is the nucleotide sequence of LTG2050_(LP-M19217-CD8 TM-41BB-CD3 zeta). SEQ ID NO: 20 is the amino acid sequence of LTG2050_(LP-M19217-CD8 TM-41BB-CD3 zeta). MLLLVTSLLLCELPHPAFLLIPEVQLVQSGAEVKKPGASVKVSCKASGYTFTSYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSDR GITATDAFDIWGQGTMVTVSSGGGGSGGGGSGGGSQSVLTQPPSVSVAPGQTAKITCGGSDIGNKNVHWYQQKPGQAPVLVVYDDYDRPSGIPERFSGSNSGDAATLTISTVEVGDEADYFCQ VWDSSGDPYWVFGGGTQLTVLGAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 21 is the nucleotide sequence of LTG2065 (LP-M19217-1-CD8 TM-41BB-CD3 Zeta). ATGCTGCTGCTGGTGACCAGCCTGCTGCTGTGCGAACTGCCGCATCCGGCGTTTCTGCTGATTCCGGAGGTCCAGCTGGT SEQ ID NO: 22 is the amino acid sequence of LTG2065_(LP-M19217-1-CD8 TM-41BB-CD3 zeta). MLLLVTSLLLCELPHPAFLLIPEVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGLINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSDR GITATDAFDIWGQGTMVTVSSGGGGSGGGGSGGGSQSVLTQPPSVSVAPGRMAKITCGGSDIGNKNVHWYQQKPGQAPVLVVYDDYDRPSGIPERFSGSNSGDAATLTISTVEVGDEADYFCQ VWDGSGDPYWMFGGGTQLTVLGAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 23 is the nucleotide sequence of LTG2066_(LP-M19217-2-CD8 TM-41BB-CD3 zeta). SEQ ID NO: 24 is the amino acid sequence of LTG2066_(LP-M19217-2-CD8 TM-41BB-CD3 zeta). MLLLVTSLLLCELPHPAFLLIPEVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGFEWMGLINPSGSSTSYAQKFQGRVTMTRDTSTSTVYMELSNLRSEDTAVYYCARSDR GITATDAFDIWGQGTMVTVSSGGGGSGGGGSGGGSQSVLTQPPSVPVAPGQTAKIICGGSDIGNKNVHWYQQKPGQAPVLVVYDDYDRPSGIPERFSGSNSGDAATLTISTVEVGDEADYFCQ VWDGSGDPYWVFGGGTQLTVLGAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 25 is the nucleotide sequence of LTG2067_(LP-M19217-7-CD8 TM-41BB-CD3 Zeta). SEQ ID NO: 26 is the amino acid sequence of LTG2067_(LP-M19217-7-CD8 TM-41BB-CD3 zeta). MLLLVTSLLLCELPHPAFLLIPEVQLVQSGAEVNKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGMINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSDR GITSTDAFDIWGQGTMVTVSSGGGGSGGGGSGGGSQSVLTQPPSVSLAPGQTAKITCGGSDIGNKNVHWYQQKPGQAPVLVVYDDYNRPSGIPERFSGSNSGDSATLTISTVEVGDEADYFCQ VWDGSGDPYWVFGGGTQLTVLGAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 27 is the nucleotide sequence of LTG2068_(LP-M19217-23-CD8 TM-41BB-CD3 Zeta). ATGCTGCTGCTGGTGACCAGCCTGCTGCTGTGCGAACTGCCGCATCCGGCGTTTCTGCTGATTCCGGAGGTCCAGCTGGTACAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCATCTGGATACACCTTCACCGGCTACTATATGCACTGGGTGCGGCAGGCCCCTGGACAAGGGCTTGAGTGGATAGGATTAATCAACCCTAGTGGTGGTAGCACAAGCTACGAACAGAAGTTCCAGGGCAGAGTCGCCATGACCAGGGACACGTCAACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGGATCGGGGAATTACCGCCACGGACGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAGGCGGAGGAGGCTCCGGGGGAGGAGGTTCCGGGGGCGGGGGTTCCCAGTCTGTGCTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGGCAGACGGCCAAGATTACCTGTGGGGGAAGTGACATTGGAGATAAAAATGTCCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTCCTGGTCGTCTATGATGATTACGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGGACGCGGCCACCCTGACGATCAGCACGGTCGAAGTCGGGGATGAGGCCGACTATTTCTGTCAGGTGTGGGACGGTATTGGTGATCCCTATTGGGTGTTCGGCGGAGGGACCCAGCTCACCGTTTTAGGTGCGGCCGCAACTACCACCCCTGCCCCTCGGCCGCCGACTCCGGCCCCAACCATCGCAAGCCAACCCCTCTCCT