Compositions and methods for treating cancer with anti-CD123 immunotherapy

Abstract

To provide methods for treating AML using an approach capable of exhibiting specific and effective antitumor effects.SOLUTION: Disclosed is a method for generating a population of RNA engineered cells comprising a step of introducing an in vitro transcribed RNA or synthetic RNA into a cell, where the RNA comprises an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR) that comprises at least one extracellular antigen binding domain comprising a CD123 antigen binding domain including a specific amino acid sequence, at least one transmembrane domain, at least one intracellular signaling domain, and at least one linker or at least one spacer domain.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 734,106, filed September 20, 2018, which is incorporated herein by reference in its entirety.

[0002] Sequence List This application includes a sequence listing, submitted electronically in ASCII format, which is incorporated herein by reference throughout. The ASCII copy, created on September 20, 2019, is named SequenceListing.txt and is 175 kilobytes in size.

[0003] Description of research or development funded by the federal government. This invention was created in the course of a Cooperative Research and Development Agreement with the National Institutes of Health, an agency of the United States Department of Health and Human Services. The U.S. government has certain rights to this invention.

[0004] Areas of this disclosure This application relates to the field of cancer, in particular to CD123 antigen-binding domains, chimeric antigen receptors (CARs) containing such CD123 antigen-binding domains, and methods of using the same. [Background technology]

[0005] background Cancer is one of the most deadly threats to human health. In the United States alone, nearly 1.3 million people are diagnosed with cancer each year, making it the second leading cause of death after cardiovascular disease and accounting for about a quarter of all deaths. Solid tumors account for the vast majority of these deaths. Despite significant progress in the medical treatment of certain cancers, the five-year overall survival rate for all cancers has improved by only about 10% in the last 20 years. Cancer, or malignant tumors, metastasize and grow rapidly without control, making them extremely difficult to treat.

[0006] AML is a devastating disease with an overall survival rate of only 26%. Younger patients tend to have a better prognosis for AML treatment, but the 5-year survival rate for older patients is likely as low as 5%. First-line treatment for AML involves multiple chemotherapy regimens (i.e., induction and intensification) that carry a high risk of toxicity. When hematopoietic stem cell transplantation is performed after the first remission, the 5-year disease-free survival rate is only 30–50% (see the World Wide Web at cancer.ca / en / cancer-information / cancer-type / leukemia-acute-myelogenous-aml / prognosis-and-survival / survival-statistics / ?region=on). Furthermore, AML patients with a high disease burden may not be candidates for bone marrow transplantation, and pre-transplant minimal residual disease is correlated with AML relapse. Current first-line induction / intensification therapy often fails to achieve MDR-negative remission sufficient to reduce tumor burden, and therefore the risk of AML recurrence after first-line therapy remains high regardless of the presence or absence of BMT (1) Biol Blood Marrow Transplant. June 2006; Vol. 12 (No. 6): pp. 691-692., Leukemia burden and outcome of allogeneic transplant in acute myelogenous leukemia., Kamble RT, Hjortsvang E, Selby GB; (2) Leuk Lymphoma. May 2015; Vol. 56 (No. 5): pp. 1353-1351. Impact of pre-transplant disease burden on the outcome of allogeneic hematopoietic stem cell transplant in refractory and relapsed acute myeloid leukemia: a single-center study. (Tian H et al.). PBDCN is a rare myeloid neoplasm classified as a subtype of AML, and may be treated as AML with induction and consolidation chemotherapy, or as ALL. BMT is often administered during the first remission. However, there are currently no ongoing clinical trials for PBDCN, and no approved first-line treatment. (See Leukemia Lymphoma Society, lls.org / leukemia / blastic-plasmacytoid-dendritic-cell-neoplasm on the World Wide Web). Therefore, a better therapeutic modality for CD123+ malignancies is urgently needed.

[0007] CAR approaches targeting CD123 are superior to chemotherapy because they may achieve higher efficacy in eliminating CD123+ tumor cells and tumor stem cells, and potentially avoid the toxicity associated with chemotherapy. Importantly, CAR T cells are expected to have a higher efficiency in removing minimal residual disease than chemotherapy, leading to better long-term treatment outcomes. Furthermore, CAR123 may be useful for tumor deburring as a bridge to transplantation, as it may help patients with high tumor burden become candidates for BMT.

[0008] CAR123 is an improvement over conventional technologies because, unlike other CAR designs that use mouse-derived ScFv, it uses a unique human ScFv (hScFv) sequence for its CAR design. Mouse-derived sequences carry an immunogenic risk, potentially inducing allergic or anaphylactic reactions in patients, leading to CAR T removal or life-threatening anaphylaxis.

[0009] Chimeric antigen receptors (CARs) are hybrid molecules containing three essential units: (1) an extracellular antigen-binding motif, (2) a ligation / 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; Vol. 2 (No. 4): e23621). The antigen-binding motif of CARs is generally constructed based on the single-chain fragment variable (scFv), the smallest binding domain of an immunoglobulin (Ig) molecule. Alternative antigen-binding motifs have also been engineered, for example, receptor ligands (i.e., IL-13 is engineered to bind to the IL-13 receptor expressed by tumors), intact immune receptors, library-derived peptides, and innate immune system effector molecules (e.g., NKG2D). Alternative cell targets for CAR expression (e.g., NK or gamma-delta T cells) are also under development (Brown CE et al. Clin Cancer Res. 2012; Vol. 18 (No. 8): pp. 2199-209; Lehner M et al. PLoS One. 2012; Vol. 7 (No. 2): e31210). Considerable effort is still required to define the most active T cell population for transduction of CAR vectors, determine optimal culture and proliferation techniques, and define the molecular details of the CAR protein structure itself.

[0010] The linking motif of the CAR can be designed to be a relatively stable structural domain, such as the constant domain of IgG, or to be an extended, flexible linker. Using a structural motif such as one derived from the constant domain of IgG, the scFv-binding domain can be extended away from the T cell membrane surface. This may be important for some tumor targets where the binding domain is particularly close to the tumor cell surface membrane (e.g., disialogangry). For Oside GD2 (Orentas et al., unpublished observations). To date, the signaling motifs used in CARs have always included the CD3-zeta chain because this core motif is a crucial signal for T cell activation. The first reported second-generation CARs featured a CD28 signaling domain and CD28 transmembrane sequence. This motif was similarly used in third-generation CARs containing the CD137(4-1BB) signaling motif (Zhao Y et al. J Immunol. 2009; Vol. 183 (No. 9): pp. 5563-74). With advances in new technologies, it is no longer necessary for T cell activation by beads linked to anti-CD3 and anti-CD28 antibodies, as well as the presence of a canonical "signal 2" derived from CD28, to be encoded by the CAR itself. Using bead activation, third-generation vectors were found not to be superior to second-generation vectors in in vitro assays, and no clear advantage over second-generation vectors was obtained in a mouse model of leukemia (Haso W, Lee DW, Shah NN, Stetler-Stevenson M, Yuan CM, Pastan IH, Dimitrov DS, Morgan RA, FitzGerald DJ, Barrett DM, Wayne AS, Mackall CL, Orentas RJ. Anti-CD22-chimeric antigen receptors targeting B cell precursor acute lymphoblastic leukemia, Blood. 2013; Vol. 121 (No. 7): pp. 1165-1174; Kochenderfer JN et al. Blood. 2012; Vol. 119 (No. 12): pp. 2709-2720). This is supported by the clinical success of CD19-specific CARs of the second-generation CD28 / CD3-zeta (Lee DW et al., American Society of Hematology Annual Meeting, New Orleans, LA; December 7-10, 2013) and CD137 / CD3-zeta signaling modes (Porter DL et al., N Engl J Med. 2011; Vol. 365 (No. 8): pp. 725-733).In addition to CD137, other tumor necrosis factor receptor superfamily members such as OX40 can also provide important sustained signaling in CAR-transduced T cells (Yvon E et al. Clin Cancer Res. 2009; Vol. 15 (No. 18): pp. 5852-5860). The culture conditions under which the CAR T cell population is cultured are equally important. [Overview of the project] [Problems that the invention aims to solve]

[0011] T cell-based immunotherapy is a new frontier in synthetic biology; multiple promoters and gene products are envisioned to guide these highly potent cells into the tumor microenvironment, where T cells can evade negative regulatory signals and mediate effective tumor death. Elimination of undesirable T cells via drug-induced dimerization of the inducible caspase 9 construct with AP1903 demonstrates one way in which a potent switch capable of controlling the T cell population can be pharmacologically initiated (Di Stasi A et al. N Engl). J Med. 2011; Vol. 365 (No. 18): pp. 1673-1673. The creation of effector T cell populations immune to the negative regulatory effects of transforming growth factor-β by decoy receptor expression further demonstrates the extent to which effector T cells can be manipulated for optimal antitumor activity (Foster AE et al. J Immunother. 2008; Vol. 31 (No. 5): pp. 500-555). Thus, CARs appear to be able to induce T cell activation in a manner similar to endogenous T cell receptors, but the major obstacles to the clinical application of this technology to date are the limited in vivo proliferation of CAR+ T cells, the rapid disappearance of cells after injection, and disappointing clinical activity. Therefore, there is an urgent and long-standing need in the field to discover novel compositions and methods for the treatment of AML using approaches that can exhibit specific and effective antitumor effects without the aforementioned drawbacks (i.e., high toxicity, insufficient efficacy). [Means for solving the problem]

[0012] The present invention addresses these needs by providing CAR compositions and therapeutic methods that can be used to treat cancer and other diseases and / or conditions. In particular, the present invention disclosed and described herein provides CARs that can be used to treat diseases, disorders or conditions associated with dysregulation of CD123 expression, wherein the CARs include a CD123 antigen-binding domain that exhibits high surface expression in transduced T cells and shows high levels of cytolysis as well as in vivo proliferation and persistence of transduced T cells.

[0013] overview Novel anti-CD123 antibodies or their antigen-binding domains, chimeric antigen receptors (CARs) containing such CD123 antigen-binding domains, host cells expressing the receptors (e.g., T cells), and nucleic acid molecules encoding the receptors are provided herein. The CARs may consist of a single molecule expressed on the surface of effector cells, or they may consist of an effector cell expression signaling module and a soluble targeting module, for example, when the soluble targeting module binds to the cell expression signaling module, forming a fully functional CAR. The CARs exhibit high surface expression on transduced T cells, high levels of cytolysis, and in vivo proliferation and persistence of transduced T cells. Methods using the disclosed CARs, host cells, and nucleic acid molecules for treating cancer in a subject are also provided.

[0014] Accordingly, in one embodiment, an isolated polynucleotide encoding a human anti-CD123 antibody or a fragment thereof is provided, comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 15, 17, 19, 23, 25, 69, 71, and 73.

[0015] In one embodiment, an isolated polynucleotide encoding a fully human anti-CD123 antibody or a fragment thereof is provided, wherein the antibody or fragment comprises a fragment selected from the group consisting of a Fab fragment, an F(ab')2 fragment, an Fv fragment, and a single-chain Fv(scFv).

