Compositions and methods for treating cancer by anti-CD19 immunotherapy

Abstract

To provide novel compositions and methods for the treatment of B-ALL and other CD19-expressing B cell malignancies using an approach that can exhibit specific and efficacious anti-tumor effect.SOLUTION: Chimeric antigen receptors containing human CD19 antigen binding domains are disclosed. Nucleic acids, recombinant expression vectors, host cells, antigen binding fragments, and pharmaceutical compositions, relating to the chimeric antigen receptors are also disclosed. Methods of treating or preventing cancer in a subject, and methods of making chimeric antigen receptor T cells are also disclosed.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 on September 15, 2017, the entire content of which is incorporated herein by reference.

[0002] Sequence Listing This application includes a Sequence Listing that has been electronically submitted 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] Description of Research or Development Sponsored by the Federal Government The present invention was made in the performance of a Cooperative Research and Development Agreement with the National Institutes of Health, an agency of the United States Department of Health and Human Services. The United States government has certain rights in the invention.

[0004] Field of the Disclosure This application relates to the field of cancer, and in particular, to CD19 antigen - binding domains, chimeric antigen receptors (CARs) that include such CD19 antigen - binding domains, and methods of using the same.

Background Art

[0005] Background Cancer is one of the most lethal threats to human health. In the United States alone, cancer affects nearly 1.3 million new patients each year, is the second most common cause of death after cardiovascular disease, and accounts for approximately one - quarter of all deaths. Solid tumors are the cause of most of these deaths. Although there have been significant advances in the medical treatment of certain cancers, the overall five - year survival rate for all cancers has improved by only about 10% over the past 20 years. Cancer, or malignancy, grows and metastasizes rapidly without control, making treatment very difficult.

[0006] CD19 is a transmembrane cell surface glycoprotein receptor of 85-95 kDa. CD19 is a member of the immunoglobulin (Ig) superfamily of proteins and contains two extracellular Ig-like domains, a transmembrane, and an intracellular signaling domain (Tedder TF, Isaacs, CM, 1989, J Immunol 143:712-171). CD19 modifies B cell receptor signaling, lowering the trigger threshold of the antigen for the B cell receptor (Carter, RH, and Fearon, DT, 1992, Science, 256:105-107), and functions in concert 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 the pro-B, pre-pre-B cell, pre-B, and early B cell stages independent of the antigen receptor, is associated with Src family protein tyrosine kinases, is tyrosine phosphorylated, and induces both intracellular calcium mobilization and inositol lipid 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 treating B cell malignancies is that CD19 is expressed in a tightly regulated manner limited to early B cell precursors at the stage of IgH gene rearrangement and mature B cells, but not in 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 treatment for B-lineage leukemias consists of remission induction with high-dose chemotherapy or radiation, followed by consolidation therapy, and may feature stem cell transplantation and additional chemotherapy courses when necessary (see the cancer.gov World Wide Web). The high toxicity associated with these treatments, as well as the risks of complications such as relapse, secondary malignancies, or GVHD, motivate the search for better alternative therapies. The expression of CD19 in malignancies of both adult and pediatric (pre-B-ALL) B cells 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 have been developed to treat B-cell leukemia and lymphoma, including bispecific antibodies that bind an anti-CD19 binding motif to a T-cell binding motif (i.e., blinatumomab, Blincyto® is indicated for the treatment of Philadelphia chromosome-negative relapsed or refractory B-cell precursor acute lymphoblastic leukemia (ALL)). To date, many of the binding moieties of CD19 used in CAR constructs utilize domains derived from murine antibodies. Many of these products are currently under consideration for approval, including those developed by Novartis and Kite Pharmaceuticals. In April 2017, Novartis announced that CTL019 (tisagenlecleucel) received Breakthrough Therapy Designation from the FDA for the treatment of adult patients with refractory or relapsed (r / r) diffuse large B-cell lymphoma (DLBCL) who have failed two or more prior therapies, and this designation was added for B-cell acute lymphoblastic leukemia (ALL). These indications are based on the Phase II JULIET trial (NCT02445248) and ELIANA trial (NCT02435849), respectively. In the JULIET trial, an overall response rate (ORR) of 45% was shown at 3 months, with 37% complete response (CR) and 8% partial response (PR). In the ELIANA trial, 82% of patients infused with the product achieved CR or CR with incomplete count recovery, and the 6-month relapse-free survival rate was 60%. Kite Pharmaceuticals' CAR-T product (KTE-C19, axicabtagene ciloleucel) received Breakthrough Therapy Designation for diffuse large B-cell lymphoma (DLBLC), transformed follicular lymphoma (TFL), and primary mediastinal B-cell lymphoma (PMBCL). In the Kite ZUMA-3 Phase II trial of KTE-C19 in r / r ALL, a CR of 73% was reported (for 2 months or more). All information is from company press releases. Regardless of whether antibodies are utilized in CAR-T therapies, a significant number of patients remain who are not helped by these therapies, and there is considerable room for improvement in treatment approaches. There is, and there is considerable room for improvement in treatment approaches.

[0009] A chimeric antigen receptor (CAR) is a hybrid molecule containing three essential units: (1) an extracellular antigen-binding motif, (2) a linker / 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 a CAR is generally based on a single-chain variable fragment (ScFv), which is the smallest binding domain of an immunoglobulin (Ig) molecule. As alternative antigen-binding motifs, for example, receptor ligands (i.e., IL-13 has been engineered to bind to the IL-13 receptor expressed by tumors), intact immunoreceptors, library-derived peptides, and innate immune system effector molecules (e.g., NKG2D) have also been engineered. Alternative cell 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 regarding defining the most active T cell population for transducing CAR vectors, determining optimal culture and expansion techniques, and defining the molecular details of the CAR protein structure itself.

[0010] The CAR linking motif can be a relatively stable structural domain such as the constant domain of IgG or can be designed to be an extended flexible linker. Using a structural motif such as one derived from the constant domain of IgG, the ScFv binding domain can be extended away from the T cell plasma membrane surface. This can 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 were characterized by the CD28 signaling domain and the 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 conjugated to anti-CD3 and anti-CD28 antibodies and the presence of the canonical “signal 2” from CD28 are no longer required to be encoded by the CAR itself. Using bead activation, third-generation vectors have not been found to be superior to second-generation vectors in in vitro assays, nor have they shown a clear benefit over second-generation vectors in a mouse model of leukemia (Haso W, Lee DW, Shah NN, Stetler-Stevenson M, Yuan CM, Pastan IH, Dimitrov DS, Morgan RA, FitzGerald DJ, Barrett DM, Wayne AS, Mackall CL, Orentas RJ. Anti-CD22-chimeric antigen receptors targeting B cell precursor acute lymphoblastic leukemia, Blood. 2013;121(7):1165–74; Kochenderfer JN et al. Blood. 2012;119(12):2709–20).This is supported by the clinical success of CD19-specific CARs of the second-generation CD28 / CD3-ζ (Lee DW et al., American Society of Hematology Annual Meeting, New Orleans, LA; December 7 - 10, 2013) and CD137 / CD3-ζ signaling formats (Porter. DL et al., N Engl J Med. 2011;365(8):725 - 33). In addition to CD137 , other tumor necrosis factor receptor superfamily members such as OX40 can also provide important persistent signals in CAR-transduced T cells (Yvon E et al. Clin Cancer Res. 2009;15(18):5852 - 60). Also important are the culture conditions in which CAR T cell populations are 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] Current challenges in the more extensive and effective application of CAR therapy for cancer relate to the lack of convincing targets. Creating binding factors to cell surface antigens is currently readily achievable, but finding cell surface antigens specific to tumors while sparing normal tissues remains a challenging task. One potential way to confer higher 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 and thus requires the alternative CAR to be bound first for the 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 the generation of a single ScFv-based CAR as an immunotherapeutic agent is the heterogeneity of tumor cells. At least one group has developed a CAR strategy for glioblastoma in which the effector cell population targets multiple antigens (HER2, IL-13Ra, EphA2) simultaneously in the hope 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 a new area in synthetic biology; multiple promoters and gene products are envisioned to direct these highly potent cells to the tumor microenvironment where T cells can escape negative regulatory signals and mediate effective tumor killing. The elimination of unwanted T cells via drug-induced dimerization with a chemical-based dimerizer such as AP1903 of an inducible caspase 9 construct demonstrates one way in which a powerful switch that can control the T cell population can be pharmacologically initiated (Di Stasi A et al., N Engl J Med. 2011;365(18):1673-83). The creation of an effector T cell population that is immune to the negative regulatory effects of transforming growth factor-β by the expression of a decoy receptor further demonstrates the extent to which effector T cells can be engineered for optimal anti-tumor 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 the endogenous T cell receptor, but 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 disappearance of cells after infusion, and off-target clinical activity. This may in part be due to some of the CAR sequences of murine origin that have been used.

[0013] The use of blinotumomab (a bispecific anti-CD19 and anti-CD3 antibody) has shown excellent results in severely ill patients who received this treatment. Nevertheless, the long-term remission rate is less than 40%, and at most only 50% of the responders can be rescued by hematopoietic stem cell transplantation (HSCT) (Gore et al., 2014, NCT01471782, and Von Stackelberg et al., 2014, NCT01471782; see summary in Benjamin, JE, Stein AS, 2016, Therapeutic Advances in Hematology 7:142-156). ) The requirements for patients to undergo HSCT after receiving bispecific antibody or CAR-T therapy to maintain a sustained response remain an area of active discussion. Although high responses, in some cases exceeding 90%, have been reported in CD19 CAR-T trials, when these trials are re-run as "treatment intent" trials, the number can be close to 70% (Davis KL, Mackall CL, 2016, Blood Advances 1:265-268). The best reported results 12 months after CAR19 treatment showed an RFS of 55% and an OS of 79% among patients who were able to receive the T cell product at the University of Pennsylvania (Maude SL, Teachey DT, Rheingold SR, Shaw PA, Aplenc R, Barrett DM, Barker CS, Callahan C, Frey NV, Farzana N, Lacey SF, Zheng A, Levine B, Melenhorst JJ, Motley L, Prter DL, June CH, Grupp SA, 2016, J Clin Oncol 34, no. 15, supplement 2016, May, 3011-3011).

Summary of the Invention

Problems to be Solved by the Invention

[0014] Accordingly, there is an urgent and long - standing need in the art to discover novel compositions and methods for treating B - ALL and other CD19 - expressing B - cell malignancies using an approach that can exhibit a specific and effective anti - tumor effect without the aforementioned drawbacks.

Means for Solving the Problems

[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 present invention disclosed and described herein provides CARs that can be used for the treatment of diseases, disorders or conditions associated with dysregulation of CD19 expression, where the CAR exhibits high surface expression in transduced T cells, exhibits a high degree of cytolysis of CD19 - expressing cells, and exhibits in vivo proliferation and persistence of the transduced T cells, and includes a CD19 antigen - binding domain.

[0016] Summary Novel anti - CD19 antibodies or antigen - binding domains thereof, chimeric antigen receptors (CARs) comprising such CD19 antigen - binding domains, host cells (e.g., T cells) expressing the receptor, and nucleic acid molecules encoding the receptor are provided herein. The CAR exhibits high surface expression in transduced T cells, a high degree of cytolysis, and in vivo proliferation and persistence of the transduced T cells. Methods of using the disclosed CARs, host cells, and nucleic acid molecules, for example, to treat cancer in a subject, are also provided.

[0017] Accordingly, in one aspect, an isolated polynucleotide encoding a human anti - CD19 antibody or a 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 a fragment thereof is provided, and the antibody or fragment thereof comprises a fragment selected from the group consisting of a Fab fragment, an 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 a fragment thereof is provided, and 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 aspect, an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR) is provided, which comprises 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 from the N-terminus to the C-terminus.

[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 antigen (antikarin) that binds to CD19.

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

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

[0026] In yet another embodiment, there is provided an isolated nucleic acid molecule 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 comprising, 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, an anti-glycolipid F77 An isolated nucleic acid molecule encoding a CAR is provided, comprising a 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 having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or any combination thereof.

[0028] In one aspect, 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, wherein an extracellular CD19 antigen-binding domain, an intracellular signaling domain, or both are 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 the 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 a 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, there is provided an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR) in which the encoded intracellular signaling domain is disposed N-terminal to the CD3 zeta intracellular domain.

[0034] In another embodiment, there is provided an isolated nucleic acid molecule encoding a CAR in which 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, there is provided an isolated nucleic acid molecule encoding a CAR in which the encoded at least one costimulatory domain comprises the functional signaling domains 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, there is provided an isolated nucleic acid molecule encoding a CAR that further comprises a leader sequence or a 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, there is provided an isolated nucleic acid molecule encoding a CAR in which 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, there is provided a CAR in which the extracellular CD19 antigen-binding domain comprises at least one single-chain variable fragment of an antibody that binds the antigen, or at least one heavy-chain variable region of an antibody that binds the antigen, or a combination thereof.

[0040] In another embodiment, provided is a CAR in which 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 combinations thereof.

[0041] In some embodiments, the CAR further encodes an extracellular antigen-binding domain comprising 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 TCR, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, 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, 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 A CAR is provided that includes an A3 TCR ScFv antigen-binding domain, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or any combination thereof.

[0043] In another embodiment, a CAR is provided that includes at least one intracellular signaling domain that includes a co-stimulatory domain and a primary signaling domain.

[0044] In yet another embodiment, a CAR is provided that includes at least one intracellular signaling domain that includes a co-stimulatory 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 includes 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 that includes 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 includes 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 encodes a CAR that includes the amino acid sequence of SEQ ID NO: 22 (LTG 2065 LP-M19217-1-CD8 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 comprises 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 comprises 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 comprises the nucleic acid sequence of SEQ ID NO: 31 (LTG It includes the 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 that includes 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 includes 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 that includes 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 aspect, the CARs disclosed herein are modified to express or contain a detectable marker for use in diagnosis, monitoring and / or predicting treatment outcomes such as progression-free survival in cancer patients, or for monitoring the progress of such treatments.

[0054] In one embodiment, the nucleic acid molecule encoding the disclosed CAR can be contained in a vector such as a viral vector. The vector is a DNA vector, an RNA vector, a plasmid vector, a cosmid vector, a herpes virus vector, a measles virus vector, a lentivirus vector, an adenovirus vector, or a retrovirus vector, or a combination thereof.

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

[0056] In yet another embodiment, the vector expressing the CAR can be further modified to include one or more operable elements by a suicide switch to control the expression of CAR T cells or to eliminate CAR-T cells. The suicide switch can include, for example, an apoptosis-inducing signaling cascade or a drug that induces cell death. In a preferred embodiment, the vector expressing the CAR can be further modified to express an enzyme such as thymidine kinase (TK) or cytosine deaminase (CD).

[0057] In another aspect, a host cell comprising a nucleic acid molecule encoding a CAR is also provided. In some embodiments, the host cell is a T cell, for example, a primary T cell obtained from a subject. In one embodiment, the host cell is a CD8+ T cell.

[0058] In yet another aspect, a pharmaceutical composition comprising an anti-tumor effective amount of a human T cell population is provided, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), the CAR comprising at least one extracellular antigen-binding domain comprising a human 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 human T cells having cancer. The cancer includes, inter alia, hematological cancers such as leukemia (e.g., chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL) or chronic myeloid leukemia (CML)), lymphoma (e.g., mantle cell lymphoma, non-Hodgkin lymphoma or Hodgkin lymphoma) or multiple myeloma, or combinations thereof.

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

[0060] In another embodiment, a pharmaceutical composition is provided, wherein the human cancer comprises oral and pharyngeal cancers (tongue, mouth, pharynx, head and neck), digestive system cancers (esophagus, stomach, small intestine, colon, rectum, anus, liver, intrahepatic bile ducts, gallbladder, pancreas), respiratory system cancers (larynx, lung and bronchi), bone and joint cancers, soft tissue cancers, skin cancers (melanoma, basal cell carcinoma and squamous cell carcinoma), pediatric tumors (neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing sarcoma), central nervous system tumors (brain tumor, astrocytoma, glioblastoma, glioma), and adult cancers including breast, reproductive system (cervix, corpus uteri, ovary, vulva, vagina, prostate, testis, penis, endometrium), urinary system (bladder, kidney and renal pelvis, ureter), eye and orbit, endocrine system (thyroid), and brain and other nervous system cancers, or any combination thereof.

[0061] In yet another embodiment, a pharmaceutical composition is provided, which comprises an anti-tumor effective amount of a population of human T cells from a human having cancer, wherein the cancer is a refractory cancer that is non-responsive to one or more chemotherapeutic agents. The cancer includes hematological cancers, myelodysplastic syndromes, pancreatic cancer, head and neck cancer, skin tumors, minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), CLL (chronic lymphocytic leukemia), CML (chronic myeloid leukemia), adult B cell malignancies including non-Hodgkin lymphoma (NHL), pediatric B cell malignancies (including B cell lineage ALL (acute lymphoblastic leukemia)), multiple myeloma, lung cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, melanoma or other hematological cancers and solid tumors, or any combination thereof.

[0062] In another aspect, a method of producing CAR-containing T cells (hereinafter "CAR T cells") is provided. This method includes transducing a vector or nucleic acid molecule encoding the disclosed CAR that specifically binds to CD19 into T cells, thereby producing CAR T cells.

