Compositions and methods for treating cancer with Anti-CD123 immunotherapy

Abstract

Chimeric antigen receptors comprising a CD123 antigen binding domain are disclosed. Nucleic acids, recombinant expression vectors, host cells, antigen binding fragments and pharmaceutical compositions relating to the chimeric antigen receptors are also disclosed. Also disclosed are methods of treating or preventing cancer in a subject, as well as methods of preparing chimeric antigen receptor T cells.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Utility Application No. 17 / 685,132, filed on March 2, 2022, and U.S. Utility Application No. 18 / 154,209, filed on January 13, 2023. The entire contents of each are incorporated herein by reference.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which is electronically submitted in the form of an XML file named Sequence_Listing. The XML file was created on February 28, 2023 and is 114,458 bytes in size. The materials in the XML file are hereby incorporated by reference in their entirety. Technical Field

[0005] The present application relates to the field of cancer, and in particular to a CD123 antigen binding domain and a chimeric antigen receptor (CAR) comprising such a CD123 antigen binding domain and a method of using the same. Background Art

[0006] Cancer is one of the most deadly threats to human health. In the United States alone, cancer affects nearly 1.3 million new patients each year and is the second leading cause of death after cardiovascular disease, accounting for about a quarter of deaths. Solid tumors are responsible for most of these deaths. Despite significant advances in the medical treatment of some cancers, the overall 5-year survival rate for all cancers has only increased by about 10% in the past 20 years. Cancer or malignant tumors grow and metastasize rapidly in an uncontrolled manner, making treatment extremely difficult.

[0007] AML is a devastating disease with an overall survival rate of only 26%. Although young patients tend to have a better prognosis for the treatment of AML, the 5-year survival in elderly patients can be as low as only 5%. The first-line treatment of AML involves multiple rounds of chemotherapy (i.e., induction, consolidation) with high toxicity risks. If hematopoietic stem cell transplantation is performed after the 1st remission, the 5-year disease-free survival rate is only 30% to 50% (http: / / www.cancer.ca / en / cancer-information / cancer-type / leukemia-acute-my elogenous-aml / prognosis-and-survival / survival-statistics / ?region=on). In addition, AML patients with a high disease burden may not be candidates for bone marrow transplantation, and minimal residual disease before transplantation is associated with AML relapse. Current first-line induction / consolidation therapy often fails to achieve MDR-negative remission to adequately reduce tumor burden, so the risk of AML relapse remains high with or without BMT after first-line therapy (1) Biol Blood Marrow Transplant. 2006 Jun; 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; 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 that is classified as a subtype of AML and is sometimes treated as AML with induction and consolidation chemotherapy, and sometimes as ALL. BMT is usually administered in first remission. However, there are currently no ongoing clinical trials for PBDCN and no approved first-line treatments (Leukemia Lymphoma Society, https: / / www.lls.org / leukemia / blastic-plasmacytoid-dendritic-cell-neoplasm).Therefore, better treatment modalities for CD123+ malignancies are urgently needed.

[0008] The CAR method targeting CD123 is superior to chemotherapy because it can achieve better efficacy in eliminating CD123+ tumor cells and tumor stem cells, and because it avoids the toxicity associated with chemotherapy. Importantly, it is expected that CART cells are more effective than chemotherapy in eliminating minimal residual disease, thereby leading to better long-term treatment prognosis. In addition, since CAR123 can help patients with high tumor burden become eligible for BMT, it can be used as a bridge for transplantation for tumor debulking (debulking).

[0009] CAR123 represents an improvement over the prior art because a unique human ScFv (hereinafter referred to as "hScFv") sequence is used in the CAR design, as opposed to the mouse-derived ScFv used in CAR designs elsewhere. Mouse-derived sequences carry the risk of immunogenicity and can induce allergic or anaphylactic reactions in patients, leading to CAR T elimination or life-threatening hypersensitivity.

[0010] Chimeric antigen receptors (CARs) are hybrid molecules containing three basic units: (1) an extracellular antigen binding motif, (2) a junction / transmembrane motif, and (3) an intracellular T cell signaling motif (Long AH, Haso WM, Orentas RJ. Lessons learned from a highly-active CD22-specific chimeric antigen receptor. Oncoimmunology. 2013; 2(4): e23621). The antigen binding motif of CAR is usually formed after the single chain variable fragment (ScFv) of the minimal binding domain of the immunoglobulin (Ig) molecule. Alternative antigen binding motifs have also been modified, such as receptor ligands (i.e., IL-13 has been modified to bind to the IL-13 receptor expressed by the tumor), complete immune receptors, library-derived peptides, and innate immune system effector molecules (e.g., NKG2D). Alternative cell targets for CAR expression (e.g., NK or γ-δ T cells) are also under development (Brown CE et al. Clin Cancer Res. 2012; 18(8): 2199–209; Lehner M et al. PLoS One. 2012; 7(2): e31210). There is still important work to be done to define the most active T cell populations for transduction with CAR vectors, determine the optimal culture and expansion techniques, and define the molecular details of the CAR protein structure itself.

[0011] The connection motif of CAR can be a relatively stable structural domain, such as the constant domain of IgG, or a flexible linker designed to be extended. Structural motifs (such as those derived from IgG constant domains) can be used to extend the ScFv binding domain away from the T cell plasma membrane surface. This can be important for some tumor targets (such as disialoganglioside GD2; Orentas et al., unpublished observations) in which the binding domain is particularly close to the surface membrane of tumor cells. So far, the signaling motif used in CAR always includes the CD3-ζ chain because the core motif is a key signal for T cell activation. The second-generation CAR reported for the first time is characterized by the CD28 signaling domain and the CD28 transmembrane sequence. This motif is also used in the third-generation CAR containing the CD137 (4-1BB) signaling motif (Zhao Y et al. J Immunol. 2009; 183 (9): 5563–74). With the advent of new technologies that utilize beads linked to anti-CD3 and anti-CD28 antibodies to activate T cells, the presence of the classic "signal 2" from CD28 no longer needs to be encoded by the CAR itself. Using bead activation, it was found that the third-generation vectors were not superior to the second-generation vectors in in vitro assays, and they did not provide a clear benefit over the second-generation vectors in mouse models of leukemia (Haso W, Lee DW, Shah NN, Stetler-Stevenson M, Yuan CM, Pastan IH, Dimitrov DS, Morgan RA, FitzGerald DJ, Barrett DM, Wayne AS, Mackall CL, Orentas RJ. Anti-CD22-chimeric antigen receptors targeting B cell precursor acute lymphoblastic leukemia. Blood. 2013; 121 (7): 1165–74; Kochenderfer JN et al. Blood. 2012; 119 (12): 2709–20). This is confirmed by the clinical success of CD19-specific CARs in the second-generation CD28 / CD3-ζ (Lee DW et al. American Society of Hematology Annual Meeting. New Orleans, LA; December 7-10, 2013) and CD137 / CD3-ζ signaling formats (Porter DL et al. N Engl J Med. 2011; 365(8):725–33).In addition to CD137, other tumor necrosis factor receptor superfamily members (such as OX40) can also provide important persistent signals in CAR-transduced T cells (Yvon E et al. Clin Cancer Res. 2009; 15(18): 5852–60). The culture conditions for culturing CAR T cell populations are also important.

[0012] T cell-based immunotherapy has become a new frontier in synthetic biology; multiple promoters and gene products are considered to direct these highly potent cells to the tumor microenvironment, where they can both escape negative regulatory signals and mediate effective tumor killing. Elimination of unwanted T cells by drug-induced dimerization of inducible caspase 9 constructs with AP1903 illustrates a way in which a powerful switch that can control T cell populations can be pharmacologically activated (Di Stasi A et al. N Engl J Med. 2011; 365(18): 1673–83). Generation of effector T cell populations immune to the negative regulatory effects of transforming growth factor-β by expression of decoy receptors further illustrates the extent to which effector T cells can be engineered to achieve optimal antitumor activity (Foster AE et al. J Immunother. 2008; 31(5): 500–5). Therefore, although CAR appears to be able to trigger T cell activation in a manner similar to endogenous T cell receptors, the main obstacle to the clinical application of this technology so far has been limited to the 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 for discovering new compositions and methods for treating AML using methods that can exhibit specific and effective anti-tumor effects without the above-mentioned disadvantages (i.e., high toxicity, insufficient efficacy).

[0013] In addition, in the past few years, cancer immunotherapy based on natural killer (NK) cells has achieved rapid development (Shimasaki, N., Jain, A. & Campana, D. NK cells for cancer immunotherapy. Nat Rev Drug Discov 19, 200–218 (2020)). In children and adult cancer patients, human haploid NK cells have been shown to be easy to adoptively transfer and expand. (Miller, J Set al. Blood 105, 3051–3057 (2005); Rubnitz, JE et al. J. Clin. Oncol. 28, 955–959 (2010)). In addition, the second-generation CD19-CAR NK cells generated in vitro are effective in killing B cell ALL (Imai, C., Iwamoto, S. & Campana, D. Blood 106, 376–383 (2005)). NK cell activity in vivo can be further enhanced by the expression of IL-15, IL-12, IL-18, or other cytokine variants that stimulate autonomous growth, cytotoxicity, and prolonged effector functions (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)). By secreting chemical attractants (such as CCL5, XCL1 and FLT3L), NK cells can attract dendritic cells to the tumor site, and thus promote a tumor microenvironment that is conducive to 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)). In addition, by controlling culture conditions, NK cells can be made more effective against tumors. For example, activation of NK cells with cytokines IL-12, IL-15 and IL-18 enhances NK cell anti-AML response (Romee, R. et al. Sci. Transl. Med. 8, 357ra123 (2016)).

[0014] It is feasible to clinically generate NK cells from umbilical cord blood hematopoietic progenitor cells (Spanholtz, J. et al. PLOS ONE6, e20740 (2011); Knorr, DA et al. Stem Cell Transl. Med. 2, 274–283 (2013)). Similarly, CAR NK cells can be obtained by differentiation from iPSC expressing CAR (Li, Y., Hermanson, DL, Moriarity, BS & Kaufman, DSCell Stem Cell 23, 181–192 (2018)). Although methods for producing efficient NK cells and CAR NK cells for cancer treatment continue to develop, significant progress has been made in this field in the past few years (Granzin, M. et al.. Oncoimmunology 5, e1219007 (2016)). In summary, these findings indicate the emerging potential of CARNK cells in cancer immunotherapy.

[0015] The present invention solves these needs by providing CAR compositions and methods of treatment that can be used to treat cancer and other diseases and / or conditions. In particular, as disclosed and described herein, the present invention provides such CAR: it can be used to treat diseases, disorders or conditions associated with CD123 expression disorders, and the CAR includes CD123 antigen binding domains, which show high surface expression on transduced T cells and NK cells, showing a high degree of cell lysis and transduced T cell in vivo expansion and persistence. Summary of the invention

[0016] Provided herein are new anti-CD123 antibodies or their antigen binding domains and chimeric antigen receptors (CARs) comprising such CD123 antigen binding domains, and host cells (e.g., T cells) expressing the receptors and nucleic acid molecules encoding the receptors.CAR may be composed of a single molecule expressed on the surface of effector cells, or CAR may include a signal transduction module and a soluble targeting module expressed by effector cells, such as when a soluble targeting module is combined with a signal transduction module expressed by a cell, to form a complete functional CAR.CAR shows high surface expression on transduced T cells, with a high degree of cell lysis and transduced T cells in vivo amplification and persistence.Also provided are methods for treating cancer in an object using disclosed CAR, host cells and nucleic acid molecules, for example.

[0017] Thus, in one aspect, 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 NO: 1, 3, 5, 7, 9, 11, 15, 17, 19, 21, 23, 25, 69, 71, 77 and 87.

[0018] In one embodiment, an isolated polynucleotide encoding a fully human anti-CD123 antibody or a fragment thereof is provided, wherein the antibody or fragment thereof comprises a fragment selected from a Fab fragment, a F(ab')2 fragment, a Fv fragment, and a single-chain Fv (ScFv).

[0019] In one embodiment, an isolated polynucleotide encoding a fully human anti-CD123 antibody or a fragment thereof is provided, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 16, 18, 20, 22, 24, 26, 70, 72, 78 and 88.

[0020] In one aspect, an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR) is provided, wherein the CAR comprises from N-terminus to C-terminus at least one CD123 antigen binding domain, at least one transmembrane domain and at least one intracellular signaling domain, wherein the at least one CD123 antigen binding domain is encoded by a nucleotide sequence comprising a nucleic acid sequence selected from SEQ ID NO: 1, 3, 5, 7, 9, 11, 15, 17, 19, 21, 23, 25, 69, 71, 77 and 87.

[0021] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded extracellular CD123 antigen binding domain comprises at least one single-chain variable fragment of an antibody that binds to CD123.

[0022] 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 to CD123.

[0023] In one embodiment, the targeting domain of CAR is independently expressed in the form of a monoclonal antibody, ScFv Fab, Fab'2 and comprises an antigen targeting domain coupled to an additional binding tag or epitope, wherein the antigen targeting domain comprises a nucleic acid sequence selected from SEQ ID NO: 1, 3, 5, 7, 9, 11, 15, 17, 19, 21, 23, 25, 69, 71, 77 and 87, and the effector cell expression component of CAR comprises a binding domain, which is specific for binding to a tag or epitope expressed on a soluble CAR module, such as the specific binding of the soluble component of CAR to the cell binding component of CAR to form a complete functional CAR structure.

[0024] In another embodiment, the targeting domain of CAR is independently expressed in the form of a monoclonal antibody, ScFv Fab, Fab'2 and comprises an antigen targeting domain and an additional ScFv, wherein the antigen targeting domain comprises a nucleic acid sequence selected from SEQ ID NO: 1, 3, 5, 7, 9, 11, 15, 17, 19, 21, 23, 25, 69, 71, 77 and 87, and the effector cell expression component of CAR comprises a tag or epitope that specifically reacts with the additional ScFv expressed on the soluble CAR module, such as the specific binding of the soluble component of CAR to the cell binding component of CAR to form a complete functional CAR structure.

[0025] In another embodiment, an isolated nucleic acid molecule encoding CAR is provided, wherein the encoded CAR extracellular CD123 antigen binding domain further comprises at least one lipocalin-based antigen binding antigen (anticalin) bound to CD123.

[0026] In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD123 antigen binding domain is connected to the transmembrane domain via a linker domain.

[0027] In another embodiment, an isolated nucleic acid molecule encoding CAR is provided, wherein the encoded CD123 extracellular antigen binding domain is preceded by a sequence encoding a leader peptide or a signal peptide.

[0028] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, the CAR comprising at least one CD123 antigen binding domain, the at least one CD123 antigen binding domain being encoded by a nucleotide sequence comprising a nucleic acid sequence selected from SEQ ID NO: 1, 3, 5, 7, 9, 11, 15, 17, 19, 21, 23, 25, 69, 71, 77 and 87, and wherein the CAR further encodes an extracellular antigen binding domain targeting an antigen, including but not limited to CD19, CD20, CD22, ROR1, mesothelin, CD33, CD38, CD138, BCMA (CD269), GPC2, GPC3, FGFR4, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, NY-ESO-1 TCR, MAGE A3 TCR, or any combination thereof.

[0029] In certain embodiments, an isolated nucleic acid molecule encoding a CAR is provided, wherein the additionally encoded extracellular antigen binding domain comprises an anti-CD19 ScFv antigen binding domain, an anti-CD20 ScFv antigen binding domain, an anti-CD22 ScFv antigen binding domain, an anti-ROR1 ScFv antigen binding domain, an anti-mesothelin ScFv antigen binding domain, an anti-CD33 ScFv antigen binding domain, an anti-CD38 ScFv antigen binding domain, an anti-CD123 (IL3RA) ScFv antigen binding domain, an anti-CD138 ScFv antigen binding domain, an anti-BCMA (CD269) ScFv antigen binding domain, an anti-GPC2 ScFv antigen binding domain, an anti-GPC3 ScFv antigen binding domain, an anti-FGFR4 ScFv antigen binding domain, an anti-c-Met ScFv antigen binding domain, an anti-PMSAScFv antigen binding domain, an anti-glycolipid F77 ScFv antigen binding domain, an anti-EGFRvIII ScFv antigen binding domain, anti-GD-2ScFv antigen binding domain, anti-NY-ESo-1 TCR ScFv antigen binding domain, anti-MAGE A3 TCR ScFv antigen binding domain, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or any combination thereof.

[0030] In one aspect, a CAR provided herein further comprises a linker or spacer domain.

[0031] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the extracellular CD123 antigen binding domain, the intracellular signaling domain, or both are connected to the transmembrane domain via a linker or a spacer domain.

[0032] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded linker domain is derived from the extracellular domain of CD8 or CD28 and is connected to the transmembrane domain.

[0033] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded CAR further comprises a transmembrane domain, wherein the transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of the alpha, beta or zeta chain of a T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, or a combination thereof.

[0034] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded intracellular signaling domain further comprises a CD3 zeta intracellular domain.

[0035] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded intracellular signaling domain is arranged at the C-terminal side relative to the CD3 zeta intracellular domain.

[0036] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded at least one intracellular signaling domain comprises a co-stimulatory domain, a primary signaling domain, or a combination thereof.

[0037] In other embodiments, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded at least one co-stimulatory domain comprises a functional signaling domain of: OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP12 and 4-1BB (CD137), or a combination thereof.

[0038] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, which further comprises a leader sequence or a signal peptide, wherein the leader or signal peptide nucleotide sequence comprises a nucleotide sequence of SEQ ID NO: 13, SEQ ID NO: 39, SEQ ID NO: 41 or SEQ ID NO: 43.

[0039] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded leader sequence comprises the amino acid sequence of SEQ ID NO: 14, SEQ ID NO: 40, SEQ ID NO: 42 or SEQ ID NO: 44.

[0040] 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.

[0041] 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.

[0042] In another embodiment, a CAR is provided, wherein the at least one transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of the α, β or ζ chain of a T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, or a combination thereof.

[0043] In some embodiments, a CAR is provided, wherein the CAR further encodes an extracellular antigen binding domain comprising CD19, CD20, CD22, ROR1, mesothelin, CD33, CD38, CD123 (IL3RA), CD138, BCMA (CD269), GPC2, GPC3, FGFR4, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, NY-ESO-1 TCR, MAGE A3 TCR, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or any combination thereof.

[0044] In one embodiment, a CAR is provided, wherein the extracellular antigen binding domain comprises an anti-CD19 ScFv antigen binding domain, an anti-CD20 ScFv antigen binding domain, an anti-CD22 ScFv antigen binding domain, an anti-ROR1 ScFv antigen binding domain, an anti-mesothelin ScFv antigen binding domain, an anti-CD33 ScFv antigen binding domain, an anti-CD38 ScFv antigen binding domain, an anti-CD123 (IL3RA) ScFv antigen binding domain, an anti-CD138 ScFv antigen binding domain, an anti-BCMA (CD269) ScFv antigen binding domain, an anti-GPC2 ScFv antigen binding domain, an anti-GPC3 ScFv antigen binding domain, an anti-FGFR4 ScFv antigen binding domain, an anti-c-Met ScFv antigen binding domain, an anti-PMSAScFv antigen binding domain, an anti-glycolipid F77 ScFv antigen binding domain, an anti-EGFRvIII ScFv antigen binding domain, anti-GD-2ScFv antigen binding domain, anti-NY-ESo-1 TCR ScFv antigen binding domain, anti-MAGE A3 TCR ScFv antigen binding domain, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or any combination thereof.

[0045] In another embodiment, a CAR is provided, wherein the extracellular antigen binding domain comprises only an immunoglobulin variable heavy chain (VH) anti-CD19 antigen binding domain, an anti-CD20 VH antigen binding domain, an anti-CD22 VH antigen binding domain, an anti-ROR1 VH antigen binding domain, an anti-mesothelin VH antigen binding domain, an anti-CD33 VH antigen binding domain, an anti-CD38 VH antigen binding domain, an anti-CD123 (IL3RA) VH antigen binding domain, an anti-CD138 VH antigen binding domain, an anti-BCMA (CD269) VH antigen binding domain, an anti-GPC2 VH antigen binding domain, an anti-GPC3 VH antigen binding domain, an anti-FGFR4 VH antigen binding domain, an anti-c-Met VH antigen binding domain, an anti-PMSAVH antigen binding domain, an anti-glycolipid F77 VH antigen binding domain, an anti-EGFRvIII VH antigen binding domain, anti-GD-2 VH antigen binding domain, anti-NY-ESO-1 TCR VH antigen binding domain, anti-MAGE A3 TCR VH antigen binding domain, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or any combination thereof.

[0046] In another embodiment, a CAR is provided, wherein the extracellular antigen binding domain comprises a protein or peptide (P) sequence capable of specifically binding to a target antigen, which may be derived from a natural or synthetic sequence comprising: an anti-CD19 P antigen binding domain, an anti-CD20 P antigen binding domain, an anti-CD22 P antigen binding domain, an anti-ROR1 P antigen binding domain, an anti-mesothelin P antigen binding domain, an anti-CD33 P antigen binding domain, an anti-CD38 P antigen binding domain, an anti-CD123 (IL3RA) P antigen binding domain, an anti-CD138 P antigen binding domain, an anti-BCMA (CD269) P antigen binding domain, an anti-GPC2 P antigen binding domain, an anti-GPC3 P antigen binding domain, an anti-FGFR4 P antigen binding domain, an anti-c-Met P antigen binding domain, an anti-PMSAP antigen binding domain, an anti-glycolipid F77 P antigen binding domain, an anti-EGFRvIII P antigen binding domain, an anti-GD-2 P antigen binding domain, an anti-NY-ESO-1 TCR P antigen binding domain, anti-MAGE A3 TCR P antigen binding domain, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or any combination thereof. In another embodiment, a CAR is provided, wherein the at least one intracellular signaling domain comprises a co-stimulatory domain and a primary signaling domain.

[0047] In another embodiment, a CAR is provided, wherein the at least one intracellular signaling domain comprises a co-stimulatory domain, wherein the co-stimulatory domain comprises a functional signaling domain of a protein selected from the group consisting of OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP12, and 4-1BB (CD137), or a combination thereof.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] In one aspect, the CAR disclosed herein is modified to express or comprise a detectable marker for diagnosis, monitoring and / or prediction of treatment outcome (e.g., progression-free survival of a cancer patient) or for monitoring the progress of such treatment.

[0065] In one embodiment, the nucleic acid molecule encoding the disclosed CAR may be contained in a vector, such as a viral vector. The vector is a DNA vector, an RNA vector, a plasmid vector, a cosmid vector, a herpes virus vector, a measles virus vector, a lentiviral vector, an adenoviral vector, an adeno-associated virus vector, a baculovirus vector, a foamy virus vector or a retroviral vector, or a combination thereof.

[0066] In certain embodiments, a lentiviral vector encoding one or more of the 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 (Vesicular Stomatitis Virus-Envelope Glycoprotein, VSV-G) pseudotyped lentiviral vector particles. In another embodiment, the pseudotyped lentiviral particles comprise baboon envelope glycoprotein pseudotype vector (BaEV-G) pseudotyped lentiviral vector particles. In another embodiment, the pseudotyped lentiviral particles comprise feline endogenous retrovirus envelope glycoprotein RD114 (RD114-G).

[0067] In certain embodiments, the vector further comprises a promoter, wherein the promoter is an inducible promoter, a tissue-specific promoter, a constitutive promoter, a suicide promoter, or any combination thereof.

[0068] In another embodiment, the vector expressing CAR may also be modified to include one or more control elements for controlling CART cell expression or eliminating CAR-T cells by suicide switch. The suicide switch may include, for example, a drug that induces apoptosis-inducing signaling cascade or induces cell death. In a preferred embodiment, the vector expressing CAR may also be modified to express an enzyme, such as thymidine kinase (thymidine kinase, TK) or cytosine deaminase (cytosinedeaminase, CD).

[0069] In another aspect, a host cell comprising a nucleic acid molecule encoding a CAR is also provided. In some embodiments, the host cell is a T cell, such as a primary T cell obtained from a subject. In one embodiment, the host cell is a CD8 + T cells.