[0016] In one embodiment, an isolated polynucleotide encoding a full human anti-CD123 antibody or a fragment thereof is provided, and the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 16, 18, 20, 22, 24, 26, 70, 72 and 74.

[0017] In one aspect, an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR) comprising at least one CD123 antigen-binding domain, at least one transmembrane domain, and at least one intracellular signaling domain encoded by a nucleotide sequence comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 15, 17, 19, 21, 23, 25, 69, 71 and 73 from the N-terminus to the C-terminus is provided.

[0018] 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.

[0019] 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.

[0020] In one embodiment, the targeting domain of the CAR is expressed separately in the form of a monoclonal antibody, ScFv Fab, Fab’2 and binds to an additional binding tag or epitope, and the antigen targeting domain comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 15, 17, 19, 21, 23, 25, 69, 71 and 73, and the effector cell expression component of the CAR is specifically directed to bind to a tag or epitope expressed on a soluble CAR module, such as forming a complete functional CAR structure by specifically binding the cell-binding component of the CAR on the soluble component of the CAR, and comprises a binding domain.

[0021] In another embodiment, the CAR targeting domain is expressed separately in the form of a monoclonal antibody, ScFv Fab, and Fab'2, and includes an antigen targeting domain comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 15, 17, 19, 21, 23, 25, 69, 71, and 73, as well as additional ScFvs, while the effector cell-expressed component of the CAR includes a tag or epitope that specifically reacts with additional ScFvs expressed on the soluble CAR module, such as by specific binding to the cell-binding component of the CAR on the soluble component of the CAR, thereby forming a complete functional CAR structure.

[0022] In yet another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded CAR extracellular CD123 antigen-binding domain further comprises at least one lipocalin-based antigen-binding antigen (anticalin) that binds to CD123.

[0023] In one embodiment, an isolated nucleic acid molecule is provided in which an encoded extracellular CD123 antigen-binding domain is connected to a transmembrane domain by a linker domain.

[0024] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded CD123 extracellular antigen-binding domain is preceded by a sequence encoding a leader or signal peptide.

[0025] In yet another embodiment, an isolated nucleic acid molecule is provided encoding a CAR comprising at least one CD123 antigen-binding domain encoded by a nucleotide sequence comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 15, 17, 19, 21, 23, 25, 69, 71, and 73, wherein the CAR further encodes an extracellular antigen-binding domain that targets antigens including, but not limited to, CD19, CD20, CD22, ROR1, mesoserine, 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.

[0026] In certain embodiments, further encoded extracellular antigen-binding domains include anti-CD19 scFV antigen-binding domains, anti-CD20 scFV antigen-binding domains, anti-CD22 scFV antigen-binding domains, anti-ROR1 scFV antigen-binding domains, anti-mesoserine scFV antigen-binding domains, anti-CD33 scFV antigen-binding domains, anti-CD38 scFV antigen-binding domains, anti-CD123(IL3RA) scFV antigen-binding domains, anti-CD138 scFV antigen-binding domains, anti-BCMA(CD269) scFV antigen-binding domains, anti-GPC2 scFV antigen-binding domains, anti-GPC3 scFV antigen-binding domains, anti-FGFR4 scFV antigen-binding domains, anti-c-Met scFV antigen-binding domains, anti-PMSA scFV antigen-binding domains, anti-glycolipid F77 scFV antigen-binding domains, anti-EGFRvIII scFV antigen-binding domains, and anti-GD-2 Isolated nucleic acid molecules encoding CARs are provided, comprising an scFV antigen-binding domain, an anti-NY-ESo-1 TCR scFV antigen-binding domain, an anti-MAGE A3 TCR scFV antigen-binding domain, or their amino acid sequences having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity, or any combination thereof.

[0027] In one embodiment, the CAR provided herein further comprises a linker or spacer domain.

[0028] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, in which an extracellular CD123 antigen-binding domain, an intracellular signaling domain, or both are connected to a transmembrane domain by a linker or spacer domain.

[0029] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded linker domain originates from the extracellular domain of CD8 or CD28 and is linked to a transmembrane domain.

[0030] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, further comprising a transmembrane domain containing a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154, or combinations thereof.

[0031] In yet another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded intracellular signaling domain further comprises a CD3 zeta intracellular domain.

[0032] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded intracellular signaling domain is located C-terminally relative to the CD3 zeta intracellular domain.

[0033] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded intracellular signaling domain comprises a co-stimulatory domain, a primary signaling domain, or a combination thereof.

[0034] In further embodiments, isolated nucleic acid molecules encoding CARs are provided, wherein at least one co-stimulatory domain to be encoded comprises the functional signaling domains of OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP12, and 4-1BB (CD137), or a combination thereof.

[0035] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, further comprising a leader sequence or a signal peptide, wherein the nucleotide sequence of the leader or signal peptide comprises the nucleotide sequence of SEQ ID NO: 13, SEQ ID NO: 39, SEQ ID NO: 41, or SEQ ID NO: 43.

[0036] In yet another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded leader sequence comprises the amino acid sequence of SEQ ID NO: 14, SEQ ID NO: 40, SEQ ID NO: 42, or SEQ ID NO: 44.

[0037] In one embodiment, a chimeric antigen receptor (CAR) is provided herein, comprising at least one CD123 antigen-binding domain, at least one transmembrane domain, and at least one intracellular signaling domain, from the N-terminus to the C-terminus.

[0038] In one embodiment, the extracellular CD123 antigen-binding domain is used to bind to an antigen with a small number of antibodies. A CAR is provided that includes at least one single-chain variable fragment, or at least one heavy-chain variable region of an antibody bound to an antigen, or a combination thereof.

[0039] In another embodiment, a CAR is provided in which at least one transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154, or a combination thereof.

[0040] In some embodiments, a CAR is provided which further encodes an extracellular antigen-binding domain comprising the amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, or any combination thereof.

[0041] In one embodiment, the extracellular antigen-binding domains include: anti-CD19 scFV antigen-binding domain, anti-CD20 scFV antigen-binding domain, anti-CD22 scFV antigen-binding domain, anti-ROR1 scFV antigen-binding domain, anti-mesoserine scFV antigen-binding domain, anti-CD33 scFV antigen-binding domain, anti-CD38 scFV antigen-binding domain, anti-CD123(IL3RA) scFV antigen-binding domain, anti-CD138 scFV antigen-binding domain, anti-BCMA(CD269) scFV antigen-binding domain, anti-GPC2 scFV antigen-binding domain, anti-GPC3 scFV antigen-binding domain, anti-FGFR4 scFV antigen-binding domain, anti-c-Met scFV antigen-binding domain, anti-PMSA scFV antigen-binding domain, anti-glycolipid F77 scFV antigen-binding domain, anti-EGFRvIII scFV antigen-binding domain, and anti-GD-2 A CAR is provided comprising an scFV antigen-binding domain, an anti-NY-ESo-1 TCR scFV antigen-binding domain, an anti-MAGE A3 TCR scFV antigen-binding domain, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity, or any combination thereof.

[0042] In another embodiment, the extracellular antigen-binding domains include: immunoglobulin variable heavy chain only (VH) anti-CD19 antigen-binding domain, anti-CD20 VH antigen-binding domain, anti-CD22 VH antigen-binding domain, anti-ROR1 VH antigen-binding domain, anti-mesoserine VH antigen-binding domain, anti-CD33 VH antigen-binding domain, anti-CD38 VH antigen-binding domain, anti-CD123 (IL3RA) VH antigen-binding domain, anti-CD138 VH antigen-binding domain, anti-BCMA (CD269) VH antigen-binding domain, anti-GPC2 VH antigen-binding domain, anti-GPC3 VH antigen-binding domain, anti-FGFR4 VH antigen-binding domain, anti-c-Met VH antigen-binding domain, anti-PMSA VH antigen-binding domain, anti-glycolipid F77 VH antigen-binding domain, anti-EGFRvIII VH antigen-binding domain, anti-GD-2 VH antigen-binding domain, and anti-NY-ESO-1 TCR. A CAR is provided that includes a VH antigen-binding domain, an anti-MAGE A3 TCR VH antigen-binding domain, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity, or any combination thereof.

[0043] In another embodiment, the extracellular antigen-binding domains include an anti-CD19 P antigen-binding domain, an anti-CD20 P antigen-binding domain, an anti-CD22 P antigen-binding domain, and an anti-ROR1 P antigen-binding domain, anti-mesoselin P antigen-binding domain, anti-CD33 P antigen-binding domain, anti-CD38 P antigen-binding domain, anti-CD123 (IL3RA) P antigen-binding domain A CAR is provided that includes a protein or peptide (P) sequence capable of specifically binding to a target antigen, which may be derived from a natural or synthetic sequence containing an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity, or any combination thereof. In another embodiment, a CAR is provided in which at least one intracellular signaling domain includes a co-stimulatory domain and a primary signaling domain.

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

[0045] In one embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 75. In another embodiment, the nucleic acid sequence encoding the CAR includes the amino acid sequence of SEQ ID NO: 76.

[0046] In another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 77. In one embodiment, the nucleic acid sequence encoding the CAR includes the amino acid sequence of SEQ ID NO: 78.

[0047] In another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 87. In one embodiment, the nucleic acid sequence encoding the CAR includes the amino acid sequence of SEQ ID NO: 88.

[0048] In another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 89. In one embodiment, the nucleic acid sequence encodes a CAR that includes the amino acid sequence of SEQ ID NO: 90.

[0049] In another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 91. In one embodiment, the nucleic acid sequence encodes a CAR that includes the amino acid sequence of SEQ ID NO: 92.

[0050] In another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 93. In one embodiment, the nucleic acid sequence encodes a CAR that includes the amino acid sequence of SEQ ID NO: 94.

[0051] In another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 95. In one embodiment, the nucleic acid sequence encodes a CAR that includes the amino acid sequence of SEQ ID NO: 96.

[0052] In another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 97. In one embodiment, the nucleic acid sequence encodes a CAR that includes the amino acid sequence of SEQ ID NO: 98.

[0053] In another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 99. In one embodiment, the nucleic acid sequence encodes a CAR that includes the amino acid sequence of SEQ ID NO: 100.

[0054] In another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 101. In one embodiment, the nucleic acid sequence encodes a CAR that includes the amino acid sequence of SEQ ID NO: 102.

[0055] In another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 103. In one embodiment, the nucleic acid sequence encodes a CAR that includes the amino acid sequence of SEQ ID NO: 104.

[0056] In another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 105. In one embodiment, the nucleic acid sequence encodes a CAR that includes the amino acid sequence of SEQ ID NO: 106.

[0057] In another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 107. In one embodiment, the nucleic acid sequence encodes a CAR that includes the amino acid sequence of SEQ ID NO: 108.

[0058] In another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 109. In one embodiment, the nucleic acid sequence encodes a CAR that includes the amino acid sequence of SEQ ID NO: 110.

[0059] In another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 111. In one embodiment, the nucleic acid sequence encodes a CAR that includes the amino acid sequence of SEQ ID NO: 112.

[0060] In one embodiment, the CAR disclosed herein is modified to express or contain a detectable marker for use in diagnosis, monitoring and / or prediction of treatment outcomes such as progression-free survival in cancer patients, or for monitoring the progression of such treatment.