[0063] In yet another aspect, a method of generating a population of RNA-engineered cells is provided, which includes introducing in vitro transcribed RNA or synthetic RNA of a nucleic acid molecule encoding the disclosed CAR into target cells to generate CAR-expressing cells.

[0064] In yet another aspect, a method of diagnosing a disease, disorder or condition associated with CD19 expression in a cell, comprising: (a) contacting the cell with a human anti-CD19 antibody or a 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 a disease, disorder or condition associated with CD19 expression.

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

[0066] In another embodiment, a method for diagnosing, prognosticating, or determining the risk of a CD19-related disease in a mammal, comprising: a) contacting a sample with a human anti-CD19 antibody or a fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, or 16; and b) detecting the presence of CD19, wherein the presence of CD19 diagnoses a CD19-related disease in the mammal, the method comprising detecting CD19 expression in a sample derived from a mammal is provided.

[0067] In another embodiment, a method for inhibiting CD19-dependent T cell inhibition, comprising contacting a cell with a human anti-CD19 antibody or a fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, or 16 is provided. In one embodiment, the cell is selected from the group consisting of tumor cells expressing CD19, tumor-associated macrophages, and any combination thereof.

[0068] In another embodiment, a method of altering the tumor microenvironment to block T cell inhibition mediated by cells expressing CD19 and inhibit tumor growth in a mammal, comprising administering to the mammal an effective amount of a composition comprising an isolated anti-CD19 antibody or a fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, and 16 is provided. In one embodiment, the cell is selected from the group consisting of tumor cells expressing CD19, tumor-associated macrophages, 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, comprising an isolated anti-CD19 antibody or a fragment thereof A method is provided that includes the step of administering to a mammal an effective amount of a composition, 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 the interaction between a first cell and a T cell, and the first cell is selected from the group consisting of tumor cells expressing CD19, tumor-associated macrophages, and any combination thereof.

[0070] In another aspect, a method of inducing anti-tumor immunity in a mammal is provided that includes the step of administering to the mammal a therapeutically effective amount of T cells transduced with a vector or nucleic acid molecule encoding the disclosed CAR.

[0071] In another embodiment, a method of treating or preventing cancer in a mammal is provided that includes the step of administering to the mammal an effective amount of one or more of the disclosed CARs to treat or prevent cancer in the mammal. The method includes administering to the subject a therapeutically effective amount of host cells expressing a disclosed CAR that specifically binds to CD19 and / or one or more of the above-described 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-described antigens in the subject.

[0072] In yet another embodiment, a method of treating a mammal having a disease, disorder, or condition associated with elevated expression of a tumor antigen is provided that includes administering to the subject a pharmaceutical composition comprising an anti-tumor effective amount of a T cell population, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), the CAR comprising at least one extracellular CD19 antigen-binding domain comprising the amino acid sequence of SEQ ID NOs: 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 the T cells are T cells of a subject having cancer.

[0073] In yet another embodiment, there is provided a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of 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 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, and at least one intracellular signaling domain, and wherein the T cells are T cells of a subject having cancer. In some embodiments of the methods described above, the at least one transmembrane domain comprises the transmembrane alpha, beta or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD19, CD22, mesothelin, CD33, CD37, CD64, CD80, CD83, CD86, CD134, CD137, CD154, TNFRSF16, TNFRSF19 or a combination thereof.

[0074] In yet another embodiment, there is provided a method for generating a persistent population of genetically engineered T cells in a human diagnosed with cancer. In one embodiment, the method comprises administering to the human T cells genetically engineered to express a CAR, the CAR comprising 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 transmembrane domain, and at least one intracellular signaling domain, and wherein the persistent population of genetically engineered T cells, or the 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 following 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 aspects and embodiments of the methods described herein, any of the cancers, diseases, disorders or conditions described above associated with elevated expression of a tumor antigen can be treated, prevented or remitted using one or more of the CARs disclosed herein.

[0077] In yet another aspect, a kit for generating the chimeric antigen receptor T cells, or a kit for preventing, treating or remitting any of the cancers, diseases, disorders or conditions associated with elevated expression of a tumor antigen in the subject, the kit comprising a container containing any one or any combination of the nucleic acid molecules, vectors, host cells or compositions disclosed above, and instructions for using the kit is provided.

[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 Description of the Drawings

[0079]

Figure 1

Figure 2A

Figure 2B

Figure 2C

Figure 2D

Figure 2E

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Figure 2G

Figure 2H

Figure 3-1

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Figure 4-2

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Mode for Carrying Out the Invention

[0080] Detailed Description Definitions As used herein, the singular forms "a", "an", and "the" refer to both the singular and plural forms unless the context clearly dictates otherwise. For example, the term "an antigen" can include one or more 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 an antigen" without excluding other elements. The phrase "and / or" means "and" or "or". It should be further understood that any and all base sizes or amino acid sizes, as well as all molecular weights or molecular mass values, given for a nucleic acid or polypeptide are approximate and provided for convenience only, unless otherwise specified. This Many methods and materials similar or equivalent to those described herein can be used, but the particularly suitable methods and materials are described below. In case of conflict, this specification, including explanations of terms, will control. Further, the materials, methods, and examples are illustrative only and not intended to be limiting. To facilitate review of the various embodiments, explanations of the following terms are provided.

[0081] The term "about", when referring to a measurable value such as an amount, a duration of time, etc., means an inclusion of a variation of ±20%, or in some instances ±10%, or in some instances ±5%, or in some instances ±1%, or in some instances ±0.1% from the specified value, such variation being appropriate for carrying out the disclosed method.

[0082] Unless otherwise noted, the technical terms in this specification are used according to their conventional usage. Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes VII, published by Oxford University Press in 1999; Kendrew et al. (eds), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd. in 1994; and Robert A. Meyers (ed), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc. in 1995; and other similar references.

[0083] The present disclosure provides a CD19 antibody or a fragment thereof, and a chimeric antigen receptor (CAR) having such a CD19 antigen-binding domain. Enhancement of the functional activity of the CAR is directly related to enhancement of the 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 cytotoxicity and cell surface expression on transduced T cells, along with increased levels of T cell proliferation and persistence in vivo of the transduced CAR-expressing T cells.

[0084] The unique ability to combine functional moieties derived from various protein domains is an important and innovative feature of chimeric antigen receptors (CARs). The selection of each of these protein domains is as important a design feature as these specific combinations. Each design domain is an essential component that can be used to engineer lymphocyte function in various CAR platforms. For example, the selection of an extracellular binding domain can enable an otherwise ineffective CAR.

[0085] The non-variable framework components of the immunoglobulin-derived protein sequences used to create the extracellular antigen-binding domain of the CAR are either completely neutral or may self-associate and put T cells into a state of metabolic exhaustion, so that the therapeutic T cells expressing this CAR become extremely ineffective. This occurs independently of the antigen-binding function of this CAR domain. Furthermore, the selection of the intracellular signaling domain(s) can also determine the activity and durability of the therapeutic lymphocyte population used in immunotherapy. While the ability to bind to the target antigen and the ability to transmit activation signals to T cells by these extracellular and intracellular domains are important aspects of CAR design, respectively, it has also become clear that the choice of the source of the extracellular antigen-binding fragment can have a significant effect on the efficacy of the CAR and thus can play a decisive role in the function and clinical utility of the CAR.

[0086] Surprisingly and unexpectedly, it has now been discovered that the functional activity of T cells expressing the CAR can also be determined by using a fully human antigen-binding domain for the CAR, rather than using a mouse-derived antigen-binding fragment that has a tendency to induce an anti-mouse immune response and CAR T elimination in the host (see the University of Pennsylvania-funded clinical trial, NCT02159716, which uses the mouse-derived SS1 ScFv sequence).

[0087] In light of this discovery, a series of CD19 binders were developed from a human scFv expression library. Since these fully human CD19 CARs are no longer of murine origin, they are 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). Thus, when these “fully human” CARs are expressed in T cells and then injected into patients, a higher therapeutic effect is expected. These CAR binders derived from human sequences can be used for the treatment of human cancers, leukemias, and lymphomas expressing 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 the CAR have a high proliferation rate in vivo, produce large amounts of cytokines, and have high cytotoxic activity against cells having the CD19 antigen to which the CAR binds on their surface. The use of a human extracellular CD19 antigen-binding domain results in the production of a CAR that functions better in vivo, while avoiding the induction of anti-CAR immunity in the host immune response and the death of the CAR T cell population. CARs expressing a fully human extracellular CD19ScFv antigen-binding domain exhibit excellent activities / characteristics, including: i) prevention of the persistence and functional deficiency of CAR T seen with murine-derived binding sequences; ii) the absence of local (i.e., intrapleural) delivery of the CAR that should be effective; and iii) the ability to generate CAR T cell designs based on both high- and low-affinity binders to CD19. This last characteristic allows, due to the higher expression of CD19 in tumors than in normal tissues, binders with lower affinity to have greater specificity for tumors than for normal tissues, thereby preventing toxicity to off-target tumors and the death of bystander cells, and thus researchers can better tune the efficacy and / or tissue specificity of CAR T products against toxicity.

[0089] Further description of CARs, antibodies and their antigen-binding fragments, conjugates, nucleotides, expression, vectors and host cells, methods of treatment using the disclosed CARs, compositions and kits, including detailed descriptions of their extracellular CD19 antigen-binding domains, transmembrane domains and intracellular domains, is provided below.

[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] A chimeric antigen receptor (CAR) is an artificially constructed hybrid protein or polypeptide containing an antigen-binding domain of an antibody (e.g., 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 towards a selected target by exploiting the antigen-binding properties of monoclonal antibodies in a non-MHC-restricted manner. Non-MHC-restricted antigen recognition confers on T cells expressing the CAR the ability to recognize antigens independent of antigen processing, thus bypassing a major mechanism of tumor escape. Further, when expressed in T cells, CARs advantageously do not dimerize with the alpha and beta chains of the endogenous T cell receptor (TCR).

[0092] As disclosed herein, the intracellular T cell signaling domains of CARs include, for example, T cell receptor signaling domains, T cell co-stimulatory signaling domains, or combinations thereof. Both may be included. The T cell receptor signaling domain refers to a part of the CAR that includes the intracellular domain of the T cell receptor, such as, by way of non-limiting example, the intracellular portion of the CD3 zeta protein. The co-stimulatory signaling domain refers to a part of the CAR that includes the intracellular domain of a co-stimulatory molecule, which is a cell surface molecule other than an antigen receptor or its ligands that is required for an efficient response of lymphocytes to an antigen.

[0093] 1. Extracellular domain In one embodiment, the CAR includes a target-specific binding element, also referred to as an antigen-binding domain or moiety. The choice of domain depends on the type and number of ligands that define the surface of the target cell. For example, the antigen-binding domain can be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state. Thus, examples of cell surface markers that can act as ligands for the antigen-binding domain in the CAR include those associated with viral, bacterial and parasitic infections, autoimmune diseases and cancer cells.

[0094] In one embodiment, the CAR can be engineered to target a desired tumor antigen by engineering a desired antigen-binding domain that specifically binds to an antigen on the tumor cell. The tumor antigen is a protein produced by the tumor cell that elicits an immune response, particularly a T cell-mediated immune response. The selection of the antigen-binding domain depends on the specific type of cancer being treated. Tumor antigens include, for example, glioma-associated antigens, carcinoembryonic antigen (CEA), beta-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2(AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, Her2 / neu, survivin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor as well as CD19. The tumor antigens disclosed herein are provided by way of example only. This list is not intended to be exclusive and further examples will be readily apparent to those skilled in the art.

[0095] In one embodiment, the tumor antigen comprises one or more antigenic cancer epitopes associated with a malignant tumor. Malignant tumors express several proteins that can function as target antigens for immune attack. These molecules include, but are not limited to, tissue-specific antigens such as MART-1, tyrosinase, and GP 100 in melanoma, and prostate acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to a group of transformation-related molecules such as the oncogene HER-2 / Neu / ErbB-2. Yet another group of target antigens are cancer fetal antigens such as carcinoembryonic antigen (CEA). In B-cell lymphoma, the tumor-specific idiotype immunoglobulin constitutes a truly tumor-specific immunoglobulin antigen unique to each individual tumor. B-cell differentiation antigens such as CD19, CD20, CD22, BCMA, ROR1, and CD37 are other candidates for target antigens in B-cell lymphoma. Some of these antigens (CEA, HER-2, CD19, CD20, idiotype) have been used, with limited success, as targets for passive immunotherapy with monoclonal antibodies.

[0096] In a preferred embodiment, the tumor antigen is CD19, and tumors associated with CD19 expression include mesothelioma of the lung, ovarian and pancreatic cancers, 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). A TSA is unique to tumor cells and does not exist on other cells in the body. A TAA is not unique to tumor cells; instead, it is expressed on normal cells under conditions that do not induce a state of immunological tolerance to the antigen. The expression of the antigen on the 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 non-responsive, or an antigen that is normally present at very low levels on normal cells but is expressed at relatively high levels on tumor cells.

[0098] Non-limiting examples of TSA or TAA include the following: differentiation antigens such as MART-1 / MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2 and tumor-specific multi-series 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, p15, p16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3 / CA 27.29 / BCAA, CA 195, CA 242, CA-50, CAM43, CD68 / P1, CO-029, FGF-5, G250, Ga733 / EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 / Mac-2 binding protein / cyclophilin C-related protein, TAAL6, TAG72, TLP and TPS.

[0099] In one embodiment, the antigen-binding domain portion of the CAR targets antigens 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 an extracellular CD19 VH-2 antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 1, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto. In one embodiment, there is provided an isolated nucleic acid molecule 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 having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 2.

[0102] In a preferred embodiment, an isolated nucleic acid molecule encoding an extracellular CD19 VH-4 antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 3, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto. In one embodiment, the encoded extracellular CD19 VH-4 antigen-binding domain has the amino acid 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, and an isolated nucleic acid molecule is provided.

[0103] In a preferred embodiment, an isolated nucleic acid molecule encoding an extracellular CD19 ScFv 9 antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 5, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto. In one embodiment, there is provided an isolated nucleic acid molecule 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 having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 6.

[0104] In a 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 having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto. In one embodiment, there is provided an isolated nucleic acid molecule 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 having 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 having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto. In one embodiment, there is provided an isolated nucleic acid molecule 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 having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 10.

[0106] 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: 11, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto. In one embodiment, there is provided an isolated nucleic acid molecule 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 having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 12.

[0107] 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: 13, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto. In one embodiment, there is provided an isolated nucleic acid molecule 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 having 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 having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto. In one embodiment, the encoded extracellular CD19 ScFv 15 antigen-binding domain has the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 16 and there is provided an isolated nucleic acid molecule containing the same.

[0109] In various embodiments of the CD19-specific CARs disclosed herein, a general schematic is shown in FIG. 1, from the N-terminus to the C-terminus, a signal or leader peptide, anti-CD19 ScFv, an extracellular linker, a CD8 transmembrane portion, 4-1BB, CD3 zeta, with the bold text representing the cloning site of the linker domain.

[0110] In one embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 19 and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO: 20 [LTG2050 LP-M19217-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in FIG. 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 having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO: 20, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto [LTG2050 LP-M19217-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in FIG. 2A)].

[0112] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 21 and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO: 22 [LTG2065 LP-M19217-1-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in FIG. 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 having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO: 22, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto [LTG2065 LP-M19217-1-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in FIG. 2B)].

[0114] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 23 and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO: 24 [LTG2066 LP-M19217-2-CD8 TM-41BB-CD3 zeta CAR amino acid sequence (shown in FIG. 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 having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO: 24, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto [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 and encodes a CAR comprising the amino acid sequence shown 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 comprises the nucleic acid sequence of SEQ ID NO: 25, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO: 26, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto [LTG2067 LP-M19217-7-CD8 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 and encodes a CAR comprising the amino acid sequence shown 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 having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and the amino acid sequence shown in SEQ ID NO: 28, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto [LTG2068 encodes a CAR comprising the LP-M19217-23-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in FIG. 2E).

[0120] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 29 and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO: 30 [(LTG2069 LP-M19217-29-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in FIG. 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 having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and the amino acid sequence shown in SEQ ID NO: 30, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto [LTG2069 encodes a CAR comprising the LP-M19217-29-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in FIG. 2F).

[0122] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 31 and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO: 32 [(LTG2070 LP-M19217-38-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in FIG. 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 having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO: 32, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto [(LTG2070 LP-M19217-38-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in FIG. 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 shown in SEQ ID NO: 34 [(LTG2071 LP-M19217-40-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in FIG. 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 having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO: 34, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto [(LTG207 1 LP-M19217-40-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in FIG. 2H))].

[0126] The surface expression of anti-CD19 CARs incorporating single-chain variable fragment (ScFv) sequences reactive with CD19 antigen is shown in Example 2 below and summarized in Table 2. The 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 a recombinant CD19-Fc peptide followed by an anti-human Fc F(ab’)2 fragment conjugated to AF647 and detected in the APC channel (see Figure 3). The ScFv-based anti-CD19 CAR constructs LTG2050, LTG2065–LTG2071 were highly expressed in human primary T cells (indicated by the gated population) compared to non-transduced T cell controls (non-gated cell population). Representative results from one donor are shown.