[0070] In another aspect, a pharmaceutical composition comprising an anti-tumor effective amount of a human T cell population is provided, wherein the T cell comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises at least one extracellular antigen binding domain, at least one linker domain, at least one transmembrane domain and at least one intracellular signaling domain, and the extracellular antigen binding domain comprises a CD123 antigen binding domain comprising an amino acid sequence selected from SEQ ID NO: 70, 72, 78 and 88, wherein the T cell is a T cell of a person suffering from cancer. The cancer particularly includes hematological cancer, such as leukemia (e.g., chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL) or chronic myelogenous leukemia (CML)), lymphoma (e.g., mantle cell lymphoma, non-Hodgkin's lymphoma or Hodgkin's lymphoma) or multiple myeloma, or a combination thereof.

[0071] In one embodiment, a pharmaceutical composition is provided, wherein at least one transmembrane domain of the CAR comprises a transmembrane domain of a protein selected from the group consisting of the alpha, beta or zeta chain of a T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, mesothelin, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, or a combination thereof.

[0072] In another embodiment, a pharmaceutical composition is provided wherein the human cancer comprises adult carcinoma, including: oral and pharynx cancer (tongue, mouth, pharynx, head and neck), digestive system cancer (esophagus, stomach, small intestine, colon, rectum, anus, liver, intrahepatic bile duct, gallbladder, pancreas), respiratory system cancer (larynx, lung and bronchus), bone and joint cancer, soft tissue cancer, skin cancer (melanoma, basal and squamous cell carcinoma), childhood tumors (neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma), tumors of the central nervous system (brain, astrocytoma, glioblastoma, glioma), as well as cancers of the breast, reproductive system (cervix, uterine body, ovary, vulva, vagina, prostate, testis, penis, endometrium), urinary system (bladder, kidney and renal pelvis, ureter), eye and orbit, endocrine system (thyroid), and brain and other nervous systems, or any combination thereof.

[0073] In another embodiment, a pharmaceutical composition comprising an anti-tumor effective amount of a human T cell population of a human suffering from cancer is provided, wherein the cancer is a refractory cancer that is not responsive to one or more chemotherapeutic agents. The cancer includes hematopoietic cancer, myelodysplastic syndrome, pancreatic cancer, head and neck cancer, skin tumors, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adult B-cell malignancies (including CLL (chronic lymphocytic leukemia), CML (chronic myeloid leukemia), non-Hodgkin's lymphoma (NHL)), minimal residual disease (MRD) in childhood B-cell malignancies (including B-lineage ALL (acute lymphoblastic leukemia)), multiple myeloma, lung cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, melanoma or other hematological cancers and solid tumors, or any combination thereof.

[0074] In another aspect, a method for preparing T cells (hereinafter referred to as "CAR-T cells") comprising CAR is provided. The method includes transducing T cells with a vector or nucleic acid molecule encoding a disclosed CAR specifically binding to CD123, thereby preparing CAR-T cells.

[0075] In another aspect, a method for producing an RNA-engineered cell population is provided, comprising introducing in vitro transcribed RNA or synthetic RNA encoding a nucleic acid molecule of the disclosed CAR into cells of a subject, thereby producing CAR cells.

[0076] In another aspect, a method for diagnosing a disease, disorder or condition associated with CD123 expression on 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 SEQ ID NO: 70, 72, 78 and 88; and b) detecting the presence of CD123, wherein the presence of CD123 is diagnostic of a disease, disorder or condition associated with CD123 expression.

[0077] 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, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), adult B-cell malignancies (including CLL (chronic lymphocytic leukemia), CML (chronic myeloid leukemia), non-Hodgkin lymphoma (NHL)), childhood B-cell malignancies (including B-lineage ALL (acute lymphocytic leukemia)) in minimal residual disease (MRD), multiple myeloma, lung cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, melanoma or other hematological cancers and solid tumors, or any combination thereof.

[0078] In another embodiment, a method for diagnosing, prognosing or determining the risk of a CD123-related disease in a mammal is provided, comprising detecting the expression of CD123 in a sample derived from the mammal, comprising: a) contacting the sample with a human anti-CD123 antibody or a fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from SEQ ID NO: 70, 72, 78 and 88; and b) detecting the presence of CD123, wherein the presence of CD123 is diagnostic of a CD123-related disease in the mammal.

[0079] In another embodiment, a method of inhibiting CD123-dependent T cell suppression is provided, comprising contacting a cell with a human anti-CD123 antibody or a fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from SEQ ID NO: 70, 72, 78 and 88. In one embodiment, the cell is selected from a tumor cell expressing CD123, a tumor-associated macrophage, and any combination thereof.

[0080] In another embodiment, a method of blocking T cell suppression mediated by cells expressing CD123 and changing the tumor microenvironment to inhibit tumor growth in a mammal is provided, comprising administering to the mammal an effective amount of a composition comprising an isolated anti-CD123 antibody or a fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from SEQ ID NO: 70, 72, 78 and 88. In one embodiment, the cell is selected from a tumor cell expressing CD123, a tumor-associated macrophage, and any combination thereof.

[0081] In another embodiment, a method of inhibiting, suppressing or preventing immunosuppression of an anti-tumor or anti-cancer immune response in a mammal is provided, comprising administering to the mammal an effective amount of a composition comprising an isolated anti-CD123 antibody or fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from SEQ ID NO: 70, 72, 78 and 88. In one embodiment, the antibody or fragment thereof inhibits the interaction between a first cell and a T cell, wherein the first cell is selected from a tumor cell expressing CD123, a tumor-associated macrophage, and any combination thereof.

[0082] In another aspect, a method for inducing anti-tumor immunity in a mammal is provided, comprising administering to the mammal a therapeutically effective amount of T cells transduced with a vector or nucleic acid molecule encoding the disclosed CAR.

[0083] In another embodiment, a method for treating or preventing cancer in a mammal is provided, comprising administering one or more disclosed CARs to the mammal in an amount effective for treating or preventing cancer in a mammal. The method includes administering to the subject a therapeutically effective amount of expression specifically binding CD123 and / or one or more of the aforementioned antigens disclosed CAR host cells under the following conditions, the conditions being sufficient to form an immune complex of the antigen binding domain on the CAR with CD123 and / or the extracellular domain of one or more of the aforementioned antigens in the subject.

[0084] In another embodiment, a method for treating a mammal suffering from a disease, disorder or condition associated with elevated tumor antigen expression is provided, the method comprising administering to a subject a pharmaceutical composition comprising an anti-tumor effective amount of a T cell population, wherein the T cell comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises at least one extracellular CD123 antigen binding domain, at least one linker or spacer domain, at least one transmembrane domain, at least one intracellular signaling domain, the extracellular CD123 antigen binding domain comprises SEQ ID NO: 70, 72, 78 or 88 amino acid sequence or any combination thereof, and wherein the T cell is a T cell of a subject suffering from cancer.

[0085] In another embodiment, a method for treating cancer in a subject in need thereof is provided, comprising administering to the subject a pharmaceutical composition comprising an anti-tumor effective amount of a T cell population, wherein the T cell comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises at least one CD123 antigen binding domain, at least one linker or spacer domain, at least one transmembrane domain, at least one intracellular signaling domain, and the CD123 antigen binding domain comprises SEQ ID NO: 70, 72, 78 or 88 amino acid sequence or any combination thereof, wherein the T cell is a T cell of a subject suffering from cancer. In some embodiments of the foregoing method, the at least one transmembrane domain comprises the following transmembrane: α, β or ζ chain of a T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, mesothelin, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, or a combination thereof.

[0086] In another embodiment, a method for producing a persistent genetically modified T cell group in a person diagnosed with cancer is provided. In one embodiment, the method includes administering to a person a T cell genetically modified to express CAR, wherein the CAR comprises at least one CD123 antigen binding domain, at least one transmembrane domain, and at least one intracellular signaling domain, and the CD123 antigen binding domain comprises SEQ ID NO:70, 72, 78 or 88 amino acid sequence or any combination thereof, wherein after administration, the persistent genetically modified T cell group or the progeny group of the T cell continues in a person for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years or 3 years.

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

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

[0089] In another aspect, a kit is provided for preparing a chimeric antigen receptor T cell as described above, or for preventing, treating or ameliorating any cancer, disease, disorder or condition associated with elevated tumor antigen expression in a subject as described above, the kit comprising a container containing any one or any combination of the nucleic acid molecules, vectors, host cells or compositions disclosed above, and instructions for using the kit.

[0090] It should be understood that CAR, host cells, nucleic acids and methods are available outside the specific aspects and embodiments described in detail herein. The foregoing features and advantages of the present disclosure will become more apparent from the detailed description below with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0091] Figures 1A to 1C Depicted are CD123 CAR structure, surface expression, and cell viability in human primary T cells. Figure 1A )Anti-CD123 CAR constructs were generated by connecting a single-chain variable fragment (scFv) sequence targeting CD123 in frame with the CD8 hinge (H) and transmembrane domain (TM), 4-1BB (CD137) co-stimulatory domain, and CD3ζ activation domain. ( Figure 1B ) In the presence of IL-2, T cells were activated with TransAct CD3 / CD28 reagents and transduced with lentiviral vectors encoding CAR123 constructs. On the 7th day of culture, live transduced T cells (7-AAD negative) were analyzed for CAR surface expression using CD123Fc and then anti-Fc-AF647. The percentage of CAR T positive population relative to non-transduced T cell controls. ( Figure 1C ) CAR T cell viability (Vi-Cell) was measured by trypan blue exclusion on days 3 and 7 of culture. CD33 CAR construct (1906) was included as a control.

[0092] Figure 2 Depicted are CD123 surface expression levels on MOLM14, KG-1a, RS4;11, 293T and A431 tumor cell lines. Representative flow cytometry histograms are shown.

[0093] Figures 3A to 3C Depicted is the analysis of tumor cell lysis induced by CAR123 constructs in vitro. Using MOL14 ( Figure 3A )、KG1a( Figure 3B ) and 293T( Figure 3C) target cell lines were subjected to luciferase-based cytotoxicity assays. CAR T cells and tumor cells were co-incubated overnight at the specified effector to target (E:T) ratios: 2.5:1, 5:1, or 10:1. The percentage of specific target lysis was assessed by luminometry.

[0094] Figures 4A to 4C Depict the CAR T cytokine release in response to leukemia cell lines.CAR T cells (listed on the x-axis) were measured by ELISA after being incubated overnight with MOLM14 leukemia lines with an E:T ratio of 10:1 for CAR T cytokine production.Bar represents the mean+SD of repeated samples.Data represent three independent experiments performed with CAR T cells from three different donors.

[0095] Figure 5A and 5B Depicted are the CAR constructs tested in two in vivo studies. Figure 5A )CD123 CAR candidate D0126 and control CAR 33LTG1906 were included in the first animal study, ( Figure 5B )CD123 CAR candidate D0131 was added to D0126 and LTG1906 in a second animal study. In both studies, tumor only (TA) groups and untransduced T cell (UTD) groups were included as controls.

[0096] Figures 6A to 6D The in vivo activity of CAR T constructs in the first animal study is depicted. NSG mice were injected iv with MOLM14-luciferase cells on day 0 and treated with 5 × 10 6 / mice were treated with CAR T cells or UTD. Six mice were studied in each CAR T treatment group and control group. ( Fig. 6A ) Representative time course bioluminescent images of tumor burden in mice. ( Figure 6B ) The time course of tumor growth based on whole-body bioluminescence (radiance) of mice was quantified and plotted individually as indicated, n=6. ( Figure 6C ) The percentage change of body weight was recorded every other day, n = 6, mean ± SEM. ( Fig.6D ) Survival curves of mice after control and CAR T treatment.

[0097] Figures 7A to 7C Depicts human T cells detected in the blood of mice during the first in vivo study. Fig. 7A ), Day 22( Figure 7B ) and day 33 ( Figure 7C), total human T cells in mouse peripheral blood were measured by volume flow cytometry and normalized using CountBright beads. All surviving mice are depicted. Results are shown as scatter plots. Lines represent group means.

[0098] Figures 8A to 8D The in vivo activity of CAR T constructs in the second in vivo study is depicted. NSG mice were injected iv with MOLM14-luciferase cells on day 0 and 5 × 10 6 / mouse T cells. Fig. 8A Representative time course bioluminescent images of tumor burden in each group are shown. Figure 8B ) Time course of tumor growth based on whole-body bioluminescence (radiance) of mice, n=6, mean±SEM. ( Figure 8C ) The percentage of body weight change was recorded every other day, n=6, mean±SEM. Fig.8D Survival curves over time for the CAR T group and the control group are shown.

[0099] Figures 9A to 9E Depicts human T cells detected in the peripheral blood of mice in a second animal study. Fig.9A ), Day 14( Fig. 9B ), Day 21( Fig. 9C ), Day 28( Fig.9D ) and day 42 ( Fig.9E ), the total number of human T cells was measured by flow cytometry and quantified with CountBright beads. The results are shown as scatter plots. The line represents the group mean.

[0100] Figures 10A to 10B Isolation of NK cells and generation of target cells targeting CD123-CAR are depicted. Fig. 10A ) Isolation and purity of NK cells. Fig. 10B ) Target cells were generated by overexpressing CD123 on RS4-11 cell line. RS4-11 transduced with CD123 were sorted and limited dilution was performed to generate homogenous RS4-11 cells expressing CD123.

[0101] Fig.11 Depict CD123-CAR conjugates expressed on transduced primary NK cells. Primary NK cells were isolated and cultured in medium containing IL-2, IL-15 and IL-1β for two days. On the 3rd day, activated NK cells were transduced with 13 different lentiviral vectors containing different CD123-CARs. CD123-CAR expression on NK cells was detected on the 8th day after transduction.

[0102] Figures 12A to 12BThe expression and cytotoxicity of CD123-CAR are depicted. Fig. 12A )NK cells were transduced with lentiviral vectors containing CD123-CAR constructs D0126 and Z32. CD123-CAR expression was determined on the 8th day after transduction. ( Fig. 12B ) The cytotoxicity of CD123-CAR-NK cells was determined using RS411-CD123 target cells. The results represent 3 independent experiments.

[0103] Figures 13A to 13B Depicts the specific killing of target cells expressing CD123 by CD123-CAR NK cells. Fig.13A )NK cells were transduced with different volumes of lentiviral vectors containing CD123-CAR. CD123-CAR expression was detected on day 8 after transduction. ( Fig. 13B ) The cytotoxicity of CD123-CAR-NK cells was determined using RS411-CD123 target cells. The ratio of effector and target for cytotoxicity experiments was 1:1.

[0104] Figures 14A to 14B The effects of CD123-CAR on NK cell expansion and viability are depicted. Fig.14A )NK cells were transduced with CD123-CAR constructs D0126 and Z32 and expanded in parallel with untransduced NK cells. ( Fig. 14B ) After transduction with CD123-CAR, NK cell viability was determined at different time points of culture. D1, D3, D5, D8 and D11 represent 1 day, 3 days, 5 days, 8 days and 11 days after transduction. UTD represents untransduced NK cells; Z32 represents CD123 conjugate Z32; D0126 represents CD123 conjugate D0126. DETAILED DESCRIPTION

[0105] definition

[0106] Unless the context clearly indicates otherwise, the nouns used in this article without quantifier modification represent one / kind or more / kind. For example, the term "antigen" includes single / kind or multiple / kind antigens, and can be considered to be equivalent to the phrase "at least one / kind antigen". The term "comprising" used in this article means "including". Therefore, "comprising antigens" means "comprising antigens" without excluding other elements. The phrase "and / or" means "and" or "or / or". It should be further understood that, unless otherwise indicated, any and all base sizes or amino acid sizes and all molecular weights or molecular mass values ​​given for nucleic acids or polypeptides are approximate, and are provided for the purpose of description. Although many methods and materials similar or equivalent to those described herein can be used, specific suitable methods and materials are described below. In the event of a conflict, this specification (including the explanation of the term) shall prevail. In addition, materials, methods and examples are only illustrative and are not intended to be restrictive. For ease of access to multiple embodiments, the following explanation of terms is provided:

[0107] The term "about" when referring to a measurable value (e.g., an amount, a duration, etc.) is meant to encompass variations of +-.20%, or in some cases +-.10%, or in some cases +-.5%, or in some cases +-.1%, or in some cases +-.0.1% from the particular value, as such variations are suitable for performing the disclosed methods.

[0108] Unless otherwise indicated, technical terms herein are used according to conventional usage. Definitions of commonly used terms in molecular biology can be found in Benjamin Lewin, Genes VII, published by Oxford University Press, 1999; Kendrew et al. (ed.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994; and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995; and other similar references.

[0109] The present disclosure provides CD123 antibodies or fragments thereof and chimeric antigen receptors (CAR) with such CD123 antigen binding domains.The enhancement of the functional activity of CAR is directly related to the enhancement of the functional activity of T cells expressing CAR.As a result of one or more of these modifications, CAR shows both high cytokine-induced cell lysis and cell surface expression on transduced T cells, as well as the persistence of the T cells expressing CAR in vivo amplification levels and transduced CAR.

[0110] The unique ability to combine functional parts derived from different protein domains is a key innovative feature of chimeric antigen receptors (CARs). The selection of each of these protein domains is a key design feature, as is the way in which its specificity is combined. Each design domain is an essential component that can be used between different CAR platforms to transform the function of lymphocytes. For example, the selection of extracellular binding domains can make otherwise ineffective CARs effective.

[0111] The constant framework components of the immunoglobulin-derived protein sequences used to establish the extracellular antigen binding domain of CAR can be completely neutral or they can self-associate and drive T cells to a state of metabolic exhaustion, thereby making the effect of the therapeutic T cells expressing the CAR much lower. This occurs independently of the antigen binding function of the CAR domain. In addition, the selection of intracellular signaling domains can also control the activity and persistence of therapeutic lymphocyte populations for immunotherapy. Although the ability to bind to target antigens and transmit activation signals to T cells through these extracellular and intracellular domains, respectively, is an important CAR design aspect, it has also become apparent that the selection of the source of the extracellular antigen binding fragment can have a significant effect on the efficacy of CAR, and thus has a limiting effect on the function and clinical utility of CAR.

[0112] Surprisingly and unexpectedly, it has now been discovered that the use of fully human antigen-binding domains in CARs rather than mouse-derived antigen-binding fragments that are prone to inducing anti-mouse immune responses and CAR T elimination in the host (see, the UPenn-sponsored clinical trial using mouse derived SS1 ScFv sequence, NCT02159716) can also determine the functional activity of CAR-expressing T cells.

[0113] CAR disclosed herein is expressed at a high level in cells. CAR-expressing cells have a high in vivo proliferation rate, produce a large amount of cytokines, and have high cytotoxic activity for cells with CD123 antigens combined with CAR on their surface. The use of human extracellular CD123 antigen-binding domains leads to the production of CARs that work better in vivo, while avoiding inducing anti-CAR immunity and killing CAR T cell groups in host immune response. CAR expressing fully human extracellular CD123ScFv antigen-binding domains shows excellent activity / characteristics, including i) preventing poor CAR T persistence and function as seen in the case of mouse-derived binding sequences; ii) making the regional (i.e., intrapleural) delivery of lacking CAR effective; and iii) it is possible to produce CAR T cell designs based on both high-affinity and low-affinity binders for CD123. The latter property allows researchers to better tune the potency versus toxicity and / or tissue specificity of CAR T products, since CD123 expression is higher on tumors than on normal tissues, lower affinity binders may have higher specificity for tumors compared to normal tissues, which may prevent on-target off tumor toxicity and bystander cell killing.

[0114] The following is a detailed description of the CAR of the present invention, including a description of its extracellular CD123 antigen binding domain, transmembrane domain and intracellular domain, as well as additional descriptions of CAR, antibodies and antigen binding fragments thereof, conjugates, nucleotides, expression, vectors and host cells, treatment methods, compositions and kits using the disclosed CAR.

[0115] A. Chimeric Antigen Receptor (CAR)

[0116] The CAR disclosed herein comprises at least one CD123 antigen binding domain capable of binding to CD123, at least one transmembrane domain and at least one intracellular domain.

[0117] Chimeric antigen receptor (CAR) is an artificially constructed hybrid protein or polypeptide, which comprises an antigen binding domain (e.g., single-chain variable fragment (ScFv)) of an antibody connected to a T cell signaling domain via a transmembrane domain. The characteristics of CAR include its ability to redirect T cell specificity and reactivity toward a selected target in a non-MHC restricted manner, as well as the antigen binding properties of monoclonal antibodies. Non-MHC restricted antigen recognition gives CAR-expressing T cells the ability to recognize antigens independently of antigen processing, thus bypassing the main tumor escape mechanism. In addition, when expressed in T cells, CAR advantageously does not dimerize with endogenous T cell receptor (TCR) α and β chains.

[0118] As disclosed herein, the intracellular T cell signaling domain of a CAR may include, for example, a T cell receptor signaling domain, a T cell co-stimulatory signaling domain, or both. A T cell receptor signaling domain refers to a portion of a CAR comprising an intracellular domain of a T cell receptor (e.g., such as, but not limited to, an intracellular portion of a CD3 ζ protein). A co-stimulatory signaling domain refers to a portion of a CAR comprising an intracellular domain of a co-stimulatory molecule, which is a cell surface molecule other than an antigen receptor or its ligand required for an effective response of a lymphocyte to an antigen.

[0119] 1. Extracellular domain

[0120] In one embodiment, CAR comprises a target-specific binding element, which is also referred to as an antigen binding domain or portion. The selection of the domain depends on the type and number of ligands that define the surface of the target cell. For example, an antigen binding domain can be selected to identify ligands that act as cell surface markers on target cells associated with a specific disease state. Therefore, some examples of cell surface markers that can act as ligands for antigen binding domains in CAR include those associated with viruses, bacteria and parasitic infections, autoimmune diseases and cancer cells.

[0121] In one embodiment, CAR can be modified to target tumor antigens by modifying the desired antigen binding domain that specifically binds to the antigen on the tumor cell. Tumor antigens are proteins produced by tumor cells that cause immune responses (especially T cell-mediated immune responses). The choice of antigen binding domain will depend on the specific type of cancer to be treated. Tumor antigens include, for example, glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostate enzyme, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, Her2 / neu, survivin and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF-1), prostate-specific antigen (PSA ... In some embodiments, the tumor antigens disclosed herein are TNF-α, ...

[0122] In one embodiment, tumor antigens include one or more antigenic cancer epitopes associated with malignant tumors. Malignant tumors express many proteins that can serve as target antigens for immune attack. These molecules include but are not limited to tissue-specific antigens, such as MART-1, tyrosinase and GP100 in melanoma, and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation-related molecules, such as oncogenes HER-2 / Neu / ErbB-2. Another group of target antigens is onco-fetal antigens, such as carcinoembryonic antigen (CEA). In B-cell lymphomas, tumor-specific idiotype immunoglobulins constitute the unique real tumor-specific immunoglobulin antigens of individual tumors. B cell differentiation antigens (such as CD19, CD20 and CD37) are other candidates for target antigens in B-cell lymphomas. Some of these antigens (CEA, HER-2, CD19, CD20, idiotype) have been used as targets for passive immunotherapy with monoclonal antibodies, but with limited success.

[0123] In a preferred embodiment, the tumor antigen is CD123, and tumors associated with CD123 expression include lung mesothelioma, ovarian cancer, and pancreatic cancer, or any combination thereof, which express high levels of the extracellular protein CD123.

[0124] The type of tumor antigen may also be a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). TSAs are unique to tumor cells and do not appear on other cells in the body. TAAs are not unique to tumor cells and are instead expressed on normal cells under conditions where a state of immune tolerance to the antigen cannot be induced. Expression of an antigen on a tumor may occur under conditions that enable the immune system to respond to the antigen. A TAA may be an antigen that is expressed on normal cells during fetal development when the immune system is immature and unable to respond, or it may be an antigen that is normally present at very low levels on normal cells but is expressed at much higher levels on tumor cells.

[0125] Some non-limiting examples of TSAs or TAAs include the following: differentiation antigens, such as MART-1 / MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2; and tumor-specific multi-lineage antigens, such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens, such as CEA; overexpressed oncogenes and mutated tumor suppressor genes, such as p53, Ras, HER-2 / neu; unique tumor antigens caused by chromosomal translocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as Epstein Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA19-9, CA72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, α-fetoprotein, β-HCG, BCA225, BTAA, CA 125, CA 15-3\CA27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associated protein, TAAL6, TAG72, TLP and TPS.

[0126] In one embodiment, the antigen binding domain portion of CAR targets antigens including but not limited to the following: CD19, CD20, CD22, ROR1, CD123, CD33, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, MY-ESO-1TCR, MAGE A3 TCR, etc.