[0061] In one embodiment, the disclosed nucleic acid molecule encoding the CAR may be contained in a vector such as a viral vector. The vector may be a DNA vector, RNA vector, plasmid vector, cosmid vector, herpesvirus vector, measles virus vector, lentivirus vector, adenovirus vector, or retrovirus vector, or a combination thereof.

[0062] In certain embodiments, the vector further includes a promoter which is an inducible promoter, a tissue-specific promoter, a constitutive promoter, a suicide promoter, or any combination thereof.

[0063] In yet another embodiment, the vector expressing CAR may be further modified to include one or more activatable elements to control the expression of CAR T cells or to eliminate CAR-T cells by a suicide switch. The suicide switch may include, for example, an apoptosis-inducible signaling cascade or a drug that induces cell death. In a preferred embodiment, the vector expressing CAR may be further modified to express an enzyme such as thymidine kinase (TK) or cytosine deaminase (CD).

[0064] In another embodiment, a host cell containing a nucleic acid molecule encoding a CAR is also provided. In some embodiments, the host cell is a T cell, for example, a primary T cell obtained from a subject. In one embodiment, the host cell is a CD8+ T cell.

[0065] In yet another embodiment, a pharmaceutical composition comprising an antitumor-effective population of human T cells, wherein the T cells comprise nucleic acid sequences encoding chimeric antigen receptors (CARs), the CARs being sequence numbers 2, 4, 6, 8, 10, 12, 16, 18, 20, 22, 24, 26, 70, 72, and 74 A pharmaceutical composition is provided comprising at least one extracellular antigen-binding domain containing a CD123 antigen-binding domain having an amino acid sequence selected from the group consisting of, at least one linker domain, at least one transmembrane domain, and at least one intracellular signaling domain, wherein the T cell is a human T cell having cancer. Cancer includes, in particular, hematological cancers, e.g., leukemia (e.g., chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), or chronic myeloid leukemia (CML)), lymphoma (e.g., mantle cell lymphoma, non-Hodgkin lymphoma, or Hodgkin lymphoma), or multiple myeloma, or a combination thereof.

[0066] In one embodiment, a pharmaceutical composition is provided in which at least one transmembrane domain of CAR comprises a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, mesoserine, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154, or a combination thereof.

[0067] In another embodiment, a pharmaceutical composition is provided that includes human cancers, including oral and pharyngeal cancers (tongue, mouth, pharynx, head and neck), gastrointestinal cancers (esophagus, stomach, small intestine, colon, rectum, anus, liver, intrahepatic bile duct, gallbladder, pancreas), respiratory cancers (larynx, lung, and bronchi), bone and joint cancers, soft tissue cancers, skin cancers (melanoma, basal cell carcinoma, and squamous cell carcinoma), pediatric tumors (neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma), central nervous system tumors (brain tumors, astrocytoma, glioblastoma, glioma), as well as adult cancers, including breast, reproductive system (cervix, uterine body, ovaries, vulva, vagina, prostate, testes, penis, endometrium), urinary system (bladder, kidneys and renal pelvis, ureters), eye and orbit, endocrine system (thyroid), and brain and other nervous system cancers, or any combination thereof.

[0068] In yet another embodiment, a pharmaceutical composition is provided comprising an antitumor effective amount of a population of human T cells of a person having cancer, wherein the cancer is a refractory cancer unresponsive to one or more chemotherapeutic agents. The cancer includes hematopoietic cancer, myelodysplastic syndrome, pancreatic cancer, head and neck cancer, skin tumors, adult B-cell malignancies including acute lymphoblastic leukemia (ALL), minimal residual disease (MRD) in acute myeloid leukemia (AML), CLL (chronic lymphocytic leukemia), CML (chronic myeloid leukemia), non-Hodgkin lymphoma (NHL), pediatric B-cell malignancies (including B-cell lineage ALL (acute lymphocytic leukemia)), multiple myeloma, lung cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, melanoma, or other hematological cancers and solid tumors, or any combination thereof.

[0069] In another embodiment, a method for producing CAR-containing T cells (hereinafter referred to as "CAR T cells") is provided. This method includes transduction of T cells with a disclosed CAR-encoding vector or nucleic acid molecule that specifically binds to CD123, thereby producing CAR T cells.

[0070] In yet another embodiment, a method is provided for generating a population of RNA-modified cells, comprising introducing in vitro transcribed RNA or synthetic RNA of a disclosed CAR-encoding nucleic acid molecule into target cells to generate CAR cells.

[0071] In yet another embodiment, a method for diagnosing a disease, disorder or condition associated with CD123 expression in cells, comprising: a) contacting cells with a human anti-CD123 antibody or a fragment thereof, wherein the antibody or fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 16, 18, 20, 22, 24, 26, 70, 72 and 74; and b) detecting the presence of CD123, C A method is provided which includes the step of diagnosing a disease, disorder, or condition associated with CD123 expression based on the presence of D123.

[0072] In one embodiment, the diseases, disorders, or conditions associated with CD123 expression include cancers such as hematopoietic cancers, myelodysplastic syndromes, pancreatic cancers, head and neck cancers, skin tumors, adult B-cell malignancies including acute lymphoblastic leukemia (ALL), minimal residual disease (MRD) in acute myeloid leukemia (AML), CLL (chronic lymphocytic leukemia), CML (chronic myeloid leukemia), and non-Hodgkin lymphoma (NHL), pediatric B-cell malignancies (including B-cell lineage ALL (acute lymphocytic leukemia)), multiple myeloma, lung cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, melanoma, or other hematological cancers and solid tumors, or any combination thereof.

[0073] In another embodiment, a method is provided for diagnosing, prognosing or determining the risk of CD123-associated disease in mammals, comprising the steps of: a) contacting a sample with a human anti-CD123 antibody or a fragment thereof, wherein the antibody or fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 16, 18, 20, 22, 24, 26, 70, 72, and 74; and b) detecting the presence of CD123, wherein the presence of CD123 diagnoses a CD123-associated disease in mammals, the method comprising detecting CD123 expression in a mammalian-derived sample.

[0074] In another embodiment, a method is provided for inhibiting CD123-dependent T cell inhibition, comprising the step of contacting cells with a human anti-CD123 antibody or a fragment thereof, wherein the antibody or fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 16, 18, 20, 22, 24, 26, 70, 72, and 74. In one embodiment, the cells are selected from the group consisting of tumor cells expressing CD123, tumor-associated macrophages, and any combination thereof.

[0075] In another embodiment, a method is provided for altering the tumor microenvironment to block T cell inhibition mediated by cells expressing CD123 and inhibit tumor growth in a mammal, comprising the step of administering an effective amount of a composition comprising an isolated anti-CD123 antibody or a fragment thereof to a mammal, wherein the antibody or fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 16, 18, 20, 22, 24, 26, 70, 72, and 74. In one embodiment, the cells are selected from the group consisting of tumor cells expressing CD123, tumor-associated macrophages, and any combination thereof.

[0076] In another embodiment, a method is provided for inhibiting, suppressing or preventing immunosuppression of an antitumor or anticancer immune response in a mammal, comprising the step of administering to a mammal an effective amount of a composition comprising an isolated anti-CD123 antibody or a fragment thereof, wherein the antibody or fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 16, 18, 20, 22, 24, 26, 70, 72, and 74. In one embodiment, the antibody or fragment inhibits the interaction between a first cell and a T cell, the first cell being selected from the group consisting of tumor cells expressing CD123, tumor-associated macrophages, and any combination thereof.

[0077] In another embodiment, a method is provided for inducing antitumor immunity in a mammal, comprising the step of administering a therapeutically effective amount of T cells transduced with a vector or nucleic acid molecule encoding a disclosed CAR to the mammal.

[0078] In another embodiment, a method for treating or preventing cancer in a mammal, one A method is provided which includes the step of administering a plurality of disclosed CARs to a mammal in an amount effective to treat or prevent cancer in the mammal. The method includes administering a therapeutically effective amount of host cells expressing a disclosed CAR that specifically binds to CD123 and / or one or more of the aforementioned antigens, under conditions sufficient to form an immune complex between the antigen-binding domain of the CAR and the extracellular domain of CD123 and / or one or more of the aforementioned antigens, in the subject.

[0079] In yet another embodiment, a method is provided for treating a mammal having a disease, disorder or condition associated with elevated expression of a tumor antigen, comprising the step of administering a pharmaceutical composition comprising an antitumor-effective amount of a population of T cells to a subject, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), the CAR comprising at least one extracellular CD123 antigen-binding domain, at least one linker or spacer domain, at least one transmembrane domain, and at least one intracellular signaling domain, comprising the amino acid sequence of SEQ ID NOs. 2, 4, 6, 8, 10, 12, 16, 18, 20, 22, 24, 26, 70, 72, or 74, or any combination thereof, and the T cells are T cells of a subject having cancer.

[0080] In yet another embodiment, a method is provided for treating cancer in a subject requiring it, comprising the step of administering to the subject a pharmaceutical composition comprising an antitumor-effective amount of a population of T cells, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), and the CAR comprises at least one CD123 antigen-binding domain, at least one linker or spacer domain, at least one transmembrane domain, and at least one intracellular signaling domain, comprising the amino acid sequence of SEQ ID NOs. 2, 4, 6, 8, 10, 12, 16, 18, 20, 22, 24, 26, 70, 72, or 74, or any combination thereof, and the T cells are T cells of a subject having cancer. In some embodiments of the method described above, at least one transmembrane domain includes the transmembrane alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, mesoserine, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154, or a combination thereof.

[0081] In yet another embodiment, a method is provided for generating a persistent population of genetically engineered T cells in a person diagnosed with cancer. In one embodiment, the method includes the step of administering T cells genetically engineered to express a CAR to a human, wherein the CAR comprises at least one CD123 antigen-binding domain including the amino acid sequence of SEQ ID NOs. 2, 4, 6, 8, 10, 12, 16, 18, 20, 22, 24, 26, 70, 72, or 74, or any combination thereof, at least one transmembrane domain, and at least one intracellular signaling domain, and the persistent population of genetically engineered T cells, or population of T cell offspring, persists in the human for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, or 3 years after administration.

[0082] In one embodiment, the descendant T cells in humans include memory T cells. In another embodiment, the T cells are autologous T cells.

[0083] 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 may be treated, prevented, or remitted using one or more of the CARs disclosed herein.

[0084] In yet another embodiment, a kit for producing the above-mentioned chimeric antigen receptor T cells, or in the above-mentioned subject, cancer, disease, disorder or condition associated with elevated expression of tumor antigens A kit is provided for preventing, treating or relieving any of the following, comprising a container containing one or any combination thereof of the nucleic acid molecules, vectors, host cells, or compositions disclosed above, and instructions for using the kit.