[0127] As shown in Example 2 and Figure 4, lentiviral vectors (LVs) expressing the following CARs were generated and tested for anti-leukemia activity, demonstrating 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 specified therein. Leukemia target cell lines with varying 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 cell lines Raji and Reh, while showing no specific lytic activity against K562 or 293T (see Figure 4). These results demonstrate the efficiency and specificity of the generated CAR constructs.

[0128] Next, the ability of anti-CD19 CAR T cells to secrete cytokines was evaluated. Tumor cells were co-incubated with CAR T cells or control T cells overnight at an effector-to-target ratio of 10:1, and the culture supernatants were analyzed by ELISA for IFN gamma, TNF alpha, and IL-2 (see Figure 5). Notably, LTG2065, LTG2066, LTG2067, LTG2068, LTG2069, LTG2070, and LTG2071 of CAR T-expressing cells produced high levels of IFN gamma, TNF alpha, and IL-2, whereas the negative control (non-transduced, UN) did not produce any obvious cytokine induction. Surprisingly, CD19 CAR LTG2050 produced significantly lower levels of induced cytokines against the tumor lines tested. The CAR, LTG1538, tested as a positive control and expressing the murine FMC63 CD19 binder (SEQ ID NO: 47), was less active than all binders tested except LTG2050. The high in vitro cell lysis function of LTG2050, which had a low cytokine production capacity, suggests that multiple CAR T functional endpoints need to be tested construct-based in the construct. Furthermore, the superiority of the LTG2065-2071 human CD19 binders over the murine FMC63 scFv was clearly demonstrated in the cytokine production assay.

[0129] While not 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 include, for example, but not by way of limitation, a) improved lateral movement within the plasma membrane allowing for more efficient signaling, b) an excellent location within plasma membrane microdomains such as lipid rafts, and a higher ability to interact with transmembrane signaling cascades associated with T cell activation, c) an excellent location within the plasma membrane due to preferential movement away from inhibitory or downregulatory interactions, e.g., not being in close proximity to or having less interaction with phosphatases such as CD45, and d) an excellent assembly into the T cell receptor signaling complex (i.e., immune synapse), or any combination thereof.

[0130] Although the present disclosure is illustrated 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-only binding domain or a subset thereof, and thus, the CD19 antigen-binding domain for use in the CARs described herein is included. In one embodiment, other nucleotide and / or amino acid variants within the CD19 variable ScFv antigen-binding domain include, for example, CD19 variable ScFv antigen-binding domain-containing variants labeled as FMC63 (SEQ ID NO: 46 and SEQ ID NO: 47 (nucleotide sequence and amino acid sequence, respectively (see Example 1))).

[0131] Depending on the desired antigen to be targeted, the CAR can be further engineered to include an appropriate antigen-binding domain specific for the desired antigen target. For example, if CD19 is the desired antigen to be targeted, an antibody against CD19 can be used as the antigen-binding domain incorporation into the CAR.

[0132] In an 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, where the nucleic acid sequence of the anti-CD33 ScFv includes the sequence shown in SEQ ID NO: 48. In one embodiment, the anti-CD33 ScFv includes 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 includes the amino acid sequence shown in SEQ ID NO: 49.

[0133] In an 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, where the nucleic acid sequence of the anti-mesothelin ScFv comprises the nucleotide sequence shown 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 shown in SEQ ID NO: 51.

[0134] In one aspect of the invention, for example, but not by way of limitation, retroviridae (e.g., human immunodeficiency virus, 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 type 1 and 2 (HSV), varicella-zoster virus, cytomegalovirus (CMV) and herpesvirus], poxviridae (e.g., variola virus, vaccinia virus and poxvirus) or antigens derived from hepatitis C virus, or CARs capable of binding to non-TSA or non-TAA comprising any combination thereof are provided.

[0135] In another aspect of the present invention, there is provided a CAR capable of binding to an antigen derived from a bacterial strain of Staphylococci, Streptococcus, Escherichia coli, Pseudomonas or Salmonella. In particular, infectious bacteria such as Helicobacter pyloris, Legionella pneumophilia, Mycobacteria sps. bacterial strains (e.g., M. tuberculosis, M. avium, M. intracellulare, M. kansaii or M. gordonea), Staphylococcus aure us, Neisseria gonorrhoeae, Neisseria meningitides, Listeria monocytogenes, Streptococcus pyogenes, Group A Streptococcus, Group B Streptococcus (Streptococcus agalactiae), Streptococcus pneumoniae or Clostridium tetani, or an antigen derived from a combination thereof, there is provided a CAR capable of binding.

[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] The transmembrane domain can be derived from either a natural source or a synthetic source. If the source is natural, the domain can be derived from any membrane-bound or transmembrane protein.

[0138] The transmembrane regions specifically used in the CARs described herein can be derived from the alpha, beta, or zeta chains 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 (i.e., can at least include those transmembrane region(s)). Alternatively, the transmembrane domain can be synthetic, in which case it predominantly contains hydrophobic residues such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan, and valine is found at each end of the synthetic transmembrane domain. Optionally, a short oligopeptide linker or polypeptide linker, preferably between 2 and 10 amino acids in length, can form the linkage 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 above transmembrane domains.

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

[0141] In one embodiment, the transmembrane domain in the CAR of the present invention is the 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 is at least one, two, or three modifications (e.g., substitutions) of the amino acid sequence of SEQ ID NO: 36, but has 20, 10, or 5 or fewer modifications (e.g., substitutions), or is a sequence having 95-99% identity to the amino acid sequence of SEQ ID NO: 36.

[0143] In some cases, the transmembrane domain of the CAR includes the CD8 alpha hinge domain. In one embodiment, the CD8 hinge domain includes the nucleic acid sequence of SEQ ID NO: 37. In one embodiment, the CD8 hinge domain includes the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 38. In another embodiment, the CD8 hinge domain includes the amino acid sequence of SEQ ID NO: 38, or a sequence having 95-99% identity thereto.

[0144] In one embodiment, there is provided an isolated nucleic acid molecule in which 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 a CAR, the spacer domain can be located between the extracellular domain and the transmembrane domain, or between the intracellular domain and the transmembrane domain. The spacer domain means any oligopeptide or polypeptide that functions to link the transmembrane domain to the extracellular domain and / or to link the transmembrane domain to the intracellular domain. The spacer domain includes up to 300 amino acids, preferably 10-100 amino acids, most preferably 25-50 amino acids.

[0146] In some embodiments, the linker, if present, may include a spacer element that increases the size of the linker and, as a result, increases 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 and include those recited in U.S. Patent No. 7,964,566, U.S. Patent No. 7,498,298, U.S. Patent No. 6,884,869, U.S. Patent No. 6,323,315, U.S. Patent No. 6,239,104, U.S. Patent No. 6,034,065, U.S. Patent No. 5,780,588, U.S. Patent No. 5,665,860, U.S. Patent No. 5,663,149, U.S. Patent No. 5,635,483, U.S. Patent No. 5,599,902, U.S. Patent No. 5,554,725, U.S. Patent No. 5,530,097, U.S. Patent No. 5,521,284, U.S. Patent No. 5,504,191, U.S. Patent No. 5,410,024, U.S. Patent No. 5,138,036, U.S. Patent No. 5,076,973, 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. 4,816,444, and U.S. Patent No. 4,486,414, as well as those recited in U.S. Patent Application Publication No. 20110212088 and U.S. Patent Application Publication No. 20110070248, each of which is incorporated herein by reference in its entirety.

[0147] The spacer domain preferably has a sequence that promotes binding of the CAR to the antigen and enhances signal transduction into the cell. Examples of amino acids 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 the amino acids that make up the spacer domain.

[0148] As the spacer domain, all or part of amino acid numbers 137 to 206 (SEQ ID NO: 39), which is the hinge region of CD8 alpha (NCBI RefSeq: NP_001759.3), amino acid numbers 135 to 195 of CD8 beta (GenBank: AAA35664.1), amino acid numbers 315 to 396 of CD4 (NCBI RefSeq: NP_000607.1), or amino acid numbers 137 to 152 of CD28 (NCBI RefSeq: NP_006130.1) can be used. Also, as the spacer domain, a part of the constant region of the antibody H chain or L chain can be used. Further, the spacer domain can be an artificially synthesized sequence.

[0149] Furthermore, in the CAR, a signal peptide sequence can be linked to the N-terminus. The signal peptide sequence is present at the N-terminus of many secreted and membrane proteins and has a length of 15 to 30 amino acids. Since many of the protein molecules mentioned above as the intracellular domain have a signal peptide sequence, these signal peptides can be used as the signal peptide for the CAR. In one embodiment, the signal peptide contains the amino acid sequence shown in SEQ ID NO: 18.

[0150] 4. Intracellular Domain The cytoplasmic domain of the CAR or, alternatively, the intracellular signaling domain 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 the specialized function of a cell. For example, the effector functions of T cells can be cytolytic activity or helper activity, including the secretion of cytokines. Thus, the term "intracellular signaling domain" refers to the portion of a protein that transmits effector function signals and instructs the cell to perform specialized functions. Usually, the entire intracellular signaling domain can be used, but often it is not necessary to use the entire chain. As long as a shortened portion of the intracellular signaling domain is used, such a shortened portion can be used in place of the intact chain as long as it transmits effector function signals. Thus, the term, intracellular signaling domain, is meant to include any shortened portion of the intracellular signaling domain that is sufficient to transmit effector function signals.

[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 signaling after antigen receptor engagement, as well as any derivatives or variants of these sequences, and any synthetic sequences having the same functional ability.

[0152] It is known that signals generated through the TCR alone are insufficient for complete activation of T cells and that secondary or co-stimulatory 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 co-stimulatory signals (secondary cytoplasmic signaling sequences).

[0153] The primary cytoplasmic signaling sequences regulate the primary activation of the TCR complex in either a stimulatory or inhibitory manner. The primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs known as immunoreceptor activation tyrosine motifs or ITAMs.

[0154] Examples of ITAMs containing primary cytoplasmic signaling sequences particularly used 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 acid numbers 51 - 164 of CD3 zeta (NCBI RefSeq:NP_932170.1), amino acid numbers 45 - 86 of Fc epsilon RI gamma (NCBI RefSeq:NP_004097.1), amino acid numbers 201 - 244 of Fc epsilon RI beta (NCBI RefSeq:NP_000130.1), amino acid numbers 139 - 182 of CD3 gamma (NCBI RefSeq:NP_000064.1), amino acid numbers 128 - 171 of CD3 delta (NCBI RefSeq:NP_000723.1), amino acid numbers 153 - 207 of CD3 epsilon (NCBI RefSeq:NP_000724.1), amino acid numbers 402 - 495 of CD5 (NCBI RefSeq:NP_055022.2), amino acid numbers 707 - 847 of 0022 (NCBI RefSeq:NP_001762.2), amino acid numbers 166 - 226 of CD79a (NCBI RefSeq:NP_001774.1), amino acid numbers 182 - 229 of CD79b (NCBI RefSeq:NP_000617.1), and peptides having the sequence of amino acid numbers 177 - 252 of CD66d (NCBI RefSeq:NP_001806.2), as well as variants thereof having the same functions as those of these peptides. It includes. Amino acid numbers based on the NCBI RefSeq ID or amino acid sequence information of GenBank described in this specification are numbered based on the full length of the precursor of each protein (including a signal peptide sequence, etc.). In one embodiment, the cytoplasmic signaling molecule in the CAR includes a cytoplasmic signaling sequence derived from CD3 zeta.

[0155] In preferred embodiments, 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 moiety and a co-stimulatory signaling region. The co-stimulatory signaling region refers to a portion of the CAR that includes the intracellular domain of a co-stimulatory molecule. Co-stimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an efficient response of lymphocytes to an antigen. Examples of such co-stimulatory 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 co-stimulatory molecules include peptides having the sequences of amino acids 236 to 351 of CD2 (NCBI RefSeq:NP_001758.2), amino acids 421 to 458 of CD4 (NCBI RefSeq:NP_000607.1), amino acids 402 to 495 of CD5 (NCBI RefSeq:NP_055022.2), amino acids 207 to 235 of CD8 alpha (NCBI RefSeq:NP_001759.3), amino acids 196 to 210 of CD83 (GenBank:AAA35664.1), amino acids 181 to 220 of CD28 (NCBI RefSeq:NP_006130.1), amino acids 214 to 255 of CD137 (4-1BB, NCBI RefSeq:NP_001552.2), amino acids 241 to 277 of CD134 (OX40, NCBI RefSeq:NP_003318.1), and amino acids 166 to 199 of ICOS (NCBI RefSeq:NP_036224.1), as well as variants thereof having the same function as those peptides. Thus, although the disclosure herein mainly exemplifies 4-1BB as a co-stimulatory signaling element, other co-stimulatory elements are within the scope of the disclosure.

[0156] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR can be linked to each other either randomly or in a specified order. Optionally, short oligopeptide linkers or polypeptide linkers, preferably between 2 and 10 amino acids in length, can form the linkage. A glycine-serine doublet provides a particularly suitable linker.

[0157] In one embodiment, the intracellular domain is designed to include the signaling domain of CD3-zeta and the signaling domain of CD28. In another embodiment, the intracellular domain is designed to include the signaling domain of CD3-zeta and the signaling domain of 4-1BB. In yet another embodiment, the intracellular domain is designed to include the signaling domain of CD3-zeta as well as the signaling domains of CD28 and 4-1BB.

[0158] In one embodiment, the intracellular domain in the CAR is designed to include the signaling domain of 4-1BB and the signaling domain of CD3-zeta, where the signaling domain of 4-1BB includes the nucleic acid sequence shown in SEQ ID NO: 40, and the signaling domain of CD3-zeta includes the nucleic acid sequence shown in SEQ ID NO: 42 and the variant nucleic acid sequence shown in SEQ ID NO: 44. In one embodiment, the intracellular domain in the CAR is designed to include the signaling domain of 4-1BB and the signaling domain of CD3-zeta, where the signaling domain of 4-1BB includes the nucleic acid sequence shown in SEQ ID NO: 40, and the signaling domain of CD3-zeta includes the nucleic acid sequence shown in SEQ ID NO: 42 , or in another embodiment, includes the CD3-zeta variant nucleic acid sequence shown in SEQ ID NO: 44.

[0159] In one embodiment, the intracellular domain in the CAR is designed to include the signaling domain of 4-1BB and the signaling domain of CD3-zeta, where the signaling domain of 4-1BB includes a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 41, and the signaling domain of CD3-zeta includes 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 include the signaling domain of 4-1BB and the signaling domain of CD3-zeta, where the signaling domain of 4-1BB includes the amino acid sequence shown in SEQ ID NO: 41, and the signaling domain of CD3-zeta includes the amino acid sequence shown in SEQ ID NO: 43 and the variant amino acid sequence shown in SEQ ID NO: 45.

[0161] 5. Further description of the CAR The functional portions of the CARs disclosed herein are also expressly included within the scope of the present invention. As used with respect to a CAR, the term "functional portion" refers to any one or more portions or fragments of the CARs disclosed herein, and such portion or fragment retains the biological activity of the CAR of which it is a part (the parent CAR). Functional portions include, for example, portions of a CAR that retain the ability to recognize target cells, or the ability to detect, treat, or prevent a disease, to a similar, the same, or a higher degree as the parent CAR. With respect to a parent CAR, a functional portion can comprise, for example, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95% or more of the parent CAR.

[0162] The functional portion may include additional amino acids at the amino terminus and / or carboxy terminus of that portion, or at both termini, and these additional amino acids are not found in the amino acid sequence of the parental CAR. Desirably, the additional amino acids do not interfere with the biological function of the functional portion, such as recognizing target cells, detecting cancer, treating or preventing cancer, etc. More desirably, the additional amino acids enhance its biological activity as compared to the biological activity of the parental CAR.

[0163] The functional variants of the CARs disclosed herein are included within the scope of the present disclosure. The term "functional variant" as used herein refers to a CAR, polypeptide or protein having substantial or significant sequence identity or similarity to a parental CAR, and this functional variant retains the biological activity of the CAR of which it is a variant. Functional variants include, for example, variants of the CARs (parental CARs) described herein that retain the ability to recognize target cells to a similar, the same, or a higher degree as the parental CAR. With respect to the parental CAR, the functional variant may have an amino acid sequence that is at least about 30%, 50%, 75%, 80%, 90%, 98% or more identical to the parental CAR.

[0164] The functional variant may include, for example, the amino acid sequence of the parental CAR having at least one conservative amino acid substitution. Alternatively, or in addition, the functional variant may include the amino acid sequence of the parental CAR having 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 may enhance the biological activity of the functional variant, such that the biological activity of the functional variant is increased as compared to the parental CAR.

[0165] Amino acid substitutions of 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 having certain physical and / or chemical properties is replaced with another amino acid having the same or similar chemical or physical properties. For example, conservative amino acid substitutions include acidic / negatively charged polar amino acids that replace another acidic / negatively charged polar amino acid (e.g., Asp or Glu), amino acids having nonpolar side chains that replace another amino acid having a nonpolar side chain (e.g., Ala, Gly, Val, He, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), basic / positively charged polar amino acids that replace another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.), uncharged amino acids having polar side chains that replace another uncharged amino acid having a polar side chain (e.g., Asn, Gin, Ser, Thr, Tyr, etc.), amino acids having beta-branched side chains that replace another amino acid having a beta-branched side chain (e.g., He, Thr, and Val), amino acids having aromatic side chains that replace another amino acid having an aromatic side chain (e.g., His, Phe, Trp, and Tyr), and the like.