[0127] In a preferred embodiment, the antigen binding domain portion of the CAR targets the extracellular CD123 antigen.

[0128] 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 having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD123 MT-16 antigen binding domain comprises the amino acid sequence of SEQ ID NO: 70, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0129] In a preferred embodiment, the 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 having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD123 MT-32 antigen binding domain comprises the amino acid sequence of SEQ ID NO: 72, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0130] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD123 Z16 antigen binding domain comprises the nucleotide sequence of SEQ ID NO: 77, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD123 Z16 antigen binding domain comprises the amino acid sequence of SEQ ID NO: 78, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0131] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD123 Z32 antigen binding domain comprises the nucleotide sequence of SEQ ID NO: 87, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD123 Z32 antigen binding domain comprises the amino acid sequence of SEQ ID NO: 88, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0132] The generation and binding properties of the specific CD123 variable heavy chain only and ScFv antigen binding fragments or antigen binders described herein are shown in Example 1.

[0133] In various embodiments of the CD123-specific CAR disclosed herein, the general scheme is Figures 1A to 1C , and comprises, from N-terminus to C-terminus, a signal or leader peptide, anti-CD123 ScFv, an extracellular linker, CD8 transmembrane, 4-1BB, CD3ζ, wherein bold text indicates a cloning site for connecting domains.

[0134] In one embodiment, the nucleic acid sequence encoding 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.

[0135] In one embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO:3, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO:4, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof.

[0136] In another embodiment, the nucleic acid sequence encoding 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.

[0137] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO:7, or a sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO:8, or a sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0138] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO:9, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO:10.

[0139] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 11, or a sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO: 12, or a sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0140] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO:15, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO:16.

[0141] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 17, or a sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO: 18, or a sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0142] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 19, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO: 20.

[0143] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO:21, or a sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO:22, or a sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0144] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO:23, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO:24.

[0145] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO:25, or a sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO:26, or a sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0146] The development of anti-CD123 CAR T cells incorporating single-chain variable fragment (ScFv) sequences reactive with the CD123 antigen is described in Examples 3 and 4 below, and the generation of NK cells expressing CD123 CAR constructs is shown in Example 5 below.

[0147] Example 3 describes the generation and in vitro evaluation of CAR T cells targeting the CD123 antigen for the treatment of AML.

[0148] Lentiviral vectors encoding CD123 CAR constructs were used to transduce CAR into human primary T cells 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 Across multiple donors, CARs D0126, D0127, D0131, D0132, D0133, and D0134 showed similar or higher surface expression than the positive CAR123 control, LTG2078; whereas CAR D0130 had slightly lower surface expression, followed by D0129 and D0128, and D0125 had the lowest expression. Figure 1C As shown in , cell viability was detected on days 3 and 7 after T cell activation. All CD123 CAR T cells showed improved or comparable viability compared to the control CAR LTG2078.

[0149] The target-specific cytotoxicity of CD123 CAR in vitro was evaluated for CD123-positive leukemia cell lines (MOLM14, KG1a, RS4; 11) and CD123-negative non-leukemia cell lines (293T and A431). CAR-T cells were co-incubated with MOLM14, KG-1a or 293T cell lines at a ratio of 2.5: 1, 5: 1 and 10: 1 effector to target. After overnight co-incubation, the culture was analyzed in a luminescent in vitro killing assay. Most of the primary T cell lines expressing CAR123 constructs lyse MOL14-CD123+, while three CD123 CAR lines D0125, D0128 and D0129 lacked target lysis ability ( Figure 3A Similarly, KG-1a-CD123+ target cells were killed by most CART constructs except D0125, D0128, and D0129 ( Figure 3B The control CD33 CAR LTG1906 showed high cytotoxicity against MOLM14 (CD33 高 ) and showed low lytic efficacy against KG1a (CD33 低), which is consistent with the CD123 expression level. In addition, no killing was observed above the background of the CD123-negative 293T cell line ( Figure 3C ), which indicates the robust target-specific cytotoxic function of all CD123 CAR constructs except CAR D0125, D0128 and D0129.

[0150] After overnight incubation of CAR T cells with the MOLM14 target line at an E:T ratio of 10, T cell homeostasis and production of proinflammatory cytokines IL-2, IFNγ, and TNFα were detected by ELISA in the culture supernatant of CD123 CAR and control constructs CAR LTG2078 and CD33 CAR LTG1906 ( Figures 4A to 4C ). By comparing each CART group incubated with target cells with the respective CART experimental group alone, and also comparing the CAR T group co-incubated with the target with the previously characterized CAR123 control LTG2078, to detect the cytokine release induced by specific targets. Although CAR123 control LTG2078 and CAR33 control LTG1906 produced (elaborate) cytokines after co-incubation with MOLM14 target cells, most of the tested CD123 CART constructs did not produce significant increases in IFNγ, TNFα or IL-2 cytokines after co-culture with MOLM14 cells overnight. An exception is CAR123D0127, which even in the absence of target cells (only T cell group) also produced IFNγ and TNFα levels, indicating tumor-independent cytokine responses. This effect is unpredictable from previous experiments, and this shows the non-obviousness of the present invention. With the exception of CAR123 D0127, the cytokine responses of the CD123 CAR constructs evaluated herein were comparable to untransduced T cell (UTD) controls, indicating a low risk of inducing cytokine-mediated adverse effects such as cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS).

[0151] NSG (NOD.Cg-Prkdc scid The MOLM14 xenograft AML model of Il2rgtm1Wjl / SzJ) mice was used to further explore the in vivo tumor rejection function of the two best CAR123 candidates, D0126 and D0131. Two animal studies using CAR T cells derived from different healthy donors were conducted, one focusing on CARD0126 ( Figure 5A ), and another compared CAR123 constructs D0126 with D0131 ( Figure 5B ). CARLTG1906, which was previously characterized and targets the CD33 antigen on MOLM14 tumor cells, was included as a comparative control.

[0152] In the first in vivo study, the CD123 CAR D0126 was compared with the previously characterized CD33CAR-T construct LTG1906, and control experimental groups of tumor only (TA) and untransduced T cells (UTD) were also included. Tumor growth kinetics were monitored over time by an in vivo imaging system (IVIS) Fig. 6A and 6B ). Since MOLM14 tumors express both CD123 and CD33 antigens, the treatment group administered with CAR D0126 targeting the CD123 antigen, and the comparison group administered with CAR LTG1906 targeting the CD33 antigen, showed robust tumor rejection compared to the tumor-only (TA) and UTD control groups. At the end of the study, five of the six mice in each group showed complete tumor rejection, and only one mouse in each group had residual tumor cells ( Figure 6B Notably, neither the CAR D0126 nor the CARLTG1906 treated groups showed weight loss ( Figure 6C ), so no CAR-related toxicity was detected in this model. Both CAR D0126 and LTG1906 mediated complete survival until study termination on day 36 (6 of 6 mice survived), while tumor-only (TA) and UTD control groups died on day 15 due to high burden of disseminated disease ( Fig.6D ). Peripheral blood was sampled from mice on days 14, 22, and 33. Human T cells were detected in all groups ( Fig. 7A , 7B , 7C). In addition, at the end of the study, CAR D0126 and LTG1906 T cells were detected in the peripheral blood of mice, indicating the high persistence of CD123 CAR candidate D0126 and comparative control CAR33LTG1906 T cells.

[0153] In the second animal study, CD123 CAR D0131 was included in addition to CAR D0126. Fig. 8A Similar to the first animal study, CAR D0126 showed strong anti-tumor efficacy, and four of the six mice showed tumor rejection. Compared with CAR123 D0126, CAR123 D0131 showed weaker anti-tumor activity ( Fig. 8Aand 8B The best survival effect was detected in the CARD0126-treated group, with four of six mice surviving to the extended study termination date of day 56 and remaining completely tumor-free ( Fig.8D Total T cells in peripheral blood were monitored in this study. As expected, two days after administration of CAR T cells or UTD, human T cells were detected in the peripheral blood of mice in all groups except the TA negative control. Fig.9A ). Over time, the amount of T cells in all CAR T groups increased, suggesting T cell expansion ( Fig. 9B ) and persisted on days 21, 28, and 42 ( Fig. 9C , 9D , 9E). On day 42 of the study, the number of T cells in the CAR123D0126 group was the highest ( Fig.9E ), indicating that T cell expansion and persistence were the strongest among the CAR constructs tested in this experiment.

[0154] In conclusion, in two in vivo studies utilizing T cells from different human donors, the CD123CAR T cell candidate D0126 effectively eliminated tumors in NSG mice transplanted with MOLM-14 cells and demonstrated potent tumor clearance, CART persistence, and prolonged survival in a MOLM14 AML xenograft mouse model ( Fig.9A ).

[0155] By transfecting primary NK cells from healthy donors with lentiviral vectors pseudotyped with baboon envelope protein (BaEV-LV), CAR NK cells targeting CD123 were generated (Example 5 below). Primary NK cells were isolated from PBMCs by magnetic separation to produce pure cell populations ( Fig. 10A ). The NK-resistant RS4-11 target cell line stably transduced with CD123 protein was used to test CD123-CAR function ( Fig. 10B ).

[0156] NK cells were activated by culturing in NK MACS medium containing IL-2 / IL-15 / IL-1β for two days and then transduced 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 ( Fig.11 ). Among the 13 CD123-CARs, Z32 and D0126 CAR constructs transduced NK cells best and produced 51.55% and 61.37% transduction efficiencies, respectively. Based on these expression results, we selected CAR constructs Z32 and D0126 for further analysis.

[0157] Activated NK cells were transduced with BaEV pseudotyped lentiviral vectors containing CD123-CAR Z32 (Z32-BaEV-LV) and BaEV pseudotyped lentiviral vectors containing D0126 (D0126-BaEV-LV). The CD123-CAR expression of Z32 and D0126 was 70.5% and 64.19%, respectively ( Fig. 12A In addition, the cytotoxicity of NK cells expressing CD123-CAR against target cells RS4-11-CD123 was tested. RS4-11 cells expressing CD123 ( Fig. 10B ) is insensitive to the natural cytotoxicity of NK cells. Therefore, untransduced NK cells cannot kill RS4;11-CD123 cells, while both CD123-CAR (Z32 and D0126) NK cells can kill RS4;11-CD123 very effectively, indicating the high functionality and specificity of the generated CD123-CAR NK cells ( Fig. 12B ).

[0158] Next, the specificity of CD123-CAR for CD123 antigen was confirmed by serial dilution. NK cells were transduced with different amounts of lentiviral vectors containing CD123-CAR. As expected, higher amounts of CD123-CAR-LV showed higher CD123-CAR expression ( Fig.13A Finally, the cytotoxicity of NK cells expressing differentially expressed CD123-CAR against RS4-11-CD123 cells was tested at the same effector-target ratio ( Fig. 13B ). The NK cells with the highest expression of CD123-CAR showed the highest killing, and the NK cells with the lowest expression of CD123-CAR showed the lowest killing, confirming the specificity of CD123-CAR for CD123 antigen. Finally, the expression of CD123-CAR had no adverse effect on the expansion and viability of NK. Primary NK cells were isolated, activated, transduced with Z32 and D0126, and then expanded for 13 days. Untransduced NK cells were used as controls. The expansion of untransduced, Z32-transduced, and D0126-transduced NK cells was 61 times, 49 times, and 42 times, respectively ( Fig.14A There was no significant difference in cell viability among untransduced, Z32-transduced, and D0126-transduced NK cells ( Fig. 14B ), indicating that CD123-CAR had no adverse effects on NK cell viability.

[0159] Together, these results indicate the successful generation of CD123-targeting CAR-T and CAR-NK cells for cancer treatment.

[0160] Without intending to be limited to any particular mechanism of action, possible reasons for the enhanced therapeutic function associated with the exemplary CARs of the present invention are believed to include, for example, but not limited to: a) improved lateral movement within the plasma membrane allowing more efficient signal transduction, b) superior location within the plasma membrane microdomains (e.g., lipid rafts), and greater ability to interact with transmembrane signaling cascades associated with T cell activation, c) superior location within the plasma membrane by preferentially moving away from inhibitory (dampening) or downregulatory interactions (e.g., lower proximity or interaction with phosphatases (e.g., CD45)), and d) superior assembly into T cell receptor signaling complexes (i.e., immune synapses), or any combination thereof.

[0161] Although the present disclosure has been illustrated with exemplary extracellular CD123 variable heavy chain only and ScFv antigen binding domains, other nucleotide and / or amino acid variants within the CD123 variable heavy chain only and ScFv antigen binding domains can be used to obtain the CD123 antigen binding domains used in the CAR described herein.

[0162] Depending on the desired antigen to be targeted, the CAR can be additionally engineered to include a suitable antigen binding domain specific for the desired antigen target. For example, if CD19 is the desired antigen to be targeted, an antibody against CD19 can be used as an antigen binding domain incorporated into the CAR.

[0163] In an exemplary embodiment, the antigen binding domain portion of CAR also targets CD19. Preferably, the antigen binding domain in CAR is an anti-CD19 ScFv, wherein the nucleic acid sequence of the anti-CD19ScFv comprises the sequence shown in SEQ ID NO:37. In one embodiment, the anti-CD19 ScFv comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:37. In another embodiment, the anti-CD19 ScFv portion of CAR comprises the amino acid sequence shown in SEQ ID NO:38.

[0164] In one aspect of the present invention, a CAR capable of binding to a non-TSA or non-TAA is provided, wherein the non-TSA or non-TAA includes, for example, but not limited to, an antigen derived from the following: Retroviridae (e.g., human immunodeficiency virus, such as HIV-1 and HIV-LP), Picornaviridae (e.g., poliovirus, hepatitis A virus, enterovirus, human coxsackievirus, rhinovirus and echovirus), Rubella virus, Coronavirus, Vesicular Stomatitis Virus, Rabies Virus, Ebola Virus, Parainfluenza Virus, Mumps Virus, Measles Virus, Respiratory Syncytial Virus, Influenza Virus, Hepatitis B Virus, Parvovirus, Adenoviridae, Herpes Viridae [e.g., Herpes simplex virus type 1 and type 2 (HSV), varicella-zoster virus, cytomegalovirus (CMV) and herpes virus], Poxviridae (e.g., smallpox virus, vaccinia virus and poxvirus) or Hepatitis C virus, or any combination thereof.

[0165] In another aspect of the present invention, a CAR is provided that is capable of binding to an antigen derived from the following bacterial strains: Staphylococci, Streptococcus, Escherichia coli, Pseudomonas, or Salmonella. In particular, CARs are provided that are capable of binding to antigens from infectious bacteria, such as Helicobacter pyloris, Legionella pneumophilia, strains of Mycobacteria sp. (e.g., M. tuberculosis, M. avium, M. intracellulare, M. kansaii, or M. gordonea), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitides, Listeria monocytogenes, Streptococcus pyogenes, Group A Streptococcus, Group B Streptococcus (Streptococcus agalactiae), and Streptococcus pyogenes. agalactiae), Streptococcus pneumoniae or Clostridium tetani, or a combination thereof.

[0166] 2. Transmembrane domain

[0167] With respect to transmembrane domains, the CAR comprises one or more transmembrane domains fused to the extracellular CD33 antigen binding domain of the CAR.

[0168] The transmembrane domain may be derived from natural or synthetic sources. When the source is natural, the domain may be derived from any membrane-bound or transmembrane protein.

[0169] The transmembrane region particularly used for the CAR described herein may be derived from the following (ie, at least comprising its transmembrane region): α, β or ζ chain of T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, mesothelin, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. Alternatively, the transmembrane domain may be synthetic, in which case it will mainly contain hydrophobic residues, such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan and valine will be present at each end of the synthetic transmembrane domain. Optionally, a short oligopeptide linker or polypeptide linker (preferably 2 to 10 amino acids in length) may form a connection between the transmembrane domain of CAR and the cytoplasmic signaling domain. Glycine-serine doublets provide particularly suitable linkers.

[0170] In one embodiment, in addition to the above-mentioned transmembrane domains, a transmembrane domain that is naturally associated with one of the domains in the CAR is used.

[0171] 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.

[0172] In one embodiment, the transmembrane domain in the CAR of the present invention is a CD8 transmembrane domain. In one embodiment, the CD8 transmembrane domain comprises a nucleic acid sequence of SEQ ID NO: 27. In one embodiment, the CD8 transmembrane domain comprises a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO: 28. In another embodiment, the CD8 transmembrane domain comprises an amino acid sequence of SEQ ID NO: 28.

[0173] In one embodiment, the encoded transmembrane domain comprises an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 20, 10 or 5 modifications (e.g., substitutions) of an amino acid sequence of SEQ ID NO:28, or a sequence with 95% to 99% identity to an amino acid sequence of SEQ ID NO:28.

[0174] In some cases, the transmembrane domain of CAR comprises a CD8.α. hinge domain. In one embodiment, the CD8 hinge domain comprises a nucleic acid sequence of SEQ ID NO:29. In one embodiment, the CD8 hinge domain comprises a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO:30. In another embodiment, the CD8 hinge domain comprises an amino acid sequence of SEQ ID NO:30, or a sequence having 95% to 99% identity thereto.

[0175] In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded linker domain is derived from the extracellular domain of CD8 and is linked to a transmembrane CD8 domain, a transmembrane CD28 domain, or a combination thereof.

[0176] In one embodiment, the transmembrane domain in the CAR of the present invention is a TNFRSF19 transmembrane domain. In one embodiment, the TNFRSF19 transmembrane domain comprises a nucleic acid sequence of SEQ ID NO:51. In one embodiment, the TNFRSF19 transmembrane domain comprises a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO:52. In another embodiment, the TNFRSF19 transmembrane domain comprises an amino acid sequence of SEQ ID NO:52.

[0177] In one embodiment, the encoded transmembrane domain comprises an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 20, 10 or 5 modifications (e.g., substitutions) of an amino acid sequence of SEQ ID NO:52, or a sequence with 95% to 99% identity to an amino acid sequence of SEQ ID NO:52.

[0178] 3. Spacer domain

[0179] In CAR, a spacer domain, also referred to as a hinge domain, may be arranged between the extracellular domain and the transmembrane domain, or between the intracellular domain and the transmembrane domain. The spacer domain means any oligopeptide or polypeptide for connecting the transmembrane domain to the extracellular domain and / or the transmembrane domain to the intracellular domain. The spacer domain comprises up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids.

[0180] In several embodiments, the linker may include a spacer element that, when present, increases the size of the linker such that the distance between the effector molecule or detectable marker and the antibody or antigen-binding fragment is increased. Exemplary spacers are known to those of ordinary skill and include those described in U.S. Pat. Nos. 7,964,5667, 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, 5,521,284, 5,504,191, 5,410,024, 5,138,036, 5,076,973, 4,986,988, 4,978,744, 4,879,278, 4,816,444 and 4,486,414 and those listed in U.S. Patent Publication Nos. 20110212088 and 20110070248, each of which is incorporated herein by reference in its entirety.

[0181] The spacer domain preferably has a sequence that promotes CAR binding to the antigen and enhances signal transduction into the cell. Some examples of amino acids that are expected to promote binding include cysteine, charged amino acids, and serine and threonine in potential glycosylation sites, and these amino acids can be used as amino acids constituting the spacer domain.

[0182] As the spacer domain, all or part of the following can be used: amino acids 118 to 178 (which is the hinge region) of CD8.α (NCBI RefSeq: NP.sub.--001759.3) (SEQ ID NO: 31), amino acids 135 to 195 of CD8.β (GenBank: AAA35664.1), amino acids 315 to 396 of CD4 (NCBI RefSeq: NP.sub.--000607.1), or amino acids 137 to 152 of CD28 (NCBI RefSeq: NP.sub.--006130.1). In addition, as the spacer domain, a part of the constant region of the antibody H chain or L chain (CH1 region or CL region, for example, a peptide having the amino acid sequence shown in SEQ ID NO: 32) can be used. In addition, the spacer domain may be an artificially synthesized sequence.

[0183] In addition, all or part of the amino acid comprising the constant region of human IgG4 (UniProt ID: P01861) may be used, comprising CH1 (amino acids 1 to 98), hinge (SEQ ID NO: 80, and corresponding nucleotides SEQ ID NO: 79) (amino acids 99 to 110), CH2 (amino acids SEQ ID NO: 82 and corresponding nucleotides SEQ ID NO: 81) (amino acids 111 to 220), and CH3 (SEQ ID NO: 84 and corresponding nucleotides SEQ ID NO: 83) (amino acids 221 to 327), or a combination thereof, such as the IgG4 hinge CH2 CH3 domain (SEQ ID NO: 86 and corresponding nucleotides SEQ ID NO: 85).

[0184] In one embodiment, the spacer domain of the CAR comprises a TNFRSF19 hinge domain comprising a nucleic acid sequence of SEQ ID NO: 53. In one embodiment, the TNFRSF19 hinge domain comprises a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO: 54. In another embodiment, the TNFRSF19 hinge domain comprises an amino acid sequence of SEQ ID NO: 54, or a sequence having 95% to 99% identity thereto.

[0185] In one embodiment, the spacer domain of the CAR comprises a truncated hinge domain of TNFRSF19 comprising a nucleic acid sequence of SEQ ID NO: 55. In one embodiment, the truncated hinge domain of TNFRSF19 comprises a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO: 56. In another embodiment, the truncated hinge domain of TNFRSF19 comprises an amino acid sequence of SEQ ID NO: 56, or a sequence having 95% to 99% identity thereto.

[0186] In one embodiment, the TNFRSF19 hinge and transmembrane domain comprises the nucleic acid sequence of SEQ ID NO: 49. In one embodiment, the TNFRSF19 hinge and transmembrane domain comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 50. In another embodiment, the TNFRSF19 hinge and transmembrane domain comprises the amino acid sequence of SEQ ID NO: 50, or a sequence with 95% to 99% identity thereof.

[0187] In one embodiment, the CD8a hinge domain is fused to a TNFRSF19 transmembrane domain comprising a nucleic acid sequence of SEQ ID NO: 57. In one embodiment, the CD8a hinge domain is fused to a TNFRSF19 transmembrane domain comprising a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO: 58. In another embodiment, the CD8a hinge domain is fused to a TNFRSF19 transmembrane domain comprising an amino acid sequence of SEQ ID NO: 58, or a sequence having 95% to 99% identity thereto.

[0188] In addition, in CAR, a signal peptide sequence (also referred to as a leader peptide) may be connected to the N-terminus. The signal peptide sequence is present at the N-terminus of many secretory proteins and membrane proteins, and is 15 to 30 amino acids in length. Since many of the protein molecules mentioned above as intracellular domains have signal peptide sequences, these signal peptides can be used as signal peptides for CAR. In one embodiment, the signal peptide comprises the amino acid sequence shown in SEQ ID NO: 14.

[0189] In one embodiment, the CD8α leader peptide comprises the nucleic acid sequence of SEQ ID NO: 43. In one embodiment, the CD8α leader peptide comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 44. In another embodiment, the CD8a hinge domain is fused to a TNFRSF19 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 44 or a sequence having 95% to 99% identity thereto.

[0190] In another embodiment, the GMCSF leader peptide comprises the nucleic acid sequence of SEQ ID NO: 39. In one embodiment, the GMCSF leader peptide comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 40. In another embodiment, the CD8a hinge domain is fused to a TNFRSF19 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 40, or a sequence having 95% to 99% identity thereof.

[0191] In another embodiment, the TNFRSF19 leader peptide comprises the nucleic acid sequence of SEQ ID NO: 41. In one embodiment, the TNFRSF19 leader peptide and the CD8α leader peptide comprise a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 42. In another embodiment, the CD8a hinge domain is fused to the TNFRSF19 transmembrane domain, which comprises the amino acid sequence of SEQ ID NO: 42 or a sequence having 95% to 99% identity thereto.

[0192] In one embodiment, the tag sequence encoding the truncated sequence of epidermal growth factor receptor (tEGFR) comprises the nucleic acid sequence of SEQ ID NO: 67. In one embodiment, tEGFR comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 68. In another embodiment, the tEGFR tag comprises the amino acid sequence of SEQ ID NO: 68, or a sequence having 95% to 99% identity thereto.

[0193] In one embodiment, the furin recognition site and downstream T2A self-cleaving peptide sequence designed for simultaneous bicistronic expression of the tag sequence and the CAR sequence comprise a nucleic acid sequence of SEQ ID NO: 65. In one embodiment, the furin and T2A sequences comprise a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO: 66. In another embodiment, the tEGFR tag comprises an amino acid sequence of SEQ ID NO: 66 or a sequence having 95% to 99% identity thereto.