[0085] It is understood that CARs, host cells, nucleic acids, and methods are useful beyond the specific embodiments and models described in detail herein. The aforementioned features and advantages of this disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying drawings. [Brief explanation of the drawing]

[0086]

Figure 1

Figure 2

Figure 3

Figure 4

[0087] Detailed explanation definition As used herein, the singular forms “a,” “an,” and “the” refer to both the singular and plural forms unless the context clearly indicates otherwise. For example, the term “one antigen” may include one or more antigens and may be considered equivalent to the phrase “at least one antigen.” As used herein, the term “comprises” means “includes.” Therefore, “comprising one antigen” means “including one antigen” without excluding other elements. The phrase “and / or” means “and” or “or.” Any and all base sizes or amino acid sizes, as well as all molecular weight or molecular mass values ​​given for nucleic acids or polypeptides, should be understood to be approximate and provided for convenience unless otherwise noted. Many methods and materials similar or equivalent to those described herein may be used, but particularly suitable methods and materials are described below. In case of any conflict, this specification, including the explanation of terms, shall prevail. Furthermore, the materials, methods, and examples are illustrative and not intended to be limiting. To facilitate further examination of each embodiment, the following definitions of terms are provided.

[0088] The term "approximately" means, when referring to measurable values ​​such as quantity or temporal duration, to include variations of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% from the specified value, such variations being appropriate for carrying out the disclosed method.

[0089] Unless otherwise specified, technical terms in this specification shall be used in accordance with their conventional usage. Definitions of general terms in molecular biology are found in Benjamin Lewin, Genes VII, 1999, published by Oxford University Press; Kendrew et al., The Encyclopedia of Molecular Biology, 1994, published by Blackwell Science Ltd.; and Robert, published by VCH Publishers, Inc. A. Meyers (ed.), Molecular Biology and Biotechnology: A Comprehensive Desk Reference, 1995; and other similar references can be found.

[0090] This disclosure provides CD123 antibodies or fragments thereof, and chimeric antigen receptors (CARs) having such CD123 antigen-binding domains. Enhancement of the functional activity of CARs is directly related to the enhancement of the functional activity of T cells expressing CARs. As a result of one or more of these modifications, CARs exhibit both high cytokine-induced cytolysis and cell surface expression in transduced T cells, along with increased T cell proliferation and sustained levels in vivo of transduced CAR-expressing T cells.

[0091] The unique ability to combine functional parts derived from various protein domains is a key and innovative feature of chimeric antigen receptors (CARs). The selection of each of these protein domains, as well as their specific combinations, are important design features. Each design domain is an essential component that can be used to manipulate lymphocyte function in various CAR platforms. For example, the selection of an extracellular binding domain can enable CARs that would otherwise be ineffective.

[0092] The immutable framework components of immunoglobulin-derived protein sequences used to create the extracellular antigen-binding domain of CARs can be completely neutral or self-associate, leading to metabolic exhaustion of T cells, thus rendering therapeutic T cells expressing this CAR extremely ineffective. This occurs independently of the antigen-binding function of the CAR domain. Furthermore, the selection of intracellular signaling domains (multiple) can also determine the activity and durability of therapeutic lymphocyte populations used in immunotherapy. While the ability to bind to target antigens and transmit activation signals to T cells, respectively, are important aspects of CAR design, it has also become clear that the selection of the source of the extracellular antigen-binding fragment has a significant effect on the efficacy of CARs and may therefore play a decisive role in their function and clinical utility.

[0093] Surprisingly and unexpectedly, it was discovered here that the functional activity of T cells expressing a CAR can also be determined by using a fully human antigen-binding domain in the CAR, rather than using a mouse-derived antigen-binding fragment (see UPenn-funded clinical trial NCT02159716, which uses a mouse-derived SS1 ScFv sequence), which tends to induce an anti-mouse immune response and CAR T elimination in the host.

[0094] The CARs disclosed herein are expressed at high levels in cells. Cells expressing CARs have a high proliferation rate in vivo, produce large amounts of cytokines, and CAR It exhibits high cytotoxic activity against cells having the CD123 antigen on its surface to which it binds. The use of the human extracellular CD123 antigen-binding domain leads to the generation of CARs that function better in vivo, while avoiding the induction of anti-CAR immunity and death of CAR T cell populations in the host immune response. CARs expressing the fully human extracellular CD123 ScFv antigen-binding domain exhibit superior activity / properties, including i) prevention of CAR T persistence and dysfunction seen in mouse-derived binding sequences; ii) absence of local (i.e., intrapleural) delivery of the CAR that should be effective; and iii) the ability to generate CAR T cell designs based on both high-affinity and low-affinity binders for CD123. This last property allows researchers to better tune the efficacy against toxicity and / or tissue specificity of CAR T products, as tumors express more CD123 than normal tissues, and binders with lower affinity can have greater specificity to tumors than normal tissues, thereby preventing toxicity to non-tumor cells and death of bystander cells.

[0095] A detailed description of the CARs of the present invention, including a description of their extracellular CD123 antigen-binding domain, transmembrane domain, and intracellular domain, is provided below, along with further descriptions of CARs, antibodies and their antigen-binding fragments, conjugates, nucleotides, expression, vectors and host cells, methods of treatment using the disclosed CARs, compositions and kits.

[0096] A. Chimeric antigen receptor (CAR) The CARs disclosed herein include at least one CD123 antigen-binding domain capable of binding to CD123, at least one transmembrane domain, and at least one intracellular domain.

[0097] Chimeric antigen receptors (CARs) are artificially constructed hybrid proteins or polypeptides containing an antigen-binding domain of an antibody (e.g., a single-chain variable fragment (scFv)) linked to a T cell signaling domain via a transmembrane domain. Features of CARs include their ability to redirect the specificity and responsiveness of T cells toward a selected target by leveraging the antigen-binding properties of monoclonal antibodies in a non-MHC-restricted manner. Non-MHC-restricted antigen recognition gives T cells expressing CARs the ability to recognize antigens independently of antigen processing, thus bypassing the primary mechanism of tumor escape. Furthermore, when expressed in T cells, CARs advantageously do not dimerize with the alpha and beta chains of the endogenous T cell receptor (TCR).

[0098] 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 costimulatory signaling domain, or both. A T cell receptor signaling domain refers to a portion of a CAR that includes the intracellular domain of a T cell receptor, for example, the intracellular portion of the CD3 zeta protein, but not limited to that. A costimulatory signaling domain refers to a portion of a CAR that includes the intracellular domain of a costimulatory molecule, which is a cell surface molecule other than the antigen receptor or its ligand, required for an efficient lymphocyte response to an antigen.

[0099] 1. Extracellular domain In one embodiment, the CAR includes a target-specific binding element, otherwise called an antigen-binding domain or subdomain. The choice of domain depends on the type and number of ligands that define the surface of the target cell. For example, the antigen-binding domain may be selected to recognize ligands that act as cell surface markers on target cells associated with a particular disease state. Thus, examples of cell surface markers that can act as ligands for the antigen-binding domain in the CAR include those associated with viruses, bacterial and parasitic infections, autoimmune diseases, and cancer cells.

[0100] In one embodiment, a CAR may be engineered to target a desired tumor antigen by manipulating a desired antigen-binding domain that specifically binds to the antigen on tumor cells. The tumor antigen is a protein produced by tumor cells that elicits an immune response, particularly a T-cell-mediated immune response. The selection of the antigen-binding domain depends on the specific type of cancer being treated. Tumor antigens include, for example, glioma-associated antigens, carcinoembryonic antigens (CEAs), beta-human chorionic gonadotropins, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, Her2 / neu, survivorbin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin-like growth factor (IGF)-I, IGF-II, IGF-I receptor, and CD123. The tumor antigens disclosed herein are for illustrative purposes only. This list is not intended to be exclusive, and further examples will be readily apparent to those skilled in the art.

[0101] In one embodiment, a tumor antigen comprises one or more antigenic cancer epitopes associated with a malignant tumor. Malignant tumors express several proteins that can function as target antigens for immune attack. These molecules include, but are not limited to, tissue-specific antigens, e.g., MART-1, tyrosinase, and GP 100 in melanoma, and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to a group of transformation-related molecules, such as the oncogene HER-2 / Neu / ErbB-2. Yet another group of target antigens are carcinoembryonic antigens, such as carcinoembryonic antigens (CEAs). In B-cell lymphoma, tumor-specific idiotype immunoglobulins constitute truly tumor-specific immunoglobulin antigens unique to individual tumors. B-cell differentiation antigens, e.g., CD19, CD20, and CD37, are other candidate target antigens in B-cell lymphoma. Some of these antigens (CEA, HER-2, CD19, CD20, idiotypes) have been used as targets for passive immunotherapy with monoclonal antibodies, with limited success.

[0102] In one preferred embodiment, the tumor antigen is CD123, and tumors associated with CD123 expression include pulmonary mesothelioma, ovarian and pancreatic cancers, or any combination thereof, that express high levels of the extracellular protein CD123.

[0103] Tumor antigens can be either tumor-specific antigens (TSAs) or tumor-associated antigens (TAAs). TSAs are unique to tumor cells and are not present on other cells throughout the body. TAAs are not unique to tumor cells and, instead, are expressed on normal cells under conditions that do not induce a state of immunological tolerance to the antigen. Antigen expression on tumors can occur under conditions that allow the immune system to respond to the antigen. TAAs may be antigens expressed on normal cells during fetal development when the immune system is immature and unresponsive, or they may be antigens that are normally present at very low levels on normal cells but expressed at considerably high levels on tumor cells.

[0104] Non-limiting examples of TSA or TAA include: differentiation antigens, e.g., MART-1 / MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2 and tumor-specific multiseries antigens, e.g., MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens, e.g., CEA; overexpressed oncogenes and mutated tumor suppressor genes, e.g., p53, Ras, HER-2 / neu; unique tumor antigens arising from chromosomal translocations; e.g., BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as Epstein-Barr virus antigen (EBVA) and human papillomavirus (HPV) antigens E6 and E7. Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3 / CA 27.29 / BCAA, CA 195, CA This includes 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 / cyclophyllin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0105] In one embodiment, the antigen-binding domain of the CAR targets antigens including, but not limited to, CD19, CD20, CD22, ROR1, CD123, CD33, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, MY-ESO-1 TCR, and MAGE A3 TCR.

[0106] In a preferred embodiment, the antigen-binding domain of the CAR targets the extracellular CD123 antigen.

[0107] In one preferred embodiment, an isolated nucleic acid molecule encoding the extracellular CD123 hScFv M12301 antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 1, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided in which the encoded extracellular CD123 hScFv M12301 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 2.

[0108] In one preferred embodiment, an isolated nucleic acid molecule encoding the extracellular CD123 hScFv M12303 antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 3, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided in which the encoded extracellular CD123 hScFv M12303 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 4.

[0109] In one preferred embodiment, an isolated nucleic acid molecule encoding the extracellular CD123 hScFv M12304 antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 5, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided in which the encoded extracellular CD123 hScFv M12304 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 6.

[0110] In a preferred embodiment, an isolated nucleic acid molecule encoding the extracellular CD123 hScFv M12305 antigen-binding domain has 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the nucleotide sequence of SEQ ID NO: 7. The sequence is included. In one embodiment, an isolated nucleic acid molecule is provided in which the encoded extracellular CD123 hScFvM12305 antigen-binding domain includes the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 8.