[0166] The 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] The CAR (including functional parts and functional variants) can be of any length, i.e., can contain any number of amino acids, provided that the CAR (or its functional part or functional variant) retains its biological activities, e.g., the ability to specifically bind to an antigen, the ability to detect diseased cells in a mammal, or the ability to treat or prevent a disease in a mammal. For example, the CAR can be about 50 to about 5000 amino acids in length, e.g., 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more amino acids in length.

[0168] CAR (including the functional portions and functional variants of the present invention) may 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, α-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-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, Ν',Ν'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, γ-diaminobutyric acid, β-diaminopropionic acid, homophenylalanine and α-tert-butylglycine.

[0169] CAR (including the functional portions and functional variants thereof) may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized, for example via disulfide bridges, or converted to an acid addition salt and / or optionally dimerized or polymerized, or conjugated.

[0170] CAR (including its functional portions and functional variants) can be obtained by methods known in the art. CAR can be made by any suitable method for making polypeptides or proteins. It can be produced by a method. Suitable methods for de novo synthesizing 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. Also, polypeptides and proteins can be recombinantly produced 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. Further, 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 (including functional portions and functional variants thereof) described herein can also be commercially synthesized by a company. In this regard, the CAR may be synthetic, recombinant, isolated, and / or purified.

[0171] B. Antibodies and Antigen-Binding Fragments One embodiment further provides a CAR, a T cell expressing the CAR, and an antibody or an antigen-binding domain or portion thereof that specifically binds to one or more of the antigens disclosed herein. As used herein, "T cell expressing a CAR" or "CAR T cell" means a T cell that expresses a CAR and has, for example, antigen specificity determined by the targeting domain derived from an antibody of the CAR.

[0172] As used herein, "antigen-binding domain" can include an antibody and an antigen-binding fragment 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 for an antigen.

[0173] A "monoclonal antibody" is an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies that make up the population are identical except for possible naturally occurring mutations that may be present in trace amounts. Monoclonal antibodies are highly specific and are directed against a single antigenic epitope. The modifier "monoclonal" indicates the characteristic of the antibody being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the 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 acids encoding the 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 the production of monoclonal antibodies are known; see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual, 2nd ed. Cold Spring Harbor P ublications, New York (2013).

[0174] Typically, an immunoglobulin has heavy (H) and light (L) chains interconnected by disulfide bonds. Immunoglobulin genes include 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) that determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA, and IgE.

[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 an antigen. In further embodiments, only the heavy chain variable region is required. For example, naturally occurring camelid antibodies consisting of only 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 those of 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. The variable regions of the light and heavy chains contain "framework" regions interrupted by three hypervariable regions, also called "complementary determining regions" or "CDRs" (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991). The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, which is the combined framework region of the constituent light and heavy chains, functions to position and align the CDRs in three-dimensional space.

[0176]

[0177] ​The CDRs are mainly responsible for binding to epitopes of antigens. The amino acid sequence boundaries of a given CDR can be readily determined using any of several well-known schemes, including those described by Kabat et al. ("Sequences of Proteins of Immunological Interest", 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991; the "Kabat" numbering scheme), Al-Lazikani et al. (JMB 273:927-948, 1997; the "Chothia" numbering scheme), and Lefranc et al. ("IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains", Dev. Comp. Immunol., 27:55-77, 2003; the "IMGT" numbering scheme). The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3 (from the N-terminus to the C-terminus), and are typically also identified by the chain on which a particular CDR is located. Thus, VH CDR3 is the CDR3 derived from the variable domain of the heavy chain of the antibody in which it is found, while VL CDR1 is the CDR1 derived from the variable domain of the light chain of the antibody in which it is found. Light chain CDRs may also be referred to as LCDR1, LCDR2, and LCDR3. Heavy chain CDRs may also be referred to as LCDR1, LCDR2, and LCDR3.

[0178] "Antigen-binding fragment" refers to a portion of a full-length antibody that retains the ability to specifically recognize a 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; diabody; linear antibody; single-chain antibody molecule (e.g., ScFv); and multispecific antibodies formed from antibody fragments. Antibody fragments include antigen-binding fragments produced by modification of the whole antibody or antigen-binding fragments synthesized de novo using recombinant DNA methodologies (see, e.g., Kontermann and Dubel (eds.), Antibody Engineering, Volumes 1-2, 2nd Edition, Springer Press, 2010).

[0179] A single-chain antibody (ScFv) is a genetically engineered molecule containing VH and VL domains of one or more antibodies (plural) genetically fused as a single-chain molecule linked by an appropriate polypeptide linker (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 the ScFv is typically not critical for the ScFv. Thus, ScFvs having both possible arrangements (VH domain-linker domain-VL domain; VL domain-linker domain-VH domain) can be used.

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

[0181] Antibodies also include genetically engineered forms such as chimeric antibodies (e.g., humanized mouse 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 Edition, W.H. Freeman & Co., New York, 1997.

[0182] Antibodies that do not exist naturally can be constructed using solid-phase peptide synthesis, can be recombinantly produced, or can be obtained, for example, by screening a combinatorial library consisting of variable heavy and variable light chains as described in Huse et al., Science 246: pp. 1275-1281 (1989), which is incorporated herein by reference. For example, these and other methods for making chimeric, humanized, CDR-grafted, single-chain, and bifunctional antibodies are well known to those skilled in the art (Winter and Harris, Immunol. Today 14: pp. 243-246 (1993); Ward et al., Nature Volume 341: pages 544 - 546 (1989); Harlow and Lane, supra, 1988; Hilyard et al., Protein Engineering: A practical approach (IRL Press 1992); Borrabeck, Antibody Engineering, 2nd Edition (Oxford University Press 1995); each of which is incorporated herein by reference )

[0183] An antibody that binds to the "same epitope as the reference antibody" refers to an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competitive assay. Conversely, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competitive assay. Antibody competition assays are known, and exemplary competitive assays are provided herein.

[0184] A "humanized" antibody or antigen - binding fragment thereof comprises human framework regions and one or more CDRs from a non - human (e.g., mouse, rat, or synthetic) antibody or antigen - binding fragment. The non - human antibody or antigen - binding fragment that provides the CDRs is called the "donor", and the human antibody or antigen - binding fragment that provides the framework is called the "acceptor". In one embodiment, all CDRs are derived from the donor immunoglobulin in the humanized immunoglobulin. The constant region need not be present, but if present, it can be substantially identical to the human immunoglobulin constant region, e.g., at least about 85 - 90%, e.g., about 95% or more identical. Thus, all parts of the humanized antibody or antigen - binding fragment are likely to be substantially identical to the corresponding parts of the native human antibody sequence, except perhaps for the CDRs.

[0185] A "chimeric antibody" is an antibody that typically contains sequences derived from two different antibodies of different species. In some examples, 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 (or originating from) the human genome and does not contain sequences from another species. In some embodiments, a human antibody contains CDRs, framework regions, and (if present) Fc regions derived from (or originating from) the human genome. Human antibodies can be identified and isolated, for example, by using technologies for creating sequences based on sequences from the human genome by phage display, or by using transgenic animals (see, for example, 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; Lonberg, 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 each other or different. For example, a naturally occurring immunoglobulin has two identical binding sites, a single-chain antibody or a Fab fragment has one binding site, while a bispecific or bifunctional antibody has two different binding sites.

[0188] Methods for testing an antibody for its 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, for example, Janeway et al., U.S. Patent Application Publication No. 2002 / 0197266 A1, and U.S. Patent No. 7,338,929, below).

[0189] In addition, the CAR, T cells expressing the CAR, antibody or antigen-binding portion thereof can be modified to include a detectable label, such as a radioisotope, fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), enzyme (e.g., alkaline phosph atase, horseradish peroxidase) and elemental particles (e.g., gold particles).

[0190] C. Conjugate The CAR, T cells expressing the CAR, or monoclonal antibody or antigen-binding fragment thereof specific for one or more of the antigens disclosed herein can be conjugated to an agent such as an effector molecule or detectable marker using any of a number of means known to those of skill in the art. Both covalent and non-covalent means can be used. Conjugates include, but are not limited to, molecules having a covalent linkage 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. Those of skill in the art will understand that various effector molecules and detectable markers can be used, including, but not limited to, chemotherapeutic agents, anti-angiogenic agents, toxins, radioactive agents, such as 125 I,[[]] 32 P,[[]] 14 C,[[]] 3 H and 35 S, as well as other labels, target moieties and ligands, etc.

[0191] The selection of a particular effector molecule or detectable marker depends on the particular target molecule or cell, and the desired biological effect. Thus, for example, the effector molecule can be a cytotoxin used to effect the death of a particular target cell (e.g., a tumor cell).

[0192] The procedure for conjugating an effector molecule or detectable marker to an antibody or antigen-binding fragment varies according to the chemical structure of the effector. Polypeptides typically contain various functional groups; for example, carboxylic acid (COOH), free amine (-NH2) or sulfhydryl (-SH) groups, which are available for reaction with appropriate functional groups on the antibody to effect binding of the effector molecule or detectable marker. Alternatively, the antibody or antigen-binding fragment is derivatized to expose or attach additional reactive functional groups. Derivatization can involve attachment of any of several known linker molecules, such as those available from Pierce Chemical Company, Rockford, IL. A linker can be any molecule used to couple an antibody or antigen-binding fragment to an effector molecule or detectable marker. A linker is capable of forming a covalent bond to both the antibody or antigen-binding fragment and the effector molecule or detectable marker. Suitable linkers are well known to those of ordinary skill in the art and include, but are not limited to, straight-chain or branched-chain carbon linkers, heterocyclic carbon linkers or peptide linkers. Where the antibody or antigen-binding fragment and the effector molecule or detectable marker are polypeptides, the linker can be attached to the constituent amino acids via their side groups (e.g., via disulfide linkage to cysteine), or to the amino and carboxyl groups of the alpha-carbon of the terminal amino acids.

[0193] In some embodiments, the linker, if present, may include a spacer element that increases the size of the linker and, as a result, increases 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 and include those recited in U.S. Patent No. 7,964,566, U.S. Patent No. 7,498,298, U.S. Patent No. 6,884,869, U.S. Patent No. 6,323,315, U.S. Patent No. 6,239,104, U.S. Patent No. 6,034,065, U.S. Patent No. 5,780,588, U.S. Patent No. 5,665,860, U.S. Patent No. 5,663,149, U.S. Patent No. 5,635,483, U.S. Patent No. 5,599,902, U.S. Patent No. 5,554,725, U.S. Patent No. 5,530,097, U.S. Patent No. 5,521,284, U.S. Patent No. 5,504,191, U.S. Patent No. 5,410,024, U.S. Patent No. 5,138,036, U.S. Patent No. 5,076,973, 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. 4,81 6,444, and U.S. Patent No. 4,486,414, as well as those recited in U.S. Patent Application Publication No. 20110212088 and U.S. Patent Application Publication No. 20110070248, each of which is incorporated herein by reference in its entirety.

[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 lysosomes or endosomes or caveolae). The linker can be a peptide linker that is cleaved by intracellular peptidases or protease enzymes including, but not limited to, lysosomal or endosomal proteases. In some embodiments, the peptide linker is at least 2 amino acids in length or at least 3 amino acids in length. However, the linker can be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length, e.g., 1-2, 1-3, 2-5, 3-10, 3-15, 1-5, 1-10, 1-15 amino acids in length. Proteases can include cathepsin B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives to yield release of the active drug inside the target cell (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). For example, a peptide linker cleavable by cathepsin B, a thiol-dependent protease, can be used (e.g., phenylalanine-leucine or glycine-phenylalanine-leucine-glycine linker). Other examples of such linkers are described, for example, in U.S. Patent No. 6,214,345, which is incorporated herein by reference. In a specific embodiment, the peptide linker cleavable by intracellular proteases is a valine-citruline linker or a phenylalanine-lysine linker (see, e.g., U.S. Patent No. 6,214,345, which describes the synthesis of doxorubicin using a valine-citruline linker).

[0195] In other embodiments, the cleavable linker is pH-sensitive, i.e., sensitive to hydrolysis at a particular pH value. Typically, the pH-sensitive linker is hydrolysable under acidic conditions. For example, acid-labile linkers that are hydrolysable in lysosomes (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amide, orthoesters, acetals, ketals, etc.) 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 pH values below about pH 5.5 or 5.0, which is the approximate pH of lysosomes. In certain embodiments, the hydrolysable linker is a thioether linker (e.g., a thioether linked to a therapeutic agent via an acylhydrazone bond) (see, for example, U.S. Patent 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 and include, for example, those that can be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene)-, SPDB and SMPT (see, for example, T horpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., Immunoconjugates: Antibody C Conjugates in Radioimagery and Therapy of (see Cancer (ed. C.W. Vogel, 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 yet 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, which is incorporated herein by reference in its entirety).

[0199] In some embodiments, the linker is resistant to cleavage in the extracellular environment. For example, when the conjugate is present in the extracellular environment (e.g., in plasma), less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 3%, or less than about 1% of the linker in the conjugate sample is cleaved. Whether the linker is resistant to cleavage in the extracellular environment can be determined, for example, by incubating a conjugate containing the linker of interest 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 conjugate are described in WO2004-010957, US Patent Application Publication No. 2006 / 0074008, US Patent Application Publication No. 20050238649, and US Patent Application Publication No. 2006 / 0024317, each of which is incorporated herein by reference in its entirety.

[0200] In some embodiments, provided are conjugates of CARs, T cells expressing CARs, antibodies or antigen-binding portions thereof, and one or more small molecule toxins, such as calicheamicin, maytansinoid, dolastatin, auristatin, trichothecin, and CC1065, and derivatives of these toxins having toxin activity.

[0201] Maytansine compounds suitable for use as maytansinoid toxins are well known in the art and can be isolated from natural sources according to known methods, or can be produced using maytansinol and maytansinol analogs prepared synthetically according to known methods (see Yu et al. (2002) PNAS 99:7968-7973). Maytansinoids are mitotic inhibitors that act by inhibiting tubulin polymerization. Maytansine was first isolated from the East African shrub Maytenus serrata (U.S. Patent No. 3,896,111). Subsequently, certain microorganisms have also been found to produce maytansinoids such as maytansinol and C-3 maytansinol esters (U.S. Patent No. 4,151,042). Synthetic maytansinol and its derivatives and analogs are disclosed, for example, in U.S. Patent No. 4,137,230; U.S. Patent No. 4,248,870; U.S. Patent No. 4,256,746; U.S. Patent No. 4,260,608; U.S. Patent No. 4,265,814; U.S. Patent No. 4,294,757; U.S. Patent No. 4,3 07,016; U.S. Patent No. 4,308,268; U.S. Patent No. 4,308,269; U.S. Patent No. 4,309,428; U.S. Patent No. 4,313,946; U.S. Patent No. 4,315,929; U.S. Patent No. 4,317,821; U.S. Patent No. 4,322,348; U.S. Patent No. 4,331,598; U.S. Patent No. 4,361,650; U.S. Patent No. 4,364,866; U.S. Patent No. 4,424,219; U.S. Patent No. 4,450,254; U.S. Patent No. 4,362,663; and U.S. Patent No. 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. Patent No. 5,208,020; U.S. Patent No. 5,416,064; U.S. Patent No. 6,441,163 and European Patent EP0 425 235 B1, the disclosures of which are expressly incorporated herein by reference.

[0202] Additional toxins can be used with the CAR, T cells expressing the CAR, an antibody or antigen-binding portion thereof. Exemplary toxins include Pseudomonas exotoxin (PE), ricin, abrin, diphtheria toxin and its subunits, ribotoxin, ribonuclease, saporin and calicheamicin, as well as 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). Envisioned toxins also include variants of these toxins (see, e.g., U.S. Patent No. 5,079,163 and U.S. Patent No. 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, Vol. 10:405 - 412, 1992). However, this toxin does not have a mechanism for specific entry into cells and thus requires conjugation to an antibody or antigen-binding fragment that recognizes a cell surface protein to be efficiently taken up by the cell.

[0204] Diphtheria toxin is isolated from Corynebacterium diphtheriae. Typically, diphtheria toxin for use in immunotoxins is mutated to reduce or eliminate non-specific toxicity. A variant known as CRM107, which has full enzymatic activity but significantly reduced non-specific toxicity, has been known since the 1970s (Laird and Groman, J. Virol. 19:220, 1976) and has been used in human clinical trials. See U.S. Patent No. 5,792,458 and U.S. Patent No. 5,208,021.

[0205] Ricinus communis (castor bean) lectin RCA60 is ricin. For examples of ricin, see U.S. Patent No. 5,079,163 and U.S. Patent No. 4,689,401. Ricinus communis agglutinin (RCA) exists in two forms, called RCA 60 and RCA 120 respectively, with molecular weights of approximately 65 kD and 120 kD (Nicholson and Blaustein, J. Biochim. Biophys. Acta 266:543, 1972). The A chain is responsible for the inactivation of protein synthesis and cell death. The B chain binds ricin to cell surface galactose residues and facilitates the transport of the A chain into the cytosol (Olsnes et al., Nature 249:627-631, 1974 and U.S. Patent No. 3,060,165).