[0194] In one embodiment, the upstream furin recognition site and T2A self-cleaving peptide sequence and the furin recognition downstream site designed for the simultaneous bicistronic expression of the tag sequence and the CAR sequence comprise the nucleic acid sequence of SEQ ID NO: 67. In one embodiment, the furin and T2A sequences comprise a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 68. In another embodiment, the tEGFR tag comprises the amino acid sequence of SEQ ID NO: 68 or a sequence having 95% to 99% identity thereto.

[0195] In one embodiment, the targeting domain of CAR is expressed separately in the form of a monoclonal antibody, ScFv Fab, Fab'2, and is contained in a binding tag or epitope, while the effector cell expression component of CAR comprises a binding domain that is specific for binding to a tag or epitope expressed on a soluble CAR module, such as the specific binding of the soluble component of CAR to the cell binding component to form a complete functional CAR structure.

[0196] 4. Intracellular domain

[0197] The cytoplasmic domain or other intracellular signaling domain of the CAR is responsible for activating at least one normal effector function of the immune cell into which the CAR has been placed. The term "effector function" refers to a specialized function of a cell. For example, the effector function of a T cell may be a cell lytic activity or an auxiliary activity, including the secretion of cytokines. Therefore, the term "intracellular signaling domain" refers to a portion of a protein that transduces an effector function signal and directs the cell to perform a specialized function. Although the entire intracellular signaling domain can usually be used, the entire chain does not have to be used in many cases. In terms of using a truncated portion of an intracellular signaling domain, such a truncated portion can be used instead of a complete chain as long as it transduces an effector function signal. Therefore, the term intracellular signaling domain is intended to include any truncated portion of an intracellular signaling domain sufficient to transduce an effector function signal.

[0198] Some preferred examples of intracellular signaling domains for CAR include cytoplasmic sequences of T cell receptors (TCRs) and co-receptors, which function together to initiate signal transduction after antigen receptor engagement; and any derivatives or variants of these sequences and any synthetic sequences having the same functional capabilities.

[0199] It is known that the signal generated by TCR alone is insufficient to fully activate T cells and that secondary or co-stimulatory signals are also required. Therefore, it can be said that T cell activation is mediated by two different types of cytoplasmic signaling sequences: those that trigger antigen-dependent primary activation through TCR (primary cytoplasmic signaling sequences) and those that act in an antigen-independent manner to provide secondary or co-stimulatory signals (secondary cytoplasmic signaling sequences).

[0200] The primary cytoplasmic signaling sequence regulates the primary activation of the TCR complex in a stimulatory manner or in an inhibitory manner. The primary cytoplasmic signaling sequence that acts in a stimulatory manner may comprise a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM.

[0201] Some examples of ITAMs comprising primary cytoplasmic signaling sequences that are particularly useful in the CARs disclosed herein include those derived from: TCRζ (CD3ζ), FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. Some specific non-limiting examples of ITAMs include peptides having the following sequences: amino acids 51 to 164 of CD3.ζ. (NCBI RefSeq: NP.sub.--932170.1), amino acids 45 to 86 of Fc.ε.RI.γ. (NCBI RefSeq: NP.sub.--004097.1), amino acids 201 to 244 of Fc.ε.RI.β. (NCBI RefSeq: NP.sub.--000130.1), amino acids 139 to 182 of CD3.γ. (NCBI RefSeq: NP.sub.--000064.1), amino acids 128 to 171 of CD3.δ. (NCBI RefSeq: NP.sub.--000723.1), amino acids 140 to 145 of CD3.ε. (NCBI RefSeq: NP.sub.--00064.1). RefSeq: NP.sub.--000724.1), amino acids 153 to 207 of CD5 (NCBI RefSeq: NP.sub.--055022.2), amino acids 707 to 847 of 0022 (NCBI RefSeq: NP.sub.--001762.2), amino acids 166 to 226 of CD79a (NCBI RefSeq: NP.sub._001774.1), amino acids 182 to 229 of CD79b (NCBI RefSeq: NP.sub._000617.1), and amino acids 177 to 252 of CD66d (NCBI RefSeq: NP.sub._001806.2), and variants thereof having the same functions as these peptides. The amino acid number based on the amino acid sequence information of the NCBI RefSeq ID or GenBank described herein is numbered based on the full length of the precursor of each protein (including the signal peptide sequence, etc.). In one embodiment, the cytoplasmic signaling molecule in the CAR comprises a cytoplasmic signaling sequence derived from CD3ζ.

[0202] In a preferred embodiment, the intracellular domain of CAR can be designed to include CD3-ζ signaling domain itself, or it can be combined with any other desired cytoplasmic domain available in the case of CAR. For example, the intracellular domain of CAR may include CD3ζ chain portion and costimulatory signaling region. Costimulatory signaling region refers to a part of CAR containing the intracellular domain of costimulatory molecules. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands required for the effective response of lymphocytes to antigens. Some examples of such costimulatory molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (lymphocyte function-associated antigen-1, LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3 and ligands specifically binding to CD83, etc. Some specific non-limiting examples of such costimulatory molecules include peptides having the following sequences: amino acids 236 to 351 of CD2 (NCBI RefSeq: NP.sub.--001758.2), amino acids 421 to 458 of CD4 (NCBI RefSeq: NP.sub.--000607.1), amino acids 402 to 495 of CD5 (NCBI RefSeq: NP.sub.--055022.2), amino acids 207 to 235 of CD8.α. (NCBI RefSeq: NP.sub.--001759.3), amino acids 196 to 210 of CD83 (GenBank: AAA35664.1), amino acids 181 to 220 of CD28 (NCBI RefSeq: NP.sub.--006130.1), amino acids 181 to 220 of CD137 (4-1BB, NCBI RefSeq: NP.sub.--001552.2) amino acids 214 to 255, amino acids 241 to 277 of CD134 (OX40, NCBI RefSeq: NP.sub.--003318.1) and amino acids 166 to 199 of ICOS (NCBI RefSeq: NP.sub.--036224.1), and variants thereof having the same functions as these peptides. Therefore, although the disclosure herein is mainly illustrated by 4-1BB as a costimulatory signaling element, other costimulatory elements are also within the scope of the disclosure.

[0203] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of CAR can be interconnected in a random or specific order. Optionally, a short oligopeptide linker or polypeptide linker (preferably 2 to 10 amino acids in length) can form a connection. Glycine-serine doublets provide particularly suitable linkers.

[0204] In one embodiment, the intracellular domain is designed to include the signaling domain of CD3-ζ and the signaling domain of CD28. In another embodiment, the intracellular domain is designed to include the signaling domain of CD3-ζ and the signaling domain of 4-1BB. In another embodiment, the intracellular domain is designed to include the signaling domain of CD3-ζ and the signaling domain of CD28 and 4-1BB.

[0205] In one embodiment, the intracellular domain in the CAR is designed to include the signaling domain of 4-1BB and the signaling domain of CD3-ζ, wherein the signaling domain of 4-1BB includes the nucleic acid sequence shown in SEQ ID NO:33 or SEQ ID NO:73 and the signaling domain of CD3-ζ includes the nucleic acid sequence shown in SEQ ID NO:35, SEQ ID NO:47, SEQ ID NO:61 or SEQ ID NO:75.

[0206] In one embodiment, the intracellular domain in the CAR is designed to include the signaling domain of 4-1BB and the signaling domain of CD3-ζ, wherein the signaling domain of 4-1BB comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 34 or SEQ ID NO: 74, respectively, and the signaling domain of CD3-ζ comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 36 or SEQ ID NO: 48, SEQ ID NO: 62, or SEQ ID NO: 76.

[0207] In one embodiment, the intracellular domain in the CAR is designed to include the signaling domain of 4-1BB and the signaling domain of CD3-ζ, wherein the signaling domain of 4-1BB comprises the amino acid sequence shown in SEQ ID NO: 34 or SEQ ID NO: 74, respectively, and the signaling domain of CD3-ζ comprises the amino acid sequence shown in SEQ ID NO: 36, SEQ ID NO: 48, SEQ ID NO: 62, or SEQ ID NO: 76, respectively.

[0208] In one embodiment, the intracellular domain in the CAR is designed to include the signaling domain of CD28 and the signaling domain of CD3-ζ, wherein the signaling domain of CD28 comprises the nucleic acid sequence shown in SEQ ID NO:45 or SEQ ID NO:59, respectively, and the signaling domain of CD3-ζ comprises the nucleic acid sequence shown in SEQ ID NO:35, SEQ ID NO:47 or SEQ ID NO:61, respectively.

[0209] In one embodiment, the intracellular domain in the CAR is designed to comprise the signaling domain of CD28 and the signaling domain of CD3-ζ, wherein the signaling domain of CD28 comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 46 or SEQ ID NO: 60, respectively, and the signaling domain of CD3-ζ comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 36, or SEQ ID NO: 48, or SEQ ID NO: 62.

[0210] In one embodiment, the intracellular domain in the CAR is designed to comprise the signaling domain of CD28 and the signaling domain of CD3-ζ, wherein the signaling domain of CD28 comprises the amino acid sequence shown in SEQ ID NO:46 or SEQ ID NO:60, respectively, and the signaling domain of CD3-ζ comprises the amino acid sequence shown in SEQ ID NO:36, SEQ ID NO:48, or SEQ ID NO:62, respectively.

[0211] 5. Additional description of CAR

[0212] The functional part of CAR disclosed herein is also clearly included within the scope of the present invention.When referring to CAR use, the term "functional part" refers to any part or fragment of one or more CAR disclosed herein, and the part or fragment retains the biological activity of the CAR (parent CAR) as part thereof. The functional part covers, for example, those CAR parts that retain the ability to identify target cells or detect, treat or prevent diseases to a similar degree or to the same degree or to a higher degree as the parent CAR. Depending on the parent CAR, the functional part may include, for example, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95% or more of the parent CAR.

[0213] The functional part may include additional amino acids at the amino or carboxyl end of the part or at both ends, and the additional amino acids are not present in the amino acid sequence of the parent CAR. Desirably, the additional amino acids do not interfere with the biological function of the functional part, such as identifying target cells, detecting cancer, treating or preventing cancer, etc. More desirably, the additional amino acids enhance the biological activity compared to the biological activity of the parent CAR.

[0214] The scope of the present disclosure includes functional variants of CAR disclosed herein. The term "functional variant" used herein refers to a CAR, polypeptide or protein having substantial or significant sequence identity or similarity with a parent CAR, and the functional variant retains the biological activity of the CAR as a variant. Functional variants encompass, for example, those variants of the CAR (parent CAR) described herein that retain the ability to recognize target cells to a similar degree, the same degree or a higher degree as the parent CAR. According to the parent CAR, the functional variant may, for example, have at least about 30%, 50%, 75%, 80%, 90%, 98% or more identity with the parent CAR in terms of amino acid sequence.

[0215] Functional variants may, for example, comprise the amino acid sequence of a parent CAR having at least one conservative amino acid substitution. As an alternative or supplement, functional variants may comprise the amino acid sequence of a parent CAR having at least one non-conservative amino acid substitution. In this case, it is preferred that non-conservative amino acid substitutions do not interfere with or inhibit the biological activity of functional variants. Non-conservative amino acid substitutions can enhance the biological activity of functional variants so that the biological activity of functional variants is improved compared to parent CAR.

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

[0217] The CAR may consist essentially of one or more specific amino acid sequences described herein, such that other components (e.g., other amino acids) do not substantially alter the biological activity of the functional variant.

[0218] CAR (including functional parts and functional variants) can have any length, that is, can contain any number of amino acids, provided that CAR (or its functional part or functional variant) retains its biological activity, for example, the ability to specifically bind to an antigen, detect diseased cells in a mammal, or treat or prevent a disease in a mammal, etc. For example, the length of CAR can be about 50 to about 5000 amino acids, for example, 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more amino acids in length.

[0219] CAR (including functional parts and functional variants of the present invention) may include synthetic amino acids to replace one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, -amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3-hydroxyproline and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxylphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, Indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, -aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, γ-diaminobutyric acid, β-diaminopropionic acid, homophenylalanine and α-tert-butylglycine.

[0220] CAR (including functional parts and functional variants) can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized (via, for example, a disulfide bridge) or converted into an acid addition salt and / or optionally dimerized or multimerized or conjugated.

[0221] CAR (including functional parts and functional variants thereof) can be obtained by methods known in the art. CAR can be prepared by any suitable method for preparing a polypeptide or protein. Suitable methods for synthesizing polypeptides and proteins from scratch are described in references, such as Chan et al., Fmoc Solid Phase Peptide Synthesis, Oxford University Press, Oxford, United Kingdom, 2000; Peptide and Protein Drug Analysis, ed. Reid, R., Marcel Dekker, Inc., 2000; Epitope Mapping, ed. Westwood et al., Oxford University Press, Oxford, United Kingdom, 2001; and U.S. Patent No. 5,449,752. In addition, the nucleic acids described herein can be used to recombinantly produce polypeptides and proteins using standard recombinant methods. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. In addition, some CARs (including functional parts and functional variants thereof) can be separated and / or purified from sources such as plants, bacteria, insects, mammals (e.g., rats, humans, etc.). Methods for separation and purification are well known in the art. Alternatively, the CARs described herein (including functional parts and functional variants thereof) can be commercially synthesized by a company. In this regard, CARs can be synthetic, recombinant, separated and / or purified.

[0222] A. Antibodies and Antigen-binding Fragments

[0223] One embodiment also provides a CAR, a T cell expressing CAR, an antibody, or an antigen binding domain or part thereof that specifically binds to one or more antigens disclosed herein. "CAR-expressing T cell" or "CART cell" used herein means a T cell expressing CAR, and has an antigen specificity determined by a targeting domain derived from, for example, an antibody of CAR.

[0224] As used herein, "antigen binding domain" may include antibodies and antigen binding fragments thereof. The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) and antigen binding fragments thereof, as long as they exhibit the desired antigen binding activity. Some non-limiting examples of antibodies include, for example, intact immunoglobulins and variants and fragments thereof known in the art that retain binding affinity for an antigen.

[0225] "Monoclonal antibody" is an antibody obtained from a group of substantially homogeneous antibodies, that is, except for naturally occurring mutations that may be present in small amounts, the individual antibodies comprising the group are identical. Monoclonal antibodies are highly specific, directed against a single antigenic epitope. The modifier "monoclonal" represents the characteristics of an antibody such as obtained from a substantially homogeneous antibody group, and is not interpreted as requiring the production of antibodies by any particular method. In some instances, a monoclonal antibody is an antibody produced by a monoclonal clone of a B lymphocyte or a cell or its progeny transfected with nucleic acid encoding a single antibody (or its antigen-binding fragment) of an antibody light chain and a variable region of a heavy chain. In some instances, a monoclonal antibody is isolated from an object. A monoclonal antibody may have a conservative amino acid substitution that has substantially no effect on antigen binding or other immunoglobulin functions. Exemplary methods for producing monoclonal antibodies are known, for example, referring to Harlow & Lane, Antibodies, A Laboratory Manual, 2nd edition. Cold Spring Harbor Publications, New York (2013).

[0226] Generally speaking, immunoglobulins have heavy chains (H) and light chains (L) interconnected by disulfide bonds. Immunoglobulin genes include κ, λ, α, γ, δ, ε and μ constant region genes and numerous immunoglobulin variable domain genes. There are two types of light chains, lambda (λ) and kappa (κ). There are five major heavy chain species (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA and IgE.

[0227] Each heavy chain and light chain comprises a constant region (or constant domain) and a variable region (or variable domain; see, e.g., Kindt et al. Kuby Immunology, 6th edition, WH Freeman and Co., page 91 (2007)). In several embodiments, the heavy chain and light chain variable regions combine to specifically bind to an antigen. In some other embodiments, only the heavy chain variable region is required. For example, naturally occurring camelid antibodies consisting only of heavy chains are functional and stable in the absence of light chains (see, e.g., Hamers-Casterman et al., Nature, 363: 446-448, 1993; Sheriff et al., Nat. Struct. Biol., 3: 733-736, 1996). Reference to "VH" or "VH" refers to the variable region of an antibody heavy chain, including an antigen-binding fragment, such as a variable region of an Fv, ScFv, dsFv or Fab. Reference to "VL" or "VL" refers to the variable domain of an antibody light chain, including the variable domain of an Fv, ScFv, dsFv or Fab.

[0228] The light and heavy chain variable regions comprise 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, USDepartment of Health and Human Services, 1991). The sequences of the framework regions of different light chains or heavy chains are relatively conserved within species. The framework region of an antibody (i.e., the combined framework region of the component light and heavy chains) is used to position and align the CDRs in three-dimensional space.

[0229] The CDR is primarily responsible for binding to the epitope of the antigen. The amino acid sequence boundaries of a given CDR can be readily determined using any of a number of well-known schemes, including those described by Kabat et al. ("Sequences of Proteins of Immunological Interest," 5th Edition. Public Health Service, National Institutes of Health, Bethesda, MD, 1991; "Kabat" numbering scheme), Al-Lazikani et al. (JMB 273, 927-948, 1997; "Chothia" numbering scheme) and Lefranc et al. ("IMGT unique numbering for immunoglobulin and 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 usually referred to as CDR1, CDR2, and CDR3 (from N-terminus to C-terminus), and are also usually identified by the chain in which the specific CDR is located. Thus, VH CDR3 is the CDR3 of the variable domain of the heavy chain of the antibody in which it is present, and VLCDR1 is the CDR1 of the variable domain of the light chain of the antibody in which it is present. Light chain CDRs are sometimes referred to as LCDR1, LCDR2, and LCDR3. Heavy chain CDRs are sometimes referred to as HCDR1, HCDR2, and HCDR3.

[0230] "Antigen binding fragments" are portions of full-length antibodies that retain the ability to specifically recognize a cognate antigen, as well as various combinations of such portions. Some non-limiting examples of antigen binding fragments include Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., ScFv); and multispecific antibodies formed from antibody fragments. Antibody fragments include antigen binding fragments produced by modifying intact antibodies or those synthesized de novo using recombinant DNA methods (see, e.g., Kontermann and Dubel (eds.), Antibody Engineering, Vols. 1-2, 2nd ed., Springer Press, 2010).

[0231] Single-chain antibodies (ScFv) are genetically engineered molecules comprising the VH and VL domains of one or more antibodies connected by a suitable polypeptide linker as a genetically fused single-chain molecule (see, e.g., Bird et al., Science, 242: 423 426, 1988; Huston et al., Proc. Natl. Acad. Sci., 85: 5879 5883, 1988; Ahmad et al., Clin. Dev. Immunol., 2012, doi: 10.1155 / 2012 / 980250; Marbry, ID Rugs, 13: 543-549, 2010). The intramolecular orientation of the VH and VL domains in ScFv is generally not critical for ScFv. Therefore, ScFvs with both possible arrangements (VH domain-linker domain-VL domain; VL domain-linker domain-VH domain) can be used.

[0232] In dsFv, the heavy and light variable chains have been mutated to introduce disulfide bonds to stabilize the association of the chains. Also included are diabodies, which are bivalent bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but a linker that is too short to allow pairing between the two domains on the same chain is used, thereby forcing the domains to pair with the complementary domains of another chain and generate two antigen binding sites (see, e.g., Holliger et al., Proc. Natl. Acad. Sci., 90: 6444 6448, 1993; Poljak et al., Structure, 2: 1121 1123, 1994).

[0233] 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.

[0234] Non-naturally occurring antibodies can be constructed using solid phase peptide synthesis, can be recombinantly produced, or can be obtained, for example, by screening a combinatorial library consisting of a variable heavy chain and a variable light chain as described in Huse et al., Science 246: 1275-1281 (1989), which is incorporated herein by reference. These and other methods for preparing, for example, chimeric, humanized, CDR-grafted, single-chain and bifunctional antibodies are well known to those skilled in the art (Winter and Harris, Immunol. Today 14: 243-246 (1993); Ward et al., Nature 341: 544-546 (1989); Harlow and Lane, supra, 1988; Hilyard et al., Protein Engineering: A practical approach (IRL Press 1992); Borrabeck, Antibody Engineering, 2nd ed. (Oxford University Press 1995); each of which is incorporated herein by reference).

[0235] An "antibody that binds to the same epitope as a reference antibody" refers to an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competition assay, and vice versa, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competition assay. Antibody competition assays are known, and exemplary competition assays are provided herein.

[0236] "Humanized" antibodies or antigen-binding fragments include human framework regions and one or more CDRs from non-human (e.g., mouse, rat, or synthetic) antibodies or antigen-binding fragments. The non-human antibody or antigen-binding fragment providing the CDR is referred to as a "donor," and the human antibody or antigen-binding fragment providing the framework is referred to as an "acceptor." In one embodiment, in a humanized immunoglobulin, all CDRs are from a donor immunoglobulin. The constant region does not need to be present, but if present, it may be substantially identical to a human immunoglobulin constant region, e.g., at least about 85% to 90%, e.g., about 95% or higher identity. Therefore, except for possible CDRs, all parts of a humanized antibody or antigen-binding fragment are substantially identical to corresponding parts of a natural human antibody sequence.

[0237] A "chimeric antibody" is an antibody comprising sequences derived from two different antibodies (which are generally from different species). In some instances, a chimeric antibody comprises one or more CDRs and / or framework regions from one human antibody and CDRs and / or framework regions from another human antibody.

[0238] "Fully human antibodies" or "human antibodies" are antibodies that contain sequences from (or derived from) the human genome, but do not contain sequences from other species. In some embodiments, human antibodies contain CDRs, framework regions, and Fc regions (if present) from (or derived from) the human genome. Human antibodies can be identified and isolated using techniques for producing antibodies based on sequences derived from the human genome, such as by phage display or using transgenic animals (see, e.g., Barbas et al. Phage display: A Laboratory Manuel. 1st edition. New York: Cold Spring Harbor Laboratory Press, 2004. Print.; Lonberg, Nat. Biotech., 23: 1117-1125, 2005; Lonenberg, Curr. Opin. Immunol., 20: 450-459, 2008).

[0239] An antibody may have one or more binding sites. If there is more than one binding site, the binding sites may be identical to each other or may be different. For example, a naturally occurring immunoglobulin has two identical binding sites, a single-chain antibody or Fab fragment has one binding site, and a bispecific or bifunctional antibody has two different binding sites.

[0240] Methods for testing the ability of an antibody to bind to any functional portion of a CAR are known in the art and include any antibody-antigen binding assay, such as, for example, radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation, and competitive inhibition assays (see, e.g., Janeway et al., infra, U.S. Patent Application Publication No. 2002 / 0197266A1 and U.S. Patent No. 7,338,929).

[0241] In addition, CAR, CAR-expressing T cells, antibodies, or antigen-binding portions thereof can be modified to include detectable labels, such as radioisotopes, fluorophores (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), enzymes (e.g., alkaline phosphatase, horseradish peroxidase), and elemental particles (e.g., gold particles).

[0242] B. Conjugates

[0243] CARs, T cells expressing CARs, or monoclonal antibodies or antigen-binding fragments thereof that are specific for one or more antigens disclosed herein can be conjugated to agents such as effector molecules or detectable markers using any number of methods known to those skilled in the art. Both covalent and non-covalent attachment methods can be used. Conjugates include, but are not limited to, molecules in which there are effector molecules or detectable markers covalently attached to antibodies or antigen-binding fragments that specifically bind to one or more antigens disclosed herein. Those skilled in the art will appreciate that a variety of effector molecules and detectable markers can be used, including but not limited to chemotherapeutic agents, anti-angiogenic agents, toxins, radioactive agents (e.g. 125 I. 32 P. 14 C. 3 H and 35 S) and other labels, target moieties and ligands, etc.

[0244] The choice of a particular effector molecule or detectable marker depends on the particular target molecule or cell and the desired biological effect. Thus, for example, the effector molecule can be a cytotoxin used to cause the death of a particular target cell (e.g., a tumor cell).