[0111] In one preferred embodiment, an isolated nucleic acid molecule encoding the extracellular CD123 hScFv M12306 antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 9, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided in which the encoded extracellular CD123 hScFv M12306 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 10.

[0112] In one preferred embodiment, an isolated nucleic acid molecule encoding the extracellular CD123 hScFv M12308 antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 11, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided in which the encoded extracellular CD123 hScFv M12308 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 12.

[0113] In one preferred embodiment, an isolated nucleic acid molecule encoding the extracellular CD123 hScFv M12309 antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 15, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided in which the encoded extracellular CD123 hScFv M12309 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 16.

[0114] In one preferred embodiment, an isolated nucleic acid molecule encoding the extracellular CD123 hScFv M12310 antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 17, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided in which the encoded extracellular CD123 hScFv M12310 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 18, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 18.

[0115] In one preferred embodiment, an isolated nucleic acid molecule encoding the extracellular CD123 hScFv M12311 antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 19, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided in which the encoded extracellular CD123 hScFv M12311 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 20, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 20.

[0116] In one preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD123 hScFv M12313 antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 21, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity. In one embodiment, the encoded extracellular CD123 hScFv M12313 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 22, or the amino acid sequence of SEQ ID NO: 22. Isolated nucleic acid molecules are provided, containing amino acid sequences that have 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the no-acid sequence.

[0117] In one preferred embodiment, an isolated nucleic acid molecule encoding the extracellular CD123 hScFv M12314 antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 23, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided in which the encoded extracellular CD123 hScFv M12314 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 24.

[0118] In one preferred embodiment, an isolated nucleic acid molecule encoding the extracellular CD123 hScFv M12315 antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 25, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided in which the encoded extracellular CD123 hScFv M12315 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 26, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 26.

[0119] In one preferred embodiment, an isolated nucleic acid molecule encoding the extracellular CD123 hScFv M12316 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 in which the encoded extracellular CD123 hScFv M12316 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 to the amino acid sequence of SEQ ID NO: 70.

[0120] In one preferred embodiment, an isolated nucleic acid molecule encoding the extracellular CD123 hScFv M12317 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 in which the encoded extracellular CD123 hScFv M12317 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 to the amino acid sequence of SEQ ID NO: 72.

[0121] In one preferred embodiment, an isolated nucleic acid molecule encoding the extracellular CD123 hScFv M12318 antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 73, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided in which the encoded extracellular CD123 hScFv M12318 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 74, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 74.

[0122] Example 1 illustrates the characteristics of generating and binding only the specific CD123 variable heavy chain described herein, and the ScFv antigen-binding fragment or antigen binder.

[0123] In various embodiments of the CD123-specific CARs disclosed herein, a general schematic is shown in Figure 1, comprising, from the N-terminus to the C-terminus, a signal or leader peptide, an anti-CD123 ScFv, an extracellular linker, a CD8 transmembrane region, 4-1BB, and a CD3 zeta, with bold text indicating the cloning site of the linking domain.

[0124] In one embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 75 and encodes a CAR that includes the amino acid sequence shown in SEQ ID NO: 76.

[0125] In one embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 75, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereof, and encodes a CAR including the amino acid sequence shown in SEQ ID NO: 76, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereof.

[0126] In another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 77 and encodes a CAR that includes the amino acid sequence shown in SEQ ID NO: 78.

[0127] In another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 77, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereof, and encodes a CAR including the amino acid sequence shown in SEQ ID NO: 78, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereof.

[0128] In another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 87 and encodes a CAR that includes the amino acid sequence shown in SEQ ID NO: 88.

[0129] In another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 87, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereof, and encodes a CAR including the amino acid sequence shown in SEQ ID NO: 88, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereof.

[0130] In yet another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 89 and encodes a CAR that includes the amino acid sequence shown in SEQ ID NO: 90.

[0131] In yet another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 89, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereof, and encodes a CAR including the amino acid sequence shown in SEQ ID NO: 90, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereof.

[0132] In yet another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 91 and encodes a CAR that includes the amino acid sequence shown in SEQ ID NO: 92.

[0133] In yet another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 91, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereof, and encodes a CAR including the amino acid sequence shown in SEQ ID NO: 92, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereof.

[0134] In yet another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 93 and encodes a CAR that includes the amino acid sequence shown in SEQ ID NO: 94.

[0135] In yet another embodiment, the nucleic acid sequence encoding CAR is the nucleic acid sequence of SEQ ID NO: 93 It encodes a CAR containing the amino acid sequence shown in SEQ ID NO: 94, or the sequence containing the amino acid sequence showing

[0136] In yet another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 95 and encodes a CAR that includes the amino acid sequence shown in SEQ ID NO: 96.

[0137] In yet another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 95, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereof, and encodes a CAR including the amino acid sequence shown in SEQ ID NO: 96, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereof.

[0138] In yet another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 97 and encodes a CAR that includes the amino acid sequence shown in SEQ ID NO: 98.

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

[0140] In yet another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 99 and encodes a CAR that includes the amino acid sequence shown in SEQ ID NO: 100.

[0141] In yet another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 99, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereof, and encodes a CAR including the amino acid sequence shown in SEQ ID NO: 100, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereof.

[0142] In yet another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 101 and encodes a CAR that includes the amino acid sequence shown in SEQ ID NO: 102.

[0143] In yet another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 101, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereof, and encodes a CAR including the amino acid sequence shown in SEQ ID NO: 102, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereof.

[0144] In yet another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 103 and encodes a CAR that includes the amino acid sequence shown in SEQ ID NO: 104.

[0145] In yet another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 103, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereof, and encodes a CAR including the amino acid sequence shown in SEQ ID NO: 104, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereof.

[0146] In yet another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 105 and encodes a CAR that includes the amino acid sequence shown in SEQ ID NO: 106.

[0147] In yet another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 105, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereof, and encodes a CAR including the amino acid sequence shown in SEQ ID NO: 106, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereof.

[0148] In yet another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 107 and encodes a CAR that includes the amino acid sequence shown in SEQ ID NO: 108.

[0149] In yet another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 107, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereof, and encodes a CAR including the amino acid sequence shown in SEQ ID NO: 108, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereof.

[0150] In yet another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 109 and encodes a CAR that includes the amino acid sequence shown in SEQ ID NO: 110.

[0151] In yet another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 109, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereof, and encodes a CAR including the amino acid sequence shown in SEQ ID NO: 110, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereof.

[0152] In yet another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 111 and encodes a CAR that includes the amino acid sequence shown in SEQ ID NO: 112.

[0153] In yet another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 111, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereof, and encodes a CAR including the amino acid sequence shown in SEQ ID NO: 112, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereof.

[0154] Surface expression of anti-CD123 CARs incorporating immunoglobulin heavy chain variable domains (VH) and single-chain fragment variable (ScFv) sequences that react with the CD123 antigen is shown in Example 2 below. Expression levels for each CAR containing ScFv or VH were determined by flow cytometry analysis of LV transducible T cells from healthy donors using recombinant CD123-Fc peptide followed by an anti-human Fc F(ab')2 fragment conjugated with AF647, and detected by APC channels (see Example 2, Figure 3). VH-based anti-CD123 CAR constructs 1905 and 1906 (black lines) were readily detected on the surface of T cells from two donors, demonstrating the reproducibility of T cell transduction. In contrast, CAR expression was not detected in negative control non-transducible T cells (gray lines) and GFP control (not shown), demonstrating the specificity of the detection method used (see Example 2, Figure 3). Similarly, ScFv-based anti-CD123 CAR constructs 1936, 1937, 1938, and 1939 were highly expressed in human primary T cells (black line) compared to untransduced T cell controls (gray line). Representative results from one donor are shown.

[0155] As shown in Example 2, lentiviral vectors (LVs) expressing the following CARs were created and tested for anti-leukemia activity, demonstrating the high cytolytic activity of the CD123 CAR. Each experimental CAR contains a CD8 hinge and transmembrane domain, a 4-1BB costimulatory domain and a CD3-zeta chain signaling domain, and a specific anti-CD123 ScFv-derived targeting domain as noted therein (Figure 1). To generate CAR T cells, lentiviral vector preparations encoding each of the novel anti-CD123 CAR constructs were created and used to transduce primary human T cells. CAR expression was determined by flow cytometry using Protein L (Figure 2). CAR constructs LTG2075, LTG2076, LTG2078, LTG2079, and LTG2088 demonstrated high CAR surface expression in the range of 35%–55% CAR+ T cells (Figure 2). Since protein L is only suitable for detecting ScFv sequences composed of kappa light chains, some sequences composed of lambda light chains that were not detected may nevertheless be expressed in CAR T cells. The potential cytotoxicity of CAR123 candidates was evaluated using a luciferase-based overnight cell death assay with either the CD123+ AML target strain MOLM-14 or the CD33- control strain 293T (Figure 3). CAR T cells (effectors) and tumor cells (targets) were co-incubated overnight at effector-to-target ratios (E:T) of 5, 10, or 20. CAR123 constructs LTG2075, LTG2076, LTG2077, LTG2078, LTG2079, LTG2082, LTG2083, LTG2085, LTG2087, and LTG2088 demonstrated potent, dose-dependent cytotoxicity against CD123+MOLM-14 target cells (Figure 3). Furthermore, negative controls consisting of non-transduced T cells (UTDs) or GFP-transduced T cells (LTG1398) did not exhibit cytotoxicity, confirming that cell death was CAR-specific (Figure 3). In comparison, no cell death activity was observed against control CD123-293 T cells, demonstrating that the cytotoxic function of CAR123 cells is target-specific (Figure 3).Therefore, the cytolytic activity of anti-CD123 CAR observed against CD123-expressing MOLM-14 tumor cells is target-specific and CAR-dependent.

[0156] Next, the ability of anti-CD123 CAR T cells to secrete cytokines was evaluated. CD123+ AML tumor cell lines MOLM-14 or Kg-1a were co-incubated overnight with CAR T cells or control T cells in an effector-to-target ratio of 10:1, and the culture supernatant was analyzed by ELISA for IFN gamma and TNF alpha (Figure 4). Notably, CAR T-expressing cells LTG2076, LTG2078, and LTG2088 produced high levels of IFN gamma and TNF alpha, while most other CAR constructs, and the negative control groups NT and 1398, did not produce any noticeable cytokine induction. These results are in contrast to the potent in vitro cytolytic function of LTG2075, LTG2077, LTG2082, LTG2083, LTG2085, and LTG2087 (see Figure 3), suggesting that cytotoxicity does not always correlate with the cytokine secretion profile and that multiple CAR T functional endpoints should be tested against each construct. This finding also suggests that it may be possible to select CAR123 constructs that efficiently kill tumors, have a lower risk of inducing cytokine release syndrome, and therefore have a better safety profile.