[0206] Ribonucleases have also been conjugated to targeting molecules for use as immunotoxins (see Suzuki et al., Nat. Biotech. 17:265-70, 19 99). 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 Pt 2:247-54, 2000. Calicheamicin was first isolated from Micromonospora echinospora and is a member of the enediyne antitumor antibiotic family that causes double-strand breaks in DNA leading to apoptosis (see, for example, Lee et al., J. Antibiot. 42:1070-87, 1989). This drug is the toxic moiety of an immunotoxin in clinical trials (see, for example, Gillespie et al., Ann. Oncol. 11:735-41, 2000).

[0207] Abrin contains a toxic lectin derived from Abrus precatorius. The toxic proteins abrin 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. Chain A inhibits protein synthesis; chain B (abrin-b) binds to D-galactose residues (see Funatsu et al., Agr. Biol. Chem. 52:1095, 1988; and Olsnes, Methods Enzymol. 50:330-335, 1978).

[0208] CARs, T cells expressing CARs, monoclonal antibodies specific for one or more of the antigens disclosed herein, antigen-binding fragments thereof may also be conjugated to a detectable marker; for example, a detectable marker detectable by ELISA, spectrophotometry, flow cytometry, microscopy or imaging diagnostic 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 conjugates, radioisotopes 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-naphthalenesulfonyl chloride, phycoerythrin, lanthanide phosphor and the like. Bioluminescent markers such as luciferase, green fluorescent protein (GFP), yellow fluorescent protein (YFP) are also used. CARs, T cells expressing CARs, antibodies or antigen-binding portions thereof may 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, a T cell expressing a CAR, an antibody or an antigen-binding portion thereof is conjugated to a detectable enzyme, it can be detected by adding further reagents used by the enzyme to produce a distinguishable reaction product. For example, in the presence of the agent horseradish peroxidase, the addition of hydrogen peroxide and diaminobenzidine results in a visually detectable colored reaction product. CARs, T cells expressing CARs, antibodies or antigen-binding portions thereof may also be conjugated to biotin and detected via an indirect measurement of the binding of avidin or streptavidin. It should be noted that avidin itself may be conjugated to an enzyme or a fluorescent label.

[0209] A CAR, a T cell expressing the CAR, an antibody or an antigen-binding portion thereof can be conjugated to a paramagnetic agent such as gadolinium. Paramagnetic agents such as superparamagnetic iron oxides are also used as labels. Antibodies can also be conjugated to lanthanides (e.g., europium and dysprosium) and manganese. An antibody or antigen-binding fragment can also be labeled with a predetermined polypeptide epitope (e.g., leucine zipper -pair sequence, binding site for a secondary antibody, metal-binding domain, epitope tag) recognized by a secondary reporter.

[0210] A CAR, a T cell expressing the CAR, an antibody or an antigen-binding portion thereof can also be conjugated to a radiolabeled amino acid. The radiolabel can be used for both diagnostic and therapeutic purposes. For example, the radiolabel can be used to detect one or more of the antigens and antigen-expressing cells disclosed herein by x-ray, luminescence spectroscopy, or other diagnostic techniques. Further, the radiolabel can be used therapeutically as a toxin for the treatment of tumors in a subject, e.g., for the treatment of 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, and fluorescent markers can be detected using photodetectors to detect the emitted illumination. Enzyme labels are typically detected by providing a substrate to the enzyme 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 Nucleic acids comprising nucleotide sequences encoding any of the CARs, antibodies or antigen-binding portions thereof (including functional portions and functional variants thereof) described herein are further provided by one embodiment of the invention. The nucleic acids of the invention may comprise nucleotide sequences encoding any of the leader sequences, antigen-binding domains, transmembrane domains and / or intracellular T cell signaling domains described herein.

[0213] In some embodiments, the nucleotide sequence may be codon-modified. Without being bound by a particular theory, codon optimization of the nucleotide sequence is thought to increase the translation efficiency of the mRNA transcript. Codon optimization of the nucleotide sequence may involve replacing native codons with other codons that encode the same amino acid but can be translated by tRNAs that are more readily available within the cell, thus increasing translation efficiency. Optimization of the nucleotide sequence may also reduce secondary mRNA structures that interfere with translation, thus increasing translation efficiency.

[0214] In embodiments of the invention, the nucleic acid may comprise a codon-modified nucleotide sequence encoding the antigen-binding domain of the CAR of the invention. In another embodiment of the invention, the nucleic acid may comprise a codon-modified nucleotide sequence encoding any of the CARs (including functional portions and functional variants thereof) described herein.

[0215] "Nucleic acid," as used herein, includes "polynucleotide," "oligonucleotide," and "nucleic acid molecule," and generally refers to a polymer of DNA or RNA that can be single-stranded or double-stranded, obtained from synthetic or natural sources (e.g., isolated and / or purified), can contain natural, non-natural, or modified nucleotides, and can contain natural, non-natural, or modified internucleotide linkages, such as phosphoroamidate linkages or phosphorothioate linkages, instead of the phosphodiester found between nucleotides of unmodified oligonucleotides. In some embodiments, the nucleic acid does not include any insertions, deletions, inversions, and / or substitutions. However, in some cases, as discussed herein, it may be appropriate for the nucleic acid to include one or more insertions, deletions, inversions, and / or substitutions.

[0216] A recombinant nucleic acid can have a sequence that does not occur naturally or can have a sequence made by an artificial combination of two otherwise separated segments of a sequence. This artificial combination is often achieved by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids by genetic engineering techniques such as those described above by Sambrook et al. 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 various 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, but are not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, methyl ester of uracil-5-oxyacetic acid, 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 companies such as Integrated DNA Technologies (Coralville, IA, USA).

[0217] The nucleic acid can include any isolated or purified nucleotide sequence encoding either a CAR or a functional portion or variant thereof. Alternatively, the nucleotide sequence can include a nucleotide sequence that degenerate with 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 any of the nucleotide sequences of the nucleic acids described herein, or a nucleotide sequence that hybridizes to any of the nucleotide sequences of the nucleic acids described herein under stringent conditions.

[0219] A nucleotide sequence that hybridizes under stringent conditions can hybridize under high stringency conditions. "High stringency conditions" means that a nucleotide sequence hybridizes specifically to a target sequence (any of the nucleotide sequences of the nucleic acids described herein) in a detectably stronger amount than non-specific hybridization. High stringency conditions include conditions for discriminating a polynucleotide having an exactly complementary sequence or a polynucleotide containing only a few scattered mismatches from random sequences that accidentally have some small regions (e.g., 3 to 10 bases) matching the nucleotide sequence. Such small regions of complementarity melt more easily than the full-length complement of 14 to 1 7 or more bases, and high stringency hybridization makes them readily distinguishable. Relatively high stringency conditions include, for example, low salt and / or high temperature conditions provided at a temperature of about 50 to 70 °C by about 0.02 to 0.1 M NaCl or equivalents. Such high stringency conditions tolerate few mismatches, if any, 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. In general, it is understood that the 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 identical, such as 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 can be incorporated into a recombinant expression vector. In this regard, one embodiment provides a recombinant expression vector comprising any of the nucleic acids. For the purposes of this specification, the term "recombinant expression vector" means that when the construct contains a nucleotide sequence encoding an mRNA, protein, polypeptide or peptide, and the vector is contacted with a cell under conditions sufficient for the mRNA, protein, polypeptide or peptide to be expressed intracellularly, it enables the expression of the mRNA, protein, polypeptide or peptide by the host cell. It refers to a genetically modified oligonucleotide or polynucleotide construct. The vector is not naturally occurring as a whole.

[0222] However, a part of the vector can be naturally occurring. The recombinant expression vector can be single-stranded or double-stranded, obtained synthetically or from a partially natural source, and can contain DNA and RNA, including but not limited to any type of nucleotide that can contain natural, non-natural or modified nucleotides. The recombinant expression vector can contain naturally occurring or non-naturally occurring nucleotide linkages, or both types of linkages. Preferably, the non-naturally occurring or modified nucleotides or nucleotide linkages do not interfere with the transcription or replication of the vector.

[0223] In one embodiment, the recombinant expression vector can be any suitable recombinant expression vector and can be used to transform or transfect any suitable host cell. Suitable vectors include those designed for propagation and growth, or expression, or both, such as plasmids and viruses. The vector can 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 λΤΙΟ, λΤΙ 1, λZapII (Stratagene), EMBL4, and λΝΜΙ 149, can also be used. Examples of plant expression vectors include pBIOl, pBI101.2, pBHOl.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 vector or a lentiviral vector. Lentiviral vectors include, in particular, vectors derived from at least a portion of the lentiviral genome, such as self-inactivating lentiviral vectors as provided by Milone et al., Mol. Ther. 17(8):1453-1464 (2009). Other examples of lentiviral vectors that can be used in the clinic include, for example, but not limited to, the LENTIVECTOR® gene delivery technology of Oxford BioMedica plc, the LENTIMAX™ vector system of Lentigen, etc. Non-clinical lentiviral vectors are also available and are 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 co-precipitation (see, e.g., Graham et al., supra), direct microinjection into cultured cells (see, e.g., Capecchi, Cell, 22:479-488 (1980)), electroporation (see, e.g., Shigekawa et al., BioTechniques, 6:742-751 (1988)), liposome-mediated gene transfer (see, e.g., Mannino et al., BioTechniques, 6:682-690 (1988)), lipid-mediated transduction (see, e.g., Feigner et al., Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)) and nucleic acid delivery using high velocity microprojectiles (see, e.g., Klein et al., Nature, 327:70-73 (1987)).

[0227] In one embodiment, the recombinant expression vector can be prepared using standard recombinant DNA techniques as described, e.g., in Sambrook et al., supra and Ausubel et al., supra. The expression vector construct, which can be circular or linear, can be prepared to contain a replication system functional in a prokaryotic or eukaryotic host cell. The replication system can be derived, for example, from ColE1, 2μ plasmid, λ, SV40, bovine papillomavirus, etc.

[0228] A recombinant expression vector may contain regulatory sequences specific to the type of host cell (e.g., bacteria, fungi, plants, or animals) into which the vector is introduced, such as transcription and translation start and stop codons, taking into account whether the vector is DNA-based or RNA-based, as necessary. The recombinant expression vector may contain restriction sites to facilitate cloning.

[0229] A recombinant expression vector may contain one or more marker genes that enable the selection of transformed or transfected host cells. Marker genes include biocide resistance, such as resistance to antibiotics, heavy metals, etc., and complementation in auxotrophic hosts to provide prototrophy. 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] A recombinant expression vector may contain a native or non-native promoter operably linked to a nucleotide sequence encoding a CAR (including its functional parts and functional variants), or to a nucleotide sequence complementary to or hybridizing to a nucleotide sequence encoding a CAR. The selection of promoters, such as strong, weak, inducible, tissue-specific, and development-specific, is within the skill of the art. Similarly, combining a nucleotide sequence with a promoter is also within the skill of the art. The promoter may be a non-viral promoter or a viral promoter, such as the cytomegalovirus (CMV) promoter, the SV40 promoter, the R SV promoter, or a promoter found in the terminal repeat sequences of the murine stem cell virus.

[0231] A recombinant expression vector may be designed for transient expression, stable expression, or both. Also, a recombinant expression vector may be prepared for constitutive expression or inducible expression.

[0232] Furthermore, the recombinant expression vector can be constructed to contain a suicide gene. As used herein, the term "suicide gene" refers to a gene that causes the death of cells expressing the suicide gene. The suicide gene can be a gene that confers sensitivity to an agent such as a drug on the cells in which the gene is expressed, or a gene that causes the death of the cells when the cells come into contact with or are exposed to the agent. 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, the herpes simplex virus (HSV) thymidine kinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase, and nitroreductase.

[0233] One embodiment further provides a host cell comprising any of the recombinant expression vectors described herein. As used herein, the term "host cell" refers to any type of cell that can contain the recombinant expression vector of the present invention. The host cell can be a eukaryotic cell, such as a plant, animal, fungus, or alga, or a prokaryotic cell, such as a bacterium or protist. The host cell can be a cultured cell or a primary cell, i.e., can be isolated directly 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, and the like. For the purpose of amplifying or replicating the recombinant expression vector, the host cell can be a prokaryotic cell, such as a DH5a cell. For the purpose of producing the 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, can originate from any type of tissue, and can be of any developmental stage, 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 the purposes of this specification, a T cell can be any T cell, e.g., a cultured T cell, e.g., a primary T cell, or a cultured T cell line, e.g., a T cell derived from Jurkat, SupTl, etc., or a T cell obtained from a mammal. When obtained from a mammal, the T cell can be obtained from a number of sources including, but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or body fluids. The T cell can also be enriched or purified. The T cell can be a human T cell. The T cell can be a T cell isolated from a human. The T cell can be any type of T cell 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., and can be at any stage of development. The T cell can be a CD8+ T cell or a CD4+ T cell.

[0235] In one embodiment, the CAR described herein can be used in suitable non-T cells. Such cells are cells having immune effector functions, e.g., NK cells and T-like cells generated from pluripotent stem cells and the like.

[0236] A population of cells comprising at least one host cell described herein is also provided by one embodiment. The population of cells can be a heterogeneous population that includes at least one other cell that does not contain any recombinant expression vectors, such as a host cell (e.g., a T cell), or a cell other than a T cell, such as a B cell, macrophage, neutrophil, erythrocyte, hepatocyte, endothelial cell, epithelial cell, muscle cell, brain cell, etc., in addition to host cells that contain any of the described recombinant expression vectors. Alternatively, the population of cells can be a substantially homogeneous population, where the population mainly includes (e.g., consists essentially of) host cells that contain recombinant expression vectors. The population can also be a clonal population of cells, where all cells of the population are clones of a single host cell that contains a recombinant expression vector, and as a result, all cells of the population contain that recombinant expression vector. In one embodiment of the invention, the population of cells is a clonal population that includes host cells that contain the recombinant expression vectors described herein.

[0237] CARs (including their functional portions and variants), nucleic acids, recombinant expression vectors, host cells (including populations thereof), and antibodies (including their antigen-binding portions) can be isolated and / or purified. For example, a preparation of purified (or isolated) host cells is a preparation in which the host cells are purer 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, the preparation of host cells 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%, can be above about 60%, about 70% or about 80%, or can be about 100%.

[0238] E. Methods of Treatment The CARs disclosed herein are contemplated to be used in methods for treating or preventing diseases in mammals. In this regard, one embodiment provides a method for treating or preventing cancer in a mammal, comprising administering to the mammal an effective amount of 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, to treat or prevent cancer in the mammal.

[0239] One embodiment further comprises a step of lymphodepleting the mammal prior to the step of administering the CARs disclosed herein. Examples of lymphodepletion can include, but are not limited to, non-myeloablative lymphodepleting chemotherapy, myeloablative lymphodepleting chemotherapy, total body irradiation, and the like.

[0240] For the purpose of the method of administering a host cell, or a population of cells, the cells can be cells that are allogeneic or autologous to the mammal. Preferably, the cells are autologous to the mammal. As used herein, "allogeneic" means 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 genetic loci. In some aspects, allogeneic materials from individuals of the same species can be genetically different enough to interact antigenically. As used herein, "autologous" means any material derived from the same individual that is later re-introduced into the individual.

[0241] The mammal referred to herein can 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 Lagomorpha, such as rabbits. The mammal can be of carnivore origin including Felines (cats) and Canines (dogs). The mammal The subject can be from Artiodactyla including Bovine (cow) and Swine (pig), or from Perissodactyla including Equine (horse). The mammal can be from Primates, New World monkeys (Ceboid) or Old World monkeys (Simoid) (monkey) or Anthropoid (human and ape). Preferably, the mammal is human.

[0242] With respect to these methods, the cancer can be any cancer including acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer (e.g., bladder carcinoma), bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, anal, anal canal or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, neck, gallbladder or pleural cancer, nasal, nasal cavity or middle ear cancer, oral cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, head and neck cancer (e.g., head and neck squamous cell carcinoma), Hodgkin lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, leukemia, liquid tumor, liver cancer, lung cancer (e.g., non-small cell lung cancer and lung adenocarcinoma), lymphoma, mesothelioma, mast cell tumor, melanoma, multiple myeloma, oropharyngeal cancer, non-Hodgkin lymphoma, B-chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia (ALL) and Burkitt 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, stomach cancer, testicular cancer, thyroid cancer and ureteral cancer.

[0243] As used herein, the terms "treat" and "prevent" and words derived therefrom do not necessarily mean 100% or complete treatment or prevention. Rather, there are various degrees of treatment or prevention that are recognized by those skilled in the art as having a potential beneficial or therapeutic effect. In this regard, the method can provide treatment or prevention of cancer in a mammal at any amount or any level.

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

[0245] Another embodiment is a method for detecting the presence of cancer in a mammal, comprising: (a) contacting a sample containing one or more cells derived from the mammal with a CAR, nucleic acid, recombinant expression vector, host cell, population of cells, antibody and / or its antigen-binding portion, or 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, such as biopsy or autopsy. A biopsy is the removal of tissue and / or cells from an individual. Such removal can be the collection of tissue and / or cells from an individual for performing experiments on the removed tissue and / or cells. The experiments can include experiments for determining whether the individual has a particular condition or disease state and / or is suffering from a particular condition or disease state. The condition or disease can be, for example, cancer.