[0245] The method for attaching an effector molecule or a detectable marker to an antibody or Fab varies according to the chemical structure of the effector. Polypeptides generally contain a variety of functional groups; for example, carboxylic acid (COOH), free amine (-NH2) or sulfhydryl (-SH) groups, which can be used to react with suitable functional groups on the antibody to cause the binding of effector molecules or detectable markers. Alternatively, the antibody or Fab is derivatized to expose or attach additional reactive functional groups. Derivatization may involve attaching any of many known linker molecules (e.g., those available from Pierce Chemical Company, Rockford, IL). A linker can be any molecule for connecting an antibody or Fab to an effector molecule or a detectable marker. A linker can form a covalent bond with both an antibody or Fab and an effector molecule or a detectable marker. Suitable linkers are well known to those skilled in the art, and include, but are not limited to, straight or branched carbon linkers, heterocyclic carbon linkers, or peptide linkers. When the antibody or antigen-binding fragment and the effector molecule or detectable marker are polypeptides, the linker can be linked to the constituent amino acids via its side groups (e.g., to cysteine ​​via a disulfide bond) or to the alpha carbon amino and carboxyl groups of the terminal amino acids.

[0246] In several embodiments, the linker may include a spacer element that, when present, increases the size of the linker such that the distance between the effector molecule or detectable marker and the antibody or antigen-binding fragment is increased. Exemplary spacers are known to those of ordinary skill in the art and include those listed in U.S. Patent Nos. 7,964,5667, 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, 5,521,28 4, 5,504,191, 5,410,024, 5,138,036, 5,076,973, 4,986,988, 4,978,744, 4,879,278, 4,816,444, and 4,486,414, and U.S. Patent Nos. 20110212088 and 20110070248, each of which is incorporated herein by reference in its entirety.

[0247] In some embodiments, the joint is cleavable under intracellular conditions, so that the cutting of the joint in the intracellular environment releases the effector molecule or detectable marker from the antibody or Fab. In other embodiments, the joint is not cleavable, and the effector molecule or detectable marker is released, for example, by antibody degradation. In some embodiments, the joint can be cut by a cutting agent present in the intracellular environment (for example, in a lysosome or endosome or caveolea). The joint can be, for example, a peptide joint, which is cut by an intracellular peptidase or protease (including but not limited to a lysosomal protease or an endosomal protease). In some embodiments, the peptide joint is at least two amino acids long or at least three amino acids long. However, the joint can be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids long, for example, 1 to 2, 1 to 3, 2 to 5, 3 to 10, 3 to 15, 1 to 5, 1 to 10, 1 to 15 amino acids long. Proteases can include cathepsins B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives, resulting in the release of active drugs in target cells (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83: 67-123). For example, a peptide linker (e.g., phenylalanine-leucine or glycine-phenylalanine-leucine-glycine linker) that can be cleaved by the sulfhydryl-dependent protease cathepsin B can be used. Other examples of such linkers are described in, e.g., U.S. Patent No 6,214,345, which is incorporated herein by reference. In a specific embodiment, the peptide linker that can be cleaved by an intracellular protease is a valine-citrulline linker or a phenylalanine-lysine linker (see, e.g., U.S. Patent No 6,214,345, which describes the synthesis of doxorubicin with a valine-citrulline linker).

[0248] In other embodiments, the cleavable linker is pH sensitive, i.e., sensitive to hydrolysis at certain pH values. Typically, pH sensitive linkers are hydrolyzable under acidic conditions. For example, acid-labile linkers (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitamides, orthoesters, acetals, ketals, etc.) that are hydrolyzable in lysosomes can be used (see, e.g., U.S. Pat. Nos. 5,122,368, 5,824,805, 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83: 67-123; Neville et al., 1989, Biol. Chem. 264: 14653-14661). Such linkers are relatively stable under neutral pH conditions (e.g., conditions in blood), but are unstable below pH 5.5 or 5.0 (the approximate pH of lysosomes). In certain embodiments, the hydrolyzable linker is a thioether linker (e.g., a thioether attached to the therapeutic agent via an acylhydrazone bond (see, e.g., U.S. Pat. No. 5,622,929).

[0249] In other embodiments, the linker is cleavable under reducing conditions (e.g., a disulfide linker). A variety of disulfide linkers are known in the art, including, for example, those that can be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio) propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio) butyrate), and SMPT (N-succinimidyl-oxycarbonyl-α-methyl-α-(2-pyridyl-dithio) toluene)-, SPDB, and SMPT. (See, e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (CW Vogeled., Oxford U. Press, 1987); Phillips et al., Cancer Res. 68:9280-9290, 2008). See also U.S. Pat. No. 4,880,935).

[0250] In other specific embodiments, the linker is a malonate linker (Johnson et al., 1995, Anticancer Res. 15: 1387-93), a maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med-Chem. 3(10): 1299-1304), or a 3'-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10): 1305-12).

[0251] In other embodiments, the linker is not cleavable and the effector molecule or detectable marker is released by degradation of the antibody (see U.S. Publication No. 2005 / 0238649, which is incorporated herein by reference in its entirety).

[0252] In several embodiments, the linker is resistant to cleavage in the extracellular environment. For example, when the conjugate is present in the extracellular environment (e.g., in plasma), no more than about 20%, no more than about 15%, no more than about 10%, no more than about 5%, no more than about 3%, or no more than about 1% of the linker is cleaved in a sample of the conjugate. It can be determined, for example, by incubating the conjugate containing the linker of interest with plasma for a predetermined period of time (e.g., 2, 4, 8, 16, or 24 hours) and then quantifying the amount of free effector molecules or detectable markers present in the plasma to determine whether the linker is resistant to cleavage in the extracellular environment. A variety of exemplary linkers that can be used in the conjugate are described in WO 2004-010957, U.S. Publication No 2006 / 0074008, U.S. Publication No 20050238649, and U.S. Publication No 2006 / 0024317, each of which is incorporated herein by reference in its entirety.

[0253] In several embodiments, conjugates of CAR, CAR-expressing T cells, antibodies, or antigen-binding portions thereof, and one or more small molecule toxins (e.g., calicheamicin, maytansinoid, dolastatin, auristatin, trichothecene, and CC1065), and derivatives of these toxins having toxin activity, are provided.

[0254] Maytansine compounds suitable for use as maytansine compounds toxin part are well known in the art, and can be separated from natural sources according to known methods, produced using genetic engineering techniques (see Yu et al. (2002) PNAS 99: 7968-7973), or maytansinol and maytansinol analogs are prepared synthetically according to known methods. Maytansine compounds are mitotic inhibitors that play a role by inhibiting tubulin polymerization. Maytansine was first separated from the East African shrub Maytenus serrata (USP No 3,896,111). Subsequently, it was found that some microorganisms also produced maytansine compounds, such as maytansinol and C-3 maytansinol esters (USP No 4,151,042). and 4,371,533, each of which is incorporated herein by reference. Conjugates comprising maytansine compounds, methods for their preparation and therapeutic uses thereof are disclosed, for example, in US Pat. Nos. 5,208,020, 5,416,064, 6,441,163 and European Patent EP 0 425 235 B1, the disclosures of which are expressly incorporated herein by reference.

[0255] Other toxins can be used with CAR, T cells expressing CAR, antibodies or their antigen binding portions.Exemplary toxins include Pseudomonas exotoxin (PE), ricin, abrin, diphtheria toxin and its subunits, nuclear toxins (ribotoxin), ribonucleases, saporin (saporin) and calicheamicin and botulinum toxin A to F. These toxins are well known in the art, and many can be easily obtained from commercial sources (such as Sigma Chemical Company, St.Louis, MO).Contemplated toxins also include variants of toxins (see, for example, referring to U.S. Patent No 5,079,163 and 4,689,401).

[0256] Saponin is a toxin derived from soapwort (Saponaria officinalis) that disrupts protein synthesis by inactivating the 60S portion of the ribosome complex (Stirpe et al., Bio / Technology, 10:405-412, 1992). However, the toxin does not have a mechanism for specific cell entry and therefore needs to be conjugated to an antibody or antigen-binding fragment that recognizes a cell surface protein that is internalized in order to be effectively taken up by the cell.

[0257] Diphtheria toxin is isolated from Corynebacterium diphtheriae. Typically, diphtheria toxins used for immunotoxins are mutated to reduce or eliminate nonspecific toxicity. A mutant known as CRM107 with full enzyme activity but significantly reduced nonspecific toxicity has been known since the 1970s (Laird and Groman, J. Virol. 19:220, 1976) and has been used in human clinical trials. See U.S. Patent No 5,792,458 and U.S. Patent No 5,208,021.

[0258] Ricin is a lectin RCA60 from Ricinus communis (castor bean). For some examples of ricin, see U.S. Pat. No. 5,079,163 and U.S. Pat. No. 4,689,401. Ricinus communis agglutinin (RCA) exists in two forms, which are denoted RCA and RCA according to their molecular weights of about 65 kD and 120 kD, respectively. 60 and RCA 120 (Nicholson & Blaustein, J. Biochim. Biophys. Acta 266:543, 1972). The A chain is responsible for inactivating protein synthesis and killing cells. The B chain binds ricin to galactose residues on the cell surface and helps the A chain to be transported into the cytosol (Olsnes et al., Nature 249:627-631, 1974 and U.S. Patent No 3,060,165).

[0259] Ribonucleases are also conjugated to targeting molecules for use as immunotoxins (see Suzuki et al., Nat. Biotech. 17:265-70, 1999). Some exemplary ribotoxins (e.g., α-sarcin and restrictocin) are discussed, for example, in Rathore et al., Gene 190:31-5, 1997; and Goyal and Batra, Biochem. 345 Pt 2:247-54, 2000. Calicheamicin was first isolated from Micromonospora echinospora and is a member of the enediyne family of antitumor antibiotics that cause double-strand breaks in DNA, leading to apoptosis (see, e.g., Lee et al., J. Antibiot. 42:1070-87, 1989). This drug is the toxic portion of an immunotoxin in clinical trials (see, eg, Gillespie et al., Ann. Oncol. 11:735-41, 2000).

[0260] Abrin comprises toxic lectins from Abrus precatorius. The toxin components abrin a, b, c and d have a molecular weight of approximately 63 kD to 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).

[0261] CARs specific for one or more antigens disclosed herein, T cells expressing CARs, monoclonal antibodies, antigen-binding fragments thereof, may also be conjugated to detectable markers; for example, detectable markers that can be detected by ELISA, spectrophotometry, flow cytometry, microscopy, or diagnostic imaging techniques (e.g., computed tomography (CT), computed axial tomography (CAT), magnetic resonance imaging (MRI), nuclear magnetic resonance imaging (NMRI), magnetic resonance tomography (MTR), ultrasound, fiber optics, and laparoscopy). Some specific non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzyme conjugates, radioactive isotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI). For example, available detectable markers include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, etc. Bioluminescent markers such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP) are also used. CAR, T cells expressing CAR, antibodies, or antigen-binding portions thereof may also be conjugated with enzymes (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, glucose oxidase, etc.) that can be used for detection. When CAR, T cells expressing CAR, antibodies, or antigen-binding portions thereof are conjugated with a detectable enzyme, it can be detected by adding additional reagents, and the enzyme uses the reagent to produce a distinguishable reaction product. For example, when the reagent horseradish peroxidase is present, hydrogen peroxide and diaminobenzidine are added to produce a visually detectable colored reaction product. CAR, CAR-expressing T cells, antibodies, or antigen-binding portions thereof, can also be conjugated to biotin and detected by indirect measurement of avidin or streptavidin binding. It should be noted that avidin itself can be conjugated to an enzyme or fluorescent label.

[0262] CAR, T cells expressing CAR, antibodies or antigen-binding portions thereof can be conjugated with paramagnetic agents (e.g., gadolinium). Paramagnetic agents (e.g., superparamagnetic iron oxide) can also be used as labels. Antibodies can also be conjugated with lanthanides (e.g., europium and dysprosium) and manganese. Antibodies or antigen-binding fragments can also be labeled with a predetermined polypeptide epitope (e.g., a leucine zipper pair sequence, a binding site of a second antibody, a metal binding domain, an epitope tag) recognized by a second reporter.

[0263] CAR, T cells expressing CAR, antibodies or antigen-binding portions thereof may also be conjugated to radiolabeled amino acids. Radiolabels can be used for both diagnostic and therapeutic purposes. For example, radiolabels can be used to detect one or more antigens and cells expressing antigens disclosed herein by x-rays, emission spectroscopy or other diagnostic techniques. In addition, radiolabels can be used therapeutically as toxins for treating tumors in subjects, such as for treating neuroblastoma. Some examples of 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.

[0264] Methods for detecting such detectable markers are well known to those skilled in the art. Thus, for example, radioactive labels can be detected using photographic film or scintillation counters, and fluorescent markers can be detected using photodetectors to detect emitted illumination. Enzyme labels are usually detected by providing a substrate to the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label.

[0265] C. Nucleotides, expression, vectors and host cells

[0266] One embodiment of the present invention also provides a nucleic acid comprising a nucleotide sequence encoding any CAR, antibody, or antigen binding portion thereof described herein (including functional portions and functional variants thereof). The nucleic acid of the present invention may include a nucleotide sequence encoding any leader sequence, antigen binding domain, transmembrane domain, and / or intracellular T cell signaling domain described herein.

[0267] In some embodiments, the nucleotide sequence can be codon-modified. Without being bound by a particular theory, it is believed that codon optimization of the nucleotide sequence improves the translation efficiency of the mRNA transcript. Codon optimization of the nucleotide sequence may involve replacing a natural codon with another codon that encodes the same amino acid but can be translated by tRNA that is more easily available in the cell, thereby improving translation efficiency. Optimization of the nucleotide sequence can also reduce secondary mRNA structures that interfere with translation, thereby improving translation efficiency.

[0268] In one embodiment of the invention, nucleic acid may include a codon-modified nucleotide sequence encoding the antigen binding domains of the CAR of the present invention. In another embodiment of the invention, nucleic acid may include a codon-modified nucleotide sequence encoding any CAR described herein (including its functional parts and functional variants).

[0269] "Nucleic acid" as used herein includes "polynucleotide", "oligonucleotide" and "nucleic acid molecule", and generally means a polymer of DNA or RNA, which can be single-stranded or double-stranded, synthesized or obtained (e.g., isolated and / or purified) from a natural source, which can contain natural, non-natural or altered nucleotides, and which can contain natural, non-natural or altered internucleotide connections, such as phosphoramidate connections or phosphorothioate connections, rather than phosphodiester between nucleotides present in unmodified oligonucleotides. In some embodiments, nucleic acid does not include any insertion, deletion, inversion and / or replacement. However, as discussed herein, in some cases for nucleic acid, it may be appropriate to include one or more insertions, deletions, inversions and / or replacements.

[0270] Recombinant nucleic acid can be a nucleic acid having a non-natural sequence or having a sequence prepared by artificially combining two otherwise separated sequence segments. This artificial combination is usually accomplished by chemical synthesis, or more commonly by artificial manipulation of separated nucleic acid segments, such as by genetic engineering techniques (such as those described in Sambrook et al. (ibid)). Nucleic acids can be constructed based on chemical synthesis and / or enzymatic ligation using methods known in the art. See, for example, Sambrook et al. (ibid) and Ausubel et al. (ibid). For example, naturally occurring nucleotides or a variety of modified nucleotides can be used to chemically synthesize nucleic acids, which are designed to improve the biological stability of the molecule or improve the physical stability of the duplex formed after hybridization (such as nucleotides substituted with thiophosphate derivatives and acridine). Some examples of modified nucleotides that can be used to generate nucleic acids include, but are not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-substituted Adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosyl quercetin, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyl adenine, uracil-5-hydroxyacetic acid (v), wybutoxosine, pseudouracil, quercetin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 3-(3-amino-3-N-2-carboxypropyl) uracil and 2,6-diaminopurine. Alternatively, one or more nucleic acids of the present invention can be purchased from a company such as Integrated DNA Technologies (Coralville, IA, USA).

[0271] The nucleic acid may comprise any isolated or purified nucleotide sequence encoding any CAR or a functional part or functional variant thereof. Alternatively, the nucleotide sequence may comprise a nucleotide sequence that is degenerate to any sequence or a combination of degenerate sequences.

[0272] One embodiment also provides an isolated or purified nucleic acid comprising a nucleotide sequence that is complementary to the nucleotide sequence of any nucleic acid described herein or a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of any nucleic acid described herein.

[0273] Nucleotide sequences hybridized under stringent conditions can hybridize under high stringency conditions. "High stringency conditions" means that the nucleotide sequence specifically hybridizes with a target sequence (nucleotide sequence of any nucleic acid described herein) in an amount that is detectably stronger than non-specific hybridization. High stringency conditions include conditions that distinguish polynucleotides with precise complementary sequences or polynucleotides containing only a few scattered mismatches from random sequences that happen to have a few small regions (e.g., 3 to 10 bases) that match the nucleotide sequence. Such complementary small regions are more easily melted than the full-length complements of 14 to 17 or more bases, and highly stringent hybridization makes them easy to distinguish. Relatively high stringency conditions include, for example, low salt and / or high temperature conditions, such as provided by about 0.02M to 0.1M NaCl or equivalents at a temperature of about 50°C to 70°C. Such high stringency conditions allow small (if any) mismatches between nucleotide sequences and templates or target chains, and are particularly suitable for detecting the expression of any CAR of the present invention. It is generally recognized that conditions can be made more stringent by adding an increased amount of formamide.

[0274] 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.

[0275] In one embodiment, nucleic acid can be incorporated into a recombinant expression vector. In this regard, one embodiment provides a recombinant expression vector comprising any nucleic acid. For purposes herein, the term "recombinant expression vector" means a genetically modified oligonucleotide or polynucleotide construct that allows host cells to express mRNA, protein, polypeptide or peptide when the construct comprises a nucleotide sequence encoding mRNA, protein, polypeptide or peptide and when the vector is contacted with a cell under conditions sufficient to allow mRNA, protein, polypeptide or peptide to be expressed in the cell. The vector as a whole is not naturally occurring.

[0276] However, portions of the vector may be naturally occurring. The recombinant expression vector may contain any type of nucleotides, including but not limited to DNA and RNA, which may be single-stranded or double-stranded, synthetic or partially obtained from a natural source, and which may contain natural, non-natural or altered nucleotides. The recombinant expression vector may contain naturally occurring or non-naturally occurring internucleotide linkages or both types of linkages. Preferably, the non-naturally occurring or altered nucleotides or internucleotide linkages do not hinder transcription or replication of the vector.

[0277] In one embodiment, the recombinant expression vector can be any suitable recombinant expression vector, and can be used for transformation or transfection of any suitable host cell. Suitable vectors include vectors designed for breeding and amplification or for expression or for both, such as plasmids and viruses. Vectors can be selected from pUC series (Fermentas Life Sciences, GlenBurnie, MD), pBluescript series (Stratagene, LaJolla, CA), pET series (Novagen, Madison, WI), pGEX series (Pharmacia Biotech, Uppsala, Sweden) and pEX series (Clontech, Palo Alto, CA).

[0278] Phage vectors can also be used, e.g. 1, λZapII (Stratagene), EMBL4 and λNMI149. Some examples of plant expression vectors include pBIOl, pBI101.2, pBHOl.3, pBI121 and pBIN19 (Clontech). Some examples of animal expression vectors include pEUK-Cl, pMAM and pMAMneo (Clontech). The recombinant expression vector can be a viral vector, such as a retroviral vector or a lentiviral vector. A lentiviral vector is a vector derived from at least a portion of a lentiviral genome, particularly including a self-inactivating lentiviral vector, such as provided in Milone et al., Mol. Ther. 17 (8): 1453-1464 (2009). Other examples of lentiviral vectors that can be used clinically include, for example, but not limited to, LENTIVECTOR.RTM. gene delivery technology from Oxford BioMedica plc, LENTIMAX.TM. vector system from Lentigen, etc. Non-clinical types of lentiviral vectors are also available and are known to those skilled in the art.

[0279] Many transfection techniques are generally known in the art (see, e.g., Graham et al., Virology, 52:456-467 (1973); Sambrook et al., supra; Davis et al., Basic Methods in Molecular Biology, Elsevier (1986); and Chu et al., Gene, 13:97 (1981).

[0280] Transfection methods include calcium phosphate coprecipitation (see, e.g., Graham et al., supra), direct microinjection into cultured cells (see, e.g., Capecchi, Cell, 22:479-488 (1980)), electroporation (see, e.g., Shigekawa et al., BioTechniques, 6:742-751 (1988)), liposome-mediated gene transfer (see, e.g., Mannino et al., BioTechniques, 6:682-690 (1988)), lipid-mediated transduction (see, e.g., Feigner et al., Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)), and nucleic acid delivery using high-speed microprojectiles (see, e.g., Klein et al., Nature, 327:70-73 (1987)).

[0281] In one embodiment, recombinant expression vectors can be prepared using standard recombinant DNA techniques as described in, for example, Sambrook et al., supra, and Ausubel et al., supra. Circular or linear expression vector constructs can be prepared to contain a replication system that is functional in a prokaryotic or eukaryotic host cell. The replication system can be derived from, for example, ColE1, 2μ plasmid, lambda, SV40, bovine papilloma virus, and the like.

[0282] Recombinant expression vectors may include regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host cell (e.g., bacteria, fungi, plants, or animals) into which the vector is to be introduced, as appropriate, and taking into account whether the vector is DNA or RNA based. Recombinant expression vectors may include restriction sites to facilitate cloning.

[0283] The recombinant expression vector may include one or more marker genes that allow selection of transformed or transfected host cells. Marker genes include biocide resistance, such as resistance to antibiotics, heavy metals, etc., providing prototrophic complementation in auxotrophic hosts, etc. Suitable marker genes for expression vectors of the present invention include, for example, neomycin / G418 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.

[0284] The recombinant expression vector may include a natural or non-natural promoter, which is operably connected to a nucleotide sequence encoding CAR (including its functional part and its functional variant) or to a nucleotide sequence complementary to or hybridized with the nucleotide sequence encoding CAR. The selection of promoters (e.g., strong, weak, inducible, tissue-specific and developmentally specific) is within the ordinary technical scope of the technician. Similarly, the combination of nucleotide sequence and promoter is also within the technical scope of the technician. Promoter can be a non-viral promoter or a viral promoter, such as a cytomegalovirus (CMV) promoter, SV40 promoter, RSV promoter or a promoter found in the long terminal repeat of a mouse stem cell virus.

[0285] The recombinant expression vector can be designed to be used for transient expression, for stable expression or for both. In addition, the recombinant expression vector can be used for constitutive expression or for inducible expression.

[0286] In addition, the recombinant expression vector can be prepared to include a suicide gene. The term "suicide gene" used herein refers to a gene that causes the cell expressing the suicide gene to die. The suicide gene can be a gene that confers sensitivity to the cell expressing the gene to an agent (e.g., a drug), and causes cell death when the cell is contacted with an agent or exposed to an agent. Suicide genes are known in the art (see, e.g., Suicide Gene Therapy: Methods and Reviews, Springer, Caroline J. (Cancer Research UK Centre for Cancer Therapeutics, Institute of Cancer Research, Sutton, Surrey, UK), Humana Press, 2004), and include, for example, herpes simplex virus (HSV) thymidine kinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase, and nitroreductase.

[0287] One embodiment also provides a host cell comprising any recombinant expression vector described herein. The term "host cell" used herein refers to any type of cell that may contain the recombinant expression vector of the present invention. The host cell may be a eukaryotic cell, such as a plant, an animal, a fungus or an algae, or may be a prokaryotic cell, such as a bacterium or a protozoan. The host cell may be a cultured cell or a primary cell (i.e., directly isolated from an organism (e.g., a human)). The host cell may be an adherent cell or a suspended cell (i.e., a cell grown in suspension). Suitable host cells are known in the art, and include, for example, DH5a Escherichia coli (E.coli) cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, etc. For the purpose of amplifying or replicating the recombinant expression vector, the host cell may be a prokaryotic cell, such as a DH5a cell. For the purpose of producing a recombinant CAR, the host cell may be a mammalian cell. The host cell may be a human cell. Although the host cell may be any cell type, may be derived from any type of tissue, and may be at any developmental stage, the host cell may be a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC). The host cell may be a T cell.

[0288] For purposes herein, T cells can be any T cells, such as cultured T cells, such as primary T cells, or T cells from cultured T cell lines (e.g., Jurkat, SupT1, etc.), or T cells obtained from mammals. If obtained from mammals, T cells can be obtained from a variety of sources, including but not limited to blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. T cells can also be enriched or purified. T cells can be human T cells. T cells can be T cells isolated from humans. T cells can be any type of T cell and can be at any developmental stage, including but not limited to CD4 + / CD8 + Double positive T cells, CD4 + Helper T cells (such as Th1 and Th2 cells), CD8 + T cells (e.g., cytotoxic T cells), tumor infiltrating cells, memory T cells, memory stem cells (i.e., Tscm), naive T cells, etc. T cells can be CD8 + T cells or CD4 + T cells.