TGCGCCCCGAAGCTTGCCGCCCGGCCGCGGGTGGAGCCGTGCATACCCGGGGGCTGGACTTTGCCTGCGATATCTACATTTGGGCCCCGCTGGCCGGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACCCTTTACTGCAAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAGCCGTTCATGCGGCCCGTGCAGACGACTCAGGAAGAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGAGGGGGGATGCGAACTGCGCGTCAAGTTCTCACGGTCCGCCGACGCCCCCGCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGG SEQ ID NO: 28 is the amino acid sequence of LTG2068_ (LP-M19217-23-CD8 TM-41BB-CD3 zeta). MLLLVTSLLLCELPHPAFLLIPEVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWIGLINPSGGSTSYEQKFQGRVAMTRDTSTSTVYMELSSLRSEDTAVYYCARSDR GITATDAFDIWGQGTMVTVSSGGGGSGGGGSGGGSQSVLTQPPSVSVAPGQTAKITCGGSDIGDKNVHWYQQKPGQAPVLVVYDDYDRPSGIPERFSGSNSGDAATLTISTVEVGDEADYFCQ VWDGIGDPYWVFGGGTQLTVLGAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 29 is the nucleotide sequence of LTG2069_(LP-M19217-29-CD8 TM-41BB-CD3 zeta). SEQ ID NO: 30 is the amino acid sequence of LTG2069_(LP-M19217-29-CD8 TM-41BB-CD3 zeta). MLLLVTSLLLCELPHPAFLLIPEVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGMINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSDR GITATDAFDIWGQGTMVTVSSGGGGSGGGGSGGGSQSVLTQPPSVSVAPGQTAKITCGGSDIGNKNAHWYQQKPGQAPVLVVYDDYDRPSGISERFSGSNSGDAATLTISTVEVGDEADYFCQ VWDGSGDPFWVFGGGTQLTVLGAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 31 is the nucleotide sequence of LTG2070_(LP-M19217-38-CD8 TM-41BB-CD3 zeta). ATGCTGCTGCTGGTGACCAGCCTGCTGCTGTGCGAACTGCCGCATCCGGCGTTTCTGCTGATTCCGGAGGTCCAGCTGGT SEQ ID NO: 32 is the amino acid sequence of LTG2070_(LP-M19217-38-CD8 TM-41BB-CD3 zeta). MLLLVTSLLLCELPHPAFLLIPEVQLVQSGAEVKRPGASVKVSCKASGYTFTSYYMHWVRQAPGRGLEWMGLINPSGGSTSYAQEFQGRVTMTRDMSTSTVYMELSSLRSEDTAVYYCARSDR GISATDAFDIWGQGTMVTVSSGGGGSGGGGSGGGSQSVLTQPPSVSVAPGQMAKITCGGSDIGNKNVHWYQQKPGQAPVLVVYDDYNRPSGIPERFSGSNSGDAATLTISTVEVGDEADYFCQ VWDGSGDPYWVFGGGTQLTDLGAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 33 is the nucleotide sequence of LTG2071_(LP-M19217-40-CD8 TM-41BB-CD3 zeta). SEQ ID NO: 34 is the amino acid sequence of LTG2071_(LP-M19217-40-CD8 TM-41BB-CD3 zeta). MLLLVTSLLLCELPHPAFLLIPEVQLVQSGAEVKRPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLVWMGLINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSDR GITATDAFDIWGQGTMVTVSSGGGGSGGGGSGGGSQSVLTQPPSVSVAPGQTAKTTCGGSDIGNKNVHWYQQKPGQAPVLVVYDDYDRPSGIPERFSGSNSGDAATLTISTVEVGDEADYVCQ VWDGSGDPYWVFGGGTQLTVLGAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 35 is the nucleotide sequence of the DNA CD8 transmembrane domain.