[0247] Regarding embodiments of a method for detecting the presence of a growth disorder, such as cancer, in a mammal, a sample containing mammalian cells can be a sample containing whole cells, their lysates, or fractions of whole cell lysates, such as nuclear or cytoplasmic fractions, whole protein fractions, or nucleic acid fractions. When the sample contains whole cells, these cells can be any cells of a mammal, such as cells of any organ or tissue containing blood cells or endothelial cells. cells or endothelial cells.

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

[0249] Also, the detection of the complex can be performed by many methods known in the art. For example, the CAR, polypeptide, protein, nucleic acid, recombinant expression vector, host cell, cell population, or antibody or antigen-binding portion thereof described herein can be labeled with a detectable label, such as the radioisotopes, fluorophores (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), enzymes (e.g., alkaline phosphatase, horseradish peroxidase), and elemental particles (e.g., gold particles) disclosed above.

[0250] Methods for testing CARs for their ability to recognize target cells and antigen specificity are known in the art. For example, Clay et al., J. Immunol., Vol. 163: 507-513 (1999) teach methods for measuring the release of cytokines (e.g., interferon-γ, granulocyte / monocyte colony stimulating factor (GM-CSF), tumor necrosis factor a (TNF-a), or interleukin 2 (IL-2)). Additionally, CAR function can be evaluated by measuring cytotoxicity, as described by Zhao et al., J. Immunol. 174: 4415-4423 (2005).

[0251] Another embodiment provides for the use of the CAR, nucleic acid, recombinant expression vector, host cell, cell population, antibody or antigen-binding portion thereof, and / or pharmaceutical composition of the present invention for the treatment or prevention of proliferative disorders such as cancer in a mammal. The cancer can be any of the cancers described herein.

[0252] Any method of administration, including local and systemic administration, can be used for the disclosed therapeutic agents. For example, topical, oral, intravascular such as intravenous, intramuscular, intraperitoneal, intranasal, intradermal, intrathecal, and subcutaneous administrations can be used. The specific mode of administration and dosing regimen are selected by the attending clinician considering the characteristics of the case (e.g., the subject, the disease, the disease state involved, and whether the treatment is prophylactic). If more than one agent or composition is being administered, one or more routes of administration can be used; for example, a chemotherapeutic agent can be administered orally, and an antibody or antigen-binding fragment or conjugate or composition can be administered intravenously. The methods of administration include injections in which CARs, CAR T cells, conjugates, antibodies, antigen-binding fragments, or compositions are provided in a non-toxic pharmaceutically acceptable carrier such as water, saline, Ringer's solution, dextrose solution, 5% human serum albumin, solid oil, ethyl oleate, or liposomes. In some embodiments, local administration of the disclosed compounds 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 an area suspected of having a tendency for tumor development. In some embodiments, sustained intratumoral (or near-tumor) release of a pharmaceutical preparation containing a therapeutically effective amount of an antibody or antigen-binding fragment can be beneficial. In other examples, a conjugate is applied topically as an eye drop to the cornea or intravitreally to the eye.

[0253] The disclosed therapeutic agents can be formulated in unit dosage forms appropriate for the precise dosing of individual administrations. Further, the disclosed therapeutic agents can be administered in a single dose or in a multiple-dose schedule. A multiple-dose schedule is a schedule in which the major course of treatment consists of more than one separate dose, for example, 1 to 10 doses, and subsequent other doses given at time intervals as needed to maintain or enhance the action of the composition. The treatment can include daily doses or multiple daily doses over a period of days to months or even years. The compound(s) can be included. Thus, the dosing regimen is also at least partially determined based on the specific requirements of the subject being treated and depends on the judgment of the practitioner administering the dose.

[0254] Typical dosages of the antibody or conjugate can range from about 0.01 to about 30 mg / kg, such as, for example, from about 0.1 to about 10 mg / kg.

[0255] In certain examples, the subject is administered a therapeutic composition comprising one or more of a conjugate, an antibody, a composition, a CAR, a CAR T cell, or an additional agent, in a multiple daily dosing schedule, such as, for example, for at least 2 consecutive days, 10 consecutive days, etc., for a period of, for example, weeks, months, or years. In one example, the subject is administered a conjugate, an antibody, a composition, or an additional agent for a period of at least 30 days, such as, for example, at least 2 months, at least 4 months, at least 6 months, at least 12 months, at least 24 months, or at least 36 months.

[0256] In some embodiments, the disclosed methods include providing to the subject a surgery, radiation therapy, and / or chemotherapy agent (e.g., continuously, substantially simultaneously, or simultaneously) in combination with the disclosed antibody, antigen-binding fragment, conjugate, CAR, or T cell expressing a CAR. Such agents and methods of treatment and therapeutic dosages are known to those of skill in the art and can be determined by a skilled clinician. Preparation and dosing schedules for additional agents can be used according to the manufacturer's instructions or can be as determined experimentally by those of skill in the art. Preparation and dosing schedules for such chemotherapy are also described in Chemotherapy Service, edited by M.C. Perry (1992), Williams & Wilkins, Baltimore, Md.

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

[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 folates (e.g., methotrexate, pemetrexed, and raltitrexed), purines (e.g., cladribine, clofarabine, fludarabine, mercaptopurine, and thioguanine), pyrimidines (e.g., capecitabine), cytarabine, fluorouracil, and gemcitabine; plant alkaloids such as podophyllum (e.g., etoposide and teniposide), taxanes (e.g., docetaxel and paclitaxel), vinca (e.g., vinblastine, vincristine, vindesine, and vinorelbine); cytotoxic / antineoplastic antibiotics such as members of the anthracycline family (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, and valrubicin), bleomycin, rifampicin, hydroxyurea, and mitomycin; topoisomerase inhibitors such as topotecan and irinotecan; monoclo -nal antibodies, such as alemtuzumab, bevacizumab, cetuximab, gemtuzumab, rituximab, panitumumab, pertuzumab and trastuzumab; tumor-affinity photosensitizing dyes, such as aminolevulinic acid, methyl aminolevulinate, sodium porfimer and verteporfin; and other agents, such as alitretinoin, altretamine, amsacrine, anagrelide, arsenic trioxide, asparaginase, axitinib, bexarotene, bevacizumab, bortezomib, celecoxib, denileukin diftitox, erlotinib, estramustine, gefitinib, hydroxycarbamide, imatinib, lapatinib, pazopanib, pentostatin, masoprocol, mitotane, pegaspargase, tamoxifen, sorafenib, sunitinib, vemurafinib, vandetanib and tretinoin, are included but not limited to these. The selection of such agents and the therapeutic dosage are known to those skilled in the art and can be determined by a skilled clinician.

[0259] Combination therapies can provide a synergistic effect and can be demonstrated to be synergistic, i.e., the effect achieved when the active ingredients are used together is greater than the sum of the effects that could result from using those compounds separately. The synergistic effect can be achieved when the active ingredients are (1) co-formulated and administered or delivered simultaneously with a unit dosage formulation in which they are combined; (2) delivered alternately or in parallel as separate formulations; or (3) by some other regimen. When delivered alternately, the synergistic effect can be achieved when the compounds are administered or delivered continuously, e.g., by different injections in separate syringes. Generally, during the alternation, each active ingredient of the effective dosage is administered continuously, i.e., sequentially, but in combination therapy, two or more active ingredients of the 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 having a tumor after anti-cancer treatment. After a sufficient amount of time has elapsed for the administered antibody or antigen-binding fragment or conjugate to form an immune complex with the antigen expressed on the respective cancer cells, the immune complex is detected. The presence (or absence) of the immune complex indicates the effectiveness of the treatment. For example, an increase in the immune complex compared to a control taken before treatment indicates that the treatment is ineffective, and a decrease in the immune complex compared to a control taken before treatment indicates that the treatment is effective.

[0261] F. Pharmaceutical Compositions Provided herein are biopharmaceutical compositions or biologic compositions (hereinafter “compositions” herein) for use in gene therapy, immunotherapy and / or cell therapy comprising, in a carrier (e.g., a pharmaceutically acceptable carrier), one or more of the disclosed CARs, or T cells expressing a CAR, antibodies, antigen-binding fragments, conjugates, CARs, or T cells expressing a CAR that specifically binds to one or more of the antigens disclosed herein. These compositions can be prepared in unit dosage forms for administration to a subject. The amount and timing of administration to achieve the desired outcome are at the discretion of the clinician performing the treatment. These compositions can be formulated for systemic (e.g., intravenous) or local (e.g., intratumoral) administration. In one example, the disclosed CARs, or T cells expressing a CAR, antibodies, antigen-binding fragments, conjugates are formulated for parenteral administration such as intravenous administration. Compositions comprising the CARs, or T cells expressing a CAR, conjugates, antibodies or antigen-binding fragments disclosed herein are used, for example, for the treatment and detection of tumors, such as, but not limited to, neuroblastoma. In some examples, these compositions are useful for the treatment or detection of cancer. Compositions comprising the CARs, or T cells expressing a CAR, conjugates, antibodies or antigen-binding fragments disclosed herein are also used, for example, for the detection of pathological angiogenesis.

[0262] For the composition for administration, CA R, or a solution of T cells expressing CAR, conjugate, antibody or antigen-binding fragment may be included. For example, various aqueous carriers such as buffered saline can be used. These solutions are sterile and generally do not contain undesirable substances. The composition can be sterilized by conventional well-known sterilization techniques. The composition may contain pharmaceutically acceptable auxiliary substances necessary to approach physiological conditions, such as pH adjusters and buffers, toxicity adjusters, adjuvant agents, etc., for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of CAR, or T cells expressing CAR, antibody or antigen-binding fragment or conjugate in these formulations can vary widely and is selected mainly based on the liquid volume, viscosity, body weight, etc., according to the specific mode of administration selected and the requirements of the subject. The actual methods for preparing such dosage forms for use in gene therapy, immunotherapy and / or cell therapy are known or will be apparent to those skilled in the art.

[0263] A typical composition for intravenous administration contains about 0.01 to about 30 mg / kg per day per subject of an antibody or antigen-binding fragment or conjugate (or the corresponding dose of CAR, or a conjugate containing T cells expressing CAR, antibody or antigen-binding fragment). The actual methods for preparing administrable compositions are known or apparent to those skilled in the art and are described in more detail in publications such as Remington’s Pharmaceutical Science, 19th Edition, Mack Publishing Company, Easton, PA (1995).

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

[0265] Controlled-release parenteral formulations can be prepared as implants, oily injections, or as particulate systems. For a general overview of protein delivery systems, see Banga, A.J., Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Technomic Publishing Company, Inc., Lancaster, PA (1995). Particulate systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain a therapeutic protein, such as a cytotoxic or a drug, as a central core. In microspheres, the therapeutic agent is dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than about 1 μm are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Capillaries have a diameter of approximately 5 μm such that only nanoparticles are administered intravenously. Microparticles typically have a diameter of approximately 100 μm and are administered subcutaneously or intramuscularly. For example, see 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, edited by A. Kydonieus, Marcel Dekker, Inc. New York, NY, pp. 315-339 (1992).

[0266] The polymer can be used for the ion-controlled release of the CARs disclosed herein, or T cells, antibodies or antigen-binding fragments or conjugate compositions expressing the CAR. 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 aspect, liposomes are used for the controlled release of lipid-encapsulated drugs and drug targeting (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA (1993)). Numerous additional systems for the controlled delivery of therapeutic proteins are known (see U.S. Patent No. 5,055,303; U.S. Patent No. 5,188,837; U.S. Patent No. 4,235,871; U.S. Patent No. 4,501,728; U.S. Patent No. 4,837,028; U.S. Patent No. 4,957,735; U.S. Patent No. 5,019,369; U.S. Patent No. 5,055,303; U.S. Patent No. 5,514,670; U.S. Patent No. 5,413,797; U.S. Patent No. 5,268,164; U.S. Patent No. 5,004,697; U.S. Patent No. 4,902,505; U.S. Patent No. 5,506,206; U.S. Patent No. 5,271,961; U.S. Patent No. 5,254,342 and U.S. Patent No. 5,534,496).

[0267] G. Kit In one aspect, kits are also provided that use the CARs disclosed herein. For example, a kit for treating a tumor in a subject or a kit for generating CAR T cells that express one or more of the CARs disclosed herein. The kit typically includes an antibody, antigen-binding fragment, conjugate, nucleic acid molecule, CAR, or T cell expressing a CAR disclosed herein. More than one of the disclosed antibodies, antigen-binding fragments, conjugates, nucleic acid molecules, CARs, or T cells expressing a CAR can be included in the kit.

[0268] The kit can include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The container can be formed from a variety of materials such as glass or plastic. The container typically holds a composition that includes one or more of the disclosed antibodies, antigen-binding fragments, conjugates, nucleic acid molecules, CARs, or T cells expressing a CAR. In some embodiments, the container can have a sterile access port (e.g., the container can be an intravenous solution bag or vial having a stopper penetrable by a hypodermic needle). The label or package insert indicates that the composition is to be used for treating a particular condition.

[0269] The label or package insert typically indicates, for example, a method of treating or preventing a tumor, or Or further comprising instructions for the use of the disclosed antibodies, antigen-binding fragments, conjugates, nucleic acid molecules, CARs or T cells expressing a CAR in a method of making CAR T cells. The package insert typically includes instructions customarily included in the commercial packaging of a therapeutic product, including information regarding indications, usage, dosage, administration, contraindications and / or warnings regarding the use of the therapeutic product. The teaching materials can be written in electronic form (e.g., computer disk or compact disk), or can be visual (e.g., video file). The kit can also include additional components to facilitate the particular application for which the kit is designed. Thus, for example, the kit can further include means for detecting a label (e.g., an enzyme substrate for an enzyme label, a filter set for detecting a fluorescent label, an appropriate secondary label such as a secondary antibody, etc.). The kit can further include buffers and other reagents customarily used for the performance of a particular method. Such kits and appropriate contents are well known to those skilled in the art.

Examples

[0270] The present invention is further illustrated by the following examples, which should in no way be construed as imposing limitations on the scope of the present invention. On the contrary, reliance must be placed on various other embodiments, modifications, and equivalents, which will readily occur to those skilled in the art after reading the description herein without departing from the spirit of the present invention and / or the appended claims.

[0271] Example 1 Isolation of CD19-Specific Antibodies from Phage Display and Yeast Displayed Fully Human ScFv Libraries Materials and Methods: a) Production of Human ScFv and CD19-Specific Antibodies A naive human ScFv (recombinant single-chain variable fragment of immunoglobulin) phage display library (approximate diversity, 10 10The uniqueness of the species (Z.Y. Zhu and D.S.Dimitrov, unpublished data) was used for the selection of ScFv against recombinant human CD19 protein (Miltenyi Biotec, unpublished). The phage-displayed ScFv10 12 The amplified library of the species was incubated with coated CD19 at 5, 3, and 1 μg in a volume of 5 × 100 μl, and equally distributed into 5 wells of a 96-well plate during the first, second, and third rounds of biopanning respectively and left at room temperature for 2 hours. After each round of incubation, the wells were washed 5 times in the first round and 10 times in subsequent rounds with phosphate-buffered saline (PBST) containing 0.05% Tween 20 to remove non-specifically bound phages. The bound phages were mixed with TG1-competent cells at 37 °C for 1 hour and the phages were amplified from the infected cells for use 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 by using an automated BioRobotics BioPick colony picking system (Genomic Solutions, Ann Arbor, MI). After the bacterial culture reached an optical density (OD600) of 0.5 at 600 nm, 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 plates were further incubated overnight at 30 °C at 250 rpm in a shaker. The phage supernatant was mixed with a 3% skim milk solution in PBS at a volume ratio of 4:1 and used in an enzyme-linked immunosorbent assay (ELISA) to identify clones of phages presenting ScFv or VH with high CD19 binding affinity. The supernatant was incubated with coated recombinant human CD19 at 50 ng per well in a 96-well plate at room temperature for 2 hours, washed 5 times with PBST, (after incubating overnight at 4 °C, It was blocked with 3% skim milk solution in PBS and washed three times with PBS containing 0.05% Tween 20. The phage bound to CD19 was detected using goat anti-M13 antibody conjugated with horseradish peroxidase. After incubation with the antibody, the wells were washed to remove non-specifically bound antibody, 3,3’,5,5’-tetramethylbenzidine (TMB) substrate was added, and the absorbance of the solution at 450 nm (A450) was measured. Clones bound to CD19 with A450 exceeding 1.0 were selected for further characterization.