[0289] In one embodiment, the CAR described herein can be used for suitable non-T cells. Such cells are those with immune effector functions, such as NK cells and T-like cells produced by pluripotent stem cells.

[0290] One embodiment also provides a cell group comprising at least one host cell described herein. The cell group can be a heterogeneous group, which includes a host cell containing any recombinant expression vector described, and in addition includes at least one other cell (e.g., host cell (e.g., T cell)) that does not contain any recombinant expression vector, or cells other than T cells, such as B cells, macrophages, neutrophils, erythrocytes, hepatocytes, endothelial cells, epithelial cells, muscle cells, brain cells, etc. Alternatively, the cell group can be a substantially homogeneous group, wherein the group mainly includes (e.g., is substantially composed of)) a host cell containing a recombinant expression vector. The group can also be a clonal group of cells, wherein all cells of the group are clones of a single host cell containing a recombinant expression vector, so that all cells of the group include the recombinant expression vector. In one embodiment of the invention, the cell group is a clonal group comprising a host cell containing a recombinant expression vector described herein.

[0291] CAR (including its functional parts and variants), nucleic acids, recombinant expression vectors, host cells (including its groups) and antibodies (including its antigen binding portions) can be separated and / or purified. For example, a purified (or separated) host cell preparation is a preparation in which the host cell is purer than cells in the natural environment in vivo. Such host cells can be produced, for example, by standard purification techniques. In some embodiments, the preparation of the host cell is purified so that the host cell accounts for at least about 50%, for example, at least about 70% of the total cell content of the preparation. For example, the purity can be at least about 50%, can be greater than about 60%, about 70% or about 80%, or can be about 100%.

[0292] D. Treatment methods

[0293] It is expected that the CAR disclosed herein can be used in a method for treating or preventing a disease in a mammal. In this regard, one embodiment provides a method for treating or preventing cancer in a mammal, comprising administering a CAR, a nucleic acid, a recombinant expression vector, a host cell, a cell population, an antibody and / or an antigen binding portion thereof and / or a pharmaceutical composition to the mammal in an amount effective to treat or prevent cancer in the mammal.

[0294] One embodiment also includes lymphodepleting the mammal prior to administering the CAR disclosed herein. Some examples of lymphodepleting include, but are not limited to, non-myeloablative lymphodepleting chemotherapy, myeloablative lymphodepleting chemotherapy, whole body irradiation, etc.

[0295] For the purpose of the method wherein administering host cells or cell groups, cells can be mammalian allogeneic or autologous cells. Preferably, cells are mammalian autologous. Allogeneic as used herein means any material derived from different animals of the same species as the individual of the introduced material. When the genes on one or more loci are different, two or more individuals are considered to be allogeneic to each other. In some aspects, allogeneic materials from individuals of the same species can be genetically different enough to interact antigenically. "Autologous" as used herein means any material derived from the same individual of the individual of the material subsequently reintroduced.

[0296] Mammals mentioned herein can be any mammals. The term "mammal" as used herein refers to any mammal, including but not limited to mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits. Mammals can be from the order Carnivora, including felines (cats) and canines (dogs). Mammals can be from the order Artiodactyla, including Bovidae (cows) and Suidae (pigs), or the order Perssodactyla, including Equine (horses). Mammals can be Primates, Ceboid or Simoid (monkeys), or the order Anthropoids (humans and apes). Preferably, mammals are humans.

[0297] With respect to the methods, the cancer can be any cancer, including any of the following: acute lymphocytic carcinoma, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer (e.g., bladder cancer), bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, anal cancer, anal canal cancer, or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervical cancer, gallbladder cancer, or pleural cancer, nasal cancer, nasal cavity cancer, or middle ear cancer, oral cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, carcinoma, fibrosarcoma, gastrointestinal carcinoid tumors, 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 cancer, omental cancer and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumors, synovial sarcoma, stomach cancer, testicular cancer, thyroid cancer and ureteral cancer.

[0298] As used herein, the terms "treat" and "prevent" and words derived therefrom do not necessarily mean 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention that one of ordinary skill in the art would consider to have a potential benefit or therapeutic effect. In this regard, the method can provide any amount or any level of treatment or prevention of cancer in a mammal.

[0299] In addition, the treatment or prevention provided by the method may include treating or preventing one or more conditions or symptoms of the disease (e.g., cancer) being treated or prevented. Moreover, for purposes herein, "prevention" may encompass delaying the onset of a disease or its symptoms or conditions.

[0300] Another embodiment provides a method for detecting the presence of cancer in a mammal, comprising: (a) contacting a sample comprising one or more cells from a mammal with a CAR, a nucleic acid, a recombinant expression vector, a host cell, a cell population, an antibody and / or an antigen binding portion thereof, or a pharmaceutical composition, thereby forming a complex, (b) and detecting the complex, wherein detection of the complex indicates the presence of cancer in the mammal.

[0301] The sample can be obtained by any suitable method (e.g., biopsy or autopsy). A biopsy is the removal of tissue and / or cells from an individual. Such removal can be the collection of tissue and / or cells from an individual in order to perform experiments on the removed tissue and / or cells. The experiment may include an experiment to determine whether the individual suffers from and / or is suffering from a certain condition or disease state. The condition or disease can be, for example, cancer.

[0302] In one embodiment of a method for detecting the presence of a proliferative disorder (e.g., cancer) in a mammal, the sample comprising mammalian cells can be a sample comprising whole cells, a lysate thereof, or a fraction of a whole cell lysate (e.g., a nuclear or cytoplasmic fraction, a whole protein fraction, or a nucleic acid fraction). If the sample comprises whole cells, the cell can be any cell of a mammal, such as a cell of any organ or tissue, including a blood cell or an endothelial cell.

[0303] For mammals, contacting can occur in vitro or in vivo. Preferably, contacting is in vitro.

[0304] In addition, the detection of the complex can be carried out by any number of methods known in the art. For example, a detectable label (e.g., a radioisotope, a fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)) disclosed above), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase) and element particles (e.g., gold particles)) can be used to label the CAR disclosed herein, the polypeptide described herein, protein, nucleic acid, recombinant expression vector, host cell, cell group or antibody or its antigen binding portion thereof.

[0305] The method for testing the ability of CAR to recognize target cells and antigen specificity is known in the art. For example, Clay et al., J.Immunol, 163: 507-513 (1999) teaches measuring cytokines (e.g., interferon-γ, granulocyte / monocyte colony stimulating factor (granulocyte / monocyte colony stimulating factor, GM-CSF), tumor necrosis factor α (tumor necrosis factor α, TNF-α) or interleukin 2 (interleukin 2, IL-2)) release method. In addition, CAR function can be evaluated by measuring the cytotoxicity of cells, such as Zhao et al., J.Immunol. 174: 4415-4423 (2005) described in.

[0306] Another embodiment provides the use of CAR, nucleic acid, recombinant expression vector, host cell, cell group, antibody or antigen binding portion thereof and / or pharmaceutical composition of the present invention for treating or preventing proliferative disorders (e.g., cancer) in mammals. Cancer can be any cancer described herein.

[0307] Any method of administration can be used for the disclosed therapeutic agent, including local administration and systemic administration. For example, surface, oral, intravascular (e.g., intravenous), intramuscular, intraperitoneal, intranasal, intradermal, intrathecal, and subcutaneous administration can be used. The specific mode of administration and dosage regimen will be selected by the attending clinician considering the specific circumstances of the case (e.g., the subject, the disease, the disease state involved, and whether the treatment is preventive). In the case where more than one agent or composition is administered, one or more routes of administration can be used; for example, a chemotherapeutic agent can be administered orally, and an antibody or antigen binding fragment or conjugate or composition can be administered intravenously. The method of administration includes injection, in which case CAR, CAR T cells, conjugates, antibodies, antigen binding fragments, or compositions are provided in, for example, the following non-toxic pharmaceutically acceptable carriers: water, saline, Ringer's solution, dextrose solution, 5% human serum albumin, fixed oil, ethyl oleate, or liposomes. In some embodiments, local administration of the disclosed compound can be used, for example, by applying an antibody or antigen binding fragment to a tissue area where a tumor is removed or an area suspected of being prone to tumors. In some embodiments, sustained intratumoral (or peritumoral) release of a pharmaceutical formulation comprising a therapeutically effective amount of an antibody or antigen-binding fragment may be beneficial. In other examples, the conjugate is applied topically to the cornea as eye drops, or intravitreally to the eye.

[0308] The disclosed therapeutic agents can be formulated into unit dosage forms suitable for individual administration of precise dosages. In addition, the disclosed therapeutic agents can be administered in single doses or multiple dose regimens. A multiple dose regimen is a regimen in which the primary course of treatment may have more than one single dose (e.g., 1 to 10 doses), and then other doses are given at subsequent time intervals as needed to maintain or enhance the effect of the composition. Treatment may involve a daily dose or multiple daily doses of a compound over a period of days to months or even years. Therefore, the dosage regimen will also be determined at least in part based on the specific needs of the subject to be treated and will depend on the judgment of the administering operator.

[0309] A typical dosage of the antibody or conjugate may be about 0.01 to about 30 mg / kg, such as about 0.1 to about 10 mg / kg.

[0310] In some specific examples, a therapeutic composition comprising one or more of a conjugate, an antibody, a composition, a CAR, a CAR T cell, or another agent is administered to a subject with multiple daily dosing regimens (e.g., at least two consecutive days, 10 consecutive days, etc., for example, for a period of weeks, months, or years). In one example, a conjugate, an antibody, a composition, or another agent is administered to a subject for at least 30 days, for example, at least 2 months, at least 4 months, at least 6 months, at least 12 months, at least 24 months, or at least 36 months.

[0311] In some embodiments, the disclosed method includes providing surgery, radiotherapy and / or chemotherapeutic agents to the subject in combination with the disclosed antibody, antigen binding fragment, conjugate, CAR or T cell expressing CAR (e.g., sequentially, substantially simultaneously or simultaneously). Such medicaments and treatment methods and therapeutic doses are known to those skilled in the art and can be determined by skilled clinicians. The preparation and dosing regimen for other medicaments can be used according to the manufacturer's instructions or determined empirically by skilled practitioners. Preparations and dosing regimens for such chemotherapy are also described in Chemotherapy Service, (1992) Ed., MCPerry, Williams & Wilkins, Baltimore, MD.

[0312] In some embodiments, the combination therapy may include administering to the subject a therapeutically effective amount of an additional cancer inhibitor. Some non-limiting examples of additional therapeutic agents that can be used with the combination therapy include microtubule binders, DNA intercalators or crosslinkers, DNA synthesis inhibitors, DNA and RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, gene regulators, and angiogenesis inhibitors. These agents (which are administered in a therapeutically effective amount) and treatments can be used alone or in combination. For example, any suitable anticancer agent or anti-angiogenic agent can be administered in combination with CAR, CAR-T cells, antibodies, antigen binding fragments, or conjugates disclosed herein. The methods and therapeutic doses of such agents are known to those skilled in the art and can be determined by skilled clinicians.

[0313] Additional chemotherapeutic agents include, but are not limited to, alkylating agents such as nitrogen mustards (e.g., chlorambucil, chlormethine, cyclophosphamide, ifosfamide, and melphalan), nitrosoureas (e.g., carmustine, fotemustine, lomustine, and streptozotocin), platinum compounds (e.g., carboplatin, cisplatin, oxaliplatin, and BBR3464), busulfan, dacarbazine, mechlorethamine, procarbazine, temozolomide, thiotepa, and uramustine; antimetabolites such as folates (e.g., methotrexate, pemetrexed, and raltitrexed), purines (e.g., cladribine, clofarabine, fludarabine, mercaptopurine, and thioguanine), pyrimidines (e.g., capecitabine), cytarabine, fluorouracil, and gemcitabine; plant alkaloids, such as podophyllotoxins (e.g., etoposide and teniposide), taxanes (e.g., docetaxel and paclitaxel), vinca alkaloids (e.g., vinblastine, vincristine, vindesine, and vinorelbine); cytotoxic / antitumor antibiotics, such as members of the anthracycline family (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, and valrubicin), bleomycin, rifampicin, hydroxyurea, and mitomycin; topoisomerase inhibitors, such as topotecan and irinotecan; monoclonal antibodies, such as alemtuzumab, bevacizumab, cetuximab, gemtuzumab, rituximab, panitumumab, pertuzumab, and trastuzumab; photosensitizers, such as aminolevulinic acid, methyl aminolevulinate, porfimer sodium, and trastuzumab. sodium) and verteporfin; and other agents, such as alitretinoin, hexamethylmelamine, amridine, anagrelide, arsenic trioxide, asparaginase, axitinib, bexarotene, bevacizumab, bortezomib, celecoxib, denileukin diftitox, erlotinib, estramustine, gefitinib, hydroxyurea, imatinib, lapatinib, pazopanib, pentostatin, masoprocol, mitotane, pegaspargase, tamoxifen, sorafenib, sunitinib, vemurafinib, vandetanib and tretinoin. The selection and therapeutic dosage of such agents are known to those skilled in the art and can be determined by a skilled clinician.

[0314] Combination therapy can provide synergy and demonstrate synergy, i.e., the effect achieved when the active ingredients are used together is greater than the sum of the effects produced by the compounds used alone. Synergy can be achieved when the active ingredients are: (1) co-formulated in a combined unit dosage formulation and administered or delivered simultaneously; (2) delivered as separate formulations in alternation or in parallel; or (3) by some other regimen. When delivered in alternation, synergy can be achieved when the compounds are administered or delivered sequentially, such as by separate injections in separate syringes. Generally, during alternation, the effective dose of each active ingredient is administered sequentially (i.e., one after the other), whereas in combination therapy, the effective doses of two or more active ingredients are administered together.

[0315] In one embodiment, an effective amount of an antibody or antigen-binding fragment or its conjugate that specifically binds to one or more antigens disclosed herein is administered to a subject with a tumor after anti-cancer treatment. After a sufficient amount of time has passed to allow the administered antibody or antigen-binding fragment or conjugate to form immune complexes with the antigens expressed on each cancer cell, immune complexes are detected. The presence (or absence) of immune complexes indicates the effectiveness of treatment. For example, an increase in immune complexes compared to a control obtained before treatment indicates that treatment is ineffective, while a decrease in immune complexes compared to a control obtained before treatment indicates that treatment is effective.

[0316] E. Biopharmaceutical Compositions

[0317] Provided herein are biopharmaceuticals or biopharmaceutical compositions (hereinafter referred to as "compositions") for gene therapy, immunotherapy and / or cell therapy, the compositions being included in a carrier (e.g., a pharmaceutically acceptable carrier) and one or more of the disclosed CARs, or T cells expressing CARs, antibodies, antigen binding fragments, conjugates, CARs, or T cells expressing CARs that specifically bind to one or more antigens disclosed herein. The composition can be prepared in a unit dose form for administration to a subject. The amount and time of administration are determined by the treating clinician to achieve the desired outcome. The composition can be formulated for systemic (e.g., intravenous) or local (e.g., intratumoral) administration. In one example, the disclosed CARs, or T cells expressing CARs, antibodies, antigen binding fragments, and conjugates are formulated for parenteral administration, such as intravenous administration. Compositions comprising CARs disclosed herein, or T cells expressing CARs, conjugates, antibodies, or antigen binding fragments are used, for example, to treat and detect tumors, such as, but not limited to, neuroblastoma. In some examples, the composition can be used to treat or detect cancer. Compositions comprising a CAR, or a T cell expressing a CAR, a conjugate, an antibody, or an antigen-binding fragment disclosed herein are also used, for example, to detect pathological angiogenesis.

[0318] The composition for administration may include a solution of CAR, or T cells expressing CAR, conjugates, antibodies or antigen binding fragments dissolved in a pharmaceutically acceptable carrier (e.g., an aqueous carrier). A variety of aqueous carriers may be used, such as buffered saline, etc. These solutions are sterile and generally do not contain undesirable substances. These compositions can be sterilized by conventional, well-known sterilization techniques. The composition may contain pharmaceutically acceptable auxiliary substances as needed to approach physiological conditions, such as pH regulators and buffers, toxicity regulators, adjuvants, etc., such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. In these preparations, the concentration of CAR, or T cells expressing CAR, antibodies or antigen binding fragments or conjugates may vary widely, and will be selected mainly based on fluid volume, viscosity, body weight, etc. according to the specific mode of administration selected and the needs of the subject. The actual method of preparing such dosage forms for gene therapy, immunotherapy and / or cell therapy is known or will be obvious to those skilled in the art.

[0319] Typical compositions for intravenous administration include about 0.01 mg / kg to about 30 mg / kg of antibody or antigen binding fragment or conjugate (or a corresponding dose of CAR, or T cells expressing CAR, or a conjugate comprising an antibody or antigen binding fragment) per subject per day. Actual methods for preparing administrable compositions will be known or apparent to those skilled in the art, and are described in more detail in publications such as Remington's Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, PA (1995).

[0320] CAR, or T cells expressing CAR, antibodies, antigen binding fragments, or conjugates can be provided in a lyophilized form and rehydrated with sterile water before administration, but they can also be provided as a sterile solution of known concentration. The solution of CAR, or T cells expressing CAR, antibodies, antigen binding fragments, or conjugates is then added to an infusion bag containing 0.9% sodium chloride (USP) and in some cases administered at a dose of 0.5 mg / kg to 15 mg / kg body weight. There is considerable experience in the art in administering antibody or antigen binding fragment or conjugate drugs; for example, since 1997, Since its approval, antibody drugs have been listed in the United States. CAR, or T cells expressing CAR, antibodies, antigen binding fragments thereof, or conjugates can be administered by slow infusion rather than by intravenous push or rapid bolus injection (bolus). In one example, a higher loading dose is administered, and the administered dose is subsequently maintained at a lower level. For example, an initial loading dose of 4 mg / kg antibody or antigen binding fragment (or a conjugate comprising an antibody or antigen binding fragment of a corresponding dose) can be infused within about 90 minutes, and then if the previous dose is well tolerated, a maintenance dose of 2 mg / kg per week is infused within about 30 minutes for 4 to 8 weeks.

[0321] Controlled release parenteral formulations can be prepared as implants, oily injections or as particle systems. For a broad overview of protein delivery systems, see Banga, AJ, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Technomic Publishing Company, Inc., Lancaster, PA, (1995). Particle systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres and nanoparticles. Microcapsules contain therapeutic proteins (e.g., cytotoxins or drugs) as cores. In microspheres, therapeutic agents are dispersed throughout the particles. Particles, microspheres and microcapsules less than about 1 μm are generally referred to as nanoparticles, nanospheres and nanocapsules, respectively. The diameter of the capillary is about 5 μm, so that only nanoparticles are administered intravenously. The diameter of the microparticle is generally about 100 μm, and is administered subcutaneously or intramuscularly. See, e.g., Kreuter, J., Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, NY, pp. 219-342 (1994); and Tice & Tabibi, Treatise on Controlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, NY, pp. 315-339, (1992).

[0322] Polymers can be used for CAR disclosed herein or T cells expressing CAR, antibodies or AFs or conjugate compositions for controlled release of ions. A variety of degradable and non-degradable polymer matrices for controlled drug delivery are known in the art (Langer, Accounts Chem. Res. 26: 537-542, 1993). For example, block copolymer poloxamer 407 exists as a viscous but flowing liquid at low temperatures, but forms a semisolid gel at body temperature. It has been shown to be an effective carrier 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 controlled release of proteins (Ijntema et al., Int. J. Pharm. 112: 215-224, 1994). In another aspect, liposomes are used for controlled release of lipid-encapsulated drugs and drug targeting (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA (1993)). Many additional systems for controlled delivery of therapeutic proteins are known (see U.S. Pat. No. 5,055,303, U.S. Pat. No. 5,188,837, U.S. Pat. No. 4,235,871, U.S. Pat. No. 4,501,728, U.S. Pat. No. 4,837,028, U.S. Pat. No. 4,957,735, U.S. Pat. No. 5,019,369, U.S. Pat. No. 5,055,303, U.S. Pat. No. 5,514,670, U.S. Pat. No. 5,413,797, U.S. Pat. No. 5,268,164, U.S. Pat. No. 5,004,697, U.S. Pat. No. 4,902,505, U.S. Pat. No. 5,506,206, U.S. Pat. No. 5,271,961, U.S. Pat. No. 5,254,342, and U.S. Pat. No. 5,534,496).

[0323] F.Medicine Box

[0324] In one aspect, a kit using the CAR disclosed herein is also provided. For example, a kit for treating a tumor in a subject or preparing a CAR T cell expressing one or more CARs disclosed herein. The kit will generally contain a disclosed antibody, antigen binding fragment, conjugate, nucleic acid molecule, CAR or T cell expressing CAR as disclosed herein. More than one disclosed antibody, antigen binding fragment, conjugate, nucleic acid molecule, CAR or T cell expressing CAR may be included in the kit.

[0325] The medicine box may include a container and a label or package insert on or attached to the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container may be formed of a variety of materials (e.g., glass or plastic). The container typically contains one or more compositions comprising the disclosed antibodies, antigen binding fragments, conjugates, nucleic acid molecules, CARs, or T cells expressing CARs. In several embodiments, the container may have a sterile inlet (e.g., the container may be an intravenous solution bag or a vial having a plug that can be pierced by a hypodermic needle). The label or package insert indicates that the composition is used to treat a specific condition.

[0326] The label or package insert will generally also include instructions for using disclosed antibodies, antigen binding fragments, conjugates, nucleic acid molecules, CARs or T cells expressing CARs, for example, in a method for treating or preventing tumors or preparing CAR T cells. The package insert generally includes instructions customarily included in the commercial packaging of therapeutic products, which include information about indications, usage, dosage, administration, contraindications and / or warnings related to the use of such therapeutic products. The illustrative material may be written in electronic form (e.g., a computer floppy disk or CD), or may be visual (e.g., a video file). The kit may also include additional components that are conducive to the specific application for which the kit is designed. Therefore, for example, the kit may additionally include tools for detecting labels (e.g., enzyme substrates for enzyme labeling, filter devices for detecting fluorescent labels, suitable secondary labels (e.g., secondary antibodies), etc.). The kit may additionally include buffers and other reagents conventionally used to practice a particular method. Such kits and suitable contents are well known to those skilled in the art.

[0327] Example

[0328] The present invention is further illustrated by the following examples, which should not be construed in any way as limiting its scope. On the contrary, it should be clearly understood that a variety of other embodiments, modifications and equivalents thereof may be adopted without departing from the spirit of the present invention and / or the scope of the appended claims, which, after reading the description herein, will themselves be suggested to those skilled in the art.

[0329] Example 1

[0330] Isolation of human CD123-specific antibodies from a fully human yeast-displayed ScFv library

[0331] This example describes the acquisition of fully human binding sequences targeting the CD123 antigen from a yeast display library.

[0332] Materials and methods:

[0333] A large yeast display human initial single-chain variable fragment (ScFv) antibody library was used to separate the anti-human CD123 antibodies described herein. The library was constructed using a collection of human antibody gene repositories from more than 60 individuals. Three rounds of magnetic-activated cell sorting (MACS) were performed to enrich human ScFv binders to recombinant human CD123-Fc. For the first round of yeast library panning, the yeast display ScFv library (5 × 10 10 5 μ g / mL CD123-Fc in 15mlPBSA (consisting of 0.1% bovine serum albumin (Bovine Serum Albumin, BSA) in Dulbecco's phosphate buffered saline (PBS) buffer) was incubated on a rotator for 1.5 hours at room temperature. After washing twice with 25mlPBSA, yeast library mixture was incubated on a rotator for 30 minutes with 100 μL protein G micro beads (Miltenyi Biotec). After washing once, the library mixture was resuspended in 50mlPBSA, and loaded on a MACS cell separation column (LS column). After washing 3 times with 10mlPBSA, the yeast display ScFv binding substances of the column were eluted twice with 2mlPBSA. These eluted yeast cells are merged and then resuspended in 50ml SDCAA culture medium (20g D-glucose, 6.7g BD DifcoTM yeast nitrogen base without amino acids in 1L water, 5g BactoTM casamino acids, 5.4gNa2.HPO4 and 8.56g NaH2PO4.H2O), and amplified at 30°C with 225rpm shaking for 20 hours.Then again in SGCAA culture medium (composed of the same components of SDCAA culture medium, but containing galactose instead of glucose) at 30°C with 225rpm under shaking induced amplification pool (pool), continue 16 hours, and be used for the next round of panning.The same process is repeated twice more to enrich CD123-Fc specific binding substances.

