Compositions and methods for treating cancer with anti-CD123 immunotherapy

Abstract

Disclosed is a chimeric antigen receptor that comprises CD123 antigen binding domain.Also disclosed are the nucleic acid, recombinant expression vector, host cell, antigen binding fragment and pharmaceutical composition related to the chimeric antigen receptor.Also disclosed are a method for treating or preventing cancer in a subject, and a method for producing chimeric antigen receptor T cells.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Utility Application No. 17 / 685,132, filed March 2, 2022, and U.S. Utility Application No. 18 / 154,209, filed January 13, 2023, the entire contents of each of which are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically as an XML file entitled Sequence_Listing. The XML file, created on Feb. 28, 2023, is 114,458 bytes in size. The contents of the XML file are incorporated herein by reference in their entirety.

[0003] Field of the Disclosure The present application relates to the field of cancer, in particular to CD123 antigen-binding domains and chimeric antigen receptors (CARs) comprising such CD123 antigen-binding domains, and methods of use thereof. [Background technology]

[0004] background Cancer is one of the most deadly threats to human health. In the United States alone, cancer affects nearly 1.3 million new cases each year, making it the second leading cause of death after cardiovascular disease, accounting for about one-quarter of all deaths. Solid tumors are responsible for most of these deaths. Although there have been significant advances in the medical treatment of certain cancers, the overall 5-year survival rate for all cancers has improved by only about 10% over the past 20 years. Cancer, or malignant tumors, metastasize and grow rapidly and uncontrolled, making them very difficult to treat.

[0005] AML is a devastating disease with an overall survival rate of only 26%. Younger patients tend to have a good prognosis for AML treatment, but older patients have a poor 5-year survival rate, perhaps only around 5%. First-line treatment for AML involves multiple chemotherapy regimens (i.e. induction, consolidation) that carry a high risk of toxicity. If hematopoietic stem cell transplantation is performed after first remission, the 5-year disease-free survival rate is only 30-50% (http: / / www.cancer.ca / en / cancer-information / cancer-type / leukemia-acute-myelogenous-aml / prognosis-and-survival / survival-statistics / ?region=on). Furthermore, AML patients with high disease burden may not be candidates for bone marrow transplantation, and minimal residual disease before transplantation has been correlated with AML relapse. Current first-line induction / consolidation therapy often fails to achieve MDR-negative remission to sufficiently reduce tumor burden, so the risk of AML relapse after first-line therapy with or without BMT remains high. (1) Biol Blood Marrow Transplant. 2006 June; Vol. 12(6): 691-2. Leukemia burden and outcome of allogeneic transplant in acute myelogenous leukemia., Kamble RT, Hjortsvang E, Selby GB; (2) Leuk Lymphoma. 2015 May; Vol. 56(5): 1353-61. Impact of pre-transplant disease burden on the outcome of allogeneic hematopoietic stem cell transplant in refractory and relapsed acute myeloid leukemia: a single-center study. Tian H et al.). PBDCN is a rare myeloid neoplasm classified as a subtype of AML and may be treated as AML with induction and consolidation chemotherapy or as ALL. BMT is often administered during first remission.However, there are currently no ongoing clinical trials and no approved first-line treatments for PBDCN (Leukemia Lymphoma Society, https: / / www.lls.org / leukemia / blastic-plasmacytoid-dendritic-cell-neoplasm). Therefore, better therapeutic modalities for CD123+ malignancies are urgently needed.

[0006] The CD123-targeting CAR approach is superior to chemotherapy as it may achieve higher efficacy in eliminating CD123+ tumor cells and tumor stem cells and avoid the toxicity associated with chemotherapy. Importantly, CAR T cells are expected to be more efficient at eliminating minimal residual disease than chemotherapy, resulting in better long-term treatment outcomes. Furthermore, CAR123 may be used for tumor debulking as a bridge to transplantation, as it may help patients with high tumor burden qualify for BMT.

[0007] CAR123 represents an improvement over the prior art because a unique human ScFv (hScFv) sequence is used in the CAR design, unlike other CAR designs that use mouse-derived ScFvs, which carry the risk of immunogenicity and can induce allergic or anaphylactic reactions in patients, leading to CAR T removal or life-threatening anaphylaxis.

[0008] Chimeric antigen receptors (CARs) are hybrid molecules that contain three essential units: (1) an extracellular antigen-binding motif, (2) a linking / transmembrane motif, and (3) an intracellular T cell signaling motif (Long AH, Haso WM, Orentas RJ. Lessons learned from a highly-active CD22-specific chimeric antigen receptor. Oncoimmunology. 2013;2(4):e23621). The antigen-binding motif of CARs is generally based on the single-chain fragment variable (scFv), the smallest binding domain of an immunoglobulin (Ig) molecule. Alternative antigen-binding motifs have also been engineered, for example, receptor ligands (i.e., IL-13 has been engineered to bind to tumor-expressed IL-13 receptors), intact immune receptors, library-derived peptides, and innate immune system effector molecules (e.g., NKG2D). Alternative cellular targets for CAR expression (e.g., NK or gamma-delta T cells) are also under development (Brown CE et al. Clin Cancer Res. 2012;18(8):2199-209; Lehner M et al. PLoS One. 2012;7(2):e31210). Considerable effort is still required to define the most active T cell populations for transduction with CAR vectors, to determine optimal culture and expansion techniques, as well as to define the molecular details of the CAR protein structure itself.

[0009] The linking motif of the CAR can be a relatively stable structural domain, such as the constant domain of IgG, or can be designed to be an extended flexible linker. A structural motif, such as that derived from the constant domain of IgG, can be used to extend the scFv binding domain away from the cell membrane surface of the T cell. This may be important for some tumor targets where the binding domain is particularly close to the tumor cell surface membrane (e.g., for disialoganglioside GD2; Orentas et al., unpublished observations). To date, the signaling motif used in CARs has always included the CD3-zeta chain, since this core motif is a key signal for T cell activation. The first reported second generation CARs featured 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 linked to anti-CD3 and anti-CD28 antibodies, as well as the presence of the canonical "signal 2" derived from CD28, is no longer required to be encoded by the CAR itself. Using bead activation, third generation vectors were found to be no better than second generation vectors in in vitro assays and offered no clear benefit over second generation vectors in mouse models of leukemia (Haso W, Lee DW, Shah NN, Stetler-Stevenson M, Yuan CM, Pastan IH, Dimitrov DS, Morgan RA, FitzGerald DJ, Barrett DM, Wayne AS, Mackall CL, Orentas RJ. Anti-CD22-chimeric antigen receptors targeting B cell precursor acute lymphoblastic leukemia, Blood. 2013;121(7):1165-74; Kochenderfer JN et al. Blood. 2012;119(12):2709-20).This is supported by the clinical success of second generation CD28 / CD3-zeta (Lee DW et al. American Society of Hematology Annual Meeting. New Orleans, LA; Dec 7-10, 2013) and CD19-specific CARs that signal in the CD137 / CD3-zeta format (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 sustained signals in CAR-transduced T cells (Yvon E et al. Clin Cancer Res. 2009; 15(18): 5852-60). The culture conditions under which the CAR T cell population is cultivated are equally important.

[0010] T cell-based immunotherapy has become an emerging 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. Elimination of unwanted T cells via drug-induced dimerization of an inducible caspase 9 construct with AP1903 demonstrates one way in which a powerful switch that can control T cell populations can be initiated pharmacologically (Di Stasi A et al. N Engl J Med. 2011;365(18):1673-83). Creation of an effector T cell population immune to the negative regulatory effects of transforming growth factor-β by expression of a decoy receptor further demonstrates the extent to which effector T cells can be engineered for optimal antitumor activity (Foster AE et al. J Immunother. 2008;31(5):500-5). Thus, although CARs appear to be able to induce T cell activation in a manner similar to endogenous T cell receptors, the major obstacles to the clinical application of this technology to date are the limited in vivo expansion of CAR+T cells, the rapid disappearance of cells after infusion, and disappointing clinical activity.Therefore, there is an urgent and long-standing need in the art to discover novel compositions and methods for the treatment of AML using approaches that can show specific and effective anti-tumor effects without the above-mentioned drawbacks (i.e., high toxicity, poor efficacy).

[0011] Moreover, natural killer (NK) cell-based cancer immunotherapy has gained momentum in recent years (Shimasaki, N., Jain, A. & Campana, D. NK cells for cancer immunotherapy. Nat Rev Drug Discov 19, 200-218 (2020)). Human haploidentical NK cells have been shown to be suitable for adoptive transfer and expansion in pediatric and adult cancer patients (Miller, JS et al., Blood 105, 3051-3057 (2005); Rubnitz, JE et al., J. Clin. Oncol. 28, 955-959 (2010)). Moreover, second generation CD19-CAR NK cells generated ex vivo were effective in killing B-cell ALL (Imai, C., Iwamoto, S. & Campana, D. Blood 106, 376-383 (2005)). In vivo NK cell activity can be further enhanced by expression of IL-15, IL-12, IL-18, or other cytokine variants that stimulate autonomous growth, cytotoxicity, and long-term effector function (Imamura, M. et al., Blood 124, 1081-1088 (2014)); Ni, J., Miller, M., Stojanovic, A., Garbi, N. & Cerwenka, AJ Exp. Med. 209, 2351-2365 (2012). NK cells can attract dendritic cells to tumor sites by secreting chemotactic factors such as CCL5, XCL1 and FLT3L, thereby promoting a tumor microenvironment favorable for tumor control by the immune system (Bottcher, JP et al., Cell 172, 1022-1037 (2018); Barry, KC et al., A natural killer-dendritic cell axis defines checkpoint therapy-responsive tumor microenvironments. Nat. Med. 24, 1178-1191 (2018)).Furthermore, NK cells can be made more effective against tumors by manipulating culture conditions: for example, NK cell activation with the cytokines IL-12, IL-15 and IL-18 enhanced NK cell anti-AML responses (Romee, R. et al., Sci. Transl. Med. 8, 357ra123 (2016)).

[0012] Clinical generation of NK cells from umbilical cord blood hematopoietic progenitors is possible (Spanholtz, J. et al., PLOS ONE 6, e20740 (2011); Knorr, DA et al., Stem Cell Transl. Med. 2, 274-283 (2013)). Similarly, CAR NK cells can be derived by differentiation from iPSCs expressing CARs (Li, Y., Hermanson, DL, Moriarity, BS & Kaufman, DS Cell Stem Cell 23, 181-192 (2018)). Methods for generating highly effective NK and CAR NK cells for cancer therapy continue to evolve, and great progress has been made in this field in the past few years (Granzin, M. et al., Oncoimmunology 5, e1219007 (2016)). Collectively, these findings demonstrate the novel potential of CAR NK cells in cancer immunotherapy. Summary of the Invention [Problem to be solved by the invention]

[0013] 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 to treat diseases, disorders or conditions associated with dysregulation of CD123 expression, wherein the CARs contain a CD123 antigen-binding domain that exhibits high surface expression on transduced T cells and NK cells, high levels of cytolysis, and in vivo proliferation and persistence of transduced T cells. [Means for solving the problem]

[0014] overview Provided herein are novel anti-CD123 antibodies or their antigen-binding domains, and chimeric antigen receptors (CARs) that include such CD123 antigen-binding domains, and host cells (e.g., T cells) that express the receptor, and nucleic acid molecules that encode the receptor. CARs may consist of a single molecule expressed on the surface of effector cells, or may consist of an effector cell-expressed signaling module and a soluble targeting module, for example, when the soluble targeting module is combined with the cell-expressed signaling module, a fully functional CAR is formed. CARs show high surface expression on transduced T cells, high cell lysis, and in vivo proliferation and persistence of transduced T cells. Methods of using the disclosed CARs, host cells, and nucleic acid molecules, for example, for treating cancer in a subject, are also provided.

[0015] Thus, in one embodiment, an isolated polynucleotide encoding a human anti-CD123 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, 15, 17, 19, 23, 25, 69, 71, 77 and 87.

[0016] In one embodiment, an isolated polynucleotide is provided encoding a fully human anti-CD123 antibody or fragment thereof, wherein the antibody or fragment thereof comprises a fragment selected from the group consisting of a Fab fragment, a F(ab')2 fragment, an Fv fragment, and a single chain Fv (scFv).

[0017] In one embodiment, an isolated polynucleotide encoding a fully human anti-CD123 antibody or fragment thereof is provided, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 16, 18, 20, 22, 24, 26, 70, 72, 78 and 88.

[0018] In one embodiment, an isolated nucleic acid molecule is provided encoding a chimeric antigen receptor (CAR) comprising, from N-terminus to C-terminus, at least one CD123 antigen-binding domain, at least one transmembrane domain, and at least one intracellular signaling domain, encoded by a nucleotide sequence comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 15, 17, 19, 21, 23, 25, 69, 71, 77, and 87.

[0019] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded extracellular CD123 antigen-binding domain comprises at least a single chain variable fragment of an antibody that binds CD123.

[0020] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded extracellular CD123 antigen-binding domain comprises at least one heavy chain variable region of an antibody that binds CD123.

[0021] In one embodiment, the targeting domain of the CAR is expressed separately in the form of a monoclonal antibody, ScFv Fab, Fab'2 and comprises an antigen targeting domain comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 15, 17, 19, 21, 23, 25, 69, 71, 77 and 87 bound to an additional binding tag or epitope, while the effector cell expression component of the CAR comprises a binding domain specifically directed to bind to a tag or epitope expressed on the soluble CAR module, such as upon specific binding to the cell binding component of the CAR on the soluble component of the CAR to form a complete functional CAR structure.

[0022] In another embodiment, the targeting domain of the CAR is expressed separately in the form of a monoclonal antibody, ScFv Fab, Fab'2 and comprises an antigen targeting domain comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 15, 17, 19, 21, 23, 25, 69, 71, 77 and 87, and an additional ScFv, while the effector cell expression component of the CAR comprises a tag or epitope that specifically reacts with an additional ScFv expressed on the soluble CAR module, such as upon specific binding to the cell binding component of the CAR on the soluble component of the CAR to form a complete functional CAR structure.

[0023] In yet another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded CAR extracellular CD123 antigen-binding domain further comprises at least one lipocalin-based antigen-binding domain (anticalin) that binds to CD123.

[0024] In one embodiment, an isolated nucleic acid molecule is provided in which the encoded extracellular CD123 antigen-binding domain is connected to the transmembrane domain by a linker domain.

[0025] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded CD123 extracellular antigen-binding domain is preceded by a sequence encoding a leader or signal peptide.

[0026] In yet another embodiment, an isolated nucleic acid molecule is provided encoding a CAR comprising at least one CD123 antigen-binding domain encoded by a nucleotide sequence comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 15, 17, 19, 21, 23, 25, 69, 71, 77, and 87, wherein the CAR further encodes an extracellular antigen-binding domain that targets an antigen including, but not limited to, CD19, CD20, CD22, ROR1, mesothelin, CD33, CD38, CD138, BCMA (CD269), GPC2, GPC3, FGFR4, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, NY-ESO-1 TCR, MAGE A3 TCR, or any combination thereof.

[0027] In certain embodiments, the further encoded extracellular antigen binding domain is an anti-CD19 scFV antigen binding domain, an anti-CD20 scFV antigen binding domain, an anti-CD22 scFV antigen binding domain, an anti-ROR1 scFV antigen binding domain, an anti-mesothelin scFV antigen binding domain, an anti-CD33 scFV antigen binding domain, an anti-CD38 scFV antigen binding domain, an anti-CD123 (IL3RA) scFV antigen binding domain, an anti-CD138 scFV antigen binding domain, an anti-BCMA (CD269) scFV antigen binding domain, an anti-GPC2 scFV antigen binding domain, an anti-GPC3 scFV antigen binding domain, an anti-FGFR4 scFV antigen binding domain, an anti-c-Met scFV antigen binding domain, an anti-PMSA scFV antigen binding domain, an anti-glycolipidF77 scFV antigen binding domain, an anti-EGFRvIII scFV antigen binding domain, an anti-GD-2 Provided is an isolated nucleic acid molecule encoding a CAR comprising an scFV antigen-binding domain, an anti-NY-ESo-1 TCR scFV antigen-binding domain, an anti-MAGE A3 TCR scFV antigen-binding domain, or an amino acid sequence thereof having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, or any combination thereof.

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

[0029] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided in which an extracellular CD123 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 a transmembrane domain.

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

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

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

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

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

[0036] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, further comprising a leader sequence or signal peptide, wherein the nucleotide sequence of the leader or signal peptide comprises the nucleotide sequence of SEQ ID NO: 13, SEQ ID NO: 39, SEQ ID NO: 41, or SEQ ID NO: 43.

[0037] In yet another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded leader sequence comprises the amino acid sequence of SEQ ID NO:14, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:44.

[0038] In one aspect, provided herein is a chimeric antigen receptor (CAR) comprising, from N-terminus to C-terminus, at least one CD123 antigen-binding domain, at least one transmembrane domain, and at least one intracellular signaling domain.

[0039] In one embodiment, a CAR is provided, wherein the extracellular CD123 antigen-binding domain comprises at least one single chain variable fragment of an antibody that binds to the antigen, or at least one heavy chain variable region of an antibody that binds to the antigen, or a combination thereof.

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

[0041] In some embodiments, CARs are provided wherein the CAR further encodes an extracellular antigen-binding domain comprising CD19, CD20, CD22, ROR1, mesothelin, CD33, CD38, CD123 (IL3RA), CD138, BCMA (CD269), GPC2, GPC3, FGFR4, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, NY-ESO-1 TCR, MAGE A3 TCR, or an amino acid sequence thereof having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, or any combination thereof.

[0042] In one embodiment, the extracellular antigen binding domain is an anti-CD19 scFV antigen binding domain, an anti-CD20 scFV antigen binding domain, an anti-CD22 scFV antigen binding domain, an anti-ROR1 scFV antigen binding domain, an anti-mesothelin scFV antigen binding domain, an anti-CD33 scFV antigen binding domain, an anti-CD38 scFV antigen binding domain, an anti-CD123 (IL3RA) scFV antigen binding domain, an anti-CD138 scFV antigen binding domain, an anti-BCMA (CD269) scFV antigen binding domain, an anti-GPC2 scFV antigen binding domain, an anti-GPC3 scFV antigen binding domain, an anti-FGFR4 scFV antigen binding domain, an anti-c-Met scFV antigen binding domain, an anti-PMSA scFV antigen binding domain, an anti-glycolipidF77 scFV antigen binding domain, an anti-EGFRvIII scFV antigen binding domain, an anti-GD-2 CARs are provided that include an scFV antigen-binding domain, an anti-NY-ESo-1 TCR scFV antigen-binding domain, an anti-MAGE A3 TCR scFV antigen-binding domain, or an amino acid sequence thereof having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, or any combination thereof.

[0043] In another embodiment, the extracellular antigen binding domain is an immunoglobulin variable heavy chain only (VH) anti-CD19 antigen binding domain, anti-CD20 VH antigen binding domain, anti-CD22 VH antigen binding domain, anti-ROR1 VH antigen binding domain, anti-mesothelin VH antigen binding domain, anti-CD33 VH antigen binding domain, anti-CD38 VH antigen binding domain, anti-CD123 (IL3RA) VH antigen binding domain, anti-CD138 VH antigen binding domain, anti-BCMA (CD269) VH antigen binding domain, anti-GPC2 VH antigen binding domain, anti-GPC3 VH antigen binding domain, anti-FGFR4 VH antigen binding domain, anti-c-Met VH antigen binding domain, anti-PMSA VH antigen binding domain, anti-glycolipid F77 VH antigen binding domain, anti-EGFRvIII VH antigen binding domain, anti-GD-2 VH antigen binding domain, anti-NY-ESO-1 TCR A CAR is provided that comprises a VH antigen-binding domain, an anti-MAGE A3 TCR VH antigen-binding domain, or an amino acid sequence thereof having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, or any combination thereof.

[0044] In another embodiment, the extracellular antigen binding domain is an anti-CD19 P antigen binding domain, an anti-CD20 P antigen binding domain, an anti-CD22 P antigen binding domain, an anti-ROR1 P antigen binding domain, an anti-mesothelin P antigen binding domain, an anti-CD33 P antigen binding domain, an anti-CD38 P antigen binding domain, an anti-CD123 (IL3RA) P antigen binding domain, an anti-CD138 P antigen binding domain, an anti-BCMA (CD269) P antigen binding domain, an anti-GPC2 P antigen binding domain, an anti-GPC3 P antigen binding domain, an anti-FGFR4 P antigen binding domain, an anti-c-Met P antigen binding domain, an anti-PMSA P antigen binding domain, an anti-glycolipid F77 P antigen binding domain, an anti-EGFRvIII P antigen binding domain, an anti-GD-2 P antigen binding domain, an anti-NY-ESO-1 TCR P antigen binding domain, an anti-MAGE A3 TCR A CAR is provided, comprising a protein or peptide (P) sequence capable of specifically binding to a target antigen, which may be derived from a natural or synthetic sequence comprising a P antigen-binding domain, or an amino acid sequence thereof having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, or any combination thereof. In another embodiment, a CAR is provided, wherein at least one intracellular signaling domain comprises a costimulatory domain and a primary signaling domain.

[0045] In yet another embodiment, a CAR is provided, wherein at least one intracellular signaling domain comprises a costimulatory domain that comprises a functional signaling domain of a protein selected from the group consisting of OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP12, and 4-1BB (CD137), or a combination thereof.

[0046] In one embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 1. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO:2.

[0047] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 3. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 4.

[0048] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 5. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO:6.

[0049] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 7. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 8.

[0050] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 9. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 10.

[0051] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 11. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 12.

[0052] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 15. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 16.

[0053] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 17. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 18.

[0054] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 19. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO:20.

[0055] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 21. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 22.

[0056] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 23. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 24.

[0057] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 25. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 26.

[0058] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 69. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 70.

[0059] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 71. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 72.

[0060] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 77. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 78.

[0061] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 87. In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO:88.

[0062] In one embodiment, the CARs disclosed herein are modified to express or contain a detectable marker for use in diagnosis, monitoring and / or prediction of treatment outcomes such as progression-free survival in cancer patients, or to monitor the progress of such treatments.

[0063] 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, an adeno-associated virus vector, a baculovirus vector, a foamy virus vector, or a retrovirus vector, or a combination thereof.

[0064] In certain embodiments, lentiviral vectors encoding one or more CARs disclosed herein can be used to produce genomic material packaged into pseudotyped lentiviral particles. In one embodiment, the pseudotyped lentiviral particles comprise vesicular stomatitis virus-envelope glycoprotein (VSV-G) pseudotyped lentiviral vector particles. In another embodiment, the pseudotyped lentiviral particles comprise Baboon envelope glycoprotein pseudotyped vector (BaEV-G) pseudotyped lentiviral vector particles. In yet another embodiment, the pseudotyped lentiviral particles comprise feline endogenous retroviral envelope glycoprotein RD114 (RD114-G).

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

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

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

[0068] In yet another aspect, a pharmaceutical composition is provided comprising an antitumor effective amount of a human T cell population, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), the CAR comprises at least one extracellular antigen binding domain comprising a CD123 antigen binding domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 70, 72, 78 and 88, 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 with cancer. Cancers include, among others, hematological cancers, such as leukemia (e.g., chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL) or chronic myelogenous leukemia (CML)), lymphoma (e.g., mantle cell lymphoma, non-Hodgkin's lymphoma or Hodgkin's lymphoma) or multiple myeloma, or a combination thereof.

[0069] In one embodiment, a pharmaceutical composition is provided, wherein at least one transmembrane domain of the CAR comprises a transmembrane domain of a protein selected from the group consisting of the alpha, beta or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, mesothelin, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, or a combination thereof.

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

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

[0072] In another aspect, a method of generating a CAR-containing T cell (hereinafter "CAR T cell") is provided, comprising transducing a T cell with a vector or nucleic acid molecule encoding a disclosed CAR that specifically binds to CD123, thereby generating a CAR T cell.

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

[0074] In yet another embodiment, a method of diagnosing a disease, disorder or condition associated with CD123 expression in a cell is provided, comprising: a) contacting the cell with a human anti-CD123 antibody or fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 70, 72, 78 and 88; and b) detecting the presence of CD123, wherein the presence of CD123 diagnoses the disease, disorder or condition associated with CD123 expression.

[0075] In one embodiment, the disease, disorder or condition associated with CD123 expression is cancer, including hematopoietic cancer, myelodysplastic syndrome, pancreatic cancer, head and neck cancer, skin tumors, adult B-cell malignancies including acute lymphoblastic leukemia (ALL), minimal residual disease (MRD) in acute myeloid leukemia (AML), CLL (chronic lymphocytic leukemia), CML (chronic myeloid leukemia), non-Hodgkin's lymphoma (NHL), pediatric B-cell malignancies (including B-cell lineage ALL (acute lymphocytic leukemia)), multiple myeloma, lung cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, melanoma or other hematological cancers and solid tumors, or any combination thereof.