[0157] While not intended to limit to any particular mechanism of action, possible reasons for the enhanced therapeutic function associated with the exemplary CARs of the present invention include, for example, a) improved lateral movement within the plasma membrane, enabling more efficient signal transduction; b) superior location within plasma membrane microdomains such as lipid rafts, and a higher ability to interact with transmembrane signaling cascades associated with T cell activation; and c) superior location within the plasma membrane due to preferential movement away from repressive or downregulatory interactions, such as CD45. This is thought to include being less in close proximity to sphatase, or having less interaction with it, as well as d) having better assembly to the T cell receptor signaling complex (i.e., the immune synapse), or any combination thereof.

[0158] While this disclosure exemplifies the use of only the exemplary extracellular CD123 variable heavy chain and the ScFv antigen-binding domain, other nucleotides and / or amino acid variants within the CD123 variable heavy chain and the ScFv antigen-binding domain may also be used to obtain the CD123 antigen-binding domain for use in the CARs described herein.

[0159] Depending on the desired antigen to be targeted, the CAR may be further manipulated to include an appropriate antigen-binding domain specific to the desired antigen target. For example, if CD19 is the desired antigen to be targeted, an antibody against CD19 may be used to incorporate the antigen-binding domain into the CAR.

[0160] In one exemplary embodiment, the antigen-binding domain portion of the CAR further targets CD19. Preferably, the antigen-binding domain in the CAR is an anti-CD19 scFV, where the nucleic acid sequence of the anti-CD19 scFV includes the sequence shown in SEQ ID NO: 37. In one embodiment, the anti-CD19 scFV includes a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 30. In another embodiment, the anti-CD19 scFV portion of the CAR includes the amino acid sequence shown in SEQ ID NO: 38.

[0161] In one aspect of the present invention, a non-TSA or non-TAA CAR is provided that contains, for example, but is not limited to, a retroviridae (e.g., human immunodeficiency virus, e.g., HIV-1 and HIV-LP), picornaviridae (e.g., poliovirus, hepatitis A virus, enterovirus, human coxsackievirus, rhinovirus and echovirus), rubella virus, coronavirus, varicella stomatitis virus, rabies virus, Ebola virus, parainfluenza virus, mumps virus, measles virus, polynuclear respiratory virus, influenza virus, hepatitis B virus, parvovirus, adenoviridae, herpesviridae [e.g., herpes simplex virus type 1 and 2 (HSV), varicella-zoster virus, cytomegalovirus (CMV), and herpesviruses], poxviridae (e.g., smallpox virus, vaccinia virus and poxvirus), or hepatitis C virus antigens, or any combination thereof.

[0162] In another aspect of the present invention, CARs capable of binding to antigens derived from bacterial strains of Staphylococci, Streptococcus, Escherichia coli, Pseudomonas, or Salmonella are provided. In particular, CARs capable of binding to antigens derived from infectious bacteria, such as Helicobacter pyloris, Legionella pneumophilia, Mycobacteria sps. (e.g., M. tuberculosis, M. avium, M. intracellulare, M. kansaii, or M. gordonea), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitides, Listeria monocytogenes, Streptococcus pyogenes, Group A Streptococcus, Group B Streptococcus (Streptococcus agalactiae), Streptococcus pneumoniae, or Clostridium tetani, or combinations thereof, are provided.

[0163] 2. Transmembrane domain Regarding the transmembrane domain, CAR has an extracellular CD123 antigen-binding domain. It contains one or more fused transmembrane domains.

[0164] The transmembrane domain may originate from either a natural or synthetic source. If the source is natural, the domain may originate from any membrane-bound or transmembrane protein.

[0165] The transmembrane domains particularly used in the CARs described herein may originate from the alpha, beta, or zeta chains of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, mesoserine, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154 (i.e., may include at least one of their transmembrane domains). Alternatively, the transmembrane domain may be synthetic, in which case it predominantly contains hydrophobic residues such as leucine and valine. Preferably, a triplicate of phenylalanine, tryptophan, and valine is found at each terminus of the synthetic transmembrane domain. Optionally, short oligopeptide linkers or polypeptide linkers, preferably between 2-amino acid and 10-amino acid lengths, may form a linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR. Glycine-serine pairs provide particularly suitable linkers.

[0166] In one embodiment, a transmembrane domain naturally associated with one of the domains in the CAR is used in addition to the aforementioned transmembrane domain.

[0167] In some cases, transmembrane domains may be selected by amino acid substitutions to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, in order to minimize interaction with other members of the receptor complex.

[0168] 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 contains the nucleic acid sequence of SEQ ID NO: 27. In one embodiment, the CD8 transmembrane domain contains a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 28. In another embodiment, the CD8 transmembrane domain contains the amino acid sequence of SEQ ID NO: 28.

[0169] In one embodiment, the encoded transmembrane domain includes an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) to the amino acid sequence of SEQ ID NO: 28, but with 20, 10, or 5 or fewer modifications (e.g., substitutions), or a sequence having 95-99% identity to the amino acid sequence of SEQ ID NO: 28.

[0170] In some cases, the transmembrane domain of the CAR includes a CD8 alpha-hinge domain. In one embodiment, the CD8 hinge domain includes the nucleic acid sequence of SEQ ID NO: 29. In one embodiment, the CD8 hinge domain includes a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 30. In another embodiment, the CD8 hinge domain includes the amino acid sequence of SEQ ID NO: 30, or a sequence having 95-99% identity thereto.

[0171] In one embodiment, an isolated nucleic acid molecule is provided in which the encoded linker domain originates from the extracellular domain of CD8 and is linked to a transmembrane CD8 domain, a transmembrane CD28 domain, or a combination thereof.

[0172] In one embodiment, the transmembrane domain in the CAR of the present invention is the TNFRSF19 transmembrane domain. In one embodiment, the TNFRSF19 transmembrane domain includes the nucleic acid sequence of SEQ ID NO: 51. In one embodiment, the TNFRSF19 transmembrane domain includes the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 52. In another embodiment, the TNFRSF19 transmembrane domain includes the amino acid sequence of SEQ ID NO: 52.

[0173] In one embodiment, the encoded transmembrane domain includes an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) of the amino acid sequence of SEQ ID NO: 52, but with 20, 10, or 5 or fewer modifications (e.g., substitutions), or a sequence having 95-99% identity with the amino acid sequence of SEQ ID NO: 52.

[0174] 3. Spacer Domain In CARs, spacer domains, also called hinge domains, may be located between the extracellular domain and the transmembrane domain, or between the intracellular domain and the transmembrane domain. A spacer domain refers to any oligopeptide or polypeptide that functions to link the transmembrane domain to the extracellular domain and / or to link the transmembrane domain to the intracellular domain. Spacer domains contain up to 300 amino acids, preferably 10-100 amino acids, and most preferably 25-50 amino acids.

[0175] In some embodiments, the linker may include a spacer element, if present, which increases the size of the linker, thereby increasing the distance between the effector molecule or detectable marker and the antibody or antigen-binding fragment. Exemplary spacers are known to those skilled in the art and include U.S. Patents 7,964,566, 7,498,298, 6,884,869, 6,323,315, 6,239,104, 6,034,065, 5,780,588, 5,665,860, 5,663,149, 5,635,483, 5,599,902, 5,554,725, 5,530,097, and 5,521 This includes U.S. Patent Nos. 284, 5,504,191, 5,410,024, 5,138,036, 5,076,973, 4,986,988, 4,978,744, 4,879,278, 4,816,444 and 4,486,414, as well as those listed in U.S. Patent Application Publication Nos. 20110212088 and U.S. Patent Application Publication Nos. 20110070248, each of which is incorporated herein by reference in its entirety.

[0176] The spacer domain preferably has a sequence that promotes CAR binding to the antigen and enhances signaling into the cell. Examples of amino acids that are expected to promote binding include cysteine, charged amino acids, and serine and threonine in the potential glycosylation site, and these amino acids can be used as amino acids constituting the spacer domain.

[0177] As spacer domains, all or part of the hinge region of CD8 alpha (NCBI RefSeq: NP_001759.3), amino acids 118-178 (SEQ ID NO: 31), CD8 beta (GenBank: AAA35664.1), amino acids 135-195, CD4 (NCBI RefSeq: NP_000607.1), or CD28 (NCBI RefSeq: NP_006130.1), amino acids 137-152, may be used. Additionally, a portion of the constant region of the antibody H or L chain (CH1 region or CL region, e.g., a peptide with the amino acid sequence shown in SEQ ID NO: 32) may be used as a spacer domain. Furthermore, the spacer domain may be an artificially synthesized sequence.

[0178] Furthermore, CH1, (amino acid numbers 1-98), Hinge, SEQ ID NO: 80, and the corresponding nucleotide SEQ ID NO: 79, (amino acid numbers 99-110), CH2, amino acid SEQ ID NO: 81 and the corresponding nucleotide SEQ ID NO: 80, (amino acid numbers 111-220), and CH3, SEQ ID NO: 84 and the corresponding nucleotide SEQ ID NO: 83, (amino acid number 221 ~327) or combinations thereof, for example, all or part of the amino acids containing the constant region of human IgG4 (UniProt ID: P01861), including the IgG4 hinge CH2 CH3 domain, SEQ ID NO: 86, and the corresponding nucleotide SEQ ID NO: 85, may be used.

[0179] In one embodiment, the CAR spacer domain includes a TNFRSF19 hinge domain containing the nucleic acid sequence of SEQ ID NO: 53. In another embodiment, the TNFRSF19 hinge domain includes a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 54. In yet another embodiment, the TNFRSF19 hinge domain includes the amino acid sequence of SEQ ID NO: 54, or a sequence having 95-99% identity thereto.

[0180] In one embodiment, the CAR spacer domain includes a TNFRSF19 truncated hinge domain containing the nucleic acid sequence of SEQ ID NO: 55. In another embodiment, the TNFRSF19 truncated hinge domain includes a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 56. In yet another embodiment, the TNFRSF19 truncated hinge domain includes the amino acid sequence of SEQ ID NO: 56, or a sequence having 95-99% identity thereto.

[0181] In one embodiment, the TNFRSF19 hinge and transmembrane domain include the nucleic acid sequence of SEQ ID NO: 49. In another embodiment, the TNFRSF19 hinge and transmembrane domain include the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 50. In yet another embodiment, the TNFRSF19 hinge and transmembrane domain include the amino acid sequence of SEQ ID NO: 50, or a sequence having 95-99% identity thereto.

[0182] In one embodiment, the CD8a hinge domain is fused to the TNFRSF19 transmembrane domain containing the nucleic acid sequence of SEQ ID NO: 57. In another embodiment, the CD8a hinge domain is fused to the TNFRSF19 transmembrane domain and contains the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 58. In yet another embodiment, the CD8a hinge domain is fused to the TNFRSF19 transmembrane domain containing the amino acid sequence of SEQ ID NO: 58, or a sequence having 95-99% identity thereto.

[0183] Furthermore, in CARs, a signal peptide sequence, also called a leader peptide, can be ligated to the N-terminus. Signal peptide sequences are present at the N-terminus of many secretory and membrane proteins and have a length of 15 to 30 amino acids. 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 CARs. In one embodiment, the signal peptide comprises the amino acid sequence shown in SEQ ID NO: 14.