[0297] atttgggccccgctggccggcacttgcggcgtgctcctgctgtcgctggtcatcaccctt tactgc SEQ ID NO: 36 is the amino acid sequence of the CD8 transmembrane domain. IWAPLAGTCGVLLLSLVITLYC SEQ ID NO: 37 is the nucleotide sequence of the DNA CD8 hinge domain.

[0298] actaccacccctgcccctcggccgccgactccggccccaaccatcgcaagccaacccctc tccttgcgccccgaagcttgccgcccggccgcgggtggagccgtgcatacccgggggctg gactttgcctgcgatatctac SEQ ID NO: 38 is the amino acid sequence of the CD8 hinge domain. TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY SEQ ID NO: 39 is the amino acid sequence of amino acids 137 to 206 of the hinge and transmembrane region of CD8.alpha (NCBI RefSeq: NP.sub.--001759.3). TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC SEQ ID NO:40 is the nucleotide sequence of the DNA signaling domain of 4-1BB.

[0299] aagaggggccggaagaagctgctttacatcttcaagcagccgttcatgcggcccgtgcag acgactcaggaagaggacggatgctcgtgcagattccctgaggaggaagaggggggatgc gaactg SEQ ID NO: 41 is the amino acid sequence of the signaling domain of 4-1BB. KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL SEQ ID NO: 42 is the nucleotide sequence of the intracellular signaling domain of CD3-zeta.

[0300] cgcgtcaagttctcacggtccgccgacgcccccgcatatcaacagggccagaatcagctc tacaacgagctgaacctgggaaggagagaggagtacgacgtgctggacaagcgacgcgga cgcgaccgggatgggggggaaaccacggcggaaaaaccctcaggaaggactgtacaac gaactccagaaagacaagatggcggaagcctactcagaaatcgggatgaagggagagcgg aggaggggaaagggtcacgacgggctgtaccagggactgagcaccgccactaaggatacc tacgatgccttgcatatgcaagcactcccaccccgg SEQ ID NO: 43 is the amino acid sequence of CD3-zeta. RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 44 is the nucleotide sequence of the intracellular signaling domain of a CD3-zeta variant.

[0301] cgcgtcaagttctcacggtccgccgacgcccccgcatataaacagggccagaatcagctc tacaacgagctgaacctgggaaggagagaggagtacgacgtgctggacaagcgacgcgga cgcgaccgggatgggggggaaaccacggcggaaaaaccctcaggaaggactgtacaac gaactccagaaagacaagatggcggaagcctactcagaaatcgggatgaagggagagcgg aggaggggaaagggtcacgacgggctgtaccagggactgagcaccgccactaaggatacc tacgatgccttgcatatgcaagcactcccaccccgg SEQ ID NO: 45 is the amino acid sequence of a CD3-zeta signaling domain mutant. RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 46 is the nucleotide sequence of ScFv CD19(FMC63).