[0076] In another embodiment, a method is provided for diagnosing, prognosing or determining the risk of a disease associated with CD123 in a mammal, comprising detecting CD123 expression in a sample from a mammal, comprising: a) contacting the sample with a human anti-CD123 antibody or fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 70, 72, 78 and 88; and b) detecting the presence of CD123, wherein the presence of CD123 diagnoses a disease associated with CD123 in the mammal.

[0077] In another embodiment, a method of inhibiting CD123-dependent T cell inhibition is provided, comprising contacting a cell with a human anti-CD123 antibody or fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 70, 72, 78 and 88. In one embodiment, the cell is selected from the group consisting of a tumor cell expressing CD123, a tumor associated macrophage, and any combination thereof.

[0078] In another embodiment, a method is provided for blocking T cell inhibition mediated by cells expressing CD123 and altering a tumor microenvironment to inhibit tumor growth in a mammal, comprising administering to the mammal an effective amount of a composition comprising an isolated anti-CD123 antibody or fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 70, 72, 78 and 88. In one embodiment, the cell is selected from the group consisting of a tumor cell expressing CD123, a tumor associated macrophage, and any combination thereof.

[0079] In another embodiment, a method is provided for inhibiting, suppressing or preventing immunosuppression of an anti-tumor or anti-cancer immune response in a mammal, comprising administering to the mammal an effective amount of a composition comprising an isolated anti-CD123 antibody or fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 70, 72, 78 and 88. In one embodiment, the antibody or fragment thereof inhibits an interaction between a first cell and a T cell, wherein the first cell is selected from the group consisting of a tumor cell expressing CD123, a tumor associated macrophage, and any combination thereof.

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

[0081] In another embodiment, a method of treating or preventing cancer in a mammal is provided, comprising administering to the mammal one or more of the disclosed CARs in an amount effective to treat or prevent cancer in the mammal. The method comprises administering to the subject a therapeutically effective amount of host cells expressing the disclosed CARs that specifically bind to CD123 and / or one or more of the above-mentioned antigens under conditions sufficient to form an immune complex between the antigen-binding domain of the CAR and the extracellular domain of CD123 and / or one or more of the above-mentioned antigens in the subject.

[0082] In yet another embodiment, a method of treating a mammal having a disease, disorder, or condition associated with elevated expression of a tumor antigen is provided, comprising administering to the subject an anti-tumor effective amount of a pharmaceutical composition comprising a population of T cells, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises at least one extracellular CD123 antigen-binding domain comprising an amino acid sequence of SEQ ID NO: 70, 72, 78, or 88, or any combination thereof, at least one linker or spacer domain, at least one transmembrane domain, at least one intracellular signaling domain, and wherein the T cells are T cells of a subject with cancer.

[0083] In yet another embodiment, a method of treating cancer in a subject in need thereof is provided, comprising administering to the subject an antitumor effective amount of a pharmaceutical composition comprising a population of T cells, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), the CAR comprises at least one CD123 antigen-binding domain comprising an amino acid sequence of SEQ ID NO: 70, 72, 78 or 88, 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 with cancer. In some embodiments of the above-mentioned method, the at least one transmembrane domain comprises a transmembrane alpha, beta or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, mesothelin, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, or a combination thereof.

[0084] In yet another embodiment, a method is provided for generating a persistent population of engineered T cells in a human diagnosed with cancer. In one embodiment, the method comprises administering to the human T cells engineered to express a CAR, wherein the CAR comprises at least one CD123 antigen binding domain, at least one transmembrane domain, and at least one intracellular signaling domain comprising an amino acid sequence of SEQ ID NO: 70, 72, 78, or 88, or any combination thereof, and wherein the persistent population of engineered T cells, or a population of progeny of the T cells, persists in the human for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, or 3 years after administration.

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

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

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

[0088] It is understood that the CAR, host cell, nucleic acid and method 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]

[0089] [Figure 1A] Figure 1 shows the structure, surface expression and cell viability of CD123 CAR in human primary T cells. (Figure 1A) Anti-CD123 CAR construct was generated by linking in-frame a single-chain fragment variable sequence (scFv) targeting CD123 to the CD8 hinge (H) and transmembrane domain (TM), 4-1BB (CD137) costimulatory domain and CD3 zeta activation domain. [Figure 1B]Figure 1B shows the structure, surface expression and cell viability of CD123 CAR in human primary T cells. T cells were activated with TransAct CD3 / CD28 reagent in the presence of IL-2 and transduced with lentiviral vector encoding the CAR123 construct. On day 7 of culture, viable transduced T cells (7-AAD negative) were assayed for CAR surface expression using CD123 Fc followed by anti-Fc-AF647. Percentage of CAR T positive population relative to non-transduced T cell control. [Figure 1C] Figure 1C shows the structure, surface expression and cell viability of CD123 CAR in human primary T cells. CAR T cell viability was measured by trypan blue exclusion (Vi-Cell) on days 3 and 7 of culture. CD33 CAR construct (1906) was included as a control. [Diagram 2] FIG. 1 shows CD123 surface expression levels in MOLM14, KG-1a, RS4;11, 293T and A431 tumor cell lines. Representative flow histograms are shown. [Figure 3A] Analysis of tumor cell lysis induced in vitro by CAR123 constructs. Luciferase-based cytotoxicity assays were performed using MOL14 (Figure 3A), KG1a (Figure 3B), and 293T (Figure 3C) target cell lines stably expressing firefly luciferase. CAR T cells and tumor cells were co-incubated overnight at the indicated effector-to-target (E:T) ratios: 2.5:1, 5:1, or 10:1. The percentage of specific target lysis was assessed by luminometry. [Figure 3B]Analysis of tumor cell lysis induced in vitro by CAR123 constructs. Luciferase-based cytotoxicity assays were performed using MOL14 (Figure 3A), KG1a (Figure 3B), and 293T (Figure 3C) target cell lines stably expressing firefly luciferase. CAR T cells and tumor cells were co-incubated overnight at the indicated effector-to-target (E:T) ratios: 2.5:1, 5:1, or 10:1. The percentage of specific target lysis was assessed by luminometry. [Figure 3C] Analysis of tumor cell lysis induced in vitro by CAR123 constructs. Luciferase-based cytotoxicity assays were performed using MOL14 (Figure 3A), KG1a (Figure 3B), and 293T (Figure 3C) target cell lines stably expressing firefly luciferase. CAR T cells and tumor cells were co-incubated overnight at the indicated effector-to-target (E:T) ratios: 2.5:1, 5:1, or 10:1. The percentage of specific target lysis was assessed by luminometry. [Figure 4A] Figure 1. CAR T cytokine release in response to leukemia cell lines. Cytokine production by CAR T cells listed on the x-axis was measured by ELISA after overnight co-culture with MOLM14 leukemia cell line at an E:T ratio of 10:1. Bars represent the mean + SD of replicate samples. Data are representative of three independent experiments performed with CAR T cells from three separate donors. [Figure 4B] Figure 1. CAR T cytokine release in response to leukemia cell lines. Cytokine production by CAR T cells listed on the x-axis was measured by ELISA after overnight co-culture with MOLM14 leukemia cell line at an E:T ratio of 10:1. Bars represent the mean + SD of replicate samples. Data are representative of three independent experiments performed with CAR T cells from three separate donors. [Figure 4C]Figure 1. CAR T cytokine release in response to leukemia cell lines. Cytokine production by CAR T cells listed on the x-axis was measured by ELISA after overnight co-culture with MOLM14 leukemia cell line at an E:T ratio of 10:1. Bars represent the mean + SD of replicate samples. Data are representative of three independent experiments performed with CAR T cells from three separate donors. [Figure 5A] Figure 5 shows the CAR constructs tested in the two in vivo studies. (Figure 5A) CD123 CAR candidate D0126 and control CAR 33 LTG1906 were included in the first animal study, (Figure 5B) CD123 CAR candidate D0131 was added to D0126 and LTG1906 in the second animal study. Tumor alone (TA) and untransduced T cell (UTD) groups were included as controls in both studies. [Figure 5B] Figure 5 shows the CAR constructs tested in the two in vivo studies. (Figure 5A) CD123 CAR candidate D0126 and control CAR 33 LTG1906 were included in the first animal study, (Figure 5B) CD123 CAR candidate D0131 was added to D0126 and LTG1906 in the second animal study. Tumor alone (TA) and untransduced T cell (UTD) groups were included as controls in both studies. [Figure 6A] Figure 6: In vivo activity of CAR T constructs in the first animal study. NSG mice were iv injected with MOLM14-luciferase cells on day 0 and treated with 5x106 / mouse T cells or UTD on day 7. Six mice were studied for CAR T treatment and control groups. (Figure 6A) Representative time-course bioluminescence images of tumor burden in mice. [Figure 6B]Figure 6: In vivo activity of CAR T constructs in the first animal study. NSG mice were injected iv with MOLM14-luciferase cells on day 0 and treated with 5x106 / mouse T cells or UTD on day 7. Six mice were studied for CAR T treatment and control groups. (Figure 6B) Time course of tumor growth based on whole-body mouse bioluminescence (radiance) was quantified and plotted individually as shown, n=6, (Figure 6C) Percentage change in body weight was recorded every other day, n=6, mean±SEM. [Figure 6C] Figure 6: In vivo activity of CAR T constructs in the first animal study. NSG mice were injected iv with MOLM14-luciferase cells on day 0 and treated with 5x106 / mouse T cells or UTD on day 7. Six mice were studied for CAR T treatment and control groups. (Figure 6B) Time course of tumor growth based on whole-body mouse bioluminescence (radiance) was quantified and plotted individually as shown, n=6, (Figure 6C) Percentage change in body weight was recorded every other day, n=6, mean±SEM. [Figure 6D] Figure 6D shows the in vivo activity of CAR T constructs in the first animal study. NSG mice were iv injected with MOLM14-luciferase cells on day 0 and treated with 5x106 / mouse T cells or UTD on day 7. Six mice were studied for the CAR T treatment and control groups. (Figure 6D) Survival curves of mice and controls after CAR T treatment. [Figure 7A] Figure 7 shows human T cells detected in the blood of mice during the first in vivo study. Total human T cells in mouse peripheral blood were measured by volumetric flow cytometry on days 14 (Figure 7A), 22 (Figure 7B), and 33 (Figure 7C) and normalized using CountBright beads. All surviving mice are shown. Results are shown as scatter plots. Lines indicate group means. [Figure 7B]Figure 7 shows human T cells detected in the blood of mice during the first in vivo study. Total human T cells in mouse peripheral blood were measured by volumetric flow cytometry on days 14 (Figure 7A), 22 (Figure 7B), and 33 (Figure 7C) and normalized using CountBright beads. All surviving mice are shown. Results are shown as scatter plots. Lines indicate group means. [Figure 7C] Figure 7 shows human T cells detected in the blood of mice during the first in vivo study. Total human T cells in mouse peripheral blood were measured by volumetric flow cytometry on days 14 (Figure 7A), 22 (Figure 7B), and 33 (Figure 7C) and normalized using CountBright beads. All surviving mice are shown. Results are shown as scatter plots. Lines indicate group means. [Figure 8A] Figure 8 shows the in vivo activity of CAR T constructs in a second in vivo study. NSG mice were injected iv with MOLM14-luciferase cells on day 0 and administered 5x106 / mouse T cells on day 7. Figure 8A shows representative bioluminescence images of tumor burden over time in each group. [Figure 8B] Figure 8B shows the in vivo activity of CAR T constructs in a second in vivo study. NSG mice were injected iv with MOLM14-luciferase cells on day 0 and 5x106 / mouse T cells were administered on day 7. (Figure 8B) Time course plot of tumor growth based on mouse whole body bioluminescence (radiance), n=6, mean±SEM. [Figure 8C] Figure 8C shows the in vivo activity of CAR T constructs in a second in vivo study. On day 0, MOLM14-luciferase cells were injected iv into NSG mice, and on day 7, 5x106 / mouse T cells were administered. (Figure 8C) Percentage of body weight change was recorded every other day, n=6, mean±SEM. [Figure 8D]Figure 8D shows the in vivo activity of CAR T constructs in a second in vivo study. On day 0, MOLM14-luciferase cells were injected iv into NSG mice, and on day 7, 5x106 / mouse T cells were administered. Figure 8D shows survival curves over time for CAR T and control groups. [Figure 9A] Figure 9 shows human T cells detected in mouse peripheral blood over the course of a second animal study. Total human T cell numbers were measured by flow cytometry and quantified with CountBright beads on days 2 (Figure 9A), 14 (Figure 9B), 21 (Figure 9C), 28 (Figure 9D), and 42 (Figure 9E). Results are shown as scatter plots. Lines indicate group means. [Figure 9B] Figure 9 shows human T cells detected in mouse peripheral blood over the course of a second animal study. Total human T cell numbers were measured by flow cytometry and quantified with CountBright beads on days 2 (Figure 9A), 14 (Figure 9B), 21 (Figure 9C), 28 (Figure 9D), and 42 (Figure 9E). Results are shown as scatter plots. Lines indicate group means. [Figure 9C] Figure 9 shows human T cells detected in mouse peripheral blood over the course of a second animal study. Total human T cell numbers were measured by flow cytometry and quantified with CountBright beads on days 2 (Figure 9A), 14 (Figure 9B), 21 (Figure 9C), 28 (Figure 9D), and 42 (Figure 9E). Results are shown as scatter plots. Lines indicate group means. [Figure 9D] Figure 9 shows human T cells detected in mouse peripheral blood over the course of a second animal study. Total human T cell numbers were measured by flow cytometry and quantified with CountBright beads on days 2 (Figure 9A), 14 (Figure 9B), 21 (Figure 9C), 28 (Figure 9D), and 42 (Figure 9E). Results are shown as scatter plots. Lines indicate group means. [Figure 9E]Figure 9 shows human T cells detected in mouse peripheral blood over the course of a second animal study. Total human T cell numbers were measured by flow cytometry and quantified with CountBright beads on days 2 (Figure 9A), 14 (Figure 9B), 21 (Figure 9C), 28 (Figure 9D), and 42 (Figure 9E). Results are shown as scatter plots. Lines indicate group means. [Figure 10A] Figure 10 shows the isolation of NK cells and generation of target cells for CD123-CAR. (Figure 10A) Isolation and purity of NK cells. (Figure 10B) Target cells were generated by overexpressing CD123 in the RS4-11 cell line. CD123-transduced RS4-11 was sorted and limiting dilution was performed to generate homogenous CD123-expressing RS4-11 cells. [Figure 10B] Figure 10 shows the isolation of NK cells and generation of target cells for CD123-CAR. (Figure 10A) Isolation and purity of NK cells. (Figure 10B) Target cells were generated by overexpressing CD123 in the RS4-11 cell line. CD123-transduced RS4-11 was sorted and limiting dilution was performed to generate homogenous CD123-expressing RS4-11 cells. [Figure 11-1] Figure 1. Expression of CD123-CAR binders on transduced primary NK cells. Primary NK cells were isolated and cultured for 2 days in medium containing IL-2, IL-15, and IL-1β. On day 3, activated NK cells were individually transduced with 13 different lentiviral vectors containing different CD123-CARs. CD123-CAR expression on NK cells was detected 8 days after transduction. [Figure 11-2] Figure 1. Expression of CD123-CAR binders on transduced primary NK cells. Primary NK cells were isolated and cultured for 2 days in medium containing IL-2, IL-15, and IL-1β. On day 3, activated NK cells were individually transduced with 13 different lentiviral vectors containing different CD123-CARs. CD123-CAR expression on NK cells was detected 8 days after transduction. [Figure 12A]Figure 12 shows the expression and cytotoxicity of CD123-CAR. (Figure 12A) NK cells were transduced with lentiviral vectors containing CD123-CAR constructs D0126 and Z32. CD123-CAR expression was measured 8 days after transduction. (Figure 12B) The cytotoxicity of CD123-CAR-NK cells was determined using RS411-CD123 target cells. Results were representative of three independent experiments. [Figure 12B] Figure 12 shows the expression and cytotoxicity of CD123-CAR. (Figure 12A) NK cells were transduced with lentiviral vectors containing CD123-CAR constructs D0126 and Z32. CD123-CAR expression was measured 8 days after transduction. (Figure 12B) The cytotoxicity of CD123-CAR-NK cells was determined using RS411-CD123 target cells. Results were representative of three independent experiments. [Figure 13A] Specific killing of CD123-CAR NK cells against target cells expressing CD123. (Figure 13A) NK cells were transduced with different doses of lentiviral vector containing CD123-CAR. Expression of CD123-CAR was detected 8 days after transduction. (Figure 13B) Cytotoxicity of CD123-CAR-NK cells was determined using RS411-CD123 target cells. The effector to target ratio used for cytotoxicity experiments was 1:1. [Figure 13B] Specific killing of CD123-CAR NK cells against target cells expressing CD123. (Figure 13A) NK cells were transduced with different doses of lentiviral vector containing CD123-CAR. Expression of CD123-CAR was detected 8 days after transduction. (Figure 13B) Cytotoxicity of CD123-CAR-NK cells was determined using RS411-CD123 target cells. The effector to target ratio used for cytotoxicity experiments was 1:1. [Figure 14A]Figure 14 shows the effect of CD123-CAR on NK cell proliferation and viability. (Figure 14A) NK cells were transduced with CD123-CAR constructs D0126 and Z32 and expanded in parallel with untransduced NK cells. (Figure 14B) NK cell viability was determined at various time points in culture after transduction with CD123-CAR. D1, D3, D5, D8, and D11 indicate 1, 3, 5, 8, and 11 days after transduction. UTD indicates untransduced NK cells; Z32 indicates CD123 binder Z32; D0126 indicates CD123 binder D0126. [Figure 14B] Figure 14 shows the effect of CD123-CAR on NK cell proliferation and viability. (Figure 14A) NK cells were transduced with CD123-CAR constructs D0126 and Z32 and expanded in parallel with untransduced NK cells. (Figure 14B) NK cell viability was determined at various time points in culture after transduction with CD123-CAR. D1, D3, D5, D8, and D11 indicate 1, 3, 5, 8, and 11 days after transduction. UTD indicates untransduced NK cells; Z32 indicates CD123 binder Z32; D0126 indicates CD123 binder D0126. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

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

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

[0093] The present disclosure provides a CD123 antibody or fragment thereof, and a chimeric antigen receptor (CAR) having such a CD123 antigen-binding domain. The enhancement of the functional activity of CAR is directly related to the enhancement of the functional activity of T cells expressing CAR. As a result of one or more of these modifications, CAR shows both high cytokine-induced cell lysis and cell surface expression on transduced T cells, together with an increased level of T cell proliferation and persistence in vivo of transduced CAR-expressing T cells.

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

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

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

[0097] The CAR disclosed herein is expressed at high levels in cells. Cells expressing CARs have high proliferation rates in vivo, produce large amounts of cytokines, and have high cytotoxicity against cells bearing the CD123 antigen on their surface to which the CAR binds. The use of human extracellular CD123 antigen-binding domains results in the generation of CARs that function better in vivo, while avoiding the induction of anti-CAR immunity in the host immune response and the death of the CAR T cell population. CARs expressing fully human extracellular CD123 ScFv antigen-binding domains show superior activity / characteristics, including: i) prevention of CAR T persistence and poor function, as seen with binding sequences derived from mice; ii) absence of local (i.e., intrapleural) delivery of CARs to be effective; and iii) ability to generate CAR T cell designs based on binders with both high and low affinity to CD123. This last property allows researchers to better tune the efficacy and / or tissue specificity of the CAR T product, since tumors have higher expression of CD123 than normal tissues, allowing lower affinity binders to have greater specificity for tumors than normal tissues, thereby preventing on-target non-tumor toxicity and bystander cell killing.

[0098] Below is a detailed description of the CARs of the present invention, including a description of their extracellular CD123 antigen-binding domain, transmembrane domain, and intracellular domain, along with further description of CARs, antibodies and antigen-binding fragments thereof, conjugates, nucleotides, expression, vectors, and host cells, methods of treatment, compositions, and kits using the disclosed CARs.

[0099] A. Chimeric Antigen Receptor (CAR) The CARs disclosed herein comprise at least one CD123 antigen-binding domain capable of binding to CD123, at least one transmembrane domain, and at least one intracellular domain.

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

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

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

[0103] In one embodiment, CAR can be engineered to target tumor antigen of interest by engineering a desired antigen-binding domain that specifically binds to the antigen on tumor cell.Tumor antigen is a protein produced by tumor cell that triggers immune response, especially T cell-mediated immune response.The choice of antigen-binding domain depends on the specific type of cancer to be treated. Tumor antigens include, for example, glioma-associated antigen, carcinoembryonic antigen (CEA), beta-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, Her2 / neu, survivin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, and CD123. The tumor antigens disclosed herein are included by way of example only: this list is not intended to be exhaustive, and further examples will be readily apparent to one of skill in the art.

[0104] In one embodiment, the tumor antigen comprises one or more antigenic cancer epitopes associated with malignant tumors. Malignant tumors express several proteins that can serve as target antigens for immune attack. These molecules include, but are not limited to, tissue-specific antigens, such as MART-1, tyrosinase and GP 100 in melanoma, and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation-related molecules, such as the oncogene HER-2 / Neu / ErbB-2. Yet another group of target antigens are carcinoembryonic antigens, such as carcinoembryonic antigen (CEA). In B-cell lymphomas, tumor-specific idiotypic immunoglobulins constitute truly tumor-specific immunoglobulin antigens that are unique to each individual tumor. B-cell differentiation antigens, such as CD19, CD20 and CD37, are other candidates for target antigens in B-cell lymphomas. Some of these antigens (CEA, HER-2, CD19, CD20, idiotype) have been used as targets for passive immunotherapy with monoclonal antibodies with limited success.

[0105] In a preferred embodiment, the tumor antigen is CD123, and tumors associated with CD123 expression include lung mesothelioma, ovarian and pancreatic cancer, or any combination thereof, which express high levels of the extracellular protein CD123.

[0106] A type of tumor antigen can 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, but instead is also expressed on normal cells under conditions that cannot induce a state of immunological tolerance to the antigen. The expression of an antigen on a tumor can occur under conditions that allow the immune system to respond to the antigen. A TAA can be an antigen that is expressed on normal cells during fetal development, when the immune system is immature and cannot respond, or an antigen that is usually present at a very low level on normal cells, but is expressed at a much higher level on tumor cells.

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

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

[0109] In a preferred embodiment, the antigen binding domain portion of the CAR targets the extracellular CD123 antigen.

[0110] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD123 MT-16 antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 69, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD123 MT-16 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 70, or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 70.

[0111] In a preferred embodiment, an isolated nucleic acid molecule encoding the extracellular CD123 MT-32 antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 71, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD123 MT-32 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 72, or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 72.

[0112] In a preferred embodiment, an isolated nucleic acid molecule encoding the extracellular CD123 Z16 antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 77, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD123 Z16 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 78, or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 78.

[0113] In a preferred embodiment, an isolated nucleic acid molecule encoding the extracellular CD123 Z32 antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 87, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD123 Z32 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 88, or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 88.

[0114] The generation and binding characteristics of the specific CD123 variable heavy chain only and ScFv antigen-binding fragments or antigen binders described herein are shown in Example 1.

[0115] In various embodiments for a CD123-specific CAR disclosed herein, a general schematic is shown in Figures 1A-1C and includes, from N-terminus to C-terminus, a signal or leader peptide, anti-CD123 ScFv, extracellular linker, CD8 transmembrane, 4-1BB, CD3 zeta, with bold text representing the cloning sites for the linking domains.

[0116] In one embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO:1 and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO:2.

[0117] In one embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 3, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, and encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 4, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.

[0118] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO:5, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO:6.

[0119] In another embodiment, the nucleic acid sequence encoding a CAR comprises the nucleic acid sequence of SEQ ID NO: 7, or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 8, or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0120] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 9, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO: 10.

[0121] In another embodiment, the nucleic acid sequence encoding a CAR comprises the nucleic acid sequence of SEQ ID NO: 11, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, and encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 12, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.

[0122] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 15, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO: 16.

[0123] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 17, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, and encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 18, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.

[0124] In yet another 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.

[0125] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 21, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, and encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 22, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.

[0126] In yet 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 set forth in SEQ ID NO: 24.

[0127] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 25, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, and encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 26, or a sequence thereof with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.

[0128] The development of anti-CD123 CAR T cells incorporating single chain fragment variable (ScFv) sequences reactive to the CD123 antigen is described in Examples 3 and 4 below, and the generation of NK cells expressing a CD123 CAR construct is shown in Example 5 below.

[0129] Example 3 describes the generation and in vitro evaluation of CAR T cells targeting the CD123 antigen for the treatment of AML.

[0130] For CAR transduction into human primary T cells, lentiviral vectors encoding CD123 CAR constructs were used at a multiplicity of infection (MOI) of 40. Different CD123 CAR constructs showed different expression levels ranging from 0 to 80% (n = 4 donors) (Figure 1B). CARs D0126, D0127, D0131, D0132, D0133, and D0134 showed surface expression equal to or higher than the positive CAR 123 control LTG2078, whereas CAR D0130 showed slightly lower surface expression, followed by D0129 and D0128, while D0125 showed the lowest expression in multiple donors. Cell viability was examined on days 3 and 7 after T cell activation, as shown in Figure 1C. All CD123 CAR T cells showed improved or equal viability compared to the control CAR LTG2078.