[0184] In one embodiment, the CD8 alpha-leader peptide contains the nucleic acid sequence of SEQ ID NO: 43. In another embodiment, the CD8 alpha-leader peptide contains the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 44. In yet another embodiment, the CD8a hinge domain is fused to the TNFRSF19 transmembrane domain containing the amino acid sequence of SEQ ID NO: 44, or a sequence having 95-99% identity.

[0185] In another embodiment, the GMCSF leader peptide includes the nucleic acid sequence of SEQ ID NO: 39. In one embodiment, the GMCSF leader peptide includes the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 40. In another embodiment, the CD8a hinge domain is fused to the TNFRSF19 transmembrane domain, which includes the amino acid sequence of SEQ ID NO: 40, or a sequence having 95-99% identity.

[0186] In another embodiment, the TNFRSF19 leader peptide is the nucleic acid sequence of SEQ ID NO: 41 In one embodiment, the TNFRSF19 leader peptide and the CD8 alpha leader peptide contain nucleic acid sequences encoding the amino acid sequence of SEQ ID NO: 42. In another embodiment, the CD8a hinge domain is fused to the TNFRSF19 transmembrane domain containing the amino acid sequence of SEQ ID NO: 42, or a sequence having 95-99% identity.

[0187] In one embodiment, the tag sequence encoding a truncated sequence of the epidermal growth factor receptor (tEGFR) includes the nucleic acid sequence of SEQ ID NO: 67. In one embodiment, tEGFR includes the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 68. In another embodiment, the tEGFR tag includes the amino acid sequence of SEQ ID NO: 68, or a sequence having 95-99% identity.

[0188] In one embodiment, the furin recognition site and downstream T2A self-cleaving peptide sequence designed for simultaneous bisistron expression of the tag sequence and CAR sequence include the nucleic acid sequence of SEQ ID NO: 65. In one embodiment, the furin and T2A sequences include nucleic acid sequences encoding the amino acid sequence of SEQ ID NO: 66. In another embodiment, the tEGFR tag includes the amino acid sequence of SEQ ID NO: 66, or a sequence having 95-99% identity.

[0189] In one embodiment, the upstream furin recognition site and T2A self-cleaving peptide sequence, as well as the downstream furin recognition site, designed for simultaneous bisistron expression of the tag sequence and CAR sequence, include the nucleic acid sequence of SEQ ID NO: 67. In one embodiment, the furin and T2A sequences include nucleic acid sequences encoding the amino acid sequence of SEQ ID NO: 68. In another embodiment, the tEGFR tag includes the amino acid sequence of SEQ ID NO: 68, or a sequence having 95-99% identity.

[0190] In one embodiment, the targeting domain of the CAR is expressed separately in the form of a monoclonal antibody, ScFv Fab, and Fab'2, and is contained in a binding tag or epitope, while the effector cell-expressed component of the CAR includes a binding domain specifically directed to bind to a tag or epitope expressed on a soluble CAR module, such as forming a complete functional CAR structure by specific binding to the cell-binding component on the soluble component of the CAR.

[0191] 4. Intracellular domains The cytoplasmic domain or, otherwise, the intracellular signaling domain of a CAR is responsible for activating at least one of the normal effector functions of the immune cell in which the CAR is placed. The term “effector function” refers to a specialized function of a cell. For example, the effector function of a T cell may be cytolytic activity or helper activity, including cytokine secretion. Thus, the term “intracellular signaling domain” refers to the portion of a protein that transmits effector function signals and directs the cell to perform its specialized function. While the entire intracellular signaling domain can usually be used, it is often not necessary to use the entire chain. Insofar as a truncated portion of the intracellular signaling domain is used, such truncated portion can be used in place of the intact chain, as long as it transmits effector function signals. Thus, the term “intracellular signaling domain” means including any truncated portion of an intracellular signaling domain that is sufficient to transmit effector function signals.

[0192] Preferred examples of intracellular signaling domains for use in CARs include cytoplasmic sequences of T cell receptors (TCRs) and co-receptors that work together to initiate signaling after antigen receptor engagement, as well as any derivatives or variants of these sequences, and any synthetic sequences having the same functional capabilities.

[0193] It is well known that the signals generated through the TCR alone are insufficient for the complete activation of T cells, and that secondary or co-stimulatory signals are also required. T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences) and those that act antigen-dependently to provide secondary or co-stimulatory signals (secondary cytoplasmic signaling sequences).

[0194] Primary cytoplasmic signaling sequences regulate the primary activation of the TCR complex either in a stimulative or inhibitory manner. Primary cytoplasmic signaling sequences that act in a stimulative manner may contain signaling motifs known as immunoreceptor-activating tyrosine motifs or ITAMs.

[0195] Examples of ITAMs containing primary cytoplasmic signaling sequences particularly used in CARs disclosed herein include those derived from TCR zeta (CD3 zeta), FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. Specific non-limiting examples of ITAM include amino acid numbers 51-164 of CD3 zeta (NCBI RefSeq: NP_932170.1), amino acid numbers 45-86 of Fc epsilon RI gamma (NCBI RefSeq: NP_004097.1), amino acid numbers 201-244 of Fc epsilon RI beta (NCBI RefSeq: NP_000130.1), amino acid numbers 139-182 of CD3 gamma (NCBI RefSeq: NP_000064.1), amino acid numbers 128-171 of CD3 delta (NCBI RefSeq: NP_000723.1), amino acid numbers 153-207 of CD3 epsilon (NCBI RefSeq: NP_000724.1), and CD5 (NCBI Amino acid numbers 402-495 of RefSeq:NP_055022.2, amino acid numbers 707-847 of 0022 (NCBI RefSeq:NP_001762.2), amino acid numbers 166-226 of CD79a (NCBI RefSeq:NP_001774.1), amino acid numbers 182-229 of CD79b (NCBI RefSeq:NP_000617.1), and CD66d (NCBI This includes peptides having sequences of amino acid numbers 177-252 (RefSeq:NP_001806.2), as well as variants of these peptides having the same function. The amino acid numbers based on NCBI RefSeq IDs or GenBank amino acid sequence information described herein are numbered based on the full length of each protein precursor (including signal peptide sequences, etc.). In one embodiment, the cytoplasmic signaling molecule in CAR includes a cytoplasmic signaling sequence derived from CD3 zeta.

[0196] In preferred embodiments, the intracellular domain of a CAR may be designed to include a CD3-zeta signaling domain, either by itself or in combination with any other desired cytoplasmic domain(s) useful in the context of the CAR. For example, the intracellular domain of a CAR may include a CD3 zeta chain portion and a costimulatory signaling region. The costimulatory signaling region refers to the portion of the CAR that includes the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that is required for an efficient lymphocyte response to an antigen. Examples of such costimulatory molecules include ligands that specifically bind to CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83. Specific non-limiting examples of such co-stimulatory molecules include amino acid numbers 236-351 of CD2 (NCBI RefSeq: NP_001758.2), amino acid numbers 421-458 of CD4 (NCBI RefSeq: NP_000607.1), amino acid numbers 402-495 of CD5 (NCBI RefSeq: NP_055022.2), amino acid numbers 207-235 of CD8 alpha (NCBI RefSeq: NP_001759.3), amino acid numbers 196-210 of CD83 (GenBank: AAA35664.1), amino acid numbers 181-220 of CD28 (NCBI RefSeq: NP_006130.1), and CD137 (4-1BB). This includes peptides having the sequences of amino acid numbers 214-255 of (NCBI RefSeq: NP_001552.2), amino acid numbers 241-277 of CD134 (OX40, NCBI RefSeq: NP_003318.1), and amino acid numbers 166-199 of ICOS (NCBI RefSeq: NP_036224.1), as well as variants of these peptides having the same functions. Therefore, while this disclosure primarily exemplifies 4-1BB as a co-stimulatory signaling element, other co-stimulatory elements are also within the scope of this disclosure.

[0197] The cytoplasmic signaling sequences within the cytoplasmic signaling region of a CAR can be linked to each other randomly or in a specified order. Optionally, short oligopeptide linkers or polypeptide linkers, preferably between 2-amino acid and 10-amino acid lengths, can form the linkage. Glycine-serine pairs provide particularly suitable linkers.

[0198] In one embodiment, the intracellular domain is designed to include a CD3-zeta signaling domain and a CD28 signaling domain. In another embodiment, the intracellular domain is designed to include a CD3-zeta signaling domain and a 4-1BB signaling domain. In yet another embodiment, the intracellular domain is designed to include a CD3-zeta signaling domain as well as CD28 and 4-1BB signaling domains.

[0199] In one embodiment, the intracellular domain in the CAR is designed to include a 4-1BB signaling domain and a CD3-zeta signaling domain, where the 4-1BB signaling domain includes the nucleic acid sequence shown in SEQ ID NO: 33, SEQ ID NO: 45, or SEQ ID NO: 59, and the CD3-zeta signaling domain includes the nucleic acid sequence shown in SEQ ID NO: 35, SEQ ID NO: 47, or SEQ ID NO: 61, respectively.

[0200] In one embodiment, the intracellular domain in the CAR is designed to include a 4-1BB signaling domain and a CD3-zeta signaling domain, where the 4-1BB signaling domain includes a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 34, SEQ ID NO: 46, or SEQ ID NO: 60, respectively, and the CD3-zeta signaling domain includes a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 36, SEQ ID NO: 48, or SEQ ID NO: 62.

[0201] In one embodiment, the intracellular domain in the CAR is designed to include a 4-1BB signaling domain and a CD3-zeta signaling domain, where the 4-1BB signaling domain includes the amino acid sequence shown in SEQ ID NO: 34, SEQ ID NO: 46, or SEQ ID NO: 60, and the CD3-zeta signaling domain includes the amino acid sequence shown in SEQ ID NO: 36, SEQ ID NO: 48, or SEQ ID NO: 62, respectively.

[0202] In one embodiment, the intracellular domain in the CAR is designed to include a CD28 signaling domain and a CD3-zeta signaling domain, where the CD28 signaling domain includes the nucleic acid sequence shown in SEQ ID NO: 45 or SEQ ID NO: 59, and the CD3-zeta signaling domain includes the nucleic acid sequence shown in SEQ ID NO: 35, SEQ ID NO: 47, or SEQ ID NO: 61, respectively.

[0203] In one embodiment, the intracellular domain in the CAR is designed to include a CD28 signaling domain and a CD3-zeta signaling domain, where the CD28 signaling domain includes a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 46 or SEQ ID NO: 60, and the CD3-zeta signaling domain includes a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 36, or SEQ ID NO: 48, or SEQ ID NO: 62.

[0204] In one embodiment, the intracellular domain in the CAR is designed to include a CD28 signaling domain and a CD3-zeta signaling domain, where the CD28 signaling domain comprises the amino acid sequence shown in SEQ ID NO: 46 or SEQ ID NO: 60, and the CD3-zeta signaling domain comprises the amino acid sequence shown in SEQ ID NO: 36, SEQ ID NO: 48, or SEQ ID NO: 62, respectively.