[0272] b) Expression and purification of the selected soluble ScFv The VH and VL of the selected clones were sequenced, and the ScFv encoded by the clones with unique sequences was expressed and purified as follows. The plasmids extracted from these clones were used for the transformation of HB2151 cells. Single colonies were selected from the plates containing the newly 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 OD of the culture reached 0.90 at 600 nm, isopropyl-β-d-thiogalactopyranoside was added at a final concentration of 0.5 mM, and the culture was further incubated at 30 °C overnight. After centrifugation at 8,000×g for 20 minutes, the bacterial pellet was collected and resuspended in PBS buffer containing 0.5 mU polymyxin B (Sigma-Aldrich, St. Louis, MO). After incubation at room temperature for 30 minutes with rotation at 50 rpm, the resuspended pellet was centrifuged at 25,000×g for 25 minutes 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 overnight at 4 °C on a 96-well plate. Purified ScFv with His and Flag tags was serially diluted and added to the wells coated with the target protein. After washing, anti-Flag antibody conjugated to HRP, diluted 1:3000, was added at RT for 1 hour. After washing, 3,3′,5,5′-tetramethylbenzidine (TMB) substrate was added and incubated at room temperature for 10 minutes, then 1 N H2SO4 was added to stop the reaction, and the O.D. was read at 450 nm to quantify the relative ability of the ScFv to bind to CD19.

[0274] d) Yeast display of the scFv library. The same ScFv starting material as for phage display was also incorporated into the yeast ScFv display system. To complement phage-based scFv analysis, a yeast library expressing a human scFv library was also screened. To enrich yeast expressing scFv that binds to both recombinant CD19-Fc and CD19 expressed on the cell surface of CHO-K1 cells, cell panning was performed with CHO-K1 transfected with CD19 cells. In the first round of panning on the cell surface, 2 days before panning, CHO-K1-CD19 cells were seeded in a 6-well plate and grown in F12K medium to 50% confluence. Then, 5×10 7The individual yeast cells were washed twice with PBSA buffer, resuspended in 3 mL of F12K medium, and then gently added dropwise to CHOK1-CD19 cells. After gently 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 yeast cells bound to CHOK1-CD19 cells from the plate. Next, the cell mix containing both yeast cells and CHOK1 cells was inoculated into 10 mL of SDCAA medium and amplified overnight at 30 °C, and then induced at 30 °C for 16 hours in SGCAA medium. In the second round of cell panning, the same protocol as above was performed, but more stringent washing conditions were used. By this panning method, the ml9217 binder was obtained. Further characterization of this binder and other binders obtained from phage display suggested that affinity maturation was necessary since the biological properties of the CAR generated from this hit were not yet optimal.

[0275] To enhance the affinity of ml9217, a yeast display ml9217 mutant scFv library was generated by using error-prone PCR, and random point mutations were introduced into the scFv gene sequence. After electroporation, the resulting mutant library was cultured overnight in SDCAA medium at 30 °C for 16 h and then switched to SGCAA medium at 30 °C for an additional 16 h. The mutant library was then sorted using MACS (immunomagnetic column, Miltenyi Biotec) with CD19-Fc as the capture antigen to downsize the library and increase the population of mutants that could bind to CD19-Fc. The pool was then double-stained with Anti-c-Myc-Alexa 488 and CD19-Fc / Anti-Hu-Fc, and the strongest binders were selected by choosing binders with the highest binding affinity similar to the c-Myc expression level. This process was then repeated two more times until the average binding affinity of the mutant pool with increased fluorescence-labeled antigen used in flow cytometry of yeast particles was generated compared to the starting construct. The binding affinity was estimated by flow cytometry of yeast pools using a reduced amount of labeled CD19. This process increased the affinity from an EC50 (effective concentration of 50% binding of labeled CD19 in yeast displaying ScFv) of 0.5 μg / ml for M19217 to an affinity < 0.01 μg / ml for affinity-matured binders (M19217-1, 19217-2, M19217-7, M19217-23, M19217-29, M19217-38, M19217-40).

[0276] Results: Due to the inherent problems of the CD19 structure, phage display candidates did not result in biologically functional CAR constructs, so the identification of ScFvs that yield biologically active binders was generated by yeast display. Based on flow cytometry analysis of 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 is 0.5 ug / ml), as well as the following affinity matured binders (EC50 < 0.01 ug / 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 receptors expressing the LTG2050, LTG2065, LTG2066, LTG2067, LTG2068, LTG2069, LTG2070, and LTG2071 human anti-CD19 binders is outlined in Example 2 below.

[0277] Example 2 CAR expressing anti-CD19 fully human binding sequences Homo sapiens CD19 (B4, CVID3, Leu-12) is a well-studied cell surface glycoprotein that is expressed in B-cell leukemia and lymphoma. At least two antibody-drug conjugates (SGN-CD19A, denintuzumab mafodotin (Seattle Genetics) and SAR3419, coltuximab ravtansine) are being evaluated in phase II clinical trials. In the phase 1 trial of SGN-19A, 35% CRc was shown in the optimal treatment group (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, due to the unexpected phase II results of SAR3419, development is 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 bispecific anti-CD3 / anti-CD19 antibody (blinatumomab) was discussed above. Considering the current status of progress of T cell-based therapies using CD19 CAR, the best approach is surely cell-based immunotherapy, and the CAR constructs shown herein are an innovative new approach for creating and implementing 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 in primary human T cells and possess specific and potent cytotoxic and cytokine functions against CD19-positive tumor cells. The CD19 CAR was designed using a CD19-binding sequence derived from ScFv candidates identified by phage display as in Example 1 and cloned into a lentiviral expression vector containing the structural and signal domains selected under the control of the EF1a promoter for characterization. In vitro, transduction efficiency, killing function, and cytokine production were tested in both model cell lines and primary human T cells. The terms used in Table 1 are outlined. The CAR construct LTG#1538 is a relevant object as the sequence from this mouse is the current binder used in commercial development (see KTE-C19, Kite Pharma, and CTL019, Novartis).

[0279]

Table 1

[0280] Materials and Methods: (a) Cell Lines The Burkitt lymphoma cell line Raji and the chronic myelogenous 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 Scientifi c. They were grown in Grand Island, NY. Single cell clones of the luciferase-expressing cell line were generated by stably transducing a lentiviral vector encoding firefly luciferase (Lentigen Technology, Inc., Gaithersburg, MD) into wild-type tumor strains, followed by cloning and selecting luciferase-positive clones.

[0281] (b) Generation of chimeric antigen receptor (CAR) - expression vector The antigen-binding domain of the CAR, ScFv, sequence was derived from a human anti-CD19 ScFv fragment. The binder sequence was ligated in-frame to the CD8a linker and transmembrane domain (UniProt sequence ID P01732, aa 138 - 206), then to the 4-1BB (CD137, aa214 - 255, UniProt sequence ID Q07011) signaling domain and the CD3 zeta signaling domain (CD247, aa52 - 163, Ref sequence ID: NP_000725.1) to generate the CAR T construct. The CAR construct sequence was cloned into a third-generation lentiviral plasmid backbone (Lentigen Technology Inc., Gaithersburg, MD). Supernatants containing the lentiviral vector (LV) were generated by transient transfection of HEK293T cells, and the vector was pelleted by centrifugation of the supernatant containing the lentiviral vector and stored at -80 °C.

[0282] (c) Primary T cell purification and transduction Human primary T cells from healthy volunteers were purified from whole blood or buffy coats (purchased from commercial providers with donor written consent) using immunomagnetic bead selection of CD4 + and CD8 + cells according to the manufacturer's protocol (Miltenyi Biotec, Bergisch Gladbach). The T cells were at a density of 0.3 - 2×10 6Cultured in TexMACS medium supplemented with 200 IU / ml IL-2 at cells / ml, activated with CD3 / CD28 MACS® GMP T Cell TransAct reagent (Miltenyi Biotec), and on day 2, transduced with a lentiviral vector encoding the CAR construct overnight in the presence of 10 μg / ml protamine sulfate (Sigma-Aldrich, St. Louis, MO), and the medium was changed on day 3. 200 IU / ml The cultures were propagated in TexMACS medium supplemented with 200 IU / ml 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). The assay range was determined using target-only wells (maximum CPS) and target-only wells with 1% Tween-20 added (minimum CPS). The percent specific lysis was calculated as (1 - (sample CPS - minimum CPS) / (maximum CPS - minimum CPS)). Supernatants were removed from co-cultures with an E:T ratio of 10:1 and analyzed by ELISA (eBioscience, San Diego, CA) for IFNγ, TNFα, and IL-2 concentrations.

[0284] (e) Flow cytometry analysis. For cell staining, 500,000 CAR T-transduced cells were harvested from the cultures and washed twice with cold AutoMACS buffer supplemented with 0.5% bovine serum albumin (Miltenyi Biotec), then 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. Negative control Non-transduced cells were used as controls. In all studies, dead cells were excluded by 7AAD staining (BD Biosciences, San Jose, CA). Cells were washed twice and resuspended in 200 μl of staining buffer prior to quantitative analysis by flow cytometry. Flow cytometry analysis was performed using a MACSQuant® 10 Analyzer (Miltenyi Biotec), and data plots were generated using FlowJo software (Ashland, OR).

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

[0286]

Table 2

[0287] T cells transduced with anti-CD19 chimeric antigen receptors demonstrated 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 was generated under the control of the human EF1a promoter as described in the Materials and Methods. Next, human primary T cells derived from healthy donors were transduced with the lentiviral vector encoding the CAR. Non-transduced cells (NT) or GFP-transduced cells derived from the same donor served as negative controls. The 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 the Materials and Methods. On days 8 - 10 of culture, the expression of anti-CD19 CAR on the T cell surface was detected with CD19-Fc peptide followed by anti-Fc-AF647 and analyzed by flow cytometry. The selected anti-CD19 CAR constructs each demonstrated surface CAR expression. When non-transduced T cells were used as the 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) Cytotoxicity assay and cytokine assay of anti-CD19 CAR To demonstrate the cytolytic function of the generated CAR T cells, in an overnight cell killing assay described in the materials and methods (Figure 4), CAR-T was combined with CD19-positive Raji-luc cells, CD19-positive Reh-luc cells, CD19-negative K562-luc, or CD19-negative 293T-luc cells at an E:T ratio of 40:1, 20:1, or 10:1. The selected constructs (LTG2050, LTG2065 - 2071) showed dose-dependent CD19-specific tumor killing. Next, the inventors measured the concentrations of the inflammatory cytokines IFN-γ, TNF-α, and IL-2 secreted by CAR T cells transduced with the CAR19 construct when challenged with CD19-positive cell tumor cells (Figure 5). To test the basal level of cytokine production, a control of CAR T cells only was included in each construct. The levels of TNF-α, IFN-γ, and IL-2 were strongly induced by T cells exposed to Raji cells. Furthermore, none of the constructs showed cytokine production above baseline in the absence of tumor cell targets. Thus, the CAR T constructs LTG2050, LTG2065 - 2071 are specific for CD19 + tumor lines. Interestingly, construct LTG2050 showed low levels of IL-2 when detected by ELISA, despite efficiently killing CD19-positive cell lines in vitro. Thus, some binders are active in soluble IgG or ScFv form and are suitable for expression on the surface of T cells in CAR T form, but are inefficient in cytokine killing or production when co-cultured with CD19 + positive tumors, so the design of the CAR and the selection of binders are not straightforward. Binders that were not expressed on the surface of T cells in the inventors' CAR expression vectors, or that did not show strong lytic activity against CD19-positive cell lines, were not included here but were frequently identified during screening. -

[0291] ​In summary, the high functionality of the novel fully human anti-CD19 CAR constructs LTG2050, LTG2065, LTG2066, LTG2067, LTG2068, LTG2069, LTG2070, and LTG2071 was demonstrated (see Table 1 below). All CAR constructs except LTG2050 are superior to the mouse FMC63 (LTG1538), which is a positive control, and are thus expected to have more potent therapeutic activity.

[0292] Each application and patent cited herein, and each document or reference cited in each application and patent (including each issued patent, “application cited documents,” during prosecution), and each PCT and foreign application or patent corresponding to and / or claiming priority from any of these applications and patents, and each document cited or referenced in each of the application cited documents, are hereby expressly incorporated herein by reference and may be used in practicing the present invention. More generally, the documents or references are cited anywhere in the text, in the list of references preceding the claims, or in the text itself, and each of these documents or references (“documents cited herein by reference”), and each document or reference cited in each of the documents cited herein by reference (including any manufacturer's specifications, instructions, etc.), are hereby expressly incorporated herein by reference.

[0293] The foregoing description of some specific embodiments provides sufficient information for others to make various adaptations and modifications, such as to specific embodiments, without departing from the general concept by applying the knowledge of the present invention, and thus such adaptations and modifications should be understood to be within the meaning and scope of equivalents of the disclosed embodiments. It is intended to be construed. It is understood that the terms or predicates used in this specification are for the purpose of description rather than limitation. In the drawings and the description, exemplary embodiments are disclosed, and although specific terms may have been used, unless otherwise stated, they are used only in a general and descriptive sense and not for purposes of limitation, and thus the claims are not so limited. Further, those skilled in the art will understand that certain steps of the methods discussed herein can be arranged in a different order or combined. Accordingly, the appended claims are not intended to be limited to the detailed embodiments disclosed herein. Those skilled in the art can understand and grasp many equivalents of the embodiments of the invention described herein using only ordinary experimentation. Such equivalents are encompassed by the following claims.