[0131] The in vitro target-specific cytotoxicity of CD123 CAR was evaluated against CD123-positive leukemic cell lines (MOLM14, KG1a, RS4;11) and CD123-negative non-leukemic cell lines (293T and A431). CAR-T cells were co-incubated with MOLM14, KG-1a or 293T cell lines at effector-to-target ratios of 2.5:1, 5:1, and 10:1. After overnight co-incubation, cultures were analyzed by a luminescence-based in vitro killing assay. Most of the primary T cell lines expressing CAR123 constructs lysed MOL14-CD123+, whereas three CD123 CAR lines, D0125, D0128, and D0129, lacked target lysis capability (Figure 3A). Similarly, KG-1a-CD123+ target cells were killed by most CAR T constructs, except for D0125, D0128, and D0129 (Figure 3B). The control CD33 CAR LTG1906 showed high cytotoxicity (CD33) against MOLM14, consistent with CD123 expression levels. High ) and showed low lytic potency against KG1a (CD33 LowFurthermore, no killing above background of the CD123-negative 293T cell line was observed (Figure 3C), demonstrating robust target-specific cytotoxicity function of all CD123 CAR constructs except for CARs D0125, D0128, and D0129.

[0132] Production of T cell homeostasis and proinflammatory cytokines IL-2, IFNγ, and TNFα by CD123 CAR and control constructs CAR LTG2078 and CD33 CAR LTG1906 was examined by ELISA in culture supernatants after overnight co-incubation of CAR T cells with MOLM14 target lines at an E:T ratio of 10 (Figures 4A-4C). Cytokine release induced by specific targets was detected by comparing each CAR T group incubated with target cells to the respective CAR T alone experimental group, and by comparing CAR T groups co-incubated with targets to the previously characterized CAR123 control LTG2078. Although CAR123 control LTG2078 and CAR33 control LTG1906 produced cytokines after co-incubation with MOLM14 target cells, most of the tested CD123 CAR T constructs did not show a significant increase in IFNγ, TNFα, or IL-2 cytokines after overnight co-culture with MOLM14 cells. One exception was CAR123 D0127, which produced IFNγ and TNFα levels even in the absence of target cells (T cell alone group), indicating a tumor-independent cytokine response. This effect was not predicted from previous experiments, demonstrating the nonobviousness of the present invention. With the exception of CAR123 D0127, the cytokine responses of the CD123 CAR constructs evaluated here were comparable to untransduced T cell (UTD) controls, suggesting a low risk of inducing cytokine-mediated side effects such as cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS).

[0133] NSG(NOD.Cg-Prkdc scidThe in vivo tumor rejection function of the two top CAR123 candidates, D0126 and D0131, was further investigated using the Il2rgtm1Wjl / SzJ) mouse MOLM14 xenograft AML model. Two animal experiments using CAR T cells derived from separate healthy donors were performed, one focused on CAR D0126 (Figure 5A) and the other comparing CAR123 constructs D0126 and D0131 (Figure 5B). As a comparison control, the previously characterized CAR LTG1906, which targets the CD33 antigen on MOLM14 tumor cells, was included.

[0134] In the first in vivo study, we compared the CD123 CAR D0126 with the previously characterized CD33 CAR-T construct LTG1906, and also included tumor alone (TA) and untransduced T cells (UTD) as control experimental groups. Tumor growth kinetics was monitored over time by an in vivo imaging system (IVIS) (Figures 6A and 6B). Because MOLM14 tumors express both CD123 and CD33 antigens, the treatment group receiving CAR D0126 targeting the CD123 antigen, and the comparison group receiving CAR LTG1906 targeting the CD33 antigen, showed robust tumor rejection compared to the tumor alone (TA) and UTD control groups. Five out of six mice in each group showed complete tumor rejection, with only one mouse per group having residual tumor cells at the end of the study (Figure 6B). Of note, no CAR-associated toxicity was detected in this model, as both CAR D0126 and CAR LTG1906-treated groups showed no weight loss (Figure 6C). Both CAR D0126 and LTG1906 resulted in complete survival until the end of the study on day 36 (6 out of 6 mice survived), while the tumor alone (TA) and UTD control groups died from high-burden disseminated disease by day 15 (Figure 6D). Peripheral blood of mice was collected on days 14, 22, and 33. Human T cells were detected in all groups (Figures 7A, 7B, 7C). Furthermore, CAR D0126 and LTG1906 T cells were detected in the peripheral blood of mice at the end of the study, demonstrating high persistence of CD123 CAR candidate D0126 and comparative control CAR33 LTG1906 T cells.

[0135] In the second animal study, in addition to CAR D0126, CD123 CAR D0131 was included. Tumor progression is shown in Figure 8A. As in the first animal study, CAR D0126 showed strong antitumor efficacy, with tumors rejected in 4 of 6 mice. CAR123 D0131 showed weaker antitumor activity compared to CAR123 D0126 (Figures 8A and 8B). The best survival effect was detected in the CAR D0126 treatment group, with 4 of 6 mice surviving and remaining completely tumor-free until day 56, the end of the extension study (Figure 8D). In this study, total T cells in the peripheral blood were monitored. As expected, human T cells were detected in the peripheral blood of mice 2 days after CAR T cell or UTD administration in all groups except the TA negative control (Figure 9A). T cell mass increased over time in all CAR T groups, suggesting T cell expansion (Figure 9B) and persistence through days 21, 28, and 42 (Figures 9C, 9D, 9E). On study day 42, the CAR123 D0126 group had the highest T cell numbers (Figure 9E), demonstrating the greatest T cell expansion and persistence of the CAR constructs tested in this experiment.

[0136] In summary, the CD123 CAR T cell candidate D0126 efficiently eliminated tumors in NSG mice implanted with MOLM-14 cells in two in vivo studies using T cells from different human donors, and demonstrated efficient tumor elimination, CAR T persistence, and extended survival in a MOLM14 AML xenograft mouse model (Figure 9A).

[0137] CD123-targeting CAR NK cells (Example 5, below) were generated by transfecting primary NK cells from healthy donors with lentiviral vectors pseudotyped with baboon envelope protein (BaEV-LV). Primary NK cells were isolated from PBMCs by magnetic separation, resulting in a pure cell population (Figure 10A). The functionality of the CD123-CAR was tested using the NK-resistant RS4-11 target cell line stably transduced with CD123 protein (Figure 10B).

[0138] NK cells were activated by culturing in NK MACS medium containing IL-2 / IL-15 / IL-1β for 2 days, followed by transduction with BaEV pseudotyped lentiviral vector (BaEV-LV), resulting in efficient transduction of primary NK cells. Transduction of NK cells with lentiviral vectors containing different CD123-CAR constructs resulted in differential expression of CD123-CAR on the surface of NK cells (Figure 11). Among the 13 CD123-CARs, the Z32 and D0126 CAR constructs were the best for transducing NK cells, with transduction efficiencies of 51.55% and 61.37%, respectively. Based on these expression results, CAR constructs Z32 and D0126 were selected for further analysis.

[0139] Activated NK cells were transduced with BaEV pseudotyped lentiviral vectors containing CD123-CAR Z32 (Z32-BaEV-LV) and D0126 (D0126-BaEV-LV). CD123-CAR expression of Z32 and D0126 was 70.5% and 64.19%, respectively (Figure 12A). Furthermore, the cytotoxicity of CD123-CAR-expressing NK cells was tested against target cells RS4-11-CD123. RS4;11 cells expressing CD123 (Figure 10B) are insensitive to the natural cytotoxicity of NK cells. The results showed that while non-transduced NK cells were unable to kill RS4;11-CD123 cells, both CD123-CAR (Z32 and D0126) NK cells killed RS4;11-CD123 very efficiently, demonstrating the high functionality and specificity of the generated CD123-CAR NK cells (Figure 12B).

[0140] Next, the specificity of CD123-CAR for the CD123 antigen was confirmed by serial dilution. NK cells were transduced with various amounts of lentiviral vectors containing CD123-CAR. As expected, the higher the amount of CD123-CAR-LV, the higher the expression of CD123-CAR (Figure 13A). Finally, the cytotoxicity of differentially expressing CD123-CAR NK cells was tested against RS4-11-CD123 cells at the same effector-target ratio (Figure 13B). The highest expressing CD123-CAR-NK cells showed the highest killing, and the lowest expressing CD123-CAR-NK cells showed the lowest killing, confirming the specificity of CD123-CAR for the CD123 antigen. Finally, the expression of CD123-CAR does not adversely affect NK proliferation and viability. Primary NK cells were isolated, activated, and expanded for 13 days after transduction with Z32 and D0126. Untransduced NK cells were used as a control. The proliferation of untransduced, Z32-transduced, and D0126-transduced NK cells was 61-, 49-, and 42-fold, respectively (Figure 14A). No significant difference in cell viability was observed between untransduced, Z32-, and D0126-transduced NK cells (Figure 14B), suggesting that CD123-CAR does not adversely affect NK cell viability.

[0141] Taken together, these results demonstrate the successful generation of CAR-T and CAR-NK cells targeting the CD123 antigen for cancer treatment.

[0142] Without intending to be limited to any particular mechanism of action, possible reasons for the enhanced therapeutic function associated with the exemplary CARs of the present invention are believed to include, for example, but not by way of limitation, a) improved lateral movement within the plasma membrane allowing for more efficient signaling, b) superior location within plasma membrane microdomains such as lipid rafts and a greater ability to interact with transmembrane signaling cascades associated with T cell activation, c) superior location within the plasma membrane due to preferential movement away from inhibitory or down-regulatory interactions, e.g., less proximity to or fewer interactions with phosphatases such as CD45, and d) superior assembly into the T cell receptor signaling complex (i.e., the immune synapse), or any combination thereof.

[0143] Although the present disclosure is exemplified using an exemplary extracellular CD123 variable heavy chain only and ScFv antigen-binding domain, other nucleotide and / or amino acid variants within the CD123 variable heavy chain only and ScFv antigen-binding domain may be used to obtain a CD123 antigen-binding domain for use in the CARs described herein.

[0144] Depending on the desired antigen to be targeted, the CAR can be further engineered to contain an appropriate antigen-binding domain specific for the desired antigen target. For example, if CD19 is the desired antigen to be targeted, an antibody against CD19 can be used as an antigen-binding domain incorporated into the CAR.

[0145] In an exemplary embodiment, the antigen-binding domain portion of the CAR further targets CD19. Preferably, the antigen-binding domain in the CAR is an anti-CD19 scFV, wherein the nucleic acid sequence of the anti-CD19 scFV comprises the sequence shown in SEQ ID NO: 37. In one embodiment, the anti-CD19 scFV comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 37. In another embodiment, the anti-CD19 scFV portion of the CAR comprises the amino acid sequence shown in SEQ ID NO: 38.

[0146] In one embodiment of the invention, a CAR is provided that is capable of binding to a non-TSA or non-TAA, including, for example and without limitation, an antigen derived from Retroviridae (e.g., human immunodeficiency viruses, e.g., HIV-1 and HIV-LP), Picornaviridae (e.g., poliovirus, Hepatitis A virus, enterovirus, human coxsackievirus, rhinovirus and echovirus), rubella virus, coronavirus, vesicular stomatitis virus, rabies virus, Ebola virus, parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus, influenza virus, Hepatitis B virus, parvovirus, Adenoviridae, Herpesviridae (e.g., herpes simplex virus type 1 and type 2 (HSV), varicella zoster virus, cytomegalovirus (CMV) and herpes virus), Poxviridae (e.g., smallpox virus, vaccinia virus and pox virus) or Hepatitis C virus, or any combination thereof.

[0147] In another aspect of the present invention, CARs are provided that can bind to antigens derived from bacterial strains of Staphylococci, Streptococcus, Escherichia coli, Pseudomonas or Salmonella. In particular, CARs are provided that can bind to antigens derived from infectious bacteria, such as Helicobacter pyloris, Legionella pneumophilia, bacterial strains of Mycobacteria sps. (e.g., M. tuberculosis, M. avium, M. intracellulare, M. kansaii or M. gordonea), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitides, Listeria monocytogenes, Streptococcus pyogenes, Group A Streptococcus, Group B Streptococcus (Streptococcus agalactiae), Streptococcus pneumoniae or Clostridium tetani, or combinations thereof.

[0148] 2. Transmembrane domain With respect to the transmembrane domain, the CAR comprises one or more transmembrane domains fused to the extracellular CD123 antigen-binding domain of the CAR.

[0149] The transmembrane domain may be derived from either natural or synthetic sources. If the source is natural, the domain may be derived from any membrane-bound or transmembrane protein.

[0150] The transmembrane regions particularly used in the CARs described herein can be derived from (i.e., at least include) the alpha, beta or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, mesothelin, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. Alternatively, the transmembrane domain can be synthetic, in which case it contains predominantly hydrophobic residues such as leucine and valine. Preferably, a phenylalanine, tryptophan and valine triplet is found at each end of the synthetic transmembrane domain. Optionally, a short oligopeptide or polypeptide linker, preferably between 2 and 10 amino acids in length, can form the link between the transmembrane domain and the cytoplasmic signaling domain of the CAR. A glycine-serine dyad provides a particularly suitable linker.

[0151] In one embodiment, a transmembrane domain naturally associated with one of the domains in the CAR is used in addition to the transmembrane domain.

[0152] In some cases, transmembrane domains may be selected by amino acid substitutions to avoid binding of such domains to transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

[0153] In one embodiment, the transmembrane domain in the CAR of the present invention is a CD8 transmembrane domain. In one embodiment, the CD8 transmembrane domain comprises the nucleic acid sequence of SEQ ID NO: 27. In one embodiment, the CD8 transmembrane domain comprises the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 28. In another embodiment, the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 28.

[0154] In one embodiment, the encoded transmembrane domain comprises an amino acid sequence having at least one, two or three alterations (e.g., substitutions) but not more than 20, 10 or 5 alterations (e.g., substitutions) of the amino acid sequence of SEQ ID NO:28, or a sequence having 95-99% identity to the amino acid sequence of SEQ ID NO:28.

[0155] In some cases, the transmembrane domain of the CAR comprises a CD8 alpha hinge domain. In one embodiment, the CD8 hinge domain comprises the nucleic acid sequence of SEQ ID NO: 29. In one embodiment, the CD8 hinge domain comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 30. In another embodiment, the CD8 hinge domain comprises the amino acid sequence of SEQ ID NO: 30, or a sequence thereof having 95-99% identity.

[0156] In one embodiment, an isolated nucleic acid molecule is provided 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.

[0157] In one embodiment, the transmembrane domain in the CAR of the present invention is a TNFRSF19 transmembrane domain. In one embodiment, the TNFRSF19 transmembrane domain comprises the nucleic acid sequence of SEQ ID NO: 51. In one embodiment, the TNFRSF19 transmembrane domain comprises the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 52. In another embodiment, the TNFRSF19 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 52.

[0158] In one embodiment, the encoded transmembrane domain comprises an amino acid sequence having at least one, two or three alterations (e.g., substitutions) but not more than 20, 10 or 5 alterations (e.g., substitutions) of the amino acid sequence of SEQ ID NO:52, or a sequence having 95-99% identity to the amino acid sequence of SEQ ID NO:52.

[0159] 3. Spacer domain In CARs, a spacer domain, also called a hinge domain, can be placed between the extracellular domain and the transmembrane domain, or between the intracellular domain and the transmembrane domain. Spacer domain refers to any oligopeptide or polypeptide that functions to link the transmembrane domain with the extracellular domain and / or to link the transmembrane domain with the intracellular domain. The spacer domain comprises up to 300 amino acids, preferably 10-100 amino acids, and most preferably 25-50 amino acids.

[0160] In some embodiments, the linker may include a spacer element, which, if present, increases the size of the linker, thereby increasing the distance between the effector molecule or detectable marker and the antibody or antigen-binding fragment. Exemplary spacers are known to those of skill in the art, including, for example, U.S. Pat. Nos. 7,964,566, 7,498,298, 6,884,869, 6,323,315, 6,239,104, 6,034,065, 5,780,588, 5,665,860, 5,663,149, 5,635,483, 5,599,902, 5,554,725, 5,530,097, 5,521, 5,532, 5,534, 5,536, 5,538, 5,539 ... No. 5,284, U.S. Pat. No. 5,504,191, U.S. Pat. No. 5,410,024, U.S. Pat. No. 5,138,036, U.S. Pat. No. 5,076,973, U.S. Pat. No. 4,986,988, U.S. Pat. No. 4,978,744, U.S. Pat. No. 4,879,278, U.S. Pat. No. 4,816,444 and U.S. Pat. No. 4,486,414, as well as those listed in U.S. Patent Application Publication No. 20110212088 and U.S. Patent Application Publication No. 20110070248, each of which is incorporated herein by reference in its entirety.

[0161] The spacer domain preferably has a sequence that promotes the binding of the CAR to the antigen and enhances signal transduction into the cell. Examples of amino acids that are predicted to promote binding include cysteine, charged amino acids, and serine and threonine in potential glycosylation sites, and these amino acids can be used as amino acids that constitute the spacer domain.

[0162] As the spacer domain, the whole or part of amino acid numbers 118 to 178 (SEQ ID NO: 31) 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) may be used. Also, as the spacer domain, a part of the constant region of the antibody H chain or L chain (CH1 region or CL region, for example, a peptide having the amino acid sequence shown in SEQ ID NO: 32) may be used. Furthermore, the spacer domain may be an artificially synthesized sequence.

[0163] Additionally, all or part of the amino acids comprising the constant region of human IgG4 (UniProt ID: P01861), including CH1, (amino acid numbers 1-98), hinge, SEQ ID NO: 80, and corresponding nucleotide SEQ ID NO: 79, (amino acid numbers 99-110), CH2, amino acid SEQ ID NO: 82 and corresponding nucleotide SEQ ID NO: 81, (amino acid numbers 111-220), and CH3, SEQ ID NO: 84 and corresponding nucleotide SEQ ID NO: 83, (amino acid numbers 221-327), or combinations thereof, such as the IgG4 hinge CH2 CH3 domain, SEQ ID NO: 86, and corresponding nucleotide SEQ ID NO: 85, may be used.

[0164] In one embodiment, the spacer domain of the CAR comprises a TNFRSF19 hinge domain comprising the nucleic acid sequence of SEQ ID NO: 53. In one embodiment, the TNFRSF19 hinge domain comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 54. In another embodiment, the TNFRSF19 hinge domain comprises the amino acid sequence of SEQ ID NO: 54, or a sequence thereof having 95-99% identity.

[0165] In one embodiment, the spacer domain of the CAR comprises a TNFRSF19 truncated hinge domain comprising the nucleic acid sequence of SEQ ID NO: 55. In one embodiment, the TNFRSF19 truncated hinge domain comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 56. In another embodiment, the TNFRSF19 truncated hinge domain comprises the amino acid sequence of SEQ ID NO: 56, or a sequence thereof having 95-99% identity.

[0166] In one embodiment, the TNFRSF19 hinge and transmembrane domain comprises the nucleic acid sequence of SEQ ID NO: 49. In one embodiment, the TNFRSF19 hinge and transmembrane domain comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 50. In another embodiment, the TNFRSF19 hinge and transmembrane domain comprises the amino acid sequence of SEQ ID NO: 50, or a sequence thereof having 95-99% identity.

[0167] In one embodiment, the CD8a hinge domain is fused to a TNFRSF19 transmembrane domain comprising the nucleic acid sequence of SEQ ID NO: 57. In one embodiment, the CD8a hinge domain is fused to a TNFRSF19 transmembrane domain comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 58. In another embodiment, the CD8a hinge domain is fused to a TNFRSF19 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 58, or a sequence thereof having 95-99% identity.

[0168] In addition, in CAR, a signal peptide sequence, also called a leader peptide, may be linked to the N-terminus. Signal peptide sequences are present at the N-terminus of many secretory proteins and membrane proteins and have a length of 15 to 30 amino acids. Many of the protein molecules referred to above as intracellular domains have signal peptide sequences, so these signal peptides can be used as signal peptides for CAR. In one embodiment, the signal peptide comprises the amino acid sequence shown in SEQ ID NO: 14.

[0169] In one embodiment, the CD8 alpha leader peptide comprises the nucleic acid sequence of SEQ ID NO: 43. In one embodiment, the CD8 alpha leader peptide comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 44. In another embodiment, the CD8a hinge domain is fused to a TNFRSF19 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 44, or a sequence thereof having 95-99% identity.

[0170] In another embodiment, the GMCSF leader peptide comprises the nucleic acid sequence of SEQ ID NO: 39. In one embodiment, the GMCSF leader peptide comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 40. In another embodiment, the CD8a hinge domain is fused to a TNFRSF19 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 40, or a sequence thereof having 95-99% identity.

[0171] In another embodiment, the TNFRSF19 leader peptide comprises the nucleic acid sequence of SEQ ID NO: 41. In one embodiment, the TNFRSF19 leader peptide and the CD8 alpha leader peptide comprise a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 42. In another embodiment, the CD8a hinge domain is fused to a TNFRSF19 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 42, or a sequence thereof having 95-99% identity.

[0172] In one embodiment, the tag sequence encoding a truncated sequence of epidermal growth factor receptor (tEGFR) comprises the nucleic acid sequence of SEQ ID NO: 67. In one embodiment, the tEGFR comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 68. In another embodiment, the tEGFR tag comprises the amino acid sequence of SEQ ID NO: 68, or a sequence thereof having 95-99% identity.

[0173] In one embodiment, the furin recognition site and downstream T2A self-cleaving peptide sequence designed for simultaneous bicistronic expression of the tag sequence and the CAR sequence comprise the nucleic acid sequence of SEQ ID NO: 65. In one embodiment, the furin and T2A sequences comprise a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 66. In another embodiment, the tEGFR tag comprises the amino acid sequence of SEQ ID NO: 66, or a sequence thereof having 95-99% identity.

[0174] In one embodiment, the upstream furin recognition site and T2A self-cleaving peptide sequence and the furin recognition downstream site designed for simultaneous bicistronic expression of the tag sequence and the CAR sequence comprise the nucleic acid sequence of SEQ ID NO: 67. In one embodiment, the furin and T2A sequences comprise a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 68. In another embodiment, the tEGFR tag comprises the amino acid sequence of SEQ ID NO: 68, or a sequence thereof having 95-99% identity.

[0175] In one embodiment, the targeting domain of the CAR is expressed separately in the form of a monoclonal antibody, ScFv Fab, Fab'2, and is contained in a binding tag or epitope, while the effector cell expressed component of the CAR contains a binding domain specifically directed to bind to a tag or epitope expressed on a soluble CAR module, such as to form a complete functional CAR structure upon specific binding to a cell binding component on the soluble component of the CAR.

[0176] 4. Intracellular domain The cytoplasmic domain or otherwise intracellular signaling domain of the CAR is responsible for activating at least one of the normal effector functions of the immune cell in which the CAR is placed. The term "effector function" refers to a specialized function of a cell. For example, the effector function of a T cell can be cytolytic activity or helper activity, including secretion of cytokines. Thus, the term "intracellular signaling domain" refers to a portion of a protein that transmits an effector function signal and instructs the cell to carry out a specialized function. Although the entire intracellular signaling domain can usually be used, it is often not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the intact chain, so long as it transmits the effector function signal. Thus, the term intracellular signaling domain is meant to include any truncated portion of the intracellular signaling domain sufficient to transmit the effector function signal.

[0177] Preferred examples of intracellular signaling domains for use in CARs include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivatives or variants of these sequences, and any synthetic sequences having the same functional capability.

[0178] It is known that signals generated through the TCR alone are insufficient for full activation of T cells, and that secondary or costimulatory signals are also required. Thus, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences) and those that act in an antigen-dependent manner to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences).

[0179] Primary cytoplasmic signaling sequences regulate the primary activation of the TCR complex in either a stimulatory or inhibitory manner. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs, or ITAMs.

[0180] Examples of ITAMs containing primary cytoplasmic signaling sequences of particular use in the CARs disclosed herein include those derived from TCR zeta (CD3 zeta), FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. Specific non-limiting examples of ITAMs include amino acids 51-164 of CD3 zeta (NCBI RefSeq: NP_932170.1), amino acids 45-86 of Fc epsilon RI gamma (NCBI RefSeq: NP_004097.1), amino acids 201-244 of Fc epsilon RI beta (NCBI RefSeq: NP_000130.1), amino acids 139-182 of CD3 gamma (NCBI RefSeq: NP_000064.1), amino acids 128-171 of CD3 delta (NCBI RefSeq: NP_000723.1), amino acids 153-207 of CD3 epsilon (NCBI RefSeq: NP_000724.1), and amino acids 153-207 of CD5 (NCBI RefSeq: NP_000724.1). The peptides include peptides having the sequences of amino acid numbers 402-495 of CD79a (NCBI RefSeq:NP_055022.2), amino acid numbers 707-847 of CD79a (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 amino acid numbers 177-252 of CD66d (NCBI RefSeq:NP_001806.2), as well as variants thereof having the same functions as these peptides. The amino acid numbers based on the NCBI RefSeq ID or GenBank amino acid sequence information described herein are numbered based on the full length of the precursor of each protein (including the signal peptide sequence, etc.). In one embodiment, the cytoplasmic signaling molecule in the CAR comprises a cytoplasmic signaling sequence derived from CD3 zeta.

[0181] In a preferred embodiment, the intracellular domain of the CAR can be designed to include a CD3-zeta signaling domain, either by itself or in combination with any other desired cytoplasmic domain(s) useful in the context of the CAR. For example, the intracellular domain of the CAR can include a CD3 zeta chain portion and a costimulatory signaling region. A costimulatory signaling region refers to a portion of the CAR that includes the intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for efficient lymphocyte response to antigens. Examples of such costimulatory molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83, and the like. Specific non-limiting examples of such costimulatory molecules include amino acids 236-351 of CD2 (NCBI RefSeq: NP_001758.2), amino acids 421-458 of CD4 (NCBI RefSeq: NP_000607.1), amino acids 402-495 of CD5 (NCBI RefSeq: NP_055022.2), amino acids 207-235 of CD8 alpha (NCBI RefSeq: NP_001759.3), amino acids 196-210 of CD83 (GenBank: AAA35664.1), amino acids 181-220 of CD28 (NCBI RefSeq: NP_006130.1), and CD137 (4-1BB, NCBI Included are peptides having sequences of amino acids 214-255 of CD134 (OX40, NCBI RefSeq:NP_001552.2), amino acids 241-277 of CD134 (OX40, NCBI RefSeq:NP_003318.1), and amino acids 166-199 of ICOS (NCBI RefSeq:NP_036224.1), as well as variants thereof having the same functions as these peptides. Thus, although the disclosure herein primarily exemplifies 4-1BB as a costimulatory signaling element, other costimulatory elements are within the scope of this disclosure.

[0182] The cytoplasmic signaling sequences in the cytoplasmic signaling portion of the CAR can be linked together randomly or in a specified order. Optionally, a short oligopeptide or polypeptide linker, preferably between 2 and 10 amino acids in length, can form the linkage. Glycine-serine duplexes provide a particularly suitable linker.

[0183] 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 and the signaling domains of CD28 and 4-1BB.

[0184] In one embodiment, the intracellular domain in the CAR is designed to comprise the signaling domain of 4-1BB and the signaling domain of CD3-zeta, where the signaling domain of 4-1BB comprises the nucleic acid sequence set forth in SEQ ID NO:33 or SEQ ID NO:73, and the signaling domain of CD3-zeta comprises the nucleic acid sequence set forth in SEQ ID NO:35, SEQ ID NO:47, SEQ ID NO:61, or SEQ ID NO:75.

[0185] In one embodiment, the intracellular domain in the CAR is designed to comprise the signaling domain of 4-1BB and the signaling domain of CD3-zeta, where the signaling domain of 4-1BB comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:34 or SEQ ID NO:74, respectively, and the signaling domain of CD3-zeta comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:36, SEQ ID NO:48, SEQ ID NO:62, or SEQ ID NO:76.

[0186] In one embodiment, the intracellular domain in the CAR is designed to comprise the signaling domain of 4-1BB and the signaling domain of CD3-zeta, where the signaling domain of 4-1BB comprises the amino acid sequence set forth in SEQ ID NO:34, or SEQ ID NO:74, respectively, and the signaling domain of CD3-zeta comprises the amino acid sequence set forth in SEQ ID NO:36, SEQ ID NO:48, SEQ ID NO:62, or SEQ ID NO:76, respectively.

[0187] In one embodiment, the intracellular domain in the CAR is designed to comprise the signaling domain of CD28 and the signaling domain of CD3-zeta, where the signaling domain of CD28 comprises the nucleic acid sequence set forth in SEQ ID NO:45 or SEQ ID NO:59, respectively, and the signaling domain of CD3-zeta comprises the nucleic acid sequence set forth in SEQ ID NO:35, SEQ ID NO:47, or SEQ ID NO:61, respectively.

[0188] In one embodiment, the intracellular domain in the CAR is designed to comprise the signaling domain of CD28 and the signaling domain of CD3-zeta, where the signaling domain of CD28 comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:46, or SEQ ID NO:60, respectively, and the signaling domain of CD3-zeta comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:36, or SEQ ID NO:48, or SEQ ID NO:62.

[0189] In one embodiment, the intracellular domain in the CAR is designed to comprise the signaling domain of CD28 and the signaling domain of CD3-zeta, where the signaling domain of CD28 comprises the amino acid sequence set forth in SEQ ID NO:46 or SEQ ID NO:60, respectively, and the signaling domain of CD3-zeta comprises the amino acid sequence set forth in SEQ ID NO:36, SEQ ID NO:48, or SEQ ID NO:62, respectively.

[0190] 5. Further description of CAR The functional portion of the CAR disclosed herein is also expressly included within the scope of the present invention. The term "functional portion" when used in relation to a CAR refers to any one or more parts or fragments of the CAR disclosed herein, which part or fragment retains the biological activity of the CAR of which it is a part (parent CAR). A functional portion includes, for example, a part of a CRA that retains the ability to recognize target cells or detect, treat or prevent disease to a similar extent, the same extent, or a higher extent than the parent CAR. With respect to a parent CAR, a functional portion may, for example, comprise about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95% or more of the parent CAR.

[0191] A functional part may contain additional amino acids at the amino or carboxy terminus of the part, or at both termini, which additional amino acids are not found in the amino acid sequence of parent CAR.Desirably, the additional amino acids do not interfere with the biological function of the functional part, such as, for example, recognizing target cells, detecting cancer, treating or preventing cancer, etc.More desirably, the additional amino acids enhance the biological activity of the functional part, compared to the biological activity of parent CAR.

[0192] The functional variants of the CARs disclosed herein are included within the scope of this disclosure. The term "functional variant" as used herein refers to a CAR, polypeptide or protein that has substantial or significant sequence identity or similarity to the parent 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 (parent CARs) described herein that retain the ability to recognize target cells to a similar degree, the same degree, or a higher degree than the parent CAR. With respect to the parent CAR, the functional variant can be, for example, at least about 30%, 50%, 75%, 80%, 90%, 98% or more identical in amino acid sequence to the parent CAR.

[0193] A functional variant may, for example, comprise the amino acid sequence of a parent CAR with at least one conservative amino acid substitution. Alternatively, or in addition, a functional variant may comprise the amino acid sequence of a parent CAR with at least one non-conservative amino acid substitution. In this case, it is preferred that the non-conservative amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. The non-conservative amino acid substitution may enhance the biological activity of the functional variant, so that the biological activity of the functional variant is increased compared to the parent CAR.

[0194] The amino acid substitutions in 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, a conservative amino acid substitution can be an acidic / negatively charged polar amino acid substituting another acidic / negatively charged polar amino acid (e.g., Asp or Glu), an amino acid with a nonpolar side chain substituting another amino acid with a nonpolar side chain (e.g., Ala, Gly, Val, He, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), a basic / positively charged polar amino acid substituting another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.), an uncharged amino acid with a polar side chain substituting another uncharged amino acid with a polar side chain (e.g., Asn, Gin, Ser, Thr, Tyr, etc.), an amino acid with a beta-branched side chain substituting another amino acid with a beta-branched side chain (e.g., He, Thr and Val), an amino acid with an aromatic side chain substituting another amino acid with an aromatic side chain (e.g., His, Phe, Trp and Tyr), etc.

[0195] A CAR can consist essentially of the specified amino acid sequence(s) described herein, such that other components, e.g., other amino acids, do not significantly alter the biological activity of the functional variant.

[0196] CARs (including functional portions and functional variants) can be of any length, i.e., comprise any number of amino acids, provided that the CARs (or functional portions or variants thereof) retain their biological activity, 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, etc. For example, CARs 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.

[0197] CARs (including functional portions and functional variants of the invention) can include synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, -amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3-hydroxyproline and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, a-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydrofuran ... These include 3-aminopropyl ...

[0198] CARs (including functional portions and functional variants) can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized, e.g., via disulfide bridges, or converted into acid addition salts and / or optionally dimerized or polymerized, or conjugated.

[0199] CAR (including its functional part and functional variant) can be obtained by methods known in the art. CAR can be produced by any suitable method of producing polypeptide or protein. Suitable methods for de novo synthesis of polypeptide and protein are described in references such as Chan et al., Fmoc Solid Phase Peptide Synthesis, Oxford University Press, Oxford, United Kingdom, 2000; Peptide and Protein Drug Analysis, Reid, R., ed., Marcel Dekker, Inc., 2000; Epitope Mapping, Westwood et al., ed., Oxford University Press, Oxford, United Kingdom, 2001; and U.S. Patent No. 5,449,752. Polypeptides and proteins can also be produced recombinantly using the nucleic acid described herein using standard recombinant methods. 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. In addition, some CARs (including functional parts and functional variants thereof) can be isolated and / or purified from sources such as plants, bacteria, insects, mammals, e.g., rats, humans, etc. Isolation and purification methods are well known in the art. Alternatively, the CARs described herein (including functional parts and functional variants thereof) can be commercially synthesized by companies. In this regard, CARs can be synthetic, recombinant, isolated, and / or purified.

[0200] A. Antibodies and Antigen-Binding Fragments One embodiment further provides a CAR, a T cell expressing a CAR, 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, a "T cell expressing a CAR" or a "CAR T cell" refers to a T cell that expresses a CAR, e.g., has an antigen specificity determined by the antibody-derived targeting domain of the CAR.

[0201] As used herein, "antigen-binding domain" can include antibodies and antigen-binding fragments thereof. The term "antibody" is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antigen-binding fragments thereof, so long as they exhibit the desired antigen-binding activity. Non-limiting examples of antibodies include, for example, intact immunoglobulins known in the art, as well as variants and fragments thereof that retain binding affinity to antigens.

[0202] A "monoclonal antibody" is an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies that make up the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and directed against a single antigenic epitope. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. In some examples, a monoclonal antibody is an antibody produced by a single clone of B lymphocytes, or by a cell transfected with nucleic acid encoding the antibody light and heavy chain variable regions of a single antibody (or antigen-binding fragment thereof), or its progeny. In some examples, a monoclonal antibody is isolated from a subject. A monoclonal antibody may have conservative amino acid substitutions that have substantially no effect on antigen binding or other immunoglobulin functions. Exemplary methods for producing monoclonal antibodies are known, see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual, 2nd ed. Cold Spring Harbor Publications, New York (2013).

[0203] Typically, immunoglobulins have heavy (H) and light (L) chains interconnected by disulfide bonds. Immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as a myriad of 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 the antibody molecule: IgM, IgD, IgG, IgA and IgE.

[0204] 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., WH 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 only of heavy chains are functional and stable in the absence of light chains (see, e.g., Hamers-Casterman et al., Nature, 363:446-448, 1993; Sheriff et al., Nat. Struct. Biol., 3:733-736, 1996). References to "VH" or "VH" refer to the variable region of an antibody heavy chain, including those of an antigen-binding fragment, e.g., Fv, scFv, dsFv or Fab. References to "VL" or "VL" refer to the variable domain of an antibody light chain, including that of an Fv, scFv, dsFv or Fab.

[0205] The variable regions of the light and heavy chains contain a "framework" region interrupted by three hypervariable regions, also called "complementarity determining regions" or "CDRs" (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, US 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 regions of the constituent light and heavy chains, functions to position and align the CDRs in three-dimensional space.

[0206] CDRs are primarily responsible for binding to an epitope of an antigen. The amino acid sequence boundaries of a given CDR can be easily determined using any of several well-known schemes, including those described in Kabat et al. ("Sequences of Proteins of Immunological Interest", 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991; "Kabat" numbering scheme), Al-Lazikani et al. (JMB 273, 927-948, 1997; "Chothia" numbering scheme), and Lefranc et al. ("IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains", Dev. Comp. Immunol., 27:55-77, 2003; "IMGT" numbering scheme). The CDRs of each chain are typically referred to as CDR1, CDR2 and CDR3 (N-terminus to C-terminus) and are typically also identified by the chain in which the particular CDR is located. Thus, a VH CDR3 is the CDR3 from the variable domain of the heavy chain of the antibody in which it is found, while a VL CDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found. The light chain CDRs are sometimes referred to as LCDR1, LCDR2 and LCDR3. The heavy chain CDRs are sometimes referred to as HCDR1, HCDR2 and HCDR3.

[0207] "Antigen-binding fragments" are portions of full-length antibodies that retain the ability to specifically recognize their cognate antigen, as well as various combinations of such portions. Non-limiting examples of antigen-binding fragments include Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. Antibody fragments include antigen-binding fragments produced by engineering whole antibodies or de novo synthesized using recombinant DNA methodology (see, e.g., Kontermann and Dubel (eds.), Antibody Engineering, Vol. 1-2, 2nd ed., Springer Press, 2010).

[0208] Single-chain antibodies (scFv) are genetically engineered molecules that contain the VH and VL domains of one or more antibodies (or antibodies) as a genetically fused single-chain molecule linked by a suitable polypeptide linker (see, for example, Bird et al., Science, 242:423-426, 1988; Huston et al., Proc. Natl. Acad. Sci., 85:5879-5883, 1988; Ahmad et al., Clin. Dev. Immunol., 2012, doi:10.1155 / 2012 / 980250; Marbry, IDrugs, 13:543-549, 2010). The intramolecular orientation of the VH and VL domains in scFvs is typically not critical for scFvs. Thus, scFvs with both possible configurations (VH domain-linker domain-VL domain; VL domain-linker domain-VH domain) can be used.

[0209] In dsFv, the variable chains of heavy and light chains are mutated to introduce disulfide bonds to stabilize the association of the chains. Also included are diabodies, which are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but use a linker that is too short to allow pairing between the two domains on the same chain, thereby pairing the domains with the complementary domains of another chain, creating two antigen binding sites (see, for example, Holliger et al., Proc. Natl. Acad. Sci., 90:6444-6448, 1993; Poljak et al., Structure, 2:1121-1123, 1994).

[0210] Antibodies also include genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies) and heteroconjugate antibodies (e.g., bispecific antibodies). See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rd ed., WH Freeman & Co., New York, 1997.

[0211] Non-naturally occurring antibodies can be constructed using solid phase peptide synthesis, can be produced recombinantly, or can be obtained by screening combinatorial libraries consisting of variable heavy and variable light chains, for example, as described in Huse et al., Science 246:1275-1281 (1989), which is incorporated herein by reference. These and other methods of making, for example, chimeric, humanized, CDR-grafted, single chain and bifunctional antibodies are well known to those of skill in the art (Winter and Harris, Immunol. Today 14:243-246 (1993); Ward et al., Nature 341:544-546 (1989); Harlow and Lane, supra, 1988; Hilyard et al., Protein Engineering: A practical approach (IRL Press 1992); Borrabeck, Antibody Engineering, 2nd Edition (Oxford University Press 1995); each of which is incorporated herein by reference).

[0212] An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks 50% or more of the binding of the reference antibody to its antigen in a competitive assay, and conversely, the reference antibody blocks 50% or more of the binding of the antibody to its antigen in a competitive assay. Antibody competition assays are known, and exemplary competition assays are provided herein.

[0213] A "humanized" antibody or antigen-binding fragment comprises a human framework region and one or more CDRs derived from a non-human (e.g., mouse, rat or synthetic) antibody or antigen-binding fragment. The non-human antibody or antigen-binding fragment providing the CDRs is referred to as the "donor" and the human antibody or antigen-binding fragment providing the framework is referred to as the "acceptor". In one embodiment, all CDRs are derived from a donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if present, can be substantially identical, e.g., at least about 85-90%, e.g., about 95% or more identical, to human immunoglobulin constant regions. Thus, all parts of a humanized antibody or antigen-binding fragment are substantially identical to the corresponding parts of a natural human antibody sequence, except possibly for the CDRs.

[0214] A "chimeric antibody" is an antibody that contains sequences from two different antibodies, typically from different species. In some instances, a chimeric antibody contains one or more CDRs and / or framework regions from one human antibody and the CDRs and / or framework regions from another human antibody.

[0215] A "fully human antibody" or "human antibody" is an antibody that contains sequences from the human genome, but does not contain sequences from another species. In some embodiments, a human antibody contains CDRs, framework regions, and (if present) Fc regions from the human genome. Human antibodies can be identified and isolated, for example, by phage display, using technology to create sequences based on sequences from the human genome, or 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; Lonenberg, Curr. Opin. Immunol., 20:450-459, 2008).

[0216] An antibody may have one or more binding sites. If there is more than one binding site, these binding sites may be identical to each other or different. For example, naturally occurring immunoglobulins have two identical binding sites, single-chain antibodies or Fab fragments have one binding site, while bispecific or bifunctional antibodies have two different binding sites.

[0217] Methods for testing antibodies for the ability to bind to any functional portion of a CAR are known in the art and include any antibody-antigen binding assay, such as radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation and competitive inhibition assays (see, e.g., Janeway et al., infra, U.S. Patent Application Publication No. 2002 / 0197266 A1, and U.S. Patent No. 7,338,929).

[0218] In addition, CARs, T cells expressing CARs, antibodies or antigen-binding portions thereof can be modified to include detectable labels, such as radioisotopes, fluorophores (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), enzymes (e.g., alkaline phosphatase, horseradish peroxidase), and elemental particles (e.g., gold particles).

[0219] B. Conjugates CARs, T cells expressing CARs, or monoclonal antibodies or antigen-binding fragments thereof specific for one or more of the antigens disclosed herein can be conjugated to agents such as effector molecules or detectable markers using a number of means known to those skilled in the art. Both covalent and non-covalent means can be used. Conjugates include, but are not limited to, molecules in which there is 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 skilled in the art will be familiar with the use of conjugated agents such as chemotherapeutic agents, antiangiogenic agents, toxins, radioactive agents, e.g. 125 I, 32 P, 14 C. 3 H and 35 It will be understood that a variety of effector molecules and detectable markers may be used, including, but not limited to, S, as well as other labels, targeting moieties and ligands.

[0220] The choice of a particular effector molecule or detectable marker will depend on the particular target molecule or cell, and the desired biological effect. Thus, for example, an effector molecule can be a cytotoxin used to effect the death of a particular target cell (e.g., a tumor cell).

[0221] The procedure for binding 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 a variety of functional groups; for example, carboxylic acid (COOH), free amine (-NH2) or sulfhydryl (-SH) groups, which are available for reaction with a suitable functional group on an antibody to result in the binding of an effector molecule or detectable marker. Alternatively, the antibody or antigen-binding fragment is derivatized to expose or bind additional reactive functional groups. The derivatization may include the attachment of any of several known linker molecules, such as those available from Pierce Chemical Company, Rockford, IL. The linker may be any molecule used to bind an antibody or antigen-binding fragment to an effector molecule or detectable marker. The linker is capable of forming a covalent bond to both the antibody or antigen-binding fragment and the effector molecule or detectable marker. Suitable linkers are well known to those skilled in the art, including, but not limited to, straight 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 linkers may be attached to the constituent amino acids through their side groups (e.g., via a disulfide linkage to a cysteine) or to the amino and carboxyl groups of the alpha carbon of the terminal amino acid.

[0222] In some embodiments, the linker may include a spacer element, which, if present, increases the size of the linker, thereby increasing the distance between the effector molecule or detectable marker and the antibody or antigen-binding fragment. Exemplary spacers are known to those of skill in the art, including, for example, U.S. Pat. Nos. 7,964,566, 7,498,298, 6,884,869, 6,323,315, 6,239,104, 6,034,065, 5,780,588, 5,665,860, 5,663,149, 5,635,483, 5,599,902, 5,554,725, 5,530,097, 5,521, 5,532, 5,534, 5,536, 5,538, 5,539 ... No. 5,284, U.S. Pat. No. 5,504,191, U.S. Pat. No. 5,410,024, U.S. Pat. No. 5,138,036, U.S. Pat. No. 5,076,973, U.S. Pat. No. 4,986,988, U.S. Pat. No. 4,978,744, U.S. Pat. No. 4,879,278, U.S. Pat. No. 4,816,444 and U.S. Pat. No. 4,486,414, as well as those listed in U.S. Patent Application Publication No. 20110212088 and U.S. Patent Application Publication No. 20110070248, each of which is incorporated herein by reference in its entirety.

[0223] 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 cleavage agent present in the intracellular environment (e.g., within a lysosome or endosome or caveolae). The linker can be, for example, a peptide linker that is cleaved by an intracellular peptidase or protease enzyme, including, but not limited to, a lysosomal or endosomal protease. In some embodiments, the peptide linker is at least 2 amino acids long or at least 3 amino acids long. However, the linker can be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids long, for example, 1-2, 1-3, 2-5, 3-10, 3-15, 1-5, 1-10, 1-15 amino acids long. Proteases may include cathepsin B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives resulting in the release of active drug inside target cells (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). For example, a peptide linker cleavable by the thiol-dependent protease cathepsin B may be used (e.g., phenylalanine-leucine or glycine-phenylalanine-leucine-glycine linker). Other examples of such linkers are described, for example, in U.S. Pat. No. 6,214,345, which is incorporated herein by reference. In a specific embodiment, the peptide linker cleavable by intracellular proteases is a valine-citrulline linker or a phenylalanine-lysine linker (see, e.g., U.S. Pat. No. 6,214,345, which describes the synthesis of doxorubicin using a valine-citrulline linker).

[0224] In other embodiments, the cleavable linker is pH sensitive, i.e., sensitive to hydrolysis at a certain pH value. Typically, pH sensitive linker is hydrolyzable under acidic conditions. For example, acid labile linker (e.g., hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal, etc.) that is hydrolyzable in lysosomes can be used (see, for example, U.S. Pat. No. 5,122,368; U.S. Pat. No. 5,824,805; U.S. Pat. No. 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661). Such linkers are relatively stable under neutral pH conditions, such as in blood, but are unstable at pH below 5.5 or 5.0, which is approximately the pH of the lysosome. In certain embodiments, the hydrolyzable linker is a thioether linker, such as a thioether attached to a therapeutic agent via an acylhydrazone bond (see, e.g., U.S. Patent No. 5,622,929).

[0225] In other embodiments, the linker is cleavable under reducing conditions (eg, a disulfide linker). A variety of disulfide linkers are known in the art, including, for example, those that can be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene)-, SPDB and SMPT (see, e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (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.

[0226] 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).

[0227] 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, the entire contents of which are incorporated herein by reference).

[0228] In some embodiments, the linker is resistant to cleavage in extracellular environment.For example, when the conjugate is present in extracellular environment (e.g., in plasma), about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 3% or less, or about 1% or less of the linker in the sample of the conjugate is cleaved.Whether the linker is resistant to cleavage in extracellular environment can be determined, for example, by incubating the conjugate containing the desired linker with plasma for a predetermined period (e.g., 2, 4, 8, 16 or 24 hours), and then quantifying the amount of free effector molecule or detectable marker present in plasma. Various exemplary linkers that may be used in the conjugates are described in WO2004-010957, U.S. Patent Application Publication No. 2006 / 0074008, U.S. Patent Application Publication No. 20050238649 and U.S. Patent Application Publication No. 2006 / 0024317, each of which is incorporated herein by reference in its entirety.

[0229] In some embodiments, conjugates of a CAR, a T cell expressing a CAR, an antibody or antigen-binding portion thereof, and one or more small molecule toxins, such as calicheamicin, maytansinoids, dolastatins, auristatins, trichothecins, and CC1065, and derivatives of these toxins that have toxin activity, are provided.

[0230] Maytansine compounds suitable for use as maytansinoid toxin moieties are well known in the art and may be isolated from natural sources according to known methods, produced using genetic engineering techniques (see Yu et al. (2002) PNAS 99:7968-7973), or synthetically prepared maytansinol and maytansinol analogs according to known methods. Maytansinoids are mitotic inhibitors that act by inhibiting tubulin polymerization. Maytansine was first isolated from the East African shrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it was discovered that certain microorganisms also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042). Synthetic maytansinol and its derivatives and analogs are described, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428; and 4,31 Nos. 3,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533, each of which is incorporated herein by reference. Conjugates containing maytansinoids, methods of making same, and their therapeutic uses are disclosed, for example, in U.S. Patent Nos. 5,208,020; 5,416,064; 6,441,163 and European Patent EP 0 425 235 B1, the disclosures of which are expressly incorporated herein by reference.

[0231] Additional toxins can be used with CAR, CAR-expressing T cells, antibodies, or antigen-binding portions thereof. Exemplary toxins include Pseudomonas exotoxin (PE), castor toxin, abrin, diphtheria toxin and its subunits, ribotoxin, ribonuclease, saporin and calicheamicin, and botulinum toxins A-F. These toxins are well known in the art, and many are readily available from commercial sources (e.g., Sigma Chemical Company, St. Louis, MO). Contemplated toxins also include variants of these toxins (see, e.g., U.S. Patent No. 5,079,163 and U.S. Patent No. 4,689,401).

[0232] Saporin is a toxin derived from Saponaria officinalis that disrupts protein synthesis by inactivating the 60S portion of the ribosomal complex (Stirpe et al., Bio / Technology 10:405-412, 1992). However, this toxin has no mechanism for specific entry into cells and therefore requires conjugation to an antibody or antigen-binding fragment that recognizes a cell surface protein that is to be internalized in order to be efficiently taken up by cells.

[0233] Diphtheria toxin is isolated from Corynebacterium diphtheriae. Typically, diphtheria toxin for use in immunotoxins is mutated to reduce or eliminate non-specific toxicity. A mutant 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.

[0234] Castor toxin is the lectin RCA60 from Ricinus communis (castor bean). For examples of castor toxins, see U.S. Patent No. 5,079,163 and U.S. Patent No. 4,689,401. Ricinus communis agglutinin (RCA) is a lectin derived from the plant Ricinus communis (castor bean), which is a lectin derived from the plant Ricinus communis. 60 and R.C.A. 120 (Nicholson and Blaustein, J. Biochim. Biophys. Acta 266:543, 1972). The A chain is responsible for inactivating protein synthesis and killing the cell. The B chain binds castor toxin to cell surface galactose residues and facilitates transport of the A chain into the cytosol (Olsnes et al., Nature 249:627-631, 1974 and U.S. Patent No. 3,060,165).

[0235] Ribonucleases have also been conjugated to targeting molecules for use as immunotoxins (see Suzuki et al., Nat. Biotech. 17:265-70, 1999). Exemplary ribotoxins, such as α-sarcin and restrictocin, are discussed, for example, in Rathore et al., Gene 190:31-5, 1997; and Goyal and Batra, Biochem. 345(2):247-54, 2000. Calicheamicin was originally isolated from Micromonospora echinospora and is a member of the enediyne antitumor antibiotic family, producing double-strand breaks in DNA that lead to apoptosis (see, for example, Lee et al., J. Antibiot. 42:1070-87, 1989). This drug is the toxic moiety of an immunotoxin undergoing clinical trials (see, eg, Gillespie et al., Ann. Oncol. 11:735-41, 2000).

[0236] Abrin includes toxic lectins from Abrus precatorius. The toxicants abrins a, b, c, and d have molecular weights of approximately 63 kD and 67 kD and are composed of two disulfide-linked polypeptide chains A and B. The A chain inhibits protein synthesis; the B chain (abrin-b) binds to D-galactose residues (see Funatsu et al., Agr. Biol. Chem. 52:1095, 1988; and Olsnes, Methods Enzymol. 50:330-335, 1978).

[0237] The CAR, T cells expressing the CAR, 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, detectable by ELISA, spectrophotometry, flow cytometry, microscopy or imaging techniques (e.g., computed tomography (CT), computed axial tomography (CAT) scan, magnetic resonance imaging (MRI), nuclear magnetic resonance imaging (NMRI), magnetic resonance tomography (MTR), ultrasound, fiber optic testing and laparoscopic testing). Specific non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzymatic linkages, radioactive isotopes and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI). For example, useful detectable markers include fluorescent compounds including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, and the like. Bioluminescent markers such as luciferase, green fluorescent protein (GFP), yellow fluorescent protein (YFP), and the like are also used. CARs, T cells expressing CARs, antibodies, or antigen-binding portions thereof, can also be conjugated to enzymes useful for detection, such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, glucose oxidase, and the like. When CARs, T cells expressing CARs, antibodies, or antigen-binding portions thereof, are conjugated to detectable enzymes, they can be detected by adding additional reagents that the enzyme uses to produce a discernible reaction product. For example, when the agent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine results in a visually detectable colored reaction product. CARs, T cells expressing CARs, antibodies or antigen-binding portions thereof can also be conjugated with biotin and detected via indirect measurement of avidin or streptavidin binding. It should be noted that avidin itself can be conjugated with an enzyme or fluorescent label.

[0238] CAR, T cells expressing CAR, antibodies or antigen-binding portions thereof can be conjugated with paramagnetic agents such as gadolinium. Paramagnetic agents such as superparamagnetic iron oxide are also used as labels. Antibodies can also be conjugated with lanthanides (e.g., europium and dysprosium) and manganese. Antibodies or antigen-binding fragments can also be labeled with a predetermined polypeptide epitope (e.g., leucine zipper pair sequence, binding site for secondary antibody, metal binding domain, epitope tag) recognized by a secondary reporter.

[0239] CAR, CAR-expressing T cells, antibodies or antigen-binding portions thereof can also be conjugated with radiolabeled amino acids. Radiolabels can be used for both diagnostic and therapeutic purposes. For example, radiolabels can be used to detect one or more of the antigens and antigen-expressing cells disclosed herein by x-ray, emission spectroscopy or other diagnostic techniques. In addition, radiolabels can be used therapeutically as toxins for the treatment of tumors in subjects, for example, 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.

[0240] Means for detecting such detectable markers are well known to those skilled in the art. Thus, for example, radiolabels can be detected using photographic film or scintillation counters, fluorescent markers can be detected using a photodetector to detect emitted illumination. 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.

[0241] C. Nucleotides, Expression, Vectors and Host Cells Further provided by one embodiment of the present invention is a nucleic acid comprising a nucleotide sequence encoding any of the CARs, antibodies or antigen-binding portions thereof (including functional portions and functional variants thereof) described herein. The nucleic acid of the present invention may comprise a nucleotide sequence encoding any of the leader sequences, antigen-binding domains, transmembrane domains and / or intracellular T cell signaling domains described herein.

[0242] In some embodiments, the nucleotide sequence can be codon modified.Without being bound by any particular theory, it is believed that the codon optimization of the nucleotide sequence increases the translation efficiency of the mRNA transcript.The codon optimization of the nucleotide sequence can include replacing the native codon with another codon that codes for the same amino acid but can be translated by the tRNA that is more readily available in the cell, thus increasing the translation efficiency.The optimization of the nucleotide sequence can also reduce the secondary mRNA structure that interferes with translation, thus increasing the translation efficiency.

[0243] In an embodiment of the invention, a nucleic acid can comprise a codon-modified nucleotide sequence encoding the antigen-binding domain of a CAR of the invention. In another embodiment of the invention, a nucleic acid can comprise a codon-modified nucleotide sequence encoding any of the CARs described herein, including functional portions and functional variants thereof.

[0244] "Nucleic acid", as used herein, includes "polynucleotide", "oligonucleotide" and "nucleic acid molecule" and generally refers to a polymer of DNA or RNA that may be single-stranded or double-stranded, synthetic or derived from a natural source (e.g., isolated and / or purified), may contain natural, non-natural or modified nucleotides, and may contain natural, non-natural or modified internucleotide linkages, such as phosphoroamidate or phosphorothioate linkages, instead of the phosphodiesters found between nucleotides of unmodified oligonucleotides. In some embodiments, the nucleic acid does not contain any insertions, deletions, inversions and / or substitutions. However, in some cases, as discussed herein, it may be appropriate for the nucleic acid to contain one or more insertions, deletions, inversions and / or substitutions.

[0245] Recombinant nucleic acids may have sequences that do not occur in nature, or sequences that are created by the artificial combination of two otherwise separated segments of sequences. This artificial combination is often achieved by chemical synthesis, or more commonly, by the artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques, such as those described in Sambrook et al., supra. Nucleic acids can be constructed based on chemical synthesis and / or enzymatic ligation reactions using procedures known in the art. See, for example, Sambrook et al., supra and Ausubel et al., supra. For example, nucleic acids can be chemically synthesized using naturally occurring nucleotides, or variously modified nucleotides (e.g., phosphorothioate derivatives and acridine-substituted nucleotides) designed to increase the biological stability of the molecule or to increase the physical stability of the duplex formed upon hybridization.Examples of modified nucleotides that can be used to generate nucleic acids include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-substituted adenines, 7-methylguanine, 8-methylguanine, 9-methylguanine, 10-methylguanine, 11-methylguanine, 12-methylguanine, 13-methylguanine, 14-methylguanine, 15-methylguanine, 16-methylguanine, 17-methylguanine, 18-methylguanine, 19-methylguanine, 20-methylguanine, 21-methylguanine, 22-methylguanine, 23-methylguanine, 24-methylguanine, 25-methylguanine, 26-methylguanine, 27-methylguanine, 28-methylguanine, 29-methylguanine, 30-methylguanine, 31-methylguanine, 32-methylguanine, 33-methylguanine, 34-methylguanine, 35-methylguanine, 36-methylguanine, 37-methylguanine, 38-methylguanine, 39-methylguanine, 39-methylguanine, 32-methylguanine, Examples of nucleic acids include, but are not limited to, uracil-5-oxyacetic acid (v), ubutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleic acids of the present invention can be purchased from a company such as Integrated DNA Technologies (Coralville, IA, USA).

[0246] The nucleic acid can include any isolated or purified nucleotide sequence encoding a CAR or any of its functional portions or variants. Alternatively, the nucleotide sequence can include a nucleotide sequence that is degenerate to any of the sequences, or a combination of degenerate sequences.

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

[0248] Nucleotide sequences that hybridize under stringent conditions may hybridize under high stringency conditions. "High stringency conditions" means that a nucleotide sequence specifically hybridizes to a target sequence (any nucleotide sequence of a nucleic acid described herein) in an amount detectably stronger than non-specific hybridization. High stringency conditions include conditions that distinguish polynucleotides with exactly complementary sequences, or polynucleotides that contain only a few scattered mismatches, from random sequences that happen to have some small regions (e.g., 3-10 bases) that match the nucleotide sequence. Such small regions of complementarity are more easily melted than full-length complements of 14-17 or more bases, and high stringency hybridization makes them easily distinguishable. Relatively high stringency conditions include low salt and / or high temperature conditions, such as those provided by about 0.02-0.1 M NaCl or equivalent at temperatures of about 50-70°C. Such high stringency conditions tolerate little, if any, mismatch between the nucleotide sequence and the template or target strand and are particularly suitable for detecting the expression of any of the CARs of the present invention. It is generally understood that conditions can be made more stringent by the addition of increasing amounts of formamide.

[0249] Also provided are nucleic acids comprising a nucleotide sequence that is at least about 70% or more, e.g., about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to any of the nucleic acids described herein.

[0250] In one embodiment, the nucleic acid may be incorporated into a recombinant expression vector. In this regard, one embodiment provides a recombinant expression vector that includes any of the nucleic acids. For the purposes of this specification, the term "recombinant expression vector" refers to a genetically modified oligonucleotide or polynucleotide construct that allows expression of an mRNA, protein, polypeptide, or peptide by a host cell when the construct includes a nucleotide sequence that codes for an mRNA, protein, polypeptide, or peptide, and when the vector is contacted with a cell under conditions sufficient for the mRNA, protein, polypeptide, or peptide to be expressed in the cell. A vector does not exist in nature as a whole.

[0251] However, some of the vectors may be naturally occurring. Recombinant expression vectors may be single-stranded or double-stranded, synthetic or partially derived from natural sources, and may contain any type of nucleotide, including but not limited to DNA and RNA, which may contain natural, non-natural or modified nucleotides. Recombinant expression vectors may contain naturally occurring or non-naturally occurring internucleotide linkages, or both types of linkages. Preferably, non-naturally occurring or modified nucleotides or internucleotide linkages do not interfere with the transcription or replication of the vector.

[0252] In one embodiment, the recombinant expression vector may be any suitable recombinant expression vector and may be used to transform or transfect any suitable host cell. Suitable vectors include those designed for propagation and propagation or for expression, or both, such as plasmids and viruses. The vector may be selected from the group consisting of pUC series (Fermentas Life Sciences, Glen Burnie, MD), pBluescript series (Stratagene, LaJolla, CA), pET series (Novagen, Madison, WI), pGEX series (Pharmacia Biotech, Uppsala, Sweden) and pEX series (Clontech, Palo Alto, CA).

[0253] Bacteriophage vectors, such as λυΤΙΟ, λυΤΙ 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 are vectors derived from at least a portion of the lentiviral genome, including, inter alia, self-inactivating lentiviral vectors as provided in Milone et al., Mol. Ther. 17(8):1453-1464 (2009). Other examples of lentiviral vectors that may be used in the clinic include, for example and without limitation, Oxford BioMedica plc's LENTIVECTOR® Gene Delivery Technology, Lentigen's LENTIMAX™ Vector System, etc. Non-clinical forms of lentiviral vectors are also available and known to those of skill in the art.

[0254] 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)).

[0255] Transfection methods include calcium phosphate coprecipitation (see, e.g., Graham et al., supra), direct microinjection into cultured cells (see, e.g., Capecchi, Cell 22:479-488 (1980)), electroporation (see, e.g., Shigekawa et al., BioTechniques 6:742-751 (1988)), liposome-mediated gene transfer (see, e.g., Mannino et al., BioTechniques 6:682-690 (1988)), lipid-mediated transduction (see, e.g., Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987)), and high velocity micropropellants. Examples of nucleic acid delivery methods include delivery of nucleic acids using microprojectiles (see, for example, Klein et al., Nature, 327:70-73 (1987)).

[0256] In one embodiment, recombinant expression vectors can be prepared using standard recombinant DNA techniques, for example, as described in Sambrook et al., supra and Ausubel et al., supra. The constructs of expression vectors, circular or linear, can be prepared to contain a functional replication system in prokaryotic or eukaryotic host cells. Replication systems can be derived from, for example, ColEl, 2μ plasmid, lambda, SV40, bovine papilloma virus, etc.

[0257] Recombinant expression vectors, taking into account whether the vector is DNA- or RNA-based, may optionally include regulatory sequences, such as transcriptional and translational initiation and termination codons, specific for the type of host cell into which the vector will be introduced (e.g., bacterial, fungal, plant or animal). Recombinant expression vectors may include restriction sites to facilitate cloning.

[0258] The recombinant expression vector may contain one or more marker genes that allow for the selection of transformed or transfected host cells. Marker genes include biocide resistance, resistance to, e.g., antibiotics, heavy metals, etc., complementation in auxotrophic hosts to provide prototrophy, etc. Suitable marker genes for the expression vector 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.

[0259] The recombinant expression vector may comprise a native or non-native promoter operably linked to the nucleotide sequence encoding the CAR (including its functional parts and functional variants) or to a nucleotide sequence that is complementary to or hybridizes with the nucleotide sequence encoding the CAR. The selection of promoters, such as strong, weak, inducible, tissue-specific and development-specific, is within the skill of the artisan. Similarly, combining a nucleotide sequence with a promoter is also within the skill of the artisan. The promoter may be a non-viral promoter or a viral promoter, such as a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, or a promoter found in the long terminal repeat of mouse stem cell virus.

[0260] Recombinant expression vectors can be designed for transient expression, stable expression, or both, and can be made for constitutive or inducible expression.

[0261] Furthermore, recombinant expression vectors can be made to contain suicide genes. As used herein, the term "suicide gene" refers to a gene that causes the cell that expresses the suicide gene to die. A suicide gene can be a gene that confers sensitivity to an agent, such as a drug, to the cell in which the gene is expressed, or a gene that causes the cell to die when the cell comes into contact with or is exposed to an 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, herpes simplex virus (HSV) thymidine kinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase and nitroreductase.

[0262] An 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 may contain a recombinant expression vector of the present invention. The host cell may be a eukaryotic cell, such as a plant, animal, fungus or algae, or a prokaryotic cell, such as a bacterium or protist. The host cell may be a cultured cell or a primary cell, i.e., directly isolated from an organism, such as a human. The host cell may 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 a recombinant expression vector, the host cell may be a prokaryotic cell, such as a DH5a cell. For the purpose of producing a recombinant CAR, the host cell may be a mammalian cell. The host cell may be a human cell. The host cell may be of any cell type, originate from any type of tissue, and be at any stage of development, but the host cell may be a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC). The host cell may be a T cell.

[0263] For purposes herein, a T cell can be any T cell, e.g., a cultured T cell, e.g., a primary T cell, or a T cell from a cultured T cell line, e.g., 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 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 and at any stage of development, 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. The T cell can be a CD8+ T cell or a CD4+ T cell.

[0264] In one embodiment, the CARs described herein can be used in suitable non-T cells, such as cells with immune effector functions, such as NK cells and T-like cells generated from pluripotent stem cells.

[0265] A population of cells comprising at least one host cell described herein is also provided by one embodiment. The population of cells may be a heterogeneous population comprising host cells comprising any of the described recombinant expression vectors in addition to at least one other cell, e.g., a host cell (e.g., a T cell), that does not comprise any recombinant expression vector, or a cell other than a T cell, e.g., a B cell, a macrophage, a neutrophil, an erythrocyte, a hepatocyte, an endothelial cell, an epithelial cell, a muscle cell, a brain cell, etc. Alternatively, the population of cells may be a substantially homogenous population, where the population comprises primarily host cells comprising (e.g., essentially consisting of) a recombinant expression vector. The population may also be a clonal population of cells, where all cells of the population are clones of a single host cell comprising a recombinant expression vector, such that all cells of the population comprise that recombinant expression vector. In one embodiment of the present invention, the population of cells is a clonal population comprising host cells comprising a recombinant expression vector described herein.

[0266] CAR (including its functional parts and variants), nucleic acids, recombinant expression vectors, host cells (including populations thereof) and antibodies (including antigen-binding portions thereof) can be isolated and / or purified. For example, a purified (or isolated) host cell preparation is one in which the host cells are more pure than the cells in their natural environment in the body. Such host cells can be produced, for example, by standard purification techniques. In some embodiments, the host cell preparation is purified such that the host cells represent at least about 50%, e.g., at least about 70%, of the total cell content of the preparation. For example, the purity can be at least about 50%, can be greater than about 60%, about 70%, or about 80%, or can be about 100%.

[0267] D. Treatment Method It is contemplated that the CAR disclosed herein can be used in a method for treating or preventing disease in a mammal. In this regard, one embodiment provides a method for treating or preventing cancer in a mammal, comprising administering to a mammal a CAR, a nucleic acid, a recombinant expression vector, a host cell, a population of cells, an antibody and / or an antigen-binding portion thereof, and / or a pharmaceutical composition in an amount effective to treat or prevent cancer in the mammal.

[0268] One embodiment further comprises lymphodepleting the mammal prior to administering the CAR 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.

[0269] For the purposes of the method in which the host cell or a population of cells is administered, the cell can be an allogeneic or autologous cell to the mammal. Preferably, the cell is autologous to the mammal. As used herein, allogeneic refers to any material that is derived from a different animal of the same species as the individual to whom the material is introduced. Two or more individuals are said to be allogeneic to each other if the genes are not identical at one or more loci. In some embodiments, allogeneic material from individuals of the same species can be genetically different enough to interact antigenically. As used herein, "autologous" refers to any material that is derived from the same individual that is subsequently reintroduced into the individual.

[0270] The mammal referred to herein may be any mammal. As used herein, the term "mammal" refers to any mammal, including but not limited to mammals of the order Rodentia, e.g., mice and hamsters, and mammals of the order Logomorpha, e.g., rabbits. The mammal may be from the order Carnivora, including Feline (cat) and Canine (dog). The mammal may be from the order Artiodactyla, including Bovine (cow) and Swine (pig), or from the order Perssodactyla, including Equine (horse). The mammal may be from the order Primates, New World Ceboid or Simoid (monkey) or Anthropoid (human and ape). Preferably, the mammal is a human.

[0271] For these methods, the cancer may be acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer (e.g., bladder carcinoma), bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, cancer of the anus, anal canal, or anorectum, eye cancer, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, The cancer may be any cancer, including any of the following: colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, head and neck cancer (e.g., head and neck squamous cell carcinoma), Hodgkin's lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, leukemia, liquid tumors, liver cancer, lung cancer (e.g., non-small cell lung cancer and lung adenocarcinoma), lymphoma, mesothelioma, mast cell tumor, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin's lymphoma, B-chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia (ALL) and Burkitt's lymphoma, ovarian cancer, pancreatic cancer, peritoneal, omental and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumors, synovial sarcoma, gastric cancer, testicular cancer, thyroid cancer and ureteral cancer.

[0272] The terms "treat" and "prevent" and words derived therefrom, as used herein, do not necessarily mean 100% or complete treatment or prevention. Rather, there are various degrees of treatment or prevention that one skilled in the art recognizes as having a potential beneficial or therapeutic effect. In this regard, the method may provide any amount or level of treatment or prevention of cancer in a mammal.

[0273] Moreover, the treatment or prevention provided by this method can include treatment or prevention of one or more conditions or symptoms of the disease, such as cancer, being treated or prevented. For purposes herein, "prevention" can also include delaying the onset of the disease, or a symptom or condition thereof.

[0274] Another embodiment provides a method of detecting the presence of cancer in a mammal, the method comprising: (a) contacting a sample comprising one or more cells from the mammal with a CAR, a nucleic acid, a recombinant expression vector, a host cell, a population of cells, an antibody and / or antigen-binding portion thereof, or a pharmaceutical composition, thereby forming a complex, and (b) detecting the complex, wherein detection of the complex indicates the presence of cancer in the mammal.

[0275] The sample can be obtained by any suitable method, for example, biopsy or autopsy. Biopsy is the removal of tissue and / or cells from an individual. Such removal can be to collect tissue and / or cells from an individual in order to carry out experiments on the removed tissue and / or cells. This experiment can include experiments to determine whether the individual has and / or is suffering from a certain condition or disease state. The condition or disease can be, for example, cancer.

[0276] With respect to embodiments of the method of detecting the presence of a proliferation disorder, e.g., cancer, in a mammal, the sample comprising mammalian cells can be a sample comprising whole cells, a lysate thereof, or a fraction of a whole cell lysate, e.g., a nuclear or cytoplasmic fraction, a whole protein fraction, or a nucleic acid fraction. When the sample comprises whole cells, these cells can be any cells of a mammal, e.g., cells of any organ or tissue, including blood cells or endothelial cells.

[0277] The contacting can occur in vitro or in vivo with respect to a mammal. Preferably, the contacting is in vitro.

[0278] Also, detection of the complex can be carried out by many methods known in the art.For example, the CAR disclosed herein, polypeptide, protein, nucleic acid, recombinant expression vector, host cell, cell population, or antibody or its antigen-binding portion described herein can be labeled with detectable label, such as radioisotope, fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), enzyme (e.g., alkaline phosphatase, horseradish peroxidase) and element particle (e.g., gold particle) as disclosed above.

[0279] 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) teaches 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)). In addition, CAR function can be evaluated by measuring cytotoxicity, as described in Zhao et al., J. Immunol. vol. 174:4415-4423 (2005).

[0280] Another embodiment provides the use of the CAR, nucleic acid, recombinant expression vector, host cell, population of cells, antibody or antigen-binding portion thereof, and / or pharmaceutical composition of the present invention to treat or prevent a proliferation disorder, such as cancer, in a mammal. The cancer can be any of the cancers described herein.

[0281] Any administration method, 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 administration can be used. The particular mode of administration and dosing regimen will be selected by the attending clinician, taking into consideration the characteristics of the case (e.g., the subject, the disease, the disease state involved, and whether the treatment is preventive). If more than one agent or composition is being administered, one or more administration routes can be used; for example, the chemotherapeutic agent can be administered orally, and the antibody or antigen-binding fragment or conjugate or composition can be administered intravenously. The administration method includes injection, in which the CAR, CAR T cell, conjugate, antibody, antigen-binding fragment or composition is provided in a non-toxic pharmacologic carrier, such as water, saline, Ringer's solution, dextrose solution, 5% human serum albumin, solid oil, ethyl oleate, or liposome. In some embodiments, local administration of the disclosed compounds may be used, for example, by applying an antibody or antigen-binding fragment to an area of ​​tissue from which a tumor has been removed, or to an area suspected of being prone to tumor development. In some embodiments, sustained intratumoral (or near-tumoral) release of a pharmaceutical preparation comprising a therapeutically effective amount of an antibody or antigen-binding fragment may be beneficial. In other examples, the conjugate is applied topically to the cornea as eye drops, or intravitreally to the eye.

[0282] The disclosed therapeutic agents may be formulated in unit dosage forms suitable for individual administration of precise dosage amounts. Furthermore, the disclosed therapeutic agents may be administered in a single dose or in a multiple dose schedule. A multiple dose schedule is a schedule in which the main course of treatment may be by more than one separate dose, for example, 1-10 doses, followed by other doses given at subsequent time intervals as needed to maintain or enhance the action of the composition. Treatment may involve a daily dose or multiple daily doses of the compound(s) over a period of days to months or even years. Thus, the dosing regime will also be determined at least in part based on the particular needs of the subject being treated and will be dependent on the judgment of the administering practitioner.

[0283] Typical dosages of antibodies or conjugates can range from about 0.01 to about 30 mg / kg, for example, from about 0.1 to about 10 mg / kg.

[0284] In certain examples, the subject is administered a therapeutic composition comprising one or more of a conjugate, antibody, composition, CAR, CAR T cell, or additional agent in a multiple daily dosing schedule, e.g., at least 2 consecutive days, 10 consecutive days, etc., for a period of, e.g., weeks, months, or years. In one example, the subject is administered a conjugate, antibody, composition, or additional agent for a period of at least 30 days, e.g., at least 2 months, at least 4 months, at least 6 months, at least 12 months, at least 24 months, or at least 36 months.

[0285] In some embodiments, the disclosed methods include providing a subject with surgery, radiation therapy and / or chemotherapy drugs (e.g., sequentially, substantially simultaneously or simultaneously) in combination with the disclosed antibodies, antigen-binding fragments, conjugates, CARs or T cells expressing CARs. Such drugs and methods of treatment and therapeutic dosages are known to those skilled in the art and can be determined by a skilled clinician. Preparation and dosing schedules for additional drugs can be used according to the manufacturer's instructions or as empirically determined by the skilled artisan. Preparation and dosing schedules for such chemotherapy are also described in Chemotherapy Service, (1992) MC Perry, ed., Williams & Wilkins, Baltimore, Md.

[0286] In some embodiments, the combination treatment may include administering to the subject a therapeutically effective amount of an additional cancer inhibitor.Non-limiting examples of additional therapeutic agents that can be used in the combination treatment include microtubule binding agents, DNA intercalators or crosslinkers, DNA synthesis inhibitors, DNA and RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, gene regulators, and angiogenesis inhibitors.These agents (administered in therapeutically effective amounts) and treatments can be used alone or in combination.For example, any suitable anti-cancer or anti-angiogenesis agent can be administered in combination with the CAR, CAR-T cells, antibodies, antigen-binding fragments, or conjugates disclosed herein.The methods and therapeutic dosages of such agents are known to those skilled in the art and can be determined by a skilled clinician.

[0287] Further chemotherapeutic agents include alkylating agents such as nitrogen mustards (e.g., chlorambucil, chlormethine, cyclophosphamide, ifosfamide, and melphalan), nitrosoureas (e.g., carmustine, fotemustine, lomustine, and streptozocin), platinum compounds (e.g., carboplatin, cisplatin, oxaliplatin, and BBR3464), busulfan, dacarbazine, mechlorethamine, procarbazine, temozolomide, thiotepa, and uramustine; antimetabolites such as folates (e.g., methotrexate, pemetrexed, and raltitrexed), purines (e.g., cladribine, clofarabine, fludarabine, mercaptopurine, and thioguanine), pyrimidines (e.g., capecitabine), cytarabine, fluorouracil, and gemcitabine; plant alkaloids such as for example, podophyllum (e.g., etoposide and teniposide), taxanes (e.g., docetaxel and paclitaxel), vincas (e.g., vinblastine, vincristine, vindesine and vinorelbine); cytotoxic / antitumor antibiotics, for example, members of the anthracycline family (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone and valrubicin), bleomycin, rifampicin, hydroxyurea and mitomycin; topoisomerase inhibitors, for example, topotecan and irinotecan; monoclonal antibodies, for example, alemtuzumab, bevacizumab, cetuximab, gemtuzumab, rituximab, panitumumab, pertuzumab and trastuzumab; tumor-affinity photochromic dyes, for example, aminolevulinic acid, methyl aminolevulinate, porfimer sodium and verteporfin;and other agents, such as, but not limited to, alitretinoin, altretamine, amsacrine, anagrelide, arsenic trioxide, asparaginase, axitinib, bexarotene, bevacizumab, bortezomib, celecoxib, denileukin diftitox, erlotinib, estramustine, gefitinib, hydroxycarbamide, imatinib, lapatinib, pazopanib, pentostatin, masoprocol, mitotane, pegaspargase, tamoxifen, sorafenib, sunitinib, vemurafenib, vandetanib and tretinoin. The choice of such agents and therapeutic dosages are known to those skilled in the art and can be determined by a skilled clinician.;

[0288] Combination therapy can provide synergistic effects and can be proven to be synergistic, i.e., the effect achieved when active ingredients are used together is greater than the sum of the effects that can result from using the compounds separately.Synergistic effects can be achieved when active ingredients are (1) co-formulated and administered or delivered simultaneously as a combined unit dosage formulation; (2) delivered alternately or in parallel as separate formulations; or (3) by some other regimen.When delivered alternately, synergistic effects can be achieved when the compounds are administered or delivered sequentially, for example, by different injections in separate syringes.Generally, during alternation, effective dosages of each active ingredient are administered sequentially, i.e., sequentially, whereas in combination therapy, effective dosages of two or more active ingredients are administered together.

[0289] In one embodiment, an effective amount of an antibody or antigen-binding fragment or conjugate thereof that specifically binds to one or more of the antigens disclosed herein is administered to a subject with a tumor after anti-cancer treatment. After a sufficient amount of time has passed for the administered antibody or antigen-binding fragment or conjugate to form an immune complex with the antigen expressed on 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 immune complexes compared to a control taken before treatment indicates that the treatment is not effective, and a decrease in immune complexes compared to a control taken before treatment indicates that the treatment is effective.

[0290] E. Biopharmaceutical Compositions Provided herein are biopharmaceutical or biologic compositions (hereinafter "compositions") for use in gene therapy, immunotherapy and / or cell therapy, comprising one or more of the disclosed CARs, or T cells expressing a CAR, antibodies, antigen-binding fragments, conjugates, CARs, or T cells expressing a CAR that specifically binds to one or more antigens disclosed herein, in a carrier (e.g., a pharma- ceutically acceptable carrier). These compositions can be prepared in unit dosage forms for administration to a subject. The amount and timing of administration to achieve a desired outcome is at the discretion of the treating clinician. These compositions can be formulated for systemic (e.g., intravenous) or local (e.g., intratumoral) administration. In one example, the disclosed CARs, or T cells expressing a CAR, antibodies, antigen-binding fragments, conjugates are formulated for parenteral administration, such as intravenous administration. Compositions comprising the disclosed CARs, or T cells expressing a CAR, conjugates, antibodies, or antigen-binding fragments are used for, e.g., treatment and detection, of tumors, e.g., but not limited to, neuroblastoma. In some examples, these compositions are useful for treating or detecting cancer. Compositions comprising the CARs disclosed herein, or T cells, conjugates, antibodies, or antigen-binding fragments expressing the CARs, are also used, for example, to detect pathological angiogenesis.

[0291] Compositions for administration may include a solution of CAR, or T cells expressing CAR, conjugates, antibodies or antigen-binding fragments dissolved in a pharma- ceutically acceptable carrier, such as an aqueous carrier. A variety of aqueous carriers may be used, such as, for example, buffered saline. These solutions are sterile and generally free of undesirable substances. The compositions may be sterilized by conventional, well-known sterilization techniques. The compositions may contain pharma- ceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, adjuvant agents, and the like, such as, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and the like. The concentration of CAR, or T cells expressing CAR, antibodies or antigen-binding fragments or conjugates, or conjugates, in these formulations may vary widely and will be selected primarily based on fluid volumes, viscosities, body weight, and the like, according to the particular mode of administration selected and the needs of the subject. The actual methods of preparing such dosage forms for use in gene therapy, immunotherapy, and / or cell therapy will be known or apparent to those skilled in the art.

[0292] A typical composition for intravenous administration will contain about 0.01 to about 30 mg / kg of antibody or antigen-binding fragment or conjugate (or a corresponding dose of CAR, or T cells expressing a CAR, or a conjugate comprising the antibody or antigen-binding fragment) per subject per day. Actual methods for preparing administrable compositions will be known or apparent to those of skill in the art and are described in more detail in publications such as Remington's Pharmaceutical Science, 19th Edition, Mack Publishing Company, Easton, PA (1995).

[0293] CAR, or T cells, antibodies, antigen-binding fragments, or conjugates expressing CAR, may be provided in lyophilized form and rehydrated with sterile water prior to administration, although they are also provided in sterile solutions of known concentrations. A solution of CAR, or T cells, antibodies, or antigen-binding fragments, or conjugates expressing CAR, 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 is available in the art in administering antibody or antigen-binding fragment and conjugate drugs; for example, antibody drugs have been commercially available in the United States since the approval of Rituxan® in 1997. CAR, or T cells, antibodies, antigen-binding fragments, and conjugates thereof expressing CAR, may be administered by slow infusion rather than by intravenous infusion or intravenous bolus. In one example, a higher loading dose is administered with a subsequent maintenance dose administered at a lower level. For example, an initial loading dose of 4 mg / kg of the antibody or antigen-binding fragment (or a corresponding dose of a conjugate comprising the antibody or antigen-binding fragment) can be infused over a period of approximately 90 minutes, followed by weekly maintenance doses of 2 mg / kg for 4-8 weeks infused over a period of 30 minutes if the previous dose was well tolerated.

[0294] Controlled release parenteral formulations can be made as implants, oily injections, or as granular systems. For a broad review of protein delivery systems, see Banga, AJ, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Technomic Publishing Company, Inc., Lancaster, PA (1995). Granular systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain a therapeutic protein, such as a cytotoxin or drug, as a central core. In microspheres, the therapeutic agent is dispersed throughout the particle. Particles smaller than about 1 μm, microspheres, and microcapsules, are commonly referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Capillaries have a diameter of approximately 5 μm, so that only nanoparticles are administered intravenously. Microparticles are typically approximately 100 μm in diameter and are administered subcutaneously or intramuscularly. See, e.g., Kreuter, J., Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, NY, pp. 219-342 (1994); and Tice and Tabibi, Treatise on Controlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, NY, pp. 315-339 (1992).

[0295] The polymers can be used for ion-controlled release of the CARs disclosed herein, or T cells, antibodies or antigen-binding fragments expressing the CARs, or conjugate compositions. Various degradable and non-degradable polymer matrices used for controlled drug delivery are known in the art (Langer, Accounts Chem. Res. 26:537-542, 1993). For example, the block copolymer polaxamer 407 exists as a viscous yet mobile liquid at low temperatures, but forms a semi-fluid gel at body temperature. It has been shown to be an effective vehicle for the formulation and sustained delivery of recombinant interleukin-2 and urease (Johnston et al., Pharm. Res. 9:425-434, 1992; and Pec et al., J. Parent. Sci. Tech. 44(2):58-65, 1990). Alternatively, hydroxyapatite has been used as a microcarrier for the controlled release of proteins (Ijntema et al., Int. J. Pharm. 112:215-224, 1994). In yet another embodiment, liposomes are used for the controlled release and drug targeting of lipid-encapsulated drugs (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA (1993)). A number of additional systems for controlled delivery of therapeutic proteins are known (see U.S. Pat. No. 5,055,303; U.S. Pat. No. 5,188,837; U.S. Pat. No. 4,235,871; U.S. Pat. No. 4,501,728; U.S. Pat. No. 4,837,028; U.S. Pat. No. 4,957,735; U.S. Pat. No. 5,019,369; U.S. Pat. No. 5,055,303; U.S. Pat. No. 5,514,670; U.S. Pat. No. 5,413,797; U.S. Pat. No. 5,268,164; U.S. Pat. No. 5,004,697; U.S. Pat. No. 4,902,505; U.S. Pat. No. 5,506,206; U.S. Pat. No. 5,271,961; U.S. Pat. No. 5,254,342 and U.S. Pat. No. 5,534,496).

[0296] F. Kit In one embodiment, a kit is also provided that uses the CAR disclosed herein.For example, a kit for treating tumor in a subject or a kit for producing CAR T cells that express one or more of the CARs disclosed herein.The kit typically includes the disclosed antibody, antigen-binding fragment, conjugate, nucleic acid molecule, CAR or T cell that expresses CAR disclosed herein.More than one of the disclosed antibody, antigen-binding fragment, conjugate, nucleic acid molecule, CAR or T cell that expresses CAR can be included in the kit.

[0297] The kit may include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The container may be formed from a variety of materials, such as glass or plastic. The container typically holds a composition comprising one or more of the disclosed antibodies, antigen-binding fragments, conjugates, nucleic acid molecules, CARs, or T cells expressing CARs. In some embodiments, the container may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). The label or package insert indicates that the composition is used to treat a particular condition.

[0298] The label or package insert typically further includes instructions for the use of the disclosed antibody, antigen-binding fragment, conjugate, nucleic acid molecule, CAR, or T cell expressing a CAR, for example, in a method of treating or preventing a tumor, or in a method of generating a CAR T cell. The package insert typically includes instructions customarily included in the commercial packaging of a therapeutic product, including information about indications, usage, dosage, administration, contraindications, and / or warnings regarding the use of the therapeutic product. The instructional material can be written in electronic form (e.g., computer diskette or compact disk), or 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 a means for detecting the 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 routinely used for the implementation of a particular method. Such kits and appropriate contents are well known to those skilled in the art.

[0299] Working Example The present invention is further illustrated by the following examples, which should not be interpreted as limiting the scope of the present invention in any way. On the contrary, various other embodiments, modifications, and equivalents thereof must be relied upon, which can easily be understood to occur to those skilled in the art after reading the description herein without departing from the spirit of the present invention and / or the scope of the appended claims.

[0300] Example 1 Isolation of human CD123-specific antibodies from a fully human yeast-displayed ScFv library This example describes the derivation of a fully human binding sequence targeting the CD123 antigen from a yeast display library.

[0301] material and method: A large yeast-displayed human naive single-chain variable fragment (ScFv) antibody library was used to isolate the anti-human CD123 antibodies described herein. The library was constructed using a collection of human antibody gene repertoires from over 60 individuals. Three rounds of magnetic activated cell sorting (MACS) were performed to enrich for human ScFv binders to recombinant human CD123-Fc. In the first round of yeast library panning, the yeast-displayed ScFv library (5 × 10 10 Yeast library mixes (100 μL protein G microbeads (Miltenyi Biotec)) were incubated with 5 μg / mL CD123-Fc in 15 ml PBSA (consisting of 0.1% bovine serum albumin (BSA) in Dulbecco's phosphate-buffered saline (PBS) buffer) for 1.5 h on a rotator at room temperature. After two washes with 25 ml PBSA, the yeast library mix was incubated with 100 μL protein G microbeads (Miltenyi Biotec) for 30 min on a rotator at room temperature. After one wash, the library mix was resuspended in 50 ml PBSA and loaded onto a MACS cell separation column (LS column). After three washes with 10 ml PBSA, yeast that presented ScFv binders to the column were then eluted twice with 2 ml PBSA. These eluted yeast cells were combined and resuspended in 50 ml of SDCAA medium (1 L water, 20 g D-glucose, 6.7 g BD Difco™ Yeast Nitrogen Base without amino acids, 5 g Bacto™ Casamino Acids, 5.4 g Na2.HPO4, 8.56 g NaH2PO4.H2O) and amplified for 20 hours with shaking at 225 rpm and 30°C. The amplified pool was then induced in SGCAA medium (consisting of the same composition of SDCAA medium but with galactose instead of glucose), shaken at 225 rpm and 30°C for another 16 hours, and used for the next round of panning. The same process was repeated two more times to enrich for CD123-Fc specific binders.

[0302] To further enrich for binders with higher affinity and better specificity, FACS-based sorting was used to isolate the strongest binders from the pool. The derived pool was incubated with 1 μg / ml CD123-Fc for 1 h at room temperature, then stained with Anti-c-Myc-Alexa 488 and goat anti-Hu-Fc PE conjugates, and the top 1% pools with the highest PE vs. FITC signal were gated and sorted. The sorted pool was amplified in SDCAA medium, yeast plasmid DNA was extracted, and transformed into bacteria for single clone DNA sequencing. Two unique sequences were identified, designated MT-16 and MT-32, and cloned into CAR constructs for expression in CAR-T or CAR-NK format constructs for further functional characterization.

[0303] Example 2 Isolation of human CD123-specific antibodies from a fully human phage-displayed InfinityOne scFv library This example describes the derivation of a fully human binding sequence targeting the CD123 antigen from a phage display library.

[0304] material and method: Generation of human phage-displayed ScFv CD123-specific antibodies For the selection of scFv specific to recombinant human CD123, a naive human scFv (recombinant single-chain fragment variable domain of immunoglobulin) phage display library (approximate diversity, 7 × 10) was constructed from peripheral blood B cells of 121 healthy donors. 10 (F. Tomszak, unpublished data). 12The amplified library of phage-displayed ScFvs was incubated with 1 μg of coated CD123 in a volume of 100 μl in one well of a 96-well plate for 2 h at room temperature during the first, second and third rounds of biopanning, respectively. After each round of incubation, the wells were washed with phosphate-buffered saline containing 0.05% Tween 20 (PBST) 10 times in the first round, 20 times in the second round and 30 times in the third round to remove non-specifically bound phages. Antigen-bound phages were eluted with 100 μl of 10 μg / ml trypsin diluted in PBS and mixed with TG1 competent cells for 1 h at 37°C, and phages were amplified from the infected cells and used in the next round of biopanning. After the third round of biopanning, 376 clones were randomly picked from the infected TG1 cells using an automated colony picking system (Molecular Devices, QPix 460) and each was inoculated into 150 μl of 2YT medium containing 100 μg / ml ampicillin and 200 mM glucose in a 96-well plate and incubated overnight at 37° C. in a shaker at 300 rpm. The next day, 10 μl of the bacterial culture was used to inoculate 150 μl of 2YT medium containing 100 μg / ml ampicillin and 50 μM isopropyl-β-d-thiogalactopyranoside in a 96-well plate and the plate was further incubated overnight at 30° C. in a shaker at 300 rpm. The scFv supernatants were mixed at a 1:1 volume ratio with 2% BSA in PBST containing horseradish peroxidase-conjugated recombinant monoclonal mouse anti-c-myc antibody diluted 1:2500, and phage clones displaying scFvs with high CD123 binding affinity were identified using enzyme-linked immunosorbent assay (ELISA). The supernatants were incubated with recombinant human CD123 coated at 30 ng per well in 384-well plates for 1 h at room temperature and washed three times with PBST (after overnight incubation at 4°C, blocked with 2% BSA in PBS containing 0.05% Tween 20 and washed three times with PBS containing 0.05% Tween 20).After incubation, 3,3',5,5'-tetramethylbenzidine (TMB) substrate was added and the solution absorbance at 450 nm (A450) was measured. Clones that bound to CD123 with a signal-to-noise ratio greater than 5 were selected for further characterization.

[0305] Flow cytometry-based binding assay Binders identified from ELISA were tested in the CD123 positive cell line MOLM-13. The CD123 negative cell lines Jeko-1 CD20KO eGFP served as negative controls. As positive control stainings, REAL270 (alpha CD123) and REAL116 (alpha CD123) antibodies, respectively, were used. Soluble scFvs were expressed as described above. Bacterial pellets were disrupted by incubation in 0.3 mL TE buffer (10 mM Tris-HCl containing 1 mM EDTA, pH 8.0 at 37°C) / well at 250 rpm for 18 h. Cultures were centrifuged at 4000 x g for 20 min at RT and the supernatants were transferred to new microtiter plates.

[0306] Cell number and viability were determined by MACSQuantX for cell staining. The cell suspension containing the required cell number was centrifuged at 300×g for 10 min at 4° C. and the supernatant was discarded. PEB buffer (1×PBS+2 mM EDTA, 0.5% BSA pH 7.4, RT) was added to resuspend the cells to a concentration of 1E+06 cells / mL. 100,000 cells per cell line were added to each well of a 96-well V-bottom plate and the plate was centrifuged at 1300×g for 2 min at 4° C. The supernatant was discarded and the cells were resuspended in 100 μL of supernatant from the periplasmic preparation and the cells were incubated on ice for 10 min. The cells were washed by adding 100 μL of PEB followed by centrifugation (1300×g for 2 min at 4° C.) twice. 50 μL of secondary antibody (anti-His-APC conjugated antibody) or REAL antibody (diluted 1:50 in PEB buffer) was added per well and the plate was incubated for 10 min at 4°C in the dark. Cells were washed twice by adding 100 μL of PEB followed by centrifugation (1300×g, 2 min at 4°C). Propidium iodide was diluted 1:100 in fixative (1×PBS+2 mM EDTA+1% PFA+0.3% MeOH+3% NaAzide) and washed cells were resuspended in 50 μL of the mixture. Signals are measured with MACSQuantX. Signals were analyzed using FlowLogic software. Statistical analysis was performed with VORTEX software.

[0307] result: Based on the results of the ELISA binding assay, 10 unique scFv clones specific for recombinant human CD123 and MOLM13 cells were identified (Table 1). The corresponding scFv sequences were incorporated as binder domains into CAR constructs for further analysis in CAR-T and CAR-NK formats.

[0308] [Table 1]

[0309] Example 3 Development of CD123-targeted CAR T cell constructs There are few treatment options for AML, and treatment-related toxicity and disease relapse after treatment are common. In addition, immunotherapies using non-human sequences, such as mouse-derived antibodies, can result in treatment rejection or side effects in patients. To develop a new CAR T treatment for AML, we designed 15 CD123-targeting CAR T constructs incorporating a fully human ScFv targeting domain and evaluated their antitumor activity.

[0310] material and method: Cell lines used to demonstrate CAR activity The acute myeloid leukemia cell line MOLM-14 was purchased from the German Collection of Microorganisms and Cell Lines (DSMZ, Braunschweig Germany). The other cell lines, myeloid leukemia line KG-1a, acute lymphoblastic leukemia line RS4;11, epidermoid carcinoma line A431, and 293T cell line, were purchased from the American Tissue Culture Collection (ATCC, Manassas, VA). The MOLM14 cell line was cultured in RPMI-1640 medium (ATCC) supplemented with 20% heat-inactivated fetal bovine serum (FBS). The KG-1a cell line was cultured in IMDM medium supplemented with 20% FBS. The A431 cell line was cultured in DMEM medium (ATCC) supplemented with 10% heat-inactivated FBS. 293T cells were cultured in Dynamis™ medium (Thermo Fisher Scientific, Grand Island, NY) containing 4 mM L-glutamine (Lonza, Morristown, NJ). Each cell line was prepared as a single-cell clone of a luciferase-expressing cell line by stably transducing a wild-type tumor line with a lentiviral vector encoding firefly luciferase (Lentigen Technology, Gaithersburg, MD).

[0311] Generation of CAR constructs and production of lentiviral vectors Human anti-CD123 chimeric antigen receptor (CAR) constructs were generated from different single chain variable fragment (ScFv) sequences targeting the extracellular domain of human CD123 / IL-3 receptor alpha. Each scFv sequence was linked in frame to CD8 hinge, 4-1BB costimulatory domain, and CD3-zeta activation domain sequences. A comparative CD33-targeting CAR sequence was generated in a similar manner, except that the heavy chain-only variable domain (VH_4) was used as the targeting domain instead of the scFv. The VH_4 sequence was linked in frame to CD8 hinge, 4-1BB costimulatory domain, and CD3-zeta activation domain sequences (Schneider, Dina, et al., A Unique Human Immunoglobulin Heavy Chain Variable Domain-Only CD33 CAR for the Treatment of Acute Myeloid Leukemia. Frontiers in Oncology 8:539. 2018 Nov. 22, doi:10.3389 / fonc.2018.00539). A leader peptide from human GMCSFR1 was included in all CAR constructs to facilitate transport to the T cell membrane. The CAR sequence was cloned into a lentiviral vector (LV) expression cassette under the control of the human EF-1α promoter (Lentigen Technology, Gaithersburg, MD). Lentiviral particles were generated by transient transfection of HEK 293T cells, pelleted by centrifugation, and stored at -80°C until transduction.

[0312] Preparation and transduction of primary T cells Primary T cells from healthy donors were isolated from leukapheresis collections (AllCells, Alameda, CA) or processed buffy coats (Oklahoma Blood Institute, Tulsa, OK) with written consent of the donors. CD4+ and CD8+ human T cells were purified by positive selection using a 1:1 mixture of CD4 and CD8 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's protocol. Purified T cells were cultured at 1 × 10 6 Cells were cultured in serum-free TexMACS medium supplemented with 30 IU / ml IL-2 at a density of 1000 cells / ml and activated with CD3 / CD28 MACS® GMP T cell TransAct reagent (Miltenyi Biotec). Additionally, activated T cells were transduced with lentiviral vector particles encoding a CAR construct on day 1. On day 3, and every 2–3 days thereafter, cultures were supplemented with fresh TexMACS medium containing 30 IU / ml IL-2 and harvested on days 8–10. Where noted, TexMACS medium supplemented with 970 IU / ml IL-7 and 90 IU / ml IL-15 was used.

[0313] CD123 surface expression on tumor cell lines CD123 surface expression was determined on arrays of tumor lines by flow cytometry using anti-CD123 antibody clone AC145 (Miltenyi Biotec, Bergisch Gladbach, Germany), with negative gating based on the cognate isotype control. CD123 surface expression density on target cell lines was assessed by QuantiBRITE phycoerythrin (PE) beads (BD Biosciences, San Jose, CA) based on the antibodies bound per cell (ABC) method according to the manufacturer's protocol. Briefly, a standard curve was generated with four different densities of PE fluorophore-conjugated beads, and tumor cells stained with PE-conjugated anti-CD123 antibodies were acquired with the same settings. Based on the standard curve, the ABC value for each tumor cell line was estimated.

[0314] Flow cytometry analysis of CAR surface expression 500,000 CAR T cells were washed twice with cold AutoMACS buffer supplemented with 0.5% bovine serum albumin (Miltenyi Biotec, Bergisch Gladbach, Germany) and stained with 2.5ug / ml CD123-Fc peptide (Novoprotein, Summit, NJ) followed by anti-Fc-AF647 conjugate (Jackson ImmunoResearch, West Grove, PA). 7-aminoactinomycin D staining (7-AAD, BD Biosciences, San Jose, CA) was added to exclude dead cells. Untransduced cells (UTD) were used as a negative control. Cells were washed twice with AutoMACS buffer supplemented with 0.5% bovine serum albumin, resuspended in 200ul staining buffer and acquired by flow cytometry. Flow cytometric analysis was performed on a MACSQuant® 10 Analyzer (Miltenyi Biotec) and data plots were generated using FlowJo software (Ashland, OR).

[0315] CAR T cell cytotoxicity and cytokine assays To assess CAR T cell-mediated cytotoxicity, 5 x 10 cells stably transduced with firefly luciferase were cultured in vitro. 3 Tumor target cells were combined with CAR T cells at the indicated effector-to-target ratios and incubated overnight at 37°C with 5% CO2. SteadyGlo reagent (Promega, Madison WI) was added to each well and the resulting luminescence was quantified as counts per second (Sample CPS). Target only wells (Max CPS) and target only wells with 1% Tween-20 (Min CPS) were used to determine the assay range. Percent specific lysis was calculated as (1-(Sample CPS-Min CPS) / (Max CPS-Min CPS)). For cytokine release analysis, 5x10 4 Effector and 5×10 3 Targets were co-cultured overnight, and supernatants from the co-cultures were removed and analyzed for IFNγ, TNFα, and IL-2 concentrations by ELISA (eBioscience, San Diego, CA). Three technical replicates were performed for each condition, and each experiment was repeated using CAR T cells generated from at least three healthy donors.

[0316] result The data in Example 3 describes the generation and in vitro evaluation of CAR T cells targeting the CD123 antigen for the treatment of AML.

[0317] A schematic of the tandem CAR construct targeting the CD123 antigen is shown in Figure 1A. CAR123 is composed of a fully human binder (InfinityOne) linked in frame to the CD8 hinge and transmembrane domains, 4-1BB costimulatory domain, and CD3 zeta activation domain. Ten scFv sequences were selected for evaluation in the CAR format based on flow cytometric binding analysis of the cognate soluble binder to target lines with and without CD123 expression. CAR variants D0125-D0134 were constructed (Table 2). The CAR sequences were further incorporated into third generation lentiviral vectors and transduced into human primary T cells in a saturating manner to generate CD123 CAR T cells under the control of the mammalian EF-1α promoter. Previously evaluated CAR control constructs targeting CD123 (LTG2078) and CD33 (LTG1906) were also included (Table 3). Untransduced T cells (UTD) from the same donor as the CAR-expressing cells were used as a negative control.

[0318] [Table 2]

[0319] [Table 3]

[0320] Lentiviral vectors encoding CD123 CAR constructs were used for CAR transduction into human primary T cells at a multiplicity of infection (MOI) of 40. CAR surface expression of transduced T cells by flow cytometry using recombinant IL3R-alpha Fc tag followed by staining with anti-Fc Alexa Flour 647. Different CD123 CAR constructs showed various expression levels ranging from 0 to 80% (n=4 donors) (Figure 1B). CARs D0126, D0127, D0131, D0132, D0133, and D0134 showed similar or higher surface expression than the positive CAR 123 control LTG2078, while CAR D0130 showed slightly lower surface expression, followed by D0129 and D0128, with D0125 showing the lowest expression in multiple donors. Cell viability was examined on days 3 and 7 after T cell activation as shown in Figure 1C. All CD123 CAR T cells showed improved or similar survival rates compared to the control CAR LTG2078.

[0321] To evaluate the target-specific cytotoxicity of CD123 CAR in vitro, leukemic (MOLM14, KG1a, RS4;11) and non-leukemic (293T and A431) lines were evaluated for surface CD123 expression by flow cytometry using a CD123-specific antibody. As shown in Figure 2, 99% MOLM14 and 66% KG-1a human AML tumor cell lines express CD123, whereas the human B-ALL cell line RS4;11 has only limited CD123 expression. In contrast, 293T and A431 do not express CD123. Therefore, MOLM14 and KG-1a lines were selected as target cell lines and 293T was selected as a negative control cell line for CAR T cell function evaluation.

[0322] Human primary T cells were transduced with lentiviral vectors encoding CAR constructs and expanded in culture until day 8. CAR-T cells were co-incubated with MOLM14, KG-1a, or 293T cell lines at effector-to-target ratios of 2.5:1, 5:1, and 10:1. After overnight co-incubation, cultures were analyzed by a luminescence-based in vitro killing assay. Most CAR123 construct-expressing primary T cell lines lysed MOL14-CD123+ with varying potency, whereas three CD123 CAR lines, D0125, D0128, and D0129, lacked target lysis capacity (Figure 3A). Similarly, KG-1a-CD123+ target cells were killed by most CAR T constructs, except for D0125, D0128, and D0129 (Figure 3B). The CD33 CAR LTG1906 showed high cytotoxicity against MOLM14 cells, consistent with CD123 expression levels (CD33 High ), but showed low lytic efficacy against KG1a (CD33 Low Furthermore, no killing above background of the CD123-negative 293T cell line was observed (Figure 3C), demonstrating the robust target-specific cytotoxic function of all CD123 CAR constructs except for CARs D0125, D0128, and D0129.

[0323] T cell homeostasis and the production of proinflammatory cytokines IL-2, IFNγ, and TNFα by CD123 CAR, as well as control constructs CAR LTG2078 and CD33 CAR LTG1906, were examined by ELISA in culture supernatants after overnight co-incubation of CAR T cells with the MOLM14 target line at an E:T ratio of 10 (Figures 4A-4C). Cytokine release induced by specific targets was detected by comparing each CAR T group incubated with target cells to the respective CAR T alone experimental group, and by comparing the target co-incubated CAR T group to the previously characterized CAR123 control LTG2078. Although CAR123 control LTG2078 and CAR33 control LTG1906 produced cytokines after co-incubation with MOLM14 target cells, most of the tested CD123 CAR T constructs did not result in a significant increase in IFNγ, TNFα, or IL-2 cytokines after overnight co-culture with MOLM14 cells. One exception was CAR123 D0127, which produced IFNγ and TNFα levels even in the absence of target cells (T cell alone group), indicating a tumor-independent cytokine response. This effect was not predicted from previous experiments, demonstrating the non-obviousness of the present invention. Except for CAR123 D0127, the cytokine responses of the CD123 CAR constructs evaluated here were comparable to untransduced T cell (UTD) controls, suggesting a low risk of inducing cytokine-mediated side effects such as cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS).

[0324] Among all CAR123 constructs, D0126 showed the highest transduction efficiency and viability, but at the same time, it showed the best cytotoxicity function against CD123+ tumor cells in this set of CAR constructs. Another CAR123 construct, D0131, also showed high CAR transduction efficiency and viability, but only moderate target cell killing activity in vitro. Therefore, CAR123 constructs D0126 and D0131 were selected for further evaluation in vivo.

[0325] Example 4 Evaluation of the antitumor function of CD123-targeted CAR T cells in a murine MOLM14 xenograft model This example describes the in vivo evaluation of CD123-targeted CAR T cells incorporating scFv sequences from the InfinityOne library.

[0326] material and method: cell line The acute myeloid leukemia cell line MOLM-14 was purchased from the German Collection of Microorganisms and Cell Lines (DSMZ, Braunschweig Germany). The MOLM14 cell line was stably transfected with the firefly luciferase gene and cultured in RPMI-1640 medium (ATCC) supplemented with 20% heat-inactivated fetal bovine serum (FBS).

[0327] In vivo analysis of CAR T function Animal studies were performed in accordance with applicable laws, regulations, and guidelines of the National Institutes of Health (NIH) and with the approval of the Animal Care and Use Committee at MI Bioresearch (Ann Arbor, MI). In this study, we investigated the function of CD123-targeting CAR T cells in NSG (NOD.Cg-Prkdcs cid Il2rg tm1Wjl On day 0, 1.0 × 10 6 MOLM-14 CD123+ AML cells were injected into the tail vein. On day 4, tumor burden was determined by IVIS bioluminescence imaging, after which mice were randomized into groups with equal mean tumor burdens, with 5.0 × 10 6 CAR T + Cells / mouse (normalized for transduction efficiency) were administered. Tumor regression was determined by bioluminescence imaging on days 14, 21, 28, 35, 42, and 49 using a Xenogen IVIS-200 instrument (Perkin Elmer, Shelton, Connecticut). Images were analyzed using Living Image version 4.1 software (Perkin Elmer), and bioluminescence signal flux for each mouse was calculated as mean radiance (cm per second). 2 The survival rate was expressed as photons per steradian (P < 0.01). Survival was recorded and analyzed at the end of the study. Peripheral blood was collected from all animals on study days 14, 21, 28, and 42 to determine the presence of CAR T cells and tumor cells. The absolute numbers of circulating CAR T cells and MOLM-14 tumor cells were determined by flow cytometry.

[0328] Flow cytometry analysis of CAR T cells and tumor cells in mouse blood Seventy microliters of mouse blood was collected on study days 14, 21, 28, and 42 and analyzed for CAR T and MOLM-14 tumor cell counts by flow cytometry. Red blood cells were then lysed with red blood cell lysing solution (BD BioScience, San Jose, CA) according to the manufacturer's instructions, and the remaining lymphocytes were stained with anti-human CD45, anti-human CD3 (Miltenyi Biotec), anti-human CD8 (Miltenyi Biotec), anti-human CD123 (Miltenyi Biotec), and 7-AAD (BD Biosciences, San Jose, CA) and then analyzed by flow cytometry. Dead cells were excluded from the analysis by 7-AAD staining. To obtain direct counts of human T cells and MOLM-14 in blood, we utilized the MACSQuant 10 volumetric function and used CountBright Absolute Counting Beads (ThermoFischer Scientific, Waltham, MA) to account for sample loss during processing, according to the manufacturer's protocol.

[0329] result: The NSG MOLM14 xenograft AML model was used to further explore the in vivo tumor rejection capabilities of the two leading CAR123 candidates, D0126 and D0131. Two animal studies using CAR T cells derived from separate healthy donors were performed, one focused on CAR D0126 (Figure 5A) and the other comparing CAR123 constructs D0126 and D0131 (Figure 5B). The previously characterized CAR LTG1906, which targets the CD33 antigen on MOLM14 tumor cells, was included as a comparison control.

[0330] In the first in vivo study, CD123 CAR D0126 was compared to the previously characterized CD33 CAR-T construct LTG1906, and tumor alone (TA) and untransduced T cells (UTD) were also included as control experimental groups. CAR-T cells were generated by transduction with lentiviral vectors encoding CAR D0126 and CAR LTG1906, followed by expansion in culture in TexMACS medium supplemented with 30 IU / ml IL2. MOLM14-Luc cells were used as target line. 1×10 6 MOLM14-Luc cells were injected intravenously (i.v.) into each NSG mouse. On day 6, tumor growth was assessed by IVIS imaging, after which mice were randomized into experimental groups. On day 7, 5 × 10 cells were injected per mouse. 6Human CAR+T cells or UTD cells were administered via tail vein injection. Tumor growth kinetics was monitored by an in vivo imaging system (IVIS) over time (Figures 6A and 6B). Because MOLM14 tumors express both CD123 and CD33 antigens, the treatment group administered CAR D0126 targeting the CD123 antigen, as well as the comparison group administered CAR LTG1906 targeting the CD33 antigen, demonstrated robust tumor rejection compared to the tumor alone (TA) and UTD control groups. At the end of the study, 5 out of 6 mice in each group demonstrated complete tumor rejection, with only one mouse in each group retaining tumor cells (Figure 6B). Notably, both CAR D0126 and CAR LTG1906 treatment groups did not show weight loss (Figure 6C), and thus, no CAR-related toxicity was detected in this model. Both CAR D0126 and LTG1906 mediated complete survival until the end of the study on day 36 (6 out of 6 mice survived), whereas the tumor alone (TA) and UTD control groups died of high burden of disseminated disease by day 15 (Figure 6D). Mouse peripheral blood was sampled on days 14, 22 and 33. Human T cells were detected in all groups (Figures 7A, 7B, 7C). Furthermore, CAR D0126 and LTG1906 T cells were detected in the peripheral blood of mice at the end of the study, thus demonstrating high persistence of CD123 CAR candidate D0126 and comparative control CAR33 LTG1906 T cells.

[0331] In the second animal study, in addition to CAR D0126, we also included CD123 CAR D0131. The CAR T cells in this study were generated from peripheral blood T cells of a different donor than those used in the first in vivo study. T cells were transduced and expanded in TexMACS medium supplemented with 970 IU / ml IL-7 and 90 IU / ml IL-15. Tumor progression in each group is shown in Figure 8A. Similar to the first animal study, CAR D0126 demonstrated strong antitumor efficacy, with tumors rejected in four out of six mice. CAR123 D0131 showed weaker antitumor activity compared to CAR123 D0126 (Figures 8A and 8B). No significant weight loss was observed in the CAR T-treated groups (Figure 8C), but mouse deaths were observed in all groups. The best survival effect was detected in the CAR D0126 treatment group, with 4 of 6 mice surviving and remaining completely tumor-free until day 56, the end of the extension study (Figure 8D). In this study, total T cells in the peripheral blood were monitored. As expected, 2 days after CAR T cell or UTD administration, human T cells were detected in the peripheral blood of mice in all groups except the TA negative control (Figure 9A). T cell abundance increased over time in all CAR T groups, suggesting T cell proliferation (Figure 9B) and persistence through days 21, 28, and 42 (Figures 9C, 9D, 9E). At day 42 of the study, the CAR123 D0126 group had the highest T cell count (Figure 9E), indicating the greatest T cell proliferation and persistence among the CAR constructs tested in this experiment.

[0332] In summary, CD123 CAR candidate D0126 efficiently eliminated tumors in NSG mice engrafted with MOLM-14 cells in two in vivo studies using T cells from different human donors, and demonstrated efficient tumor elimination, CAR T persistence, and extended survival in a MOLM14 AML xenograft mouse model (Figure 9A). Thus, CAR123 D0126 was identified as a prime candidate for the development of CD123-targeted CAR T therapy for the treatment of CD123-positive malignancies.

[0333] Example 5 Development of CD123-targeted CAR NK cells This example describes the generation of CAR NK cells by lentiviral transduction.

[0334] material and method: Lentiviral vector constructs and production Each CD123-CAR was composed of a CD123 scFv binder, CD8 hinge and transmembrane domains, a 4-1BB transactivation domain, and a CD3 zeta signaling domain. Constructs were cloned into a third generation lentiviral plasmid backbone (Lentigen) under the control of the human EF-1α promoter. Supernatants containing lentiviral vectors (LVs) were generated by transient transfection of HEK 293T cells as previously described (Kuroda H et al., J Virol Methods. (2009) 157:113-21). For pseudotyping of lentiviral vectors, a modified BaEV envelope glycoprotein was used as previously described (Girard-Gagnepain A et al., Blood. (2014) 124:1221-31). Supernatants containing LVs were stored at -80°C and titers were determined on NK-92 cells.

[0335] Primary NK cell isolation To isolate NK cells from the buffy coat, peripheral blood mononuclear cells (PBMCs) were prepared by standard density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare). Resting NK cells were enriched from PBMCs by removing non-NK cell populations using an NK cell isolation kit for human cells (Miltenyi Biotec).

[0336] Cell culture and transduction NK cells were cultured at 10 in NK MACS medium containing 5% human AB serum, 500 U / mL IL-2 (Miltenyi Biotec), 10 ng / mL IL-15 (Miltenyi Biotec), and 10 ng / mL IL-1β (Miltenyi Biotec). 6 After 2 days of culture, NK cells were transduced as previously described (Bari R, Granzin M, et al., Front Immunol. (2019) 10:2001).

[0337] Briefly, for transduction, 5 × 10 NK cells were cultured in 200 μL of serum-free culture medium containing 10 μg / mL Vectofusin-1 and up to 50 μL of LV supernatant. 5 The NK cells were suspended at 0.5 million cells / mL. After 2 h of spinoculation at 400g, the cells were cultured with LV in a cell culture incubator for 24 h. The cell culture medium was then replaced with fresh complete cell culture medium containing 5% human AB serum, 500 U / mL IL-2, and 10 ng / mL IL-15. From day 3 post-transduction onwards, the transduction efficiency was determined by flow cytometry. The transduced NK cells were centrifuged, counted and adjusted to a cell number of 0.5 million cells / mL in fresh complete NK cell culture medium (5% human AB serum, 500 U / mL IL-2 and 10 ng / mL IL-15) every 3 days for long-term culture.

[0338] result: Primary NK cells were efficiently transduced with CD123-CAR using baboon envelope glycoprotein-pseudotyped lentiviral vectors.

[0339] We generated 13 CD123-CAR constructs containing CD123 binder, CD8 hinge and transmembrane domains, 4-1BB transactivation domain, and CD3 zeta signaling domain (Table 4). These CD123-CAR constructs were cloned into a third generation lentiviral plasmid backbone (Lentigen) under the control of the human EF-1α promoter. In our previous publication, we showed that modified baboon envelope glycoprotein pseudotyped lentiviral vectors (BaEV) can efficiently transduce NK cells (Bari R et al., Front Immunol. (2019) 10:2001). All of the 13 listed lentiviral vectors (LV) containing CD123-CAR were pseudotyped with BaEV, and the viral vectors were generated by transient transfection of HEK 293T cells.

[0340] [Table 4]

[0341] Primary NK cells were isolated from PBMCs by magnetic separation to obtain a pure cell population (Figure 10A). Most cell lines, especially acute myeloid leukemia (AML) cells, are not suitable for testing the cytotoxicity of CAR-NK cells because they are sensitive to the natural cytotoxicity of NK cells. However, the RS4-11 cell line is known to be insensitive to the natural cytotoxicity of NK cells. Therefore, many NK cell laboratories, including ours, routinely use RS4-11 as a target cell to test the function of CAR-NK cells. To use RS4-11 as a target cell to test the function of CD123-CAR, a daughter RS4-11 cell line stably expressing CD123 was generated (Figure 10B).

[0342] NK cells were cultured in NK MACS medium containing IL-2 / IL-15 / IL-1β for 2 days and then transduced with BaEV pseudotyped lentiviral vector (BaEV-LV), resulting in efficient transduction and activation of primary NK cells. Transduction of NK cells with lentiviral vectors containing different CD123-CAR constructs resulted in different expression of CD123-CAR on the surface of NK cells (Figure 11). Among the 13 CD123-CARs, Z32 and D0126 binders were the best for transducing NK cells, with transduction efficiencies of 51.55% and 61.37%, respectively. Based on these expression results, CAR constructs Z32 and D0126 were selected for further analysis.

[0343] CD123-CAR NK cells efficiently and specifically kill target cells expressing CD123 Activated NK cells were transduced with BaEV pseudotyped lentiviral vectors containing CD123-CAR Z32 (Z32-BaEV-LV) and D0126 (D0126-BaEV-LV). CD123-CAR expression of Z32 and D0126 was 70.5% and 64.19%, respectively (Figure 12A). Furthermore, the cytotoxicity of CD123-CAR-expressing NK cells was tested against target cells RS4-11-CD123. RS4;11 cells expressing CD123 (Figure 10B) are insensitive to the natural cytotoxicity of NK cells. The results showed that non-transduced NK cells were unable to kill RS4;11-CD123 cells, whereas both CD123-CAR (Z32 and D0126) NK cells killed RS4;11-CD123 very efficiently, demonstrating the high functionality and specificity of the generated CD123-CAR NK cells (Figure 12B).

[0344] Next, the specificity of CD123-CAR for the CD123 antigen was confirmed by serial dilution. NK cells were transduced with different amounts of lentiviral vectors containing CD123-CAR. As expected, the higher the amount of CD123-CAR-LV, the higher the expression of CD123-CAR (Figure 13A). Finally, the cytotoxicity of differentially expressing CD123-CAR NK cells was tested against RS4-11-CD123 cells at the same effector-target ratio (Figure 13B). The highest expressing CD123-CAR-NK cells showed the highest killing, and the lowest expressing CD123-CAR-NK cells showed the lowest killing, confirming the specificity of CD123-CAR for the CD123 antigen.

[0345] Expression of CD123-CAR does not adversely affect NK proliferation and viability Primary NK cells were isolated, activated, transduced with Z32 and D0126, and then expanded for 13 days. Untransduced NK cells were used as a control. The proliferation of untransduced, Z32-transduced, and D0126-transduced NK cells was 61-fold, 49-fold, and 42-fold, respectively (Figure 14A). The experiment was started with equal NK cell numbers for each condition. It is possible that some of the NK cells lost during the transduction process could explain the difference in cell proliferation between untransduced and transduced cells. However, the difference in proliferation between NK cells transduced with lentiviral vectors encoding Z32- and D0126-CAR was negligible. The viability of NK cells on days 3, 5, 8, and 11 (Figure 5B) was also checked. There was no significant difference in cell viability between untransduced, Z32-transduced, and D0126-transduced NK cells (Figure 14B), suggesting that CD123-CAR does not adversely affect NK cell viability.

[0346] Reference to sequence listing This application contains a Sequence Listing that has been submitted electronically to the U.S. Patent and Trademark Office via a PDF file titled "Sequence Listing." The Sequence Listing is incorporated by reference.