[0334] To further enrich for binders with higher affinity and better specificity, FACS-based sorting was used to separate the strongest binders from the pool. The induced pool was incubated with 1 μg / ml CD123-Fc for 1 hour at room temperature and subsequently stained with anti-c-Myc-Alexa 488 and goat anti-Hu-Fc PE conjugates, and the top 1% of the pool with the highest PE relative to the FITC signal was gated and sorted. The sorted pool was amplified in SDCAA medium, and yeast plasmid DNA was extracted and transformed into bacteria for monoclonal DNA sequencing. Two unique sequences were identified and named MT-16 and MT-32, which were cloned into CAR constructs for expression in CAR-T or CAR-NK form constructs for expression in CAR-T or CAR-NK form for further functional characterization.

[0335] Example 2

[0336] Isolation of human CD123-specific antibodies from a fully human phage-displayed InfinityOne scFv library

[0337] This example describes the acquisition of fully human binding sequences targeting the CD123 antigen from a phage display library.

[0338] Materials and methods:

[0339] Generation of human phage-displayed ScFv CD123-specific antibodies

[0340] The initial human scFv (recombinant single-chain variable fragment of immunoglobulin) phage display library constructed from peripheral blood B cells of 121 healthy donors (approximate diversity, 7×10 10 Unique specificity (F. Tomszak, unpublished data) was used to select scFvs specific for recombinant human CD123. During the first, second and third rounds of bio-panning, 10 12The amplified library of phage display ScFv was incubated at room temperature for 2 hours in one hole of a 96-well plate with 1 μg coated CD123 (100 μl volume). After each round of incubation, the wells were washed with phosphate buffered saline (PBST) containing 0.05% Tween 20, 10 times in the first round, 20 times in the second round, and 30 times in the third round to remove non-specifically bound phage. Antigen-bound phages were eluted with 100 μl of 10 μg / ml trypsin diluted in PBS, and mixed with TG1 competent cells for 1 hour at 37°C, and phages were amplified from infected cells and used for the next round of bio-panning. After the third round of bio-panning, 376 clones were randomly picked from infected TG1 cells, and each clone was inoculated into 150 μl 2YT medium containing 100 μg / ml ampicillin and 200 mM glucose in a 96-well plate by using an automated colony picking system (Molecular Devices, QPix 460), and incubated overnight at 37° C. in a shaker at 300 rpm. The next day, 10 μl of bacterial culture was used to inoculate 150 μl 2YT medium containing 100 μg / ml ampicillin and 50 μM isopropyl-β-d-thiogalactoside in a 96-well plate, and the plate was further incubated overnight at 30° C. in a shaker at 300 rpm. The scFv supernatant was mixed with 2% BSA in PBST containing 1:2500 diluted horseradish peroxidase-conjugated recombinant monoclonal mouse anti-c-myc antibody in a volume ratio of 1:1, and used for enzyme-linked immunosorbent assay (ELISA) to identify clones of phage display scFv with high CD123 binding affinity. The supernatant was incubated with 30ng / well coated recombinant human CD123 in 384-well plates at room temperature for 1 hour and washed three times with PBST (after overnight incubation at 4°C, it was 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 (A450) at 450nm was measured. Clones that bound to CD123 with a signal-to-noise ratio >5 were selected for further characterization.

[0341] Flow cytometry-based binding assay.

[0342] The binding substances identified from ELISA are tested on CD123 positive cell line MOLM-13. CD123 negative cell line Jeko-1 CD20KO eGFP is used as negative control. For positive control staining, REAL270 (αCD123) and REAL116 (αCD123) antibodies are used respectively. Soluble scFv is expressed as described above. Bacterial precipitate is destroyed by incubation at 37 ℃ with 250rpm for 18 hours with 0.3mLTE buffer (10mM Tris-HCl containing 1mMEDTA, pH 8.0 at 37 ℃) / well. Culture is centrifuged at 4000 × g for 20 minutes at RT, and supernatant is transferred to a new microtiter plate.

[0343] For cell staining, cell number and viability were determined on MACSQuantX. A cell suspension containing the desired number of cells was centrifuged at 300 × g for 10 minutes at 4 ° C, and the supernatant was discarded. The cells were resuspended to a concentration of 1E+06 cells / mL by adding PEB buffer (1 × PBS+2mMEDTA, 0.5% BSA pH 7.4 at room temperature). 100,000 cells / cell line were added to each well of a 96-well V-bottom plate, and the plate was centrifuged at 1300 × g for 2 minutes at 4 ° C. The supernatant was discarded, the cells were resuspended in 100 μL of supernatant from a periplasmatic preparation, and the cells were incubated on ice for 10 minutes. The cells were washed by adding 100 μL PEB, and then centrifuged (1300 × g, 4 ° C for 2 minutes) twice. 50 microliters of secondary antibody (anti-His-APC conjugated antibody) or REAL antibody (diluted 1:50 in PEB buffer) were added to each well, and the plate was incubated at 4°C in the dark for 10 minutes. The cells were washed by adding 100 μL PEB and then centrifuged (1300×g, 4°C for 2 minutes) twice. Propidium iodide was diluted 1:100 in a fixing solution (1×PBS+2mM EDTA+1% PFA+0.3% MeOH+3% sodium azide), and the washed cells were resuspended in 50 μL of the mixture. Signals were measured on MACSQuantX. Signals were analyzed using FlowLogic software. Statistical analysis was performed using VORTEX software.

[0344] result:

[0345] Based on the results of ELISA binding assay, 10 unique scFv clones specific for recombinant human CD123 and MOLM13 cells were identified (Table 1). The corresponding scFv sequence was incorporated into the CAR construct as a binding domain for further analysis of CAR-T and CAR-NK forms.

[0346] Table 1.

[0347] ScFv Name 1 MB31-A01 2 MB31-C01 3 MB35-E02 4 MB36-A05 5 MB40-F08 6 MB40-H08 7 MB42-D03 8 MB42-E02 9 MB42-E12 10 MB44-H01

[0348] Example 3

[0349] Development of CD123-targeting CAR T cell constructs

[0350] There are few treatment options for AML, and treatment-related toxicity and disease relapse after treatment are common. In addition, immunotherapy using non-human sequences (e.g., mouse-derived antibodies) can lead to treatment rejection or adverse reactions in patients. In order to develop new CAR T therapies for AML, 15 CAR T cells incorporating fully human ScF were designed. v The CD123-targeting CAR T constructs with the targeting domain were constructed and the anti-tumor activity was evaluated.

[0351] Materials and methods:

[0352] Cell lines used to demonstrate CAR activity

[0353] Acute myeloid leukemia cell line MOLM-14 was purchased from the German Collection of Microorganisms and Cell Lines (DSMZ, Braunschweig Germany). 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 (American Tissu e The MOLM14 cell line was cultured in RPMI-1640 medium (ATCC) supplemented with 20% heat-inactivated fetal bovine serum (FBS). The KG-1a line was cultured in IMDM medium supplemented with 20% FBS. The A431 line was cultured in DMEM medium (ATCC) supplemented with 10% heat-inactivated FBS. 293T cells were cultured in Dynamis(R) containing 4 mM L-glutamine (Lonza, Morristown, NJ). TM Culture medium (ThermoFisher Scientific, Grand Island, NY) Each cell line was prepared as a single cell clone of a luciferase-expressing cell line by stably transducing a wild-type tumor cell line with a lentiviral vector encoding firefly luciferase (Lentigen Technology, Inc., Gaithersburg, MD).

[0354] Generation of CAR constructs and production of lentiviral vectors

[0355] Human anti-CD123 chimeric antigen receptor (CAR) constructs are produced by multiple single-chain variable fragments (ScFv) sequences of the extracellular domains of targeting human CD123 / IL-3 receptor α. Each scFv sequence is connected in frame with CD8 hinge, 4-1BB costimulatory domain and CD3-ζ activation domain sequence. The CAR sequence of comparator targeting CD33 is produced in a similar manner, except that only variable domain (VH_4) of heavy chain is used instead of scFv as targeting domain. VH_4 sequence is connected in frame with CD8 hinge, 4-1BB costimulatory domain and CD3-ζ activation domain sequence (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 vol.8539.22Nov.2018, doi: 10.3389 / fonc.2018.00539). The leader peptide derived from human GMCSFR1 is included in all CAR constructs to promote transport to T cell membranes. Under the control of human EF-1α promoter (Lentigen Technology Inc., Gaithersburg, MD), the CAR sequence is cloned into a lentiviral vector (LV) expression cassette. Lentiviral particles are produced by transient transfection of HEK 293T cells, precipitated by centrifugation, and stored at -80 ° C until transduction.

[0356] Preparation and transduction of primary T cells

[0357] Healthy donor primary T cells were isolated from leukapheresis collections (AllCells, Alameda, CA) or from processed buffy coats (Oklahoma Blood Institute, Tulsa, OK) with written consent from the donors. CD4-positive and CD8-positive 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. The purified T cells were incubated at 1×10 6 The cells were cultured at a density of 10 cells / ml and stained with CD3 / CD28 GMP T cell TransAct reagent (Miltenyi Biotec) was used for activation. In addition, activated T cells were transduced with lentiviral vector particles encoding CAR constructs on day 1. On day 3, and every 2 to 3 days thereafter, the culture was supplemented with fresh TexMACS medium containing 30 IU / ml IL-2 until harvested on day 8 to 10. It is noted that TexMACS medium supplemented with 970 IU / ml IL-7 and 90 IU / ml IL-15 was used.

[0358] Surface expression of CD123 on tumor cell lines

[0359] Anti-CD123 antibody clone AC145 (Miltenyi Biotec, Bergisch Gladbach, Germany) was used to determine the CD123 surface expression in a series of tumor cell lines by flow cytometry, and negative gates were set based on homologous isotype controls. Based on the antibody (antibody bound per cell, ABC) method for each cell combination, the CD123 surface expression density on the target cell line was evaluated according to the manufacturer's scheme by QuantiBRITE phycoerythrin (PE) beads (BD Biosciences, San Jose, CA). In short, beads conjugated with PE fluorophores at four different densities were used to generate a standard curve, and tumor cells stained with anti-CD123 antibodies conjugated with PE were obtained under the same settings. The ABC values ​​of each tumor cell line were calculated based on the standard curve.

[0360] Flow cytometric analysis of CAR surface expression

[0361] 500,000 CAR T cells were washed twice in 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) and then stained with anti-Fc-AF647 conjugate (Jackson ImmunoResearch, West Grove, PA). 7-Aminoactinomycin D staining (7-Aminoactinomycin D, 7-AAD, BD Biosciences, San Jose, CA) was added to exclude dead cells. Untransduced cells (UTD) were used as negative controls. The cells were washed twice with AutoMACS buffer supplemented with 0.5% bovine serum albumin, resuspended in 200ul staining buffer, and collected by flow cytometry. Flow cytometric analysis was performed on a 10 analyzer (Miltenyi Biotec), and data graphs were generated using FlowJo software (Ashland, OR).

[0362] CAR T cell cytotoxicity and cytokine assays

[0363] To evaluate CAR T cell-mediated cytotoxicity, 5 × 10 cells stably transduced with firefly luciferase were cultured in 3 Tumor target cells were combined with CAR T cells at a specified effector to target ratio and incubated overnight at 37 ° C and 5% CO2. SteadyGlo reagent (Promega, Madison WI) was added to each well, and the resulting luminescence was quantified as counts per second (sample CPS). The wells with only the target (maximum CPS) and the wells with only the target plus 1% Tween-20 (minimum CPS) were used to determine the range of the assay. The specific lysis percentage was calculated as follows: (1-(sample CPS-minimum CPS) / (maximum CPS-minimum CPS)). For cytokine release analysis, 5×10 4 effectors and 5×10 3 The target cells were co-cultured overnight, and the supernatant was removed from the co-culture and analyzed for the concentrations of IFNγ, TNFα, and IL-2 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.

[0364] result

[0365] The data in Example 3 describe the generation and in vitro evaluation of CAR T cells targeting the CD123 antigen for the treatment of AML.

[0366] Figure 1AThe schematic diagram of the tandem CAR construct targeting CD123 antigen is shown. CAR 123 is composed of a fully human conjugate (InfinityOne), connected in frame with CD8 hinge and transmembrane domain, 4-1BB costimulatory domain and CD3ζ activation domain. Based on the flow cytometry binding analysis of homologous soluble conjugates and target systems with and without CD123 expression, 10 scFv sequences were selected for evaluation in the form of CAR. CAR variants D0125-D0134 (Table 2) were constructed. The CAR sequence is further incorporated into the third generation lentiviral vector and transduced into human primary T cells under saturation to produce CD123CAR T cells under the control of mammalian EF-1α promoter. The CAR control constructs targeting CD123 (LTG2078) and CD33 (LTG1906) evaluated previously are also included (Table 3). Untransduced T cells derived from the same donor as the cells (UTD) expressing CAR are used as negative controls.

[0367] Table 2.CD123CAR constructs

[0368] Construct number FWf Construct name D0125 CD123(MB31-A01) EF-1a-CD123MB31-A01CD8BB7 D0126 CD123(MB31-C01) EF-1a-CD123MB31-C01CD8BBz D0127 CD123(MB35-E02) EF-1a-CD123MB35-E02CD8BBz D0128 CD123(MB36-A05) EF-1a-CD123MB36-A05CD8BBz D0129 CD123(MB40-F08) EF-1a-CD123MB40-F08CD8BBz D0130 CD123(MB40-H08) EF-1a-CD123MB40-H08CD8BBz D0131 CD123(MB42-D03) EF-1a-CD123MB42-D03CD8BBz D0132 CD123(MB42-E02) EF-1a-CD123MB42-E02CD8BBz D0133 CD123(MB42-E12) EF-1a-CD123MB42-E12CD8BBz D0134 CD123(MB44-H01) EF-1a-CD123MB44-H01CD8BBz

[0369] Table 3. Single-target CAR comparison

[0370] Construct number scFv Construct name LTG2078 M12306 EF-1αCD123CD8BBz LTG1906 CD33_4 EF-1αCD33CD8BBz

[0371] The lentiviral vector encoding the CD123CAR construct was used to transduce the CAR into human primary T cells at a multiplicity of infection (MOI) of 40. Using recombinant IL3R-αFc labeled, followed by anti-Fc Alexa Flour 647 staining, the CAR surface expression of transduced T cells was performed by flow cytometry. Different CD123 CAR constructs showed different expression levels, ranging from 0 to 80% (n = 4 donors), Figure 1B Across multiple donors, CARs D0126, D0127, D0131, D0132, D0133, and D0134 showed similar or higher surface expression than the positive CAR 123 control, LTG2078; whereas CAR D0130 had slightly lower surface expression, followed by D0129 and D0128, and D0125 had the lowest expression. Figure 1C As shown in, cell viability was detected on the 3rd and 7th days after T cell activation. Compared with the control CAR LTG2078, all CD123CART cells showed improved or comparable viability.

[0372] To evaluate the in vitro target-specific cytotoxicity of CD123 CAR, surface CD123 expression of leukemia lines (MOLM14, KG1a, RS4;11) and non-leukemia lines (293T and A431) was evaluated by flow cytometry using CD123-specific antibodies. Figure 2 As shown in , 99% of MOLM14 and 66% of KG-1a human AML tumor cell lines expressed CD123, while the human B-ALL line RS;411 had only limited CD123 expression. In contrast, 293T and A431 did not have CD123 expression. Therefore, MOLM14 and KG-1a were selected as target cell lines, and 293T was selected as a negative control cell line for CAR T cell function evaluation.

[0373] Human primary T cells were transduced with a lentiviral vector encoding a CAR construct and expanded in culture to the 8th day. CAR-T cells were co-incubated with MOLM14, KG-1a or 293T cell lines at a ratio of 2.5: 1, 5: 1 and 10: 1 effector to target. After overnight co-incubation, the culture was analyzed in a luminescent in vitro killing assay. Most of the primary T cell lines expressing the CAR123 construct lyse MOL14-CD123+ with different potencies, while the three CD123 CAR lines D0125, D0128 and D0129 lacked target cleavage ability ( Figure 3A ). Similarly, KG-1a-CD123+ target cells were killed by most CAR T constructs except D0125, D0128, and D0129 ( Figure 3B CD33 CAR LTG1906 exhibits high cytotoxicity against MOLM14 (CD33 高 ) and showed low lytic efficacy against KG1a (CD33 低 ), which is consistent with the CD123 expression level. In addition, no killing was observed above the background of the CD123-negative 293T cell line ( Figure 3C ), which indicates the robust target-specific cytotoxic function of all CD123 CAR constructs except CAR D0125, D0128 and D0129.

[0374] After overnight incubation of CAR T cells with the MOLM14 target line at an E:T ratio of 10, T cell homeostasis and production of proinflammatory cytokines IL-2, IFNγ, and TNFα were detected by ELISA in the culture supernatant of CD123 CAR and control constructs CAR LTG2078 and CD33 CAR LTG1906 ( Figures 4A to 4C). By comparing each CART group incubated with target cells with the respective CART experimental group alone, and also comparing the CAR T group co-incubated with the target with the previously characterized CAR123 control LTG2078, to detect the cytokine release induced by specific targets. 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 produce significant increases in IFNγ, TNFα or IL-2 cytokines after co-culture with MOLM14 cells overnight. An exception is CAR123 D0127, which even in the absence of target cells (only T cell group) also produced IFNγ and TNFα levels, indicating tumor-independent cytokine responses. This effect is unpredictable from previous experiments, and this shows the non-obviousness of the present invention. With the exception of CAR123 D0127, the cytokine responses of the CD123 CAR constructs evaluated herein were comparable to untransduced T cell (UTD) controls, indicating a low risk of inducing cytokine-mediated adverse effects such as cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS).

[0375] Among all CAR123 constructs, D0126 showed the highest transduction efficiency and viability, as well as the best cytotoxic function for CD123+ tumor cells in this group of CAR constructs. Another CAR123 construct D0131 also showed high CAR transduction efficiency and viability, but showed moderate in vitro target cell killing activity. Therefore, CAR123 constructs D0126 and D0131 were selected for further in vivo evaluation.

[0376] Example 4

[0377] Evaluation of the anti-tumor function of CAR T cells targeting CD123 in the mouse MOLM14 xenograft model

[0378] This example describes the in vivo evaluation of CD123-targeted CART cells incorporating scFv sequences derived from the InfinityOne library.

[0379] Materials and methods:

[0380] Cell lines

[0381] 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 transduced with the firefly luciferase gene and cultured in RPMI-1640 medium (ATCC) supplemented with 20% heat-inactivated fetal bovine serum (FBS).

[0382] In vivo analysis of CAR T function

[0383] Animal experiments were performed in accordance with applicable laws, regulations, and guidelines of the National Institutes of Health (NIH) and approved by the Animal Care and Use Committee of MI Bioresearch (Ann Arbor, MI). cid Iq tm1Wjl The function of CAR T cells targeting CD123 was evaluated in S / SzJ mice. 6 MOLM-14CD 123 + AML cells were injected into the tail vein of 6- to 8-week-old female NSG mice (6 / group). Tumor burden was determined by IVIS bioluminescence imaging on day 4, and mice were subsequently randomized into groups with the same average tumor burden and administered 5.0 × 10 6 CAR T + The cells / mouse were normalized for transduction efficiency. Tumor regression was determined by bioluminescence imaging using a Xenogen IVIS-200 instrument (PerkinElmer, Shelton, Connecticut) on days 14, 21, 28, 35, 42, and 49. Images were analyzed using Living Image version 4.1 software (Perkin Elmer), and the bioluminescent signal flux for each mouse was expressed as the mean radiance (photons / second / cm 2 / steradian). Survival was recorded and analyzed at the end of the study. To determine the presence of CAR T and tumor cells, peripheral blood was collected from all animals on study days 14, 21, 28, and 42. The absolute number of blood CAR T cells and MOLM-14 tumor cells was determined by flow cytometry.

[0384] Flow cytometric analysis of CAR T and tumor cells in mouse blood

[0385] 70 microliters of mouse blood were collected in the study on the 14th, 21st, 28th and 42nd days, and CAR T and MOLM-14 tumor cell numbers were analyzed by flow cytometry. Then erythrocytes were lysed according to the manufacturer's instructions using erythrocyte lysis solution (BD BioScience, San Jose, CA), 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 analysis by 7-AAD staining. In order to obtain the direct counting of human T cells and MOLM-14 in blood, MACSQuant 10 volume functions were used, and CountBright absolute counting beads (ThermoFischer Scientific, Waltham, MA) were used to calculate the sample loss during the treatment according to the manufacturer's scheme.

[0386] result:

[0387] The NSG MOLM14 xenograft AML model was used to further explore the in vivo tumor rejection function of the two best CAR123 candidates, D0126 and D0131. Two animal studies using CAR T cells derived from different healthy donors were conducted, one focusing on CAR D0126 ( Figure 5A ), and another compared CAR123 constructs D0126 with D0131 ( Figure 5B ). A previously characterized CAR, LTG1906, targeting the CD33 antigen on MOLM14 tumor cells, was included as a comparative control.

[0388] In the first in vivo study, CD123 CAR D0126 was compared with the previously characterized CD33 CAR-T construct LTG1906, and control experimental groups of tumor only (TA) and untransduced T cells (UTD) were also included. CAR-T cells were generated by transduction with lentiviral vectors encoding CAR D0126 and lentiviral vectors encoding CAR LTG1906 and then cultured and expanded in TexMACS medium supplemented with 30 IU / ml IL2. MOLM14-Luc cells were used as the target line. 1×10 6 MOLM14-Luc cells were injected intravenously (iv) into each NSG mouse. Tumor growth was evaluated by IVIS imaging on day 6, and mice were then randomly divided into experimental groups. On day 7, 5×10 6Personal CAR+T cells or UTD cells / mouse. Tumor growth dynamics were monitored over time by in vivo imaging system (IVIS) ( Fig. 6A and 6B ). Since MOLM14 tumors express both CD123 and CD33 antigens, compared with tumor-only (TA) and UTD control groups, the treatment group administered with CAR D0126 targeting CD123 antigen, and the comparison group administered with CAR LTG1906 targeting CD33 antigen, showed robust tumor rejection. At the end of the study, five of the six mice in each group showed complete tumor rejection, and only one mouse in each group had residual tumor cells ( Figure 6B Notably, neither the CARD0126 nor the CARLTG1906 treated groups showed weight loss ( Figure 6C ), so no CAR-related toxicity was detected in this model. Both CAR D0126 and LTG1906 mediated complete survival until study termination on day 36 (6 of 6 mice survived), while tumor-only (TA) and UTD control groups died on day 15 due to high burden of disseminated disease ( Fig.6D ). Peripheral blood was sampled from mice on days 14, 22, and 33. Human T cells were detected in all groups ( Fig. 7A , 7B , 7C). In addition, at the end of the study, CAR D0126 and LTG1906 T cells were detected in the peripheral blood of mice, indicating high persistence of CD123 CAR candidate D0126 and comparative control CAR33 LTG1906 T cells.

[0389] In the second animal study, CD123 CAR D0131 was included in addition to CAR D0126. The CAR T cells in this study were generated from peripheral blood T cells from a different donor than that used in the first in vivo study. T cells were transduced and expanded with TexMACS medium supplemented with 970 IU / ml IL-7 and 90 IU / ml IL-15. Tumor progression in each group was Fig. 8A Similar to the first animal study, CAR D0126 showed strong anti-tumor efficacy, and four of the six mice showed tumor rejection. Compared with CAR123 D0126, CAR123 D0131 showed weaker anti-tumor activity ( Fig. 8A and 8B Although no significant weight loss was observed in the CAR T-treated group ( Figure 8C), but mouse deaths were observed in all groups. The best survival effect was detected in the CAR D0126-treated group, with four of six mice surviving to the extended study termination day, day 56, and remaining completely tumor-free ( Fig.8D Total T cells in peripheral blood were monitored in this study. As expected, two days after administration of CAR T cells or UTD, human T cells were detected in the peripheral blood of mice in all groups except the TA negative control. Fig.9A ). Over time, the amount of T cells in all CAR T groups increased, suggesting T cell expansion ( Fig. 9B ) and persisted on days 21, 28, and 42 ( Fig. 9C , 9D , 9E). On day 42 of the study, the number of T cells in the CAR123 D0126 group was the highest ( Fig.9E ), indicating that T cell expansion and persistence were the strongest among the CAR constructs tested in this experiment.