[0302] gacattcagatgactcagaccacctcttccttgtccgcgtcactgggagacagagtgaccat ctcgtgtcgcgcaagccaggatatctccaagtacctgaactggtaccaacagaagcccga cgggactgtgaagctgctgatctaccacacctcacgcctgcacagcggagtgccaagcag attctccggctccggctcgggaaccgattactcgcttaccattagcaacctcgagcagga ggacatcgctacctacttctgccagcaaggaaataccctgccctacaccttcggcggagg aaccaaattggaaatcaccggcggaggaggctccgggggaggaggttccgggggcggggg ttccgaagtgaagctccaggagtccggccccggcctggtggcgccgtcgcaatcactctc tgtgacctgtaccgtgtcgggagtgtccctgcctgattacggcgtgagctggattcggca gccgccgcggaagggcctggaatggctgggtgtcatctggggatccgagactacctacta caactcggccctgaagtcccgcctgactatcatcaaagacaactcgaagtcccaggtctt tctgaagatgaactccctgcaaactgacgacaccgccatctattactgtgctaagcacta ctactacggtggaagctatgctatggactactgggggcaaggcacttcggtgactgtgtc aagc SEQ ID NO: 47 is the amino acid sequence of ScFv CD19(FMC63). DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGG SEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS SEQ ID NO: 48 is the nucleotide sequence of anti-CD33 ScFv (LTG1936). CAGGTGCAGCTGGTGCAATCTGGGGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAGGATCTCCTGTAAGGGTTCTGGATTCAGTTTTCCCACCTACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGACTAGTTGGAGATGGCTACAATACGGGGGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAGGAGGTGGCGGGTCTGGTGGTGGCGGTAGCGGTGGTGGCGGATCCGATATTGTGATGACCCACACTCCACTCTCTCTGTCCGTCACCCCTGGACAGCCGGCCTCCATCTCCTGCAAGTCTAGTCAGAGCCTCCTGCATAGTAATGGAAAGACCTATTTGTATTGGTACCTGCAGAAGCCAGGCCAGCCTCCACAGCTCCTGATCTATGGAGCTTCCAACCGGTTCTCTGGAGTGCCAGACAGGTTCAGTGGCAGCGGGTCAGGGACAGATTTCACACTGAAAATCAGCCGGGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAAGTATACAGCTTCCTATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA SEQ ID NO: 49 is the amino acid sequence of anti-CD33 ScFv (LTG1936). QVQLVQSGAEVKKPGESLRISCKGSGFSFPTYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARLVGDGYNTGAFDIWGQGTMVTVSSGG GGSGGGGSGGGGSDIVMTHTPLSVTPGQPASISCKSSQSLLHSNGKTYLYWYLQKPGQPPQLLIYGASNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYYCMQSIQLPITFGQGTRLEIK SEQ ID NO: 50 is the nucleotide sequence of the anti-mesothelin ScFv (LTG1904). GAGGTCCAGCTGGTACAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTAGTTGGAATAGTGGTAGCATAGGCTATGCGGACTCTGTGAAGGGCCG ATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGATTTATCGTCAGTGGCTGGACCCTTTAACTACTGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGGAGGTGGCGGGTCTGGTGGAGGCGGTAGCGGCGGTGGCG GATCCTCTTCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAGGATCACATGCCAAGGAGACAGCCTCAGAAGCTATTATGCAAGCTGGTACCAGCAGAAGCCAGGACAGGCCCCTGTACTTGTCATCTATGGTAAAAACAACCGGCC CTCAGGGATCCCAGACCGATTCTCTGGCTCCAGCTCAGGAAACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAGGATGAGGCTGACTATTACTGTAACTCCCGGGACAGCAGTGGTAACCATCTGGTATTCGGCGGAGGCACCCAGCTGACCGTCCTCGGT SEQ ID NO: 51 is the amino acid sequence of anti-mesothelin ScFv (LTG1904). EVQLVQSGGGLVQPGGSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDLSSVAGPFNYWGQGTLVTVSSGG GGSGGGGSGGGGSSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHLVFGGGTQLTVLG

Claims

1. An isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), comprising at least one extracellular antigen-binding domain comprising a CD19 antigen-binding domain encoded by a nucleotide sequence comprising SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, or 15, at least one transmembrane domain, and at least one intracellular signaling domain.

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

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

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

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

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

7. 7. The isolated nucleic acid molecule of claim 6, wherein the leader nucleotide sequence comprises a nucleotide sequence comprising SEQ ID NO: 17, which encodes the leader amino acid sequence of SEQ ID NO:

18.

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 CD19 antigen-binding domain comprises a nucleic acid sequence comprising SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.

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

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

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

13. The encoded at least one costimulatory domain is 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 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 CD19 antigen-binding domain comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16, at least one transmembrane domain, and at least one intracellular signaling domain.

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

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

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

19. The CAR of claim 18, wherein the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 35, 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 CD19 antigen-binding domain comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, or 16, 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 or CD28 and is linked to a transmembrane domain.

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

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

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

25. DNA vector, RNA vector, plasmid vector, cosmid vector, herpes 25. The vector of claim 24, selected from the group consisting of a viral vector, a measles viral vector, a lentiviral vector, an adenoviral vector, or a retroviral vector, or a combination thereof.

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

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

28. A cell comprising the vector of claim 24.

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

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

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

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

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

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

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

36. 1. A pharmaceutical composition comprising an anti-tumor effective amount of a population of human T cells, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), the CAR comprising at least one extracellular antigen-binding domain, including a CD19 antigen-binding domain comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, or 16, at least one linker domain, at least one transmembrane domain, and at least one intracellular signaling domain, and the T cells are from a human with cancer.

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

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

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

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

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

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

44. 1. A method of treating a mammal having a disease, disorder, or condition associated with elevated expression of a tumor antigen, comprising 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), the CAR comprising at least one extracellular antigen-binding domain, the CD19 antigen-binding domain comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, or 16, at least one linker or spacer domain, at least one transmembrane domain, and at least one intracellular signaling domain, and wherein the T cells are 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), the CAR comprising at least one extracellular antigen-binding domain, the CD19 antigen-binding domain comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, or 16, at least one linker or spacer domain, at least one transmembrane domain, and at least one intracellular signaling domain, and wherein the T cells are from 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. 10. 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 for producing a chimeric antigen receptor-expressing cell according to claim 47, wherein the cell is a T cell or a cell population comprising a T cell.

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