[0205] 5. Further description of CAR Functional parts of CARs disclosed herein are also expressly included within the scope of the invention. The term “functional part,” when used in relation to a CAR, means any part or fragment of one or more CARs disclosed herein, which retain the biological activity of the CAR from which it is a part (parent CAR). A functional part includes, for example, a part of a CRA that retains the ability to recognize target cells or to detect, treat, or prevent disease to a similar, equal, or greater degree than that of the parent CAR. With respect to a parent CAR, a functional part may constitute, for example, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more of the parent CAR.

[0206] The functional moiety may contain additional amino acids at its amino-terminus, carboxy-terminus, or both, which are not found in the amino acid sequence of the parent CAR. Preferably, the additional amino acids do not interfere with the biological function of the functional moiety, such as recognizing target cells, detecting cancer, or treating or preventing cancer. More preferably, the additional amino acids enhance the biological activity of the functional moiety compared to that of the parent CAR.

[0207] Functional variants of CARs disclosed herein are included within the scope of this disclosure. The term “functional variant” as used herein means a CAR, polypeptide, or protein having substantial or significant sequence identity or similarity to the parent CAR, wherein the functional variant retains the biological activity of the CAR from which it is a variant. Functional variants include, for example, variants of the CARs described herein (parent CARs) that retain the ability to recognize target cells to a similar, identical, or greater degree than the parent CAR. With respect to the parent CAR, a functional variant may, for example, have amino acid sequence identity with respect to the parent CAR by at least about 30%, 50%, 75%, 80%, 90%, 98%, or more.

[0208] A functional variant may, for example, include the amino acid sequence of a parent CAR having at least one conserved amino acid substitution. Alternatively, the functional variant may further include the amino acid sequence of a parent CAR having at least one non-conserved amino acid substitution. In this case, it is preferable that the non-conserved amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. The non-conserved amino acid substitution may enhance the biological activity of the functional variant, resulting in an increased biological activity of the functional variant compared to the parent CAR.

[0209] The amino acid substitutions of CARs are preferably conservative amino acid substitutions. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid having certain physical and / or chemical properties is replaced by another amino acid having the same or similar chemical or physical properties. For example, conservative amino acid substitutions include: an acidic / negatively charged polar amino acid substituting another acidic / negatively charged polar amino acid (e.g., Asp or Glu); an amino acid with a nonpolar side chain substituting another amino acid with a nonpolar side chain (e.g., Ala, Gly, Val, He, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.); a basic / positively charged polar amino acid substituting another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.); and another amino acid with a polar side chain. These may include uncharged amino acids having polar side chains that substitute for uncharged amino acids (e.g., Asn, Gin, Ser, Thr, Tyr, etc.), amino acids having beta-branched side chains that substitute for other amino acids having beta-branched side chains (e.g., He, Thr, and Val), and amino acids having aromatic side chains that substitute for other amino acids having aromatic side chains (e.g., His, Phe, Trp, and Tyr).

[0210] CARs may essentially consist of one or more of the specified amino acid sequences described herein, and as a result, other components, such as other amino acids, do not significantly alter the biological activity of the functional variant.

[0211] CARs (including functional parts and functional variants) can be of any length, i.e., they can contain any number of amino acids, provided that CARs (or their functional parts or functional variants) retain their biological activity, such as the ability to specifically bind to an antigen, the ability to detect diseased cells in mammals, or the ability to treat or prevent disease in mammals. For example, CARs can be approximately 50 to 5000 amino acid long, e.g., 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or longer.

[0212] CAR (including the functional parts and functional variants of the present invention) may include synthetic amino acids instead of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, -amino n-decanoic acid, homoserine, S-acetylaminomethylcysteine, trans-3-hydroxyproline and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, and 1,2,3,4-tetrahydroxy It contains diisoquinoline-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.

[0213] CARs (including functional moieties and functional variants) can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized via disulfide crosslinks, etc., or converted to acid addition salts and / or optionally dimerized, polymerized, or conjugated.

[0214] CARs (including their functional parts and functional variants) can be obtained by methods known in the art. CARs can be produced by any suitable method for producing polypeptides or proteins. Suitable methods for the de novo synthesis of polypeptides and proteins are described in reference to Chan et al., Fmoc Solid Phase Peptide Synthesis, Oxford University Press, Oxford, United Kingdom, 2000; Peptide and Protein Drug Analysis, edited by Reid, R., Marcel Dekker, Inc., 2000; Epitope Mapping, edited by Westwood et al., Oxford University Press, Oxford, United Kingdom, 2001; and U.S. Patent No. 5,449,752. Furthermore, polypeptides and proteins can be synthesized using standard recombination methods, and the nucleic acids described herein can be synthesized. Recombinant production is possible using the following methods. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. Furthermore, some CARs (including functional moieties and their functional variants) can be isolated and / or purified from sources such as plants, bacteria, insects, mammals, e.g., rats, humans. Methods for isolation and purification are well known in the art. Alternatively, CARs (including functional moieties and their functional variants) described herein can be commercially synthesized by companies. In this regard, CARs may be synthetic, recombinant, isolated, and / or purified.

[0215] B. Antibodies and antigen-binding fragments One embodiment further provides a CAR, a CAR-expressing T cell, and an antibody or its antigen-binding domain or portion that specifically binds to one or more of the antigens disclosed herein. As used herein, “CAR-expressing T cell” or “CAR T cell” means a T cell that expresses a CAR and has antigen specificity determined, for example, by the antibody-derived targeting domain of the CAR.

[0216] As used herein, “antigen-binding domain” may include an antibody and its antigen-binding fragments. The term “antibody” is used herein in its 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 their antigen-binding fragments, as long as they exhibit the desired antigen-binding activity. Non-limiting examples of antibodies include, for example, intact immunoglobulins known in the art, as well as their variants and fragments that retain binding affinity to an antigen.

[0217] A "monoclonal antibody" is an antibody obtained from a substantially homogeneous population of antibodies; that is, the individual antibodies constituting the population are identical except for possible naturally occurring mutations that may exist in trace amounts. Monoclonal antibodies are highly specific and target a single antigenic epitope. The modifier "monoclonal" indicates the characteristic of the antibody being obtained from a substantially homogeneous population of antibodies, and should not be interpreted as requiring antibody production by any particular method. In some cases, monoclonal antibodies are antibodies produced by a single clone of B lymphocytes, or antibodies produced by cells transfected with nucleic acids encoding the light and heavy chain variable regions of a single antibody (or its antigen-binding fragment), or by their offspring. In some cases, monoclonal antibodies are isolated from the subject. Monoclonal antibodies may have conserved amino acid substitutions that substantially have no effect on antigen binding or other immunoglobulin functions. Exemplary methods for producing monoclonal antibodies are publicly known; see, for example, Harlow & Lane, Antibodies, A Laboratory Manual, 2nd edition, Cold Spring Harbor Publications, New York (2013).

[0218] Typically, immunoglobulins have heavy (H) and light (L) chains interconnected by disulfide bonds. Immunoglobulin genes include kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable domain genes. Two types of light chains exist: lambda (λ) and kappa (κ). There are five major heavy chain classes (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE.

[0219] Each heavy chain and light chain comprises a constant region (or constant domain) and a variable region (or variable domain; see, for example, Kindt et al., Kuby Immunology, 6th edition, WHFreeman and Co., p. 91 (2007)). In some embodiments, the variable regions of the heavy chain and light chain combine to specifically bind to the antigen. In further embodiments, only the variable region of the heavy chain is required. For example, naturally occurring camelid antibodies consisting only of heavy chains are functional and stable in the absence of the light chain. This is definitive (see, for example, Hamers-Casterman et al., Nature, Vol. 363: pp. 446-448, 1993; Sheriff et al., Nat. Struct. Biol., Vol. 3: pp. 733-736, 1996). A reference to "VH" or "VH" refers to the variable region of the antibody heavy chain, including antigen-binding fragments, e.g., those of Fv, scFv, dsFv, or Fab. A reference to "VL" or "VL" refers to the variable domain of the antibody light chain, including those of Fv, scFv, dsFv, or Fab.

[0220] The variable regions of the light and heavy chains contain a "framework" region interrupted by three hypervariable regions, also called "complementarity-determining regions" or "CDRs" (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, USD Department of Health and Human Services, 1991). The sequences of different light or heavy chain framework regions are relatively conserved within a species. The antibody framework region, which is the combined framework region of the constituent light and heavy chains, functions to position and align the CDRs in three-dimensional space.

[0221] CDRs are primarily responsible for binding antigens to epitopes. The amino acid sequence boundaries of a given CDR are described in 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 Vol. 273, pp. 927-948, 1997; "Chothia" numbering scheme), and Lefranc et al. ("IMGT unique numbering for immunoglobulin and T cell receptor"). The CDRs can be readily determined using one of several well-known schemes, including the one described in “Variable Domains and Ig Superfamily V-like Domains,” Dev. Comp. Immunol., Vol. 27: pp. 55–77, 2003 (the “IMGT” numbering scheme). The CDRs of each chain are typically called CDR1, CDR2, and CDR3 (from N-terminus to C-terminus) and are also typically identified by the chain on which a particular CDR is located. Thus, VH CDR3 is the CDR3 derived from the variable domain of the heavy chain of the antibody in which it is found, while VL CDR1 is the CDR1 derived from the variable domain of the light chain of the antibody in which it is found. Light chain CDRs are sometimes called LCDR1, LCDR2, and LCDR3. Heavy chain CDRs are sometimes called HCDR1, HCDR2, and HCDR3.

[0222] An "antigen-binding fragment" is a portion of a full-length antibody that retains the ability to specifically recognize a congener antigen, as well as various combinations of such portions. Non-limiting examples of antigen-binding fragments include Fv, Fab, Fab', Fab'-SH, F(ab')2; diabody; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. Antibody fragments include antigen-binding fragments produced by modification of the entire antibody, or antigen-binding fragments synthesized de novo using recombinant DNA methodologies (see, e.g., Kontermann and Dubel (eds.), Antibody Engineering, vols. 1-2, 2nd edition, Springer Press, 2010).

[0223] Single-chain antibodies (scFv) are genetically engineered molecules containing the VH and VL domains of one or more antibodies linked by a suitable polypeptide linker as a single-chain molecule (see, e.g., Bird et al., Science, vol. 242: pp. 423-426, 1988; Huston et al., Proc. Natl. Acad. Sci., vol. 85: pp. 5879-5883, 1988; Ahmad et al., Clin. Dev. Immunol., 2012, doi:10.1155 / 2012 / 980250; Marbry, IDrugs, vol. 13: pp. 543-549, 2010). The intramolecular orientation of the VH and VL domains in scFv is typically not definitive for the scFv. Therefore, scFv can be used with both possible configurations (VH domain - linker domain - VL domain; VL domain - linker domain - VH domain).

[0224] In dsFv, the variable chains of the heavy and light chains are mutated to introduce disulfide bonds to stabilize chain association. Diabodies are also included, which are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but use a linker that is too short to allow pairing between the two domains on the same chain, thereby pairing that domain with a complementary domain on another chain and creating two antigen-binding sites (see, e.g., Holliger et al., Proc. Natl. Acad. Sci., vol. 90: pp. 6444-6448, 1993; Poljak et al., Structure, vol. 2: pp. 1121-1123, 1994).

[0225] Antibodies also include genetically modified forms such as chimeric antibodies (e.g