[0347] Sequences of the present disclosure The nucleic acid and amino acid sequences listed below are shown using standard abbreviations for nucleotide bases, and one-letter or three-letter codes for amino acids, as defined in 37 C.FR1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included by any reference to the shown strand. In the accompanying sequence listing: SEQ ID NO:1 Nucleotide sequence of CAR D0125 CD123 MB31-A01 CD8 BBz SEQ ID NO:2 Amino acid sequence of CAR D0125 CD123 MB31-A01 CD8 BBz MLLLVTSLLLCELPHPAFLLIPQIQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGSGEL LYASYYYYYMDVWGKGTTVTVSSGGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPELMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEAD YYCSSYTSSSTPVVFGGGTKLTVLAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCS CRFPEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 3 Nucleotide sequence of CAR D0126 CD123 MB31-C01 CD8 BBz SEQ ID NO: 4: Amino acid sequence of CAR D0126 CD123 MB31-C01 CD8 BBz MLLLVTSLLLCELPHPAFLLIPEVQLLESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTGLL WFGTRNYYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLVIYDVSNRPSGLSNRFSGSKSGNTASLTISGLQAEDEA DYYCNSYAGSGSWVFGGGTKLTVLAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCS CRFPEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 5 Nucleotide sequence of CAR D0127 CD123 MB35-E02 CD8 BBz SEQ ID NO:6: Amino acid sequence of CAR D0127 CD123 MB35-E02 CD8 BBz MLLLVTSLLLCELPHPAFLLIPQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARHGG MATMLPYGAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIRLTQSPSSLSASVGDRVTITCRASQGISSYLNWYQQKPGKAPKLLIYAASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQSYSTSLTFGGGTKVEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 7 Nucleotide sequence of CAR D0128 CD123 MB36-A05 CD8 BBz SEQ ID NO: 8 Amino acid sequence of CAR D0128 CD123 MB36-A05 CD8 BBz MLLLVTSLLLCELPHPAFLLIPQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGGR NSYYYYYMDVWGKGTTVTVSSGGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRPSGVSNRFSGSKSSGNTASLTISGLQAEDEADY YCSSYTSSSPVVFGGGTKLTVLAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 9 Nucleotide sequence of CAR D0129 CD123 MB40-F08 CD8 BBz SEQ ID NO: 10 Amino acid sequence of CAR D0129 CD123 MB40-F08 CD8 BBz MLLLVTSLLLCELPHPAFLLIPQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGSGE LLYASYYYYYMDVWGKGTTVTVSSGGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEA DYYCSSYTSSSPLVFGTGTKVTVLAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCS CRFPEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 11 Nucleotide sequence of CAR D0130 CD123 MB40-H08 CD8 BBz SEQ ID NO: 12 Amino acid sequence of CAR D0130 CDAR123 MB40-H08 CD8 BBz MLLLVTSLLLCELPHPAFLLIPQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARNEWY SYYYYYMGVWGKGTTVTVSSGGGGSGGGGSGGGGSQSALTQPASVGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYY CSSYTSSSTLVVFGGGTKLTVLAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO:13 Nucleotide sequence of leader / signal peptide sequence atgctgctgctggtgaccagcctgctgctgtgcgaactgccgcatccggcgtttctgctgattccg SEQ ID NO: 14: Amino acid sequence of leader / signal peptide sequence MLLLVTSLLLCELPHPAFLLIP SEQ ID NO: 15 Nucleotide sequence of CAR D0131 CD123 MB42-D03 CD8 BBz SEQ ID NO: 16: Amino acid sequence of CAR D0131 CD123 MB42-D03 CD8 BBz MLLLVTSLLLCELPHPAFLLIPQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARGAY YDFWSSYSWFDPWGQGTLVTVSSGGGGSGGGGSGGGGSSYVLTQPPSVSVAPKTARITCGGNSIGSKSVQWYQQKPGQAPVLVIYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYY CQVWDSSSDVVFGGGTKLTVLAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 17 Nucleotide sequence of CAR D0132 CD123 MB42-E02 CD8 BBz SEQ ID NO: 18: Amino acid sequence of CAR D0132 CD123 MB42-E02 CD8 BBz MLLLVTSLLLCELPHPAFLLIPQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARGAY YDFWSGYSWFDPWGQGTLVTVSSGGGGSGGGGSGGGGSQSVLTQPPSVSVAPGQTARIPCGGNNIGSKGVHWYQQKSGQAPVMVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYY CQVWDSSGDLVLFGGGTKLTVLAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 19 Nucleotide sequence of CAR D0133 CD123 MB42-E12 CD8 BBz SEQ ID NO: 20: Amino acid sequence of CAR D0133 CD123 MB42-E12 CD8 BBz MLLLVTSLLLCELPHPAFLLIPQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARGAY YDFWSSYSWFDPWGQGTLVTVSSGGGGSGGGGSGGGGSSYVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYY CQVWDSSSDHVVFGGGTKLTVLAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 21 Nucleotide sequence of CAR D0134 CD123 MB44-H01 CD8 BBz SEQ ID NO: 22 Amino acid sequence of CAR D0134 CD123 MB44-H01 CD8 BBz MLLLVTSLLLCELPHPAFLLIPQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQRFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGLGT SYYYYYMDVWGKGTTVTVSSGGGGSGGGGSGGGGSQSVLTQPPSASGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYY CSSYTSSSTPVVFGGGTKLTVLAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 23 Nucleotide sequence of CAR LTG2078 CD123 M12306 CD8 BBz SEQ ID NO: 24: Amino acid sequence of CAR LTG2078 CD123 M12306 CD8 BBz MLLLVTSLLLCELPHPAFLLIPEVQLVQSGSELKKPGASVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCASTAR RGWDTAGPLDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTIACRASQTISRYLNWYQQKPGKAPKLLIYAASSLQSGVSSRFSGSGSGTEFTLTISSLQPEDFATY FCQQTYSPPITFGQGTRLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 25 Nucleotide sequence of CAR LTG1906 CD33_4 CD8 BBz SEQ ID NO: 26: Amino acid sequence of CAR LTG1906 CD33_4 CD8 BBz MLLLVTSLLLCELPHPAFLLIPEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPRQGLEWVANIKQDGSEKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTATYYCAKENVDWGQGTLVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDF ACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 27 DNA CD8 transmembrane domain nucleotide sequence atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc accctttact gc SEQ ID NO: 28: Amino acid sequence of the CD8 transmembrane domain Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys SEQ ID NO: 29 Nucleotide sequence of DNA CD8 hinge domain accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg gacttcgcct gtgat SEQ ID NO: 30 Amino acid sequence of the CD8 hinge domain Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Amino acid sequence of the hinge region at amino acid positions 118 - 178 of SEQ ID NO: 31 CD8.alpha (NCBI RefSeq: NP_001759.3) Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Amino acid sequence of SEQ ID NO: 32 human IgG CL sequence Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala Pro Thr Glu Cys Ser SEQ ID NO: 33 Nucleotide sequence of the DNA signaling domain of 4-1BB aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggagaggtgt gaactg SEQ ID NO: 34: Amino acid sequence of the signaling domain of 4-1BB Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu SEQ ID NO: 35 Nucleotide sequence of the DNA signaling domain of CD3-zeta agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc tacgacgccc ttcacatgca ggccctgccc cctcgc CD3 Photo Information Photo Information Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp With Leu His Met Gln Ala Leu Pro Pro Arg Characteristics of 37 ScFv CD19 gataccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcaccatcagttgca gggcaagtca ggacattagt aaatatttta attggtacca gcagaaacca gatgactg ttaaactcct gatctaccat acatcagat tacaccagtcag aggtcagg aggtc The shapes eat eat. ccattagcaa cctggagcaa gaagatattg ccacttactt ttgccaacag ggtaatacgc ttccgtacac gttcggaggg gggaccaagc tggagatcac aggtggcggt ggctcgggcg gtggtgggtc gggtggcggc ggatctgagg tgaaactgca ggagtcagga cctggcctgg tggcgccctc acagagcctg tccgtcacat gcactgtctc aggggtctca ttacccgact atggtgtaag ctggattcgc cagcctccac gaaagggtct ggagtggctg ggagtaatat ggggtagtga aaccacatac tataattcag ctctcaaatc cagactgacc atcatcaagg acaactccaa gagccaagtt ttcttaaaaa tgaacagtct gcaaactgat gacacagcca tttactactg tgccaaacat tattactacg gtggtagcta tgctatggac tactggggcc aaggaacctc agtcaccgtc tcctca Amino acid sequence of SEQ ID NO: 38 ScFv CD19 Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser SEQ ID NO:39 Nucleotide sequence of GMCSF leader peptide ATGCTGCTGCTGGTGACCAGCCTGCTGCTGTGCGAACTGCCGCATCCGGCGTTTCTGCTGATTCCG SEQ ID NO: 40 Amino acid sequence of GMCSF leader peptide MLLLVTSLLLCELPHPAFLLIP SEQ ID NO: 41 Nucleotide sequence of TNFRSF19 leader peptide GGCTCTGAAAGTGCTGTTGGAACAAGAAAAGACCTTCTTCACCTTGCTCGTGTTGCTGGGGTACCTGTCCTGCAAAGTCACCTGT SEQ ID NO: 42 Amino acid sequence of TNFRSF19 leader peptide MALKVLLEQEKTFFTLLVLLGYLSCKVTC SEQ ID NO: 43 Nucleotide sequence of CD8 alpha leader peptide atggcgctgccggtgaccgcgctgctgctgccgctggcgctgctgctgcatgcggcgcgc ccg SEQ ID NO: 44: Amino acid sequence of CD8 alpha leader peptide MALPVTALLLPLALLLHAARP SEQ ID NO: 45 Nucleotide sequence of CD28 costimulatory domain CGGTCGAAGAGGTCCAGACTCTTGCACTCCGACTACATGAACATGACTCCTAGAAGGCCCGGACCCACTAGAAAGCACTACCAGCCGTACGCCCCTCCTCGGGATTTCGCCGCATACCGG TCC SEQ ID NO: 46: Amino acid sequence of the CD28 costimulatory domain RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS SEQ ID NO: 47 Nucleotide sequence of CD3 zeta activation domain AGAGTGAAGTTCAGCCGCTCAGCCGATGCACCGGCCTACCAGCAGGGACAGAACCAGCTCTACAACGAGCTCAACCTGGGTCGGCGGGAAGAATATGACGTGCTGGACAAACGGCGCGGCAGAGATCCGGAGATGGGGGGAAAGCCGAGGAGGAAGAACCCTCAAGAG GGCCTGTACAACGAACTGCAGAAGGACAAGATGGCGGAAGCCTACTCCGAGATCGGCATGAAGGGAGAACGCCGGAGAGGGAAGGGTCATGACGGACTGTACCAGGGCCTGTCAACTGCCACTAAGGACACTTACGATGCGCTCCATATGCAAGCTTTGCCCCCGCGG SEQ ID NO: 48 Amino acid sequence of CD3 zeta activation domain RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 49 Nucleotide sequence of TNFRSF19 hinge and transmembrane domain (transmembrane domain underlined) GCGGCCGCGGTCGGATTCCAAGACATGGAATGCGTGCCCTGCGGCGACCCGCCACCTCCTTACGAGCCGCACTGCGCATCGAAGGTCAACCTCGTGAAGATCGCGAGCACCGCGTCCTCACCCCGGGATACTGCTCTG GCCGCCGTGATTTGTTCCGCCTTGGCCACCGTGCTTCTGGCCCTGCTGATCCTCTGTGTGATC SEQ ID NO: 50 Amino acid sequence of TNFRSF19 hinge and transmembrane domain (transmembrane domain underlined) AAAVGFQDMECVPCGDPPPPY EPHCASKVNLVKIASTASS PRDT AL AAVICSALATVLLALLILCVI SEQ ID NO: 51 Nucleotide sequence of TNFRSF19 transmembrane domain GCCGCCGTGATTTGTTCCGCCTTGGCCACCGTGCTTCTGGCCCTGCTGATCCTCTGTGTGATC SEQ ID NO: 52 Amino acid sequence of TNFRSF19 transmembrane domain AAVICSALATVLLALLILCVI SEQ ID NO: 53 Nucleotide sequence of TNFRSF19 hinge domain GCGGCCGCGGTCGGATTCCAAGACATGGAATGCGTGCCCTGCGGCGACCCGCCACCTCCTTACGAGCCGCACTGCGCATCGAAGGTCAACCTCGTGAAGATCGCGAGCACCGCGTCCTCACCCCGGGATACTGCTCTG SEQ ID NO: 54: Amino acid sequence of TNFRSF19 hinge domain AAAVGFQDMECVPCGDPPPPY EPHCASKVNLVKIASTASSPR DTAL SEQ ID NO: 55 Nucleotide sequence of truncated TNFRSF19 hinge domain TACGAGCCTCACTGCGCCAGCAAAGTCAACTTGGTGAAGATCGCGAGCACTGCCTCGTCCCCTCGGGACACTGCTCTGGC SEQ ID NO: 56: Amino acid sequence of truncated TNFRSF19 hinge domain YEPHCASKVNLVKIASTASSP RDTAL SEQ ID NO: 57 Nucleotide sequence of CD8a hinge domain fused to TNFRSF19 transmembrane domain (transmembrane sequence underlined) GCGGCCGCGCCCGCCCCTCGGCCCCCGACTCCTGCCCGACGATCGCTTCCCAACCTCTCTCGCTGCGCCCGGAAGCATGCCGGCCCGCCGCCGGTGGCGCTGTCCACACTCGCGGACTGGACTTTGATACCGCACTG GCGGCCGTGATCTGTAGCGCCCTGGCCACCGTGCTGCTGGCGCTGCTCATCCTTTGCGTGATCTACTGCAAGCGGCAGCCTAGG SEQ ID NO: 58 Amino acid sequence of the CD8a hinge domain fused to the TNFRSF19 transmembrane domain (transmembrane sequence underlined) AAAPAPRPPTPAPTIASQPLS LRPEACRPAAGGAVHTRGLDF DTAL AAVICSALATVLLALLILCVI YCKRQPR SEQ ID NO: 59 Nucleotide sequence of CD28 costimulatory domain CGGTCGAAGAGGTCCAGACTCTTGCACTCCGACTACATGAACATGACTCCTAGAAGGCCCGGACCCACTAGAAAGCACTACCAGCCGTACGCCCCTCCTCGGGATTTCGCCGCATACCGGTCC SEQ ID NO: 60 Amino acid sequence of the CD28 costimulatory domain RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS SEQ ID NO:61 Nucleotide sequence of CD3 zeta version 2 cgcgtgaaatttagccgcagcgcggatgcgccggcgtatcagcagggccagaaccagctg tataacgaactgaacctgggccgccgcgaagaatatgatgtgctggataaacgccgcggc cgcgatccggaaatgggcggcaaaccgcgccgcaaaaacccgcaggaaggcctgtataac gaactgcagaaagataaaatggcggaagcgtatagcgaaattggcatgaaaggcgaacgc cgccgcggcaaaggccatgatggcctgtatcagggcctgagcaccgcgaccaaagatacc tatgatgcgctgcatatgcaggcgctgccgccgcgc SEQ ID NO: 62 Amino acid sequence of CD3 zeta version 2 RVKFSRSADAPAYQQGQNQLY NELNLG RREEYDVLDKRRGRDPEMGGK PRRKNP QEGLYNELQKDKMAEAYSEIG MKGERRRGKGHDGLYQGLSTA TKDTYDALHMQALPPR SEQ ID NO: 63 Nucleotide sequence of furin P2A furin CGCGCGAAACGCAGCGGCAGCGGCGCGACCAACTTTAGCCTGCTGAAACAGGCGGGCGAT GTGGAAGAAAACCCGGGCCCGCGAGCAAAGAGG SEQ ID NO: 64 Amino acid sequence of Furin P2A furin (furin sequence underlined) RAKR SGSGATNFSLLKQAGDVEENPGP RAKR SEQ ID NO: 65 Nucleotide sequence of furin T2A AGAGCTAAACGCTCTGGGTCTGGTGAAGGACGAGGTAGCCTTCTTACGTGCGGAGACGTGGAGGAAAACCCAGGACCC SEQ ID NO: 66 Amino acid sequence of furin T2A (furin sequence underlined) RAKR SGSGEGRGSLLTCGDVEENPGP SEQ ID NO: 67 Nucleotide sequence of truncated EGFR (tEGFR) tag SEQ ID NO: 68: Amino acid sequence of truncated EGFR (tEGFR) tag RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCK ATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM SEQ ID NO: 69 Nucleotide sequence of CD123 binder MT-16 CAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCCACGAGCACAGCCTACACGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGCCCGGTTGGGAGGAGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAGGAGGTGGCGGGTCTGGTGGAGGCGGTAGCGGTGGTGGCGGATCCCAGTCTGTGCTGACGCAGCCGCCCTCAGTGTCTGCGGCCCCAGGACAGAAGGTCACCATCTCCTGCTCTGGAGGCAGCTCCAACATTGGCAATCATTATGTGTCCTGGTATCAGCAGCTCCCAGGAGCAGCCCCCAAACTCCTCATTTATGACGATAATAAGCGACCCTCAGGGATTCCTGACCGATTCTCTGGCTCCAGGTCTGGCACGTCAGCCACCCTGGGCATCACCGGACTCCAGAGTGGGGACGAGGCCGATTATTACTGCGGAGCATGGGATAGTAGTCTTGCTGCTCATGTCTTCGGAACTGGGACCAAGGTCACCGTCCTAGGT SEQ ID NO: 70 Amino acid sequence of CD123 binder MT-16 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYTELSSLRSEDTAVYYCARARLGGAFDIWGQGTMVTVSSGGGG SGGGGSGGGGSQSVLTQPPSVSAAPGQKVTISSCSGGSSNIGNHYVSWYQQLPGAAPKLLIYDDNKRPSGIPDRFSGSRSGTSATLGITGLQSGDEADYYCGAWDSSLAAHVFGTGTKVTVLG SEQ ID NO: 71 Nucleotide sequence of CD123 binder MT-32 CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTATAATGATTATGCAGTATCTGTGAAAAGTCGAATAACCATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACATGGCTGTGTATTACTGTGCAAGAGGCGTTGATAGTAGCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGAGGTGGCGGGTCTGGTGGAGGCGGTAGCGGTGGTGGCGGATCCCAGTCTGTCGTGACGCAGCCGCCCTCAGTGTCTGCGGCCCCAGGACAGAGCGTCACCATCTCCTGTTCTGGAAGCAGTTCCACCGTTGGCGATAATTATGTGTCCTGGTACCAGCAACTCCCAGGAACAGCCCCCAAACTCCTCATTTTTGACGATTATAAACGACCCTCAGGGGTTCCTGACCGATTCTCTGGCTCCCAGTCTGGCACCTCAGCCTCCCTGGTCATCACTGGTCTCCAGGCAGAAGATGAGGCTGATTATTACTGCCAGTCTTATGACAGCAGCCTGAGTGGTTATGTCTTCGGGCCTGGGACCAAGGTCACCGTCCTAGGT SEQ ID NO: 72 Amino acid sequence of CD123 binder MT-32 QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDMAVYYCARGVDSSFDYWGQGTLVTVSSGGG GSGGGGSGGGGSQSVVTQPPSVSAAPGQSVTISCSGSSSTVGDNYVSWYQQLPGTAPKLLIFDDYKRPSGVPDRFSGSQSGTSLVITGLQAEDEADYYCQSYDSSLSGYVFGPGTKVTVLG SEQ ID NO: 73 Nucleotide sequence of the DNA signaling domain of 4-1BB AAGCGCGGACGGAAGAAACTCTTGTACATCTTCAAGCAGCCGTTCATGCGCCCTGTGCAAACCACCCAAGAAGAGGACGGGTGCTCCTGCCGGTTCCCGGAAGAGGAAGAGGGCGGCTGCGAACTG SEQ ID NO: 74: Amino acid sequence of the DNA signaling domain of 4-1BB KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL SEQ ID NO: 75 Nucleotide sequence of the DNA signaling domain of CD3z CGCGTGAAGTTTTCCCGGTCCGCCGACGCTCCGGCGTACCAGCAGGGGCAAAACCAGCTGTACAACGAACTTAACCTCGGTCGCCGGGAAGAATATGACGTGCTGGACAAGCGGCGGGGAAGAGATCCCGAGATGGGTGGAAAGCCGCGGCGGAAGAACCCTCAGGAG GGCTTGTACAACGAGCTGCAAAAGGACAAAATGGCCGAAGCCTACTCCGAGATTGGCATGAAGGGAGAGCGCAGACGCGGGAAGGGACACGATGGACTGTACCAGGGACTGTCAACCGCGACTAAGGACACTTACGACGCCCTGCACATGCAGGCCCTGCCCCCGCGC SEQ ID NO: 76 Amino acid sequence of the DNA signaling domain of CD3z RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 77 Nucleotide sequence of CAR123 Z16 SEQ ID NO: 78 Amino acid sequence of CAR123 Z16 MLLLVTSLLLCELPHPAFLLIPQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYTELSSLRSEDTAVYYCARARL GGAFDIWGQGTMVTVSSGGGGSGGGGSGGGSQSVLTQPPSVSAAPGQKVTISSCSGGSSNIGNHYVSWYQQLPGAAPKLLIYDDNKRPSGIPDRFSGSRSGTSATLGITGLQSGDEADYYCGA WDSSLAAHVFGTGTKVTVLGAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 79 Nucleotide sequence of human IgG4 hinge GAGAGCAAATACGGGCCGCCATGTCCCCCGTGTCCG SEQ ID NO: 80 Amino acid sequence of human IgG4 hinge ESKYGPPCPPCP SEQ ID NO: 81 Nucleotide sequence of human IgG4 CH2 domain GCACCACCAGTTGCTGGCCCTAGTGTCTTCTTGTTCCCTCCCAAGCCCAAAGACACCTTGATGATTTCCAGAACTCCTGAGGTTACCTGCGTTGTCGTAGATGTTTCTCAGGAGGACCCAGAGGTCCAATTTAACTGGTACGTTGATGGGGTGGAAGTTCACA ATGCGAAGACAAAGCCGCGGGAAGAACAATTTCAGTCCACTTACCGGGTTGTCAGCGTTCTGACGGTATTGCATCAAGACTGGCTTAATGGAAAGGAATATAAGTGTAAGGTGTCCAACAAAGGTTTGCCGAGCAGTATTGAGAAGACCATATCAAAGGCGAAG SEQ ID NO: 82 Amino acid sequence of human IgG4 CH2 domain APPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV DGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKA K SEQ ID NO: 83 Nucleotide sequence of human IgG4 CH3 domain GGGCAGCCGCGCGAGCCACAAGTTTACACTTTGCCGCCATCTCAAGAGGAAATGACTAAAAACCAGGTATCCTTGACATGCCTCGTAAAAGGATTTTATCCATCTGATATTGCTGTGGAATGGGAGTCTAACGGGCAGCCGGAAAATAATTACAAAACTA CACCACCTGTGCTCGATTCAGATGGAAGTTTCTTTCCTTTACAGTAGACTTACGGTGGACAAATCTAGGTGGCAGGAAGGGAATGTGTTTAGTTGTAGTGTAATGCACGAGGCACTTCATAACCACTATACACAGAAGTCACTGAGTTTGAGTCTTGGCAAA SEQ ID NO: 84 Amino acid sequence of human IgG4 CH3 domain GQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO: 85 Nucleotide sequence of human IgG4 hinge CH2 CH3 domain GAGAGCAAATACGGGCCGCCATGTCCCCCGTGTCCGGCACCACCAGTTGCTGGCCCTAGTGTCTTCTTGTTCCCTCCCAAGCCCAAAGACACCTTGATGATTTCCAGAACTCCTGAGGTTACCTGCGTTGTCGTAGATGTTTCTCAGGAGGACCCAGAGGTCCAATTTAAC TGGTACGTTGATGGGGTGGAAGTTCACAATGCGAAGACAAAGCCGCGGGAAGAACAATTTCAGTCCACTTACCGGGTTGTCAGCGTTCTGACGGTATTGCATCAAGACTGGCTTAATGGAAAGGAATATAAGTGTAAGGTGTCCAACAAAGGTTTGCCGAGCAGTATTGAG AAGACCATATCAAAGGCGAAGGGGCAGCCGCGCGAGCCACAAGTTTACACTTTGCCGCCATCTCAAGAGGAAATGACTAAAAACCAGGTATCCTTGACATGCCTCGTAAAAGGATTTTATCCATCTGATATTGCTGTGGAATGGGAGTCTAACGGGCAGCCGGAAAATAAT TACAAAACTACACCACCTGTGCTCGATTCAGATGGAAGTTTCTTCCTTTACAGTAGACTTACGGTGGACAAATCTAGGTGGCAGGAAGGGAATGTGTTTAGTTGTAGTGTAATGCACGAGGCACTTCATAACCACTATACACAGAAGTCACTGAGTTTGAGTCTTGGCAAA SEQ ID NO: 86 Amino acid sequence of human IgG4 hinge CH2 CH3 domain ESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIE KTISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO: 87 Nucleotide sequence of CAR123 Z32 SEQ ID NO: 88: Amino acid sequence of CAR123 Z32 MLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDMAVYYCAR GVDSSFDYWGQGTLVTVSSGGGGSGGGGSGGGSQSVVTQPPSVSAAPGQSVTISCGSGSSSTVGDNYVSWYQQLPGTAPKLLIFDDYKRPSGVPDRFSGSQSGTSASLVITGLQAEDEADYYCQ SYDSSLSGYVFGPGTKVTVLGAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

Claims

1. An isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR) comprising amino acid residues 23 to 274 of the amino acid sequence of SEQ ID NO:

4.

2. A chimeric antigen receptor (CAR) encoded by the isolated nucleic acid molecule described in claim 1.

3. A pharmaceutical composition comprising an anti-tumor effective amount of a population of human T cells, wherein the T cells comprise a nucleic acid sequence encoding the chimeric antigen receptor (CAR) of claim 2, and the T cells are T cells of a human having cancer.

4. A vector comprising the nucleic acid molecule described in claim 1.

5. The vector described in claim 4, selected from the group consisting of a DNA vector, an RNA vector, a plasmid vector, a cosmid vector, a herpes virus vector, a measles virus vector, a lentivirus vector, an adenovirus vector, or a retrovirus vector.

6. A cell containing the vector of claim 4.

7. An isolated nucleic acid molecule described in claim 1, wherein CAR comprises the amino acid sequence of SEQ ID NO:

4.

8. Use of a T cell population for the manufacture of a pharmaceutical composition for treating cancer, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), the CAR comprising at least one extracellular antigen-binding domain, including a CD123 antigen-binding domain comprising amino acid residues 23 to 274 of the amino acid sequence of SEQ ID NO: 4, at least one linker or spacer domain, at least one transmembrane domain, and at least one intracellular signaling domain, and the T cells are T cells of a subject with cancer.

9. The use of claim 8, wherein the at least one transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of T cell receptor alpha, T cell receptor beta, T cell receptor zeta chain, CD8, CD28, CD3 epsilon, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.

10. The use described in claim 8, wherein at least one extracellular antigen-binding domain, at least one intracellular signaling domain, or both, are connected to at least one transmembrane domain by a linker or spacer domain.

11. The use of claim 10, wherein at least one linker or spacer domain is derived from the extracellular domain of CD8, TNFRSF19, or CD28 and is linked to at least one transmembrane domain.

12. The use described in claim 8, wherein the nucleic acid sequence encoding the extracellular antigen-binding domain comprises a nucleic acid sequence having a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to amino acid residues 23 to 274 of the amino acid sequence of SEQ ID NO:

4.

13. The use described in claim 8, wherein the CAR comprises the amino acid sequence of SEQ ID NO:

4.

14. The use described in claim 8, wherein at least one intracellular signaling domain further comprises a CD3 zeta intracellular domain.

15. The use described in claim 8, wherein at least one intracellular signaling domain comprises a costimulatory domain, a primary signaling domain, or any combination thereof.

16. The use of claim 15, wherein at least one costimulatory domain comprises a functional signaling domain of OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP12 and 4-1BB (CD137), or any combination thereof.

17. The use described in claim 8, wherein the cancer is a hematological cancer.

18. The use described in claim 17, wherein the hematological cancer is leukemia, lymphoma, or multiple myeloma.

19. The use of claim 18, wherein the leukemia is acute myeloid leukemia (AML), blastic plasmacytoid dendritic cell neoplasm (BPDCN), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), acute lymphoblastic T-cell leukemia (T-ALL), or acute lymphoblastic B-cell leukemia (B-ALL).

20. The use described in claim 18, wherein the lymphoma is mantle cell lymphoma, non-Hodgkin's lymphoma or Hodgkin's lymphoma.

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