[0294] The arrangements of the present disclosure The nucleic acid and amino acid sequences listed below are shown using standard abbreviations for nucleotide bases and the 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 to be included by reference to the strand shown. 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). EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSDRGITATDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSQSVLTQPPSVSVAPGQTAKITCGGSDIGNKNVHWYQQKPGQAPVLVVYDDYDRPSGIPERFSGSNSGDAATLTISTVEVGDEADYFCQVWDSSGDPYWVFGGGTQLTVLG SEQ ID NO: 3 is the nucleotide sequence of the CD19-reactive ScFv2-binding domain (LTG2065). GAGGTCCAGCTGGTACAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCAGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATTAATCAACCCTAGTGGTGGTAGCACAAGCTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGGATCGGGGAATTACCGCCACGGACGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAGGCGGAGGAGGCTCCGGGGGAGGAGGTTCCGGGGGCGGGGGTTCCCAGTCTGTGCTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGGCGGATGGCCAAGATTACCTGTGGGGGAAGTGACATTGGAAATAAAAATGTCCACTGGTATCAGCAGAAGCCAGGCCAGGCCCCTGTCCTGGTTGTCTATGATGATTACGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGGACGCGGCCACCCTGACGATCAGCACGGTCGAAGTCGGGGATGAGGCCGACTATTTCTGTCAGGTGTGGGACGGTAGTGGTGATCCTTATTGGATGTTCGGCGGAGGGACCCAGCTCACCGTTTTAGGT SEQ ID NO: 4 is the amino acid sequence of the CD19-reactive ScFv2 binding domain (LTG2065). EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGLINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSDRGITATDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSQSVLTQPPSVSVAPGRMAKITCG GSDIGNKNVHWYQQKPGQAPVLVVYDDYDRPSGIPERFSGSNSGDAATLTISTVEVGDEADYFCQVWDGSGDPYWMFGGGTQLTVLG SEQ ID NO: 5 is the nucleotide sequence of the CD19-reactive ScFv3-binding domain (LTG2066). GAGGTCCAGCTGGTACAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCCGGATACACCTTCACCAGCTACTACATGCACTGGGTGCGACAGGCCCCTGGACAAGGGTTTGAGTGGATGGGATTAATCAACCCTAGTGGTAGTAGCACAAGCTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAACCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGGATCGGGGAATTACCGCCACGGACGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAGGCGGAGGAGGCTCCGGGGGAGGAGGTTCCGGGGGCGGGGGTTCCCAGTCTGTGCTGACTCAGCCACCCTCGGTGCCAGTGGCCCCAGGGCAGACGGCCAAGATTATCTGTGGGGGAAGTGACATTGGAAATAAAAATGTCCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTCCTGGTCGTCTATGATGACTACGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGGACGCGGCCACCCTGACGATCAGCACGGTTGAAGTCGGGGATGAGGCCGACTATTTCTGTCAGGTGTGGGACGGTAGTGGTGATCCTTATTGGGTGTTCGGCGGAGGGACCCAGCTCACCGTCTTAGGT SEQ ID NO: 6 is the amino acid sequence of the CD19-reactive ScFv3-binding domain (LTG2066). EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGFEWMGLINPSGSSTSYAQKFQGRVTMTRDTSTSTVYMELSNLRSEDTAVYYCARSDRGITATDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSQSVLTQPPSVPVAPGQTAKIICGGSDIGNKNVHWYQQKPGQAPVLVVYDDYDRPSGIPERFSGSNSGDAATLTISTVEVGDEADYFCQVWDGSGDPYWVFGGGTQLTVLG SEQ ID NO: 7 is the nucleotide sequence of the CD19-reactive ScFv4-binding domain (LTG2067). GAGGTCCAGCTGGTACAGTCTGGAGCTGAGGTGAACAAGCCTGGTGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCAGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATGATCAACCCTAGTGGTGGTAGCACAAGCTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGGATCGGGGAATTACCTCCACGGACGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAGGCGGAGGAGGCTCCGGGGGAGGAGGTTCCGGGGGCGGGGGTTCCCAGTCTGTGCTGACTCAGCCACCCTCGGTGTCATTGGCCCCAGGGCAGACGGCCAAGATTACCTGTGGGGGAAGTGACATTGGAAATAAAAATGTCCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTCCTAGTCGTCTATGATGATTACAACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGGACTCAGCCACCCTGACGATCAGCACGGTCGAAGTCGGGGATGAGGCCGACTATTTCTGTCAGGTGTGGGACGGTAGTGGTGATCCTTATTGGGTGTTCGGCGGAGGGACCCAGCTCACCGTTTTAGGT SEQ ID NO: 8 is the amino acid sequence of the CD19-reactive ScFv4 binding domain (LTG2067). EVQLVQSGAEVNKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGMINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSDRGITSTDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSQSVLTQPPSVSLAPGQTAKITCGGSDIGNKNVHWYQQKPGQAPVLVVYDDYNRPSGIPERFSGSNSGDSATLTISTVEVGDEADYFCQVWDGSGDPYWVFGGGTQLTVLG SEQ ID NO: 9 is the nucleotide sequence of the CD19-reactive ScFv5-binding domain (LTG2068). GAGGTCCAGCTGGTACAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCATCTGGATACACCTTCACCGGCTACTATATGCACTGGGTGCGGCAGGCCCCTGGACAAGGGCTTGAGTGGATAGGATTAATCAACCCTAGTGGTGGTAGCACAAGCTACGAACAGAAGTTCCAGGGCAGAGTCGCCATGACCAGGGACACGTCAACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGGATCGGGGAATTACCGCCACGGACGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAGGCGGAGGAGGCTCCGGGGGAGGAGGTTCCGGGGGCGGGGGTTCCCAGTCTGTGCTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGGCAGACGGCCAAGATTACCTGTGGGGGAAGTGACATTGGAGATAAAAATGTCCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTCCTGGTCGTCTATGATGATTACGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGGACGCGGCCACCCTGACGATCAGCACGGTCGAAGTCGGGGATGAGGCCGACTATTTCTGTCAGGTGTGGGACGGTATTGGTGATCCCTATTGGGTGTTCGGCGGAGGGACCCAGCTCACCGTTTTAGGT SEQ ID NO: 10 is the amino acid sequence of the CD19-reactive ScFv5 binding domain (LTG2068). EVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWIGLINPSGGSTSYEQKFQGRVAMTRDTSTSTVYMELSSLRSEDTAVYYCARSDRGITATDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSQSVLTQPPSVSVAPGQTAKITCGGSDIGDKNVHWYQQKPGQAPVLVVYDDYDRPSGIPERFSGSNSGDAATLTISTVEVGDEADYFCQVWDGIGDPYWVFGGGTQLTVLG Sequence number 11 is the nucleotide sequence of the CD19-reactive ScFv6-binding domain (LTG2069). GAGGTCCAGCTAGTACAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCAGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATGATCAACCCTAGTGGTGGTAGCACAAGCTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGGATCGGGGAATTACCGCCACGGACGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAGGCGGAGGAGGCTCCGGGGGAGGAGGTTCCGGGGGCGGGGGTTCCCAGTCTGTGCTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGGCAGACGGCCAAGATTACCTGTGGGGGAAGTGACATTGGAAATAAAAATGCCCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTCCTGGTCGTCTATGATGATTACGACCGGCCCTCAGGGATCTCTGAGCGATTCTCTGGCTCCAACTCTGGGGACGCGGCCACCCTGACGATCAGCACGGTCGAAGTCGGGGATGAGGCCGACTATTTCTGTCAGGTGTGGGACGGTAGTGGTGATCCTTTTTGGGTGTTCGGCGGAGGGACCCAGCTCACCGTTTTAGGT Accession number 12 is the amino acid sequence of the CD19-reactive ScFv6 binding domain (LTG2069). EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGMINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSDRGITATDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSQSVLTQPPSVSVAPGQTAKITCGGSDIGNKNAHWYQQKPGQAPVLVVYDDYDRPSGISERFSGSNSGDAATLTISTVEVGDEADYFCQVWDGSGDPFWVFGGGTQLTVLG Sequence number 13 is the nucleotide sequence of the CD19-reactive ScFv7 binding domain (LTG2070). GAGGTCCAGCTGGTACAGTCTGGAGCTGAGGTGAAGAGGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCAGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACGAGGGCTTGAGTGGATGGGATTAATCAACCCTAGTGGTGGTAGCACAAGCTACGCACAGGAGTTCCAGGGCAGAGTCACCATGACCAGGGACATGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGGATCGGGGAATTAGCGCCACGGACGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAGGCGGAGGAGGCTCCGGGGGAGGAGGTTCCGGGGGCGGGGGTTCCCAGTCTGTGCTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGGCAGATGGCCAAGATTACCTGTGGGGGAAGTGACATTGGAAATAAAAATGTCCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTCCTGGTCGTCTATGATGATTACAACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGGACGCGGCCACCCTGACGATCAGCACGGTCGAAGTCGGGGATGAGGCCGACTATTTCTGTCAGGTATGGGACGGTAGTGGTGATCCTTATTGGGTGTTCGGCGGAGGGACCCAGCTCACCGATTTAGGT SEQ ID NO: 14 is the amino acid sequence of the CD19-reactive ScFv7 binding domain (LTG2070). EVQLVQSGAEVKRPGASVKVSCKASGYTFTSYYMHWVRQAPGRGLEWMGLINPSGGSTSYAQEFQGRVTMTRDMSTSTVYMELSSLRSEDTAVYYCARSDRGISATDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSQSVLTQPPSVSVAPGQMAKITCGGSDIGNKNVHWYQQKPGQAPVLVVYDDYNRPSGIPERFSGSNSGDAATLTISTVEVGDEADYFCQVWDGSGDPYWVFGGGTQLTDLG Sequence number 15 is the nucleotide sequence of the CD19-reactive ScFv8-binding domain (LTG2071). GAGGTCCAGCTGGTACAGTCTGGAGCTGAGGTGAAGAGGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCAGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGTGTGGATGGGATTAATCAACCCTAGTGGTGGCAGCACAAGCTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGGATCGGGGAATTACCGCCACGGACGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAGGCGGAGGAGGCTCCGGGGGAGGAGGTTCCGGGGGCGGGGGTTCCCAGTCTGTGCTGACTCAGCCACCCTCGGTCTCAGTGGCCCCAGGGCAGACGGCCAAGACTACCTGTGGG GGAAGTGACATTGGAAATAAAAATGTCCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTCCTGGTCGTCTATGATGATTACGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGGACGCGGCCACCCTGACGATCAGCACGGTCGAAGTCGGGGATGAGGCCGACTATGTCTGTCAGGTGTGGGACGGTAGTGGTGATCCTTATTGGGTGTTCGGCGGAGGGACCCAGCTCACCGTTTTAGGT SEQ ID NO:16 is the amino acid sequence of the CD19-reactive ScFv8-binding domain (LTC2071). EVQLVQSGAEVKRPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLVWMGLINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSDRGITATDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSQSVLTQPPSVSVAPGQTAKTTCGGSDIGNKNVHWYQQKPGQAPVLVVYDDYDRPSGIPERFSGSNSGDAATLTISTVEVGDEADYVCQVWDGSGDPYWVFGGGTQLTVLG

[0295] SEQ ID NO:17 is the nucleotide sequence of the leader / signal peptide sequence (LP). atgctgctgctggtgaccagcctgctgctgtgcgaactgccgcatccggcgtttctgctgattccg

[0296] SEQ ID NO:18 is the amino acid sequence of the leader / signal peptide sequence (LP). 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). MLLLVTSLLLCELPHPAFLLIPEVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSDRGITATDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSQSVLTQPPSVSVAPGQTAKITCGGSDIGNKNVHWYQQKPGQAPVLVVYDDYDRPSGIPERFSGSNSGDAATLTISTVEVGDEADYFCQVWDSSGDPYWVFGGGTQLTVLGAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 21 is the nucleotide sequence of LTG2065 (LP-M19217-1-CD8 TM-41BB-CD3 zeta). ATGCTGCTGCTGGTGACCAGCCTGCTGCTGTGCGAACTGCCGCATCCGGCGTTTCTGCTGATTCCGGAGGTCCAGCTGGT Sequence number 22 is the amino acid sequence of LTG2065_ (LP-M19217-1-CD8 TM-41BB-CD3 zeta). MLLLVTSLLLCELPHPAFLLIPEVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGLINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSDRGITATDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSQSVLTQPPSVSVAPGRMAKITCGGSDIGNKNVHWYQQKPGQAPVLVVYDDYDRPSGIPERFSGSNSGDAATLTISTVEVGDEADYFCQVWDGSGDPYWMFGGGTQLTVLGAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Sequence number 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). MLLLVTSLLLCELPHPAFLLIPEVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGFEWMGLINPSGSSTSYAQKFQGRVTMTRDTSTSTVYMELSNLRSEDTAVYYCARSDRGITATDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSQSVLTQPPSVPVAPGQTAKIICGGSDIGNKNVHWYQQKPGQAPVLVVYDDYDRPSGIPERFSGSNSGDAATLTISTVEVGDEADYFCQVWDGSGDPYWVFGGGTQLTVLGAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 25 is the nucleotide sequence of LTG2067_(LP-M19217-7-CD8 TM-41BB-CD3 zeta). Sequence number 26 is the amino acid sequence of LTG2067_ (LP-M19217-7-CD8 TM-41BB-CD3 zeta). MLLLVTSLLLCELPHPAFLLIPEVQLVQSGAEVNKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGMINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSDRGITSTDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSQSVLTQPPSVSLAPGQTAKITCGGSDIGNKNVHWYQQKPGQAPVLVVYDDYNRPSGIPERFSGSNSGDSATLTISTVEVGDEADYFCQVWDGSGDPYWVFGGGTQLTVLGAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Sequence number 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). MLLLVTSLLLCELPHPAFLLIPEVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWIGLINPSGGSTSYEQKFQGRVAMTRDTSTSTVYMELSSLRSEDTAVYYCARSDRGITATDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSQSVLTQPPSVSVAPGQTAKITCGGSDIGDKNVHWYQQKPGQAPVLVVYDDYDRPSGIPERFSGSNSGDAATLTISTVEVGDEADYFCQVWDGIGDPYWVFGGGTQLTVLGAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 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). MLLLVTSLLLCELPHPAFLLIPEVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGMINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSDRGITATDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSQSVLTQPPSVSVAPGQTAKITCGGSDIGNKNAHWYQQKPGQAPVLVVYDDYDRPSGISERFSGSNSGDAATLTISTVEVGDEADYFCQVWDGSGDPFWVFGGGTQLTVLGAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 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). MLLLVTSLLLCELPHPAFLLIPEVQLVQSGAEVKRPGASVKVSCKASGYTFTSYYMHWVRQAPGRGLEWMGLINPSGGSTSYAQEFQGRVTMTRDMSTSTVYMELSSLRSEDTAVYYCARSDRGISATDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSQSVLTQPPSVSVAPGQMAKITCGGSDIGNKNVHWYQQKPGQAPVLVVYDDYNRPSGIPERFSGSNSGDAATLTISTVEVGDEADYFCQVWDGSGDPYWVFGGGTQLTDLGAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 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). MLLLVTSLLLCELPHPAFLLIPEVQLVQSGAEVKRPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLVWMGLINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSDRGITATDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSQSVLTQPPSVSVAPGQTAKTTCGGSDIGNKNVHWYQQKPGQAPVLVVYDDYDRPSGIPERFSGSNSGDAATLTISTVEVGDEADYVCQVWDGSGDPYWVFGGGTQLTVLGAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0297] SEQ ID NO: 35 is the nucleotide sequence of the DNA CD8 transmembrane domain. atttgggccccgctggccggcacttgcggcgtgctcctgctgtcgctggtcatcaccctt tactgc SEQ ID NO: 36 is the amino acid sequence of the CD8 transmembrane domain. IWAPLAGTCGVLLLSLVITLYC

[0298] SEQ ID NO: 37 is the nucleotide sequence of the DNA CD8 hinge domain. 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 the hinge and transmembrane regions of amino acids 137 to 206 of CD8.alpha (NCBI RefSeq: NP_001759.3). TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC

[0299] SEQ ID NO: 40 is the nucleotide sequence of the DNA signaling domain of 4-1BB. aagaggggccggaagaagctgctttacatcttcaagcagccgttcatgcggcccgtgcag acgactcaggaagaggacggatgctcgtgcagattccctgaggaggaagaggggggatgc gaactg SEQ ID NO: 41 is the amino acid sequence of the signaling domain of 4-1BB. KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0300] SEQ ID NO: 42 is the nucleotide sequence of the intracellular signaling domain of CD3-zeta. cgcgtcaagttctcacggtccgccgacgcccccgcatatcaacagggccagaatcagctc tacaacgagctgaacctgggaaggagagaggagtacgacgtgctggacaagcgacgcgga cgcgacccggagatgggggggaaaccacggcggaaaaaccctcaggaaggactgtacaac gaactccagaaagacaagatggcggaagcctactcagaaatcgggatgaagggagagcgg aggaggggaaagggtcacgacgggctgtaccagggactgagcaccgccactaaggatacc tacgatgccttgcatatgcaagcactcccaccccgg SEQ ID NO: 43 is the amino acid sequence of CD3-zeta. RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0301] SEQ ID NO: 44 is the nucleotide sequence of the intracellular signaling domain of a CD3-zeta mutant. cgcgtcaagttctcacggtccgccgacgcccccgcatataaacagggccagaatcagctc tacaacgagctgaacctgggaaggagagaggagtacgacgtgctggacaagcgacgcgga cgcgacccggagatgggggggaaaccacggcggaaaaaccctcaggaaggactgtacaac gaactccagaaagacaagatggcggaagcctactcagaaatcgggatgaagggagagcgg aggaggggaaagggtcacgacgggctgtaccagggactgagcaccgccactaaggatacc tacgatgccttgcatatgcaagcactcccaccccgg SEQ ID NO: 45 is the amino acid sequence of a CD3-zeta signal transduction domain mutant. RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0302] SEQ ID NO: 46 is the nucleotide sequence of ScFv CD19 (FMC63). 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). DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS SEQ ID NO: 48 is the nucleotide sequence of anti-CD33 ScFv (LTG1936). CAGGTGCAGCTGGTGCAATCTGGGGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAGGATCTCCTGTAAGGGTTCTGGATTCAGTTTTCCCACCTACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGACTAGTTGGAGATGGCTACAATACGGGGGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAGGAGGTGGCGGGTCTGGTGGTGGCGGTAGCGGTGGTGGCGGATCCGATATTGTGATGACCCACACTCCACTCTCTCTGTCCGTCACCCCTGGACAGCCGGCCTCCATCTCCTGCAAGTCTAGTCAGAGCCTCCTGCATAGTAATGGAAAGACCTATTTGTATTGGTACCTGCAGAAGCCAGGCCAGCCTCCACAGCTCCTGATCTATGGAGCTTCCAACCGGTTCTCTGGAGTGCCAGACAGGTTCAGTGGCAGCGGGTCAGGGACAGATTTCACACTGAAAATCAGCCGGGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAAGTATACAGCTTCCTATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA Sequence number 49 is the amino acid sequence of anti-CD33 ScFv (LTG1936). QVQLVQSGAEVKKPGESLRISCKGSGFSFPTYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARLVGDGYNTGAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIVMTHTPLSLSVTPGQPASISCKSSQSLLHSNGKTYLYWYLQKPGQPPQLLIYGASNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQSIQLPITFGQGTRLEIK SEQ ID NO: 50 is the nucleotide sequence of anti-mesothelin ScFv (LTG1904). GAGGTCCAGCTGGTACAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTAGTTGGAATAGTGGTAGCATAGGCTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGATTTATCGTCAGTGGCTGGACCCTTTAACTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGGAGGTGGCGGGTCTGGTGGAGGCGGTAGCGGCGGTGGCG GATCCTCTTCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAGGATCACATGCCAAGGAGACAGCCTCAGAAGCTATTATGCAAGCTGGTACCAGCAGAAGCCAGGACAGGCCCCTGTACTTGTCATCTATGGTAAAAACAACCGGCCCTCAGGGATCCCAGACCGATTCTCTGGCTCCAGCTCAGGAAACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAGGATGAGGCTGACTATTACTGTAACTCCCGGGACAGCAGTGGTAACCATCTGGTATTCGGCGGAGGCACCCAGCTGACCGTCCTCGGT SEQ ID NO: 51 is the amino acid sequence of anti-mesothelin ScFv (LTG1904). EVQLVQSGGGLVQPGGSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDLSSVAGPFNYWGQGTLVTVSSGGGGSGGGGSGGGGSSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHLVFGGGTQLTVLG

Claims

**Claim 1** A method for generating a population of RNA-engineered cells, comprising the step of introducing in vitro transcribed RNA or synthetic RNA into cells in vitro, wherein the RNA comprises an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR) consisting of the amino acid sequence of SEQ ID NO: 22, 24, 26, 28, 30, 32 or 34. **Claim 2** Use of isolated cells for the manufacture of a pharmaceutical composition for effecting anti-tumor immunity in a mammal, wherein the isolated cells comprise a vector comprising a nucleic acid molecule encoding a chimeric antigen receptor (CAR) consisting of the amino acid sequence of SEQ ID NO: 22, 24, 26, 28, 30, 32 or 34. **Claim 3** Use of a CAR for the manufacture of a pharmaceutical composition for treating or preventing cancer in a mammal, wherein the CAR consists of the amino acid sequence of SEQ ID NO: 22, 24, 26, 28, 30, 32 or 34.

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