[0390] In conclusion, in two in vivo studies utilizing T cells from different human donors, the CD123CAR candidate D0126 effectively eliminated tumors in NSG mice transplanted with MOLM-14 cells and demonstrated potent tumor clearance, CAR T persistence, and prolonged survival in a MOLM14 AML xenograft mouse model ( Fig.9A ). Therefore, CAR123 D0126 was identified as the lead candidate for the development of CD123-targeted CAR T therapy for the treatment of CD123-positive malignancies.

[0391] Example 5

[0392] Development of CD123-targeted CAR NK cells

[0393] This example describes the generation of CAR NK cells by lentiviral transduction.

[0394] Materials and methods:

[0395] Lentiviral vector construction and production

[0396] Each CD123-CAR consists of a CD123 scFv binder, a CD8 hinge and transmembrane domain, a 4-1BB transactivation domain, and a CD3ζ signaling domain. Under the control of the human EF-1α promoter, the construct was cloned into a third-generation lentiviral plasmid backbone (Lentigen). As previously described, HEK 293T cells were transiently transfected to produce a supernatant containing a lentiviral vector (LV) (Kuroda Het al., J Virol Methods. (2009) 157: 113–21). In order to pseudotype the lentiviral vector, a modified BaEV envelope glycoprotein was used as previously described (Girard-Gagnepain A et al., Blood. (2014) 124: 1221–31). The supernatant containing LV was stored at -80 ° C and the titer on NK-92 cells was determined.

[0397] Primary NK cell isolation

[0398] To isolate NK cells from buffy coats, peripheral blood mononuclear cells (PBMC) were prepared by standard density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare). Resting NK cells were enriched from PBMCs by depletion of non-NK cell populations using the NK Cell Isolation Kit for Human Cells (Miltenyi Biotec).

[0399] Cell culture and transduction

[0400] NK cells were cultured at 10 °C 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).

[0401] Briefly, NK cells were cultured at 5 × 10 510 μ L LV supernatant. After 2 hours of spinoculation at 400 g, cells were cultured in a cell culture incubator with LV for 24 hours. The cell culture medium was then replaced with a fresh complete cell culture medium containing 5% human AB serum, 500 U / mL IL-2, and 10 ng / mL IL-15. From the 3rd day after transduction, transduction efficiency was determined by flow cytometry. Every 3 days, the transduced NK cells were centrifuged, counted, and in fresh complete NK cell culture medium (5% human AB serum, 500 U / ml IL-2, and 10 ng / mL IL-15), the cell number was adjusted to 500,000 cells / mL for long-term culture. Result:

[0402] Primary NK cells were efficiently transduced with CD123-CAR using a baboon envelope glycoprotein-pseudotyped lentiviral vector.

[0403] We have produced 13 CD123-CAR constructs, which contain CD123 binders, CD8 hinge and transmembrane domains, 4-1BB transactivation domains, and CD3ζ signaling domains (Table 4). Under the control of the human EF-1α promoter, these CD123-CAR constructs were cloned into the third-generation lentiviral plasmid backbone (Lentigen). We have shown in earlier publications that modified baboon envelope glycoprotein pseudotype lentiviral vectors (BaEV) can effectively transduce NK cells (BariR et al., Front Immunol. (2019) 10: 2001). All 13 listed CD123-CAR-containing lentiviral vectors (LV) were pseudotyped with BaEV, and viral vectors were produced by transient transfection of HEK 293T cells.

[0404] Table 4. List of CD123CAR and conjugates

[0405] Construct number FWf Construct name SsFv source library Z16 CD123(Z16) EF-1a-CD123Z16-CD8BBz yeast LTG2078, control CD123(Z23) EF-1a-CD123Z23-CD8BBz yeast Z32 CD123(Z32) EF-1a-CD123Z32-CD8BBz yeast D0125 CD123(MB31-A01) EF-1a-CD123MB31-A01CD8BBz Bacteriophage D0126 CD123(MB31-C01) EF-1a-CD123MB31-C01CD8BBz Bacteriophage D0127 CD123(MB35-E02) EF-1a-CD123MB35-E02CD8BBz Bacteriophage D0128 CD123(MB36-A05) EF-1a-CD123MB36-A05CD8BBz Bacteriophage D0129 CD123(MB40-F08) EF-1a-CD123MB40-F08CD8BBz Bacteriophage D0130 CD123(MB40-H08) EF-1a-CD123MB40-H08CD8BBz Bacteriophage D0131 CD123(MB42-D03) EF-1a-CD123MB42-D03CD8BBz Bacteriophage D0132 CD123(MB42-E02) EF-1a-CD123MB42-E02CD8BBz Bacteriophage D0133 CD123(MB42-E12) EF-1a-CD123MB42-E12CD8BBz Bacteriophage D0134 CD123(MB44-H01) EF-1a-CD123MB44-H01CD8BBz Bacteriophage

[0406] Primary NK cells were isolated from PBMCs by magnetic separation to generate pure cell populations ( Fig. 10A). Most cell lines, especially acute myeloid leukemia (AML) cells, are sensitive to the natural cytotoxicity of NK cells and are therefore not suitable for testing the cytotoxicity of CAR-NK cells. However, the RS4-11 cell line is known to be insensitive to the natural cytotoxicity of NK cells. Therefore, many NK cell research laboratories, including ours, often use RS4-11 as target cells to test CAR-NK cell function. In order to use RS4-11 as a target cell to test CD123-CAR function, a sub-RS4-11 cell line stably expressing CD123 was generated ( Fig. 10B ).

[0407] NK cells were activated by culturing in NK MACS medium containing IL-2 / IL-15 / IL-1β for two days and then transduced 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 ( Fig.11 ). Among the 13 CD123-CARs, the Z32 and D0126 conjugates had the best effect in transducing NK cells and produced a transduction efficiency of 51.55% and 61.37%, respectively. Based on these expression results, we selected the CAR constructs Z32 and D0126 for further analysis.

[0408] CD123-CAR NK cells effectively and specifically kill target cells expressing CD123.

[0409] Activated NK cells were transduced with BaEV pseudotyped lentiviral vectors containing CD123-CAR Z32 (Z32-BaEV-LV) and BaEV pseudotyped lentiviral vectors containing D0126 (D0126-BaEV-LV). The CD123-CAR expression of Z32 and D0126 was 70.5% and 64.19%, respectively ( Fig. 12A In addition, the cytotoxicity of NK cells expressing CD123-CAR against target cells RS4-11-CD123 was tested. RS4-11 cells expressing CD123 ( Fig. 10B ) is insensitive to the natural cytotoxicity of NK cells. Therefore, untransduced NK cells cannot kill RS4;11-CD123 cells, while both CD123-CAR (Z32 and D0126) NK cells can kill RS4;11-CD123 very effectively, indicating the high functionality and specificity of the generated CD123-CAR NK cells ( Fig. 12B ).

[0410] Next, the specificity of CD123-CAR for CD123 antigen was confirmed by serial dilution. NK cells were transduced with different amounts of lentiviral vectors containing CD123-CAR. As expected, higher amounts of CD123-CAR-LV showed higher CD123-CAR expression ( Fig.13A Finally, the cytotoxicity of NK cells expressing differentially expressed CD123-CAR against RS4-11-CD123 cells was tested at the same effector-target ratio ( Fig. 13B ). NK cells with the highest expression of CD123-CAR showed the highest killing, and NK cells with the lowest expression of CD123-CAR showed the lowest killing, confirming the specificity of CD123-CAR for CD123 antigen.

[0411] The expression of CD123-CAR had no adverse effects on the expansion and viability of NK cells.

[0412] Primary NK cells were isolated, activated, transduced with Z32 and D0126, and then expanded for 13 days. Untransduced NK cells were used as controls. The expansion of untransduced, Z32-transduced, and D0126-transduced NK cells was 61-fold, 49-fold, and 42-fold, respectively ( Fig.14A ). At the beginning of the experiment, the number of NK cells in each condition was the same. Some NK cells lost during the transduction process may explain the difference in cell expansion between untransduced and transduced cells. However, the difference in expansion 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 was also examined ( Figure 5B There was no significant difference in cell viability among untransduced, Z32-transduced, and D0126-transduced NK cells ( Fig. 14B ), indicating that CD123-CAR had no adverse effects on NK cell viability.

[0413] References to sequence listings

[0414] This application contains a Sequence Listing that has been electronically submitted to the United States Patent and Trademark Office via a PDF file entitled “Sequence Listing.” This Sequence Listing is incorporated by reference.

[0415] Sequence of the present disclosure

[0416] The nucleic acid and amino acid sequences listed below are shown using standard letter abbreviations for nucleotide bases and single-letter or three-letter codes for amino acids as defined in 37 C.FR 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included by any reference to the strand shown. In the attached sequence listing:

[0417] SEQ ID NO:1 Nucleotide sequence of CARD0125 CD123 MB31-A01 CD8 BBz

[0418]

[0419] SEQ ID NO:2 Amino acid sequence of CAR D0125 CD123 MB31-A01 CD8 BBz

[0420]

[0421] SEQ ID NO:3 Nucleotide sequence of CAR D0126 CD123 MB31-C01 CD8 BBz

[0422]

[0423]

[0424] SEQ ID NO:4 Amino acid sequence of CAR D0126 CD123 MB31-C01 CD8 BBz

[0425]

[0426] SEQ ID NO:5 Nucleotide sequence of CAR D0127 CD123 MB35-E02 CD8 BBz

[0427]

[0428] SEQ ID NO:6 Amino acid sequence of CAR D0127 CD123 MB35-E02 CD8 BBz

[0429]

[0430] SEQ ID NO:7 Nucleotide sequence of CAR D0128 CD123 MB36-A05 CD8 BBz

[0431]

[0432]

[0433] SEQ ID NO:8 Amino acid sequence of CAR D0128 CD123 MB36-A05 CD8 BBz

[0434]

[0435] SEQ ID NO:9 Nucleotide sequence of CAR D0129 CD123 MB40-F08 CD8 BBz

[0436]

[0437]

[0438] SEQ ID NO:10 Amino acid sequence of CAR D0129 CD123 MB40-F08 CD8 BBz

[0439]

[0440] SEQ ID NO:11 Nucleotide sequence of CARD0130 CD123 MB40-H08 CD8 BBz

[0441]

[0442] SEQ ID NO:12 Amino acid sequence of CAR D0130 CDAR123 MB40-H08 CD8 BBz

[0443]

[0444] SEQ ID NO: 13 Nucleotide sequence of leader / signal peptide sequence

[0445]

[0446] SEQ ID NO:14 Amino acid sequence of leader / signal peptide sequence

[0447]

[0448] SEQ ID NO:15 Nucleotide sequence of CAR D0131 CD123 MB42-D03 CD8 BBz

[0449]

[0450] SEQ ID NO:16 Amino acid sequence of CAR D0131 CD123 MB42-D03 CD8 BBz

[0451]

[0452] SEQ ID NO: 17 Nucleotide sequence of CAR D0132 CD123 MB42-E02 CD8 BBz

[0453]

[0454]

[0455] SEQ ID NO:18 Amino acid sequence of CAR D0132 CD123 MB42-E02 CD8 BBz

[0456]

[0457] SEQ ID NO: 19 Nucleotide sequence of CAR D0133 CD123 MB42-E12 CD8 BBz

[0458]

[0459] SEQ ID NO:20 Amino acid sequence of CAR D0133 CD123 MB42-E12 CD8 BBz

[0460]

[0461] SEQ ID NO:21 Nucleotide sequence of CAR D0134 CD123 MB44-H01 CD8 BBz

[0462]

[0463]

[0464] SEQ ID NO:22 Amino acid sequence of CAR D0134 CD123 MB44-H01 CD8 BBz

[0465]

[0466] SEQ ID NO:23 Nucleotide sequence of CAR LTG2078 CD123 M12306 CD8 BBz

[0467]

[0468] SEQ ID NO:24 Amino acid sequence of CAR LTG2078 CD123 M12306 CD8 BBz

[0469]

[0470] SEQ ID NO:25 Nucleotide sequence of CAR LTG1906 CD33_4CD8 BBz

[0471]

[0472] SEQ ID NO:26 Amino acid sequence of CAR LTG1906 CD33_4CD8 BBz

[0473]

[0474] SEQ ID NO:27 Nucleotide sequence of DNA CD8 transmembrane domain

[0475]

[0476] SEQ ID NO:28 Amino acid sequence of CD8 transmembrane domain

[0477]

[0478] SEQ ID NO: 29 Nucleotide sequence of DNA CD8 hinge domain

[0479]

[0480] SEQ ID NO:30 Amino acid sequence of CD8 hinge domain

[0481]

[0482] SEQ ID NO:31 Amino acid sequence of the hinge region of amino acids 118 to 178 of CD8.α. (NCBI RefSeq: NP.sub.--001759.3)

[0483]

[0484] SEQ ID NO:32 Amino acid sequence of human IgG CL sequence

[0485]

[0486] SEQ ID NO:33 Nucleotide sequence of the DNA signaling domain of 4-1BB

[0487]

[0488] SEQ ID NO:34 Amino acid sequence of the signaling domain of 4-1BB

[0489]

[0490] SEQ ID NO:35 Nucleotide sequence of the DNA signaling domain of CD3-ζ

[0491]

[0492] SEQ ID NO:36 Amino acid sequence of CD3ζ

[0493]

[0494] SEQ ID NO:37 Nucleotide sequence of ScFv CD 19

[0495]

[0496] SEQ ID NO:38 Amino acid sequence of ScFv CD 19

[0497]

[0498] SEQ ID NO: 39 Nucleotide sequence of GMCSF leader peptide

[0499]

[0500] SEQ ID NO:40 Amino acid sequence of GMCSF leader peptide

[0501]

[0502] SEQ ID NO:41 Nucleotide sequence of TNFRSF19 leader peptide

[0503]

[0504] SEQ ID NO:42 Amino acid sequence of TNFRSF19 leader peptide

[0505]

[0506] SEQ ID NO:43 Nucleotide sequence of CD8α leader peptide

[0507]

[0508] SEQ ID NO:44 Amino acid sequence of CD8α leader peptide

[0509]

[0510] SEQ ID NO:45 Nucleotide sequence of CD28 costimulatory domain

[0511]

[0512] SEQ ID NO:46 Amino acid sequence of CD28 co-stimulatory domain

[0513]

[0514] SEQ ID NO:47 Nucleotide sequence of CD3ζ activation domain

[0515]

[0516] SEQ ID NO:48 Amino acid sequence of CD3ζ activation domain

[0517]

[0518] SEQ ID NO:49 Nucleotide sequence of hinge and transmembrane domain of TNFRSF19 (transmembrane domain is underlined)

[0519]

[0520] SEQ ID NO:50 Amino acid sequence of hinge and transmembrane domain of TNFRSF19 (transmembrane domain is underlined)

[0521]

[0522] SEQ ID NO:51 Nucleotide sequence of TNFRSF19 transmembrane domain

[0523]

[0524] SEQ ID NO:52 Amino acid sequence of TNFRSF19 transmembrane domain

[0525]

[0526] SEQ ID NO:53 Nucleotide sequence of TNFRSF19 hinge domain

[0527]

[0528] SEQ ID NO:54 Amino acid sequence of TNFRSF19 hinge domain

[0529]

[0530] SEQ ID NO:55 Nucleotide sequence of truncated TNFRSF19 hinge domain

[0531]

[0532] SEQ ID NO:56 Amino acid sequence of truncated TNFRSF19 hinge domain

[0533]

[0534] SEQ ID NO:57 Nucleotide sequence of CD8a hinge domain fused to TNFRSF19 transmembrane domain (transmembrane sequence is underlined)

[0535]

[0536] SEQ ID NO:58 Amino acid sequence of the CD8a hinge domain fused to the TNFRSF19 transmembrane domain (transmembrane sequence is underlined)

[0537]

[0538] SEQ ID NO:59 Nucleotide sequence of CD28 co-stimulatory domain

[0539]

[0540] SEQ ID NO:60 Amino acid sequence of CD28 co-stimulatory domain

[0541]

[0542] SEQ ID NO:61 Nucleotide sequence of CD3ζ form 2

[0543]

[0544] SEQ ID NO:62 Amino acid sequence of CD3ζ form 2

[0545]

[0546] SEQ ID NO:63 Nucleotide sequence of furin P2A furin

[0547]

[0548] SEQ ID NO:64 Amino acid sequence of Furin P2A Furin (Furin sequence is underlined)

[0549]

[0550] SEQ ID NO:65 Nucleotide sequence of Furin T2A

[0551]

[0552] SEQ ID NO:66 Amino acid sequence of Furin T2A (Furin sequence is underlined)

[0553]

[0554] SEQ ID NO:67 Nucleotide sequence of truncated EGFR (tEGFR) tag

[0555]

[0556] SEQ ID NO:68 Amino acid sequence of truncated EGFR (tEGFR) tag

[0557]

[0558] SEQ ID NO:69 Nucleotide sequence of CD123 binder MT-16

[0559]

[0560]

[0561] SEQ ID NO:70 Amino acid sequence of CD123 binder MT-16

[0562]

[0563] SEQ ID NO:71 Nucleotide sequence of CD123 binder MT-32

[0564]

[0565] SEQ ID NO:72 Amino acid sequence of CD123 binder MT-32

[0566]

[0567] SEQ ID NO:73 Nucleotide sequence of the DNA signaling domain of 4-1BB

[0568]

[0569] SEQ ID NO:74 Amino acid sequence of the DNA signaling domain of 4-1BB

[0570]

[0571] SEQ ID NO:75 Nucleotide sequence of the DNA signaling domain of CD3z

[0572]

[0573] SEQ ID NO:76 Amino acid sequence of the DNA signaling domain of CD3z

[0574]

[0575] SEQ ID NO: 77 Nucleotide sequence of CAR123 Z16

[0576]

[0577] SEQ ID NO:78 Amino acid sequence of CAR123 Z16

[0578]

[0579] SEQ ID NO: 79 Nucleotide sequence of human IgG4 hinge

[0580]

[0581] SEQ ID NO: 80 Amino acid sequence of human IgG4 hinge

[0582]

[0583] SEQ ID NO: 81 Nucleotide sequence of human IgG4 CH2 domain

[0584]

[0585] SEQ ID NO:82 Amino acid sequence of human IgG4 CH2 domain

[0586]

[0587] SEQ ID NO: 83 Nucleotide sequence of human IgG4 CH3 domain

[0588]

[0589] SEQ ID NO:84 Amino acid sequence of human IgG4 CH3 domain

[0590]

[0591] SEQ ID NO: 85 Nucleotide sequence of human IgG4 hinge CH2 CH3 domain

[0592]

[0593] SEQ ID NO:86 Amino acid sequence of human IgG4 hinge CH2 CH3 domain

[0594]

[0595] SEQ ID NO:87 Nucleotide sequence of CAR123 Z32

[0596]

[0597] SEQ ID NO:88 Amino acid sequence of CAR123 Z32

[0598]

Claims

1. An isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the chimeric antigen receptor (CAR) comprises at least one extracellular antigen binding domain, at least one transmembrane domain and at least one intracellular signaling domain, wherein the extracellular antigen binding domain comprises a CD123 antigen binding domain encoded by a nucleotide sequence comprising SEQ ID NO: 69, 71, 77 or 87.

2. The isolated nucleic acid molecule of claim 1, wherein the encoded at least one CD123 antigen binding domain comprises at least one single-chain variable fragment of an antibody that binds to CD123.

3. The isolated nucleic acid molecule of claim 1, wherein the encoded at least one CD123 antigen binding domain comprises at least one heavy chain variable region of an antibody that binds to CD123.

4. The isolated nucleic acid molecule of claim 1, wherein the encoded at least one CD123 antigen binding domain, the at least one intracellular signaling domain, or both are connected to the transmembrane domain via a linker or spacer domain.

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

6. the isolated nucleic acid molecule of claim 1, wherein before the encoded extracellular CD123 antigen binding domain is a leading nucleotide sequence encoding a leading peptide.

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

44.

8. The isolated nucleic acid molecule of claim 1, wherein the transmembrane domain comprises a transmembrane domain of a protein comprising: the alpha, beta or zeta chain of a T cell receptor, CD8, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD83, CD86, CD134, CD137, CD154 and TNFRSF19, or any combination thereof.

9. the isolated nucleic acid molecule of claim 1, wherein the nucleic acid sequence encoding the extracellular CD123 antigen binding domain comprises a nucleic acid sequence containing SEQ ID NO:69, 71, 77 or 87 or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

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

11. The isolated nucleic acid molecule of claim 10, wherein the encoded at least one intracellular signaling domain is arranged on the C-terminal side relative to the CD3ζ intracellular domain.

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

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

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

15. The CAR of claim 14, comprising at least one extracellular antigen binding domain, at least one transmembrane domain and at least one intracellular signaling domain, wherein the extracellular antigen binding domain comprises a CD123 antigen binding domain comprising an amino acid sequence of SEQ ID NO: 70, 72, 78 or 88.

16. The CAR of claim 15, wherein the CD123 antigen binding domain comprises at least one single-chain variable fragment of an antibody that binds to CD123.

17. The CAR of claim 15, wherein the CD123 antigen binding domain comprises at least one heavy chain variable region of an antibody that binds to CD123.

18. The CAR of claim 15, wherein the transmembrane domain comprises a transmembrane domain of a protein comprising: the α, β or ζ chain of a T cell receptor, CD8, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154 and TNFRSF19, or any combination thereof.

19. The CAR of claim 18, wherein the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:

28.

20. The CAR of claim 15, wherein the at least one extracellular antigen binding domain and the at least one intracellular signaling domain or both are connected to the transmembrane domain via a linker or a spacer domain, and the extracellular antigen binding domain comprises a CD123 antigen binding domain comprising an amino acid sequence of SEQ ID NO: 70, 72, 78 or 88.

21. The CAR of claim 20, wherein the linker or spacer domain is derived from the extracellular domain of CD8, TNFRSF19, IgG4 or CD28 and is connected to the transmembrane domain.

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

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

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

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

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

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

28. A cell comprising the vector of claim 24.

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

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

31. The cell of claim 28, wherein the cell is a human cell.

32. A method for preparing cells, comprising transducing T cells with the vector of claim 24.

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

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

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

36. A pharmaceutical composition comprising an anti-tumor effective amount of a human T cell population, wherein the T cell comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises at least one extracellular antigen binding domain, at least one linker domain, at least one transmembrane domain, and at least one intracellular signaling domain, and the extracellular antigen binding domain comprises a CD123 antigen binding domain comprising an amino acid sequence of SEQ ID NO: 70, 72, 78 or 88, and wherein the T cell is a T cell of a person suffering from cancer.

37. The pharmaceutical composition of claim 36, wherein the at least one transmembrane domain comprises a transmembrane domain of a protein comprising: the alpha, beta or zeta chain of a T cell receptor, CD8, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, or any combination thereof.

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

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

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

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

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

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

44. A method for treating a mammal suffering from a disease, disorder or condition associated with elevated expression of a tumor antigen, the method comprising administering to a subject a pharmaceutical composition comprising an anti-tumor effective amount of a population of T cells, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises at least one extracellular antigen binding domain, at least one linker or spacer domain, at least one transmembrane domain, at least one intracellular signaling domain, the extracellular antigen binding domain comprising a CD123 antigen binding domain comprising an amino acid sequence of SEQ ID NO: 70, 72, 78 or 88, wherein the T cells are T cells of a subject suffering from cancer.

45. A method for treating cancer in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising an anti-tumor effective amount of a T cell population, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises at least one extracellular antigen binding domain, at least one linker or spacer domain, at least one transmembrane domain, and at least one intracellular signaling domain, and the extracellular antigen binding domain comprises a CD123 antigen binding domain comprising an amino acid sequence of SEQ ID NO: 70, 72, 78 or 88, wherein the T cells are T cells of a subject suffering from cancer.

46. ​​The method of claim 44 or 45, wherein the at least one transmembrane domain comprises a transmembrane domain of a protein comprising: the α, β or ζ chain of the T cell receptor, CD8, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, or any combination thereof.

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

48. The method for producing a cell expressing a chimeric antigen receptor according to claim 47, wherein the cell is a T cell or a cell population containing a T cell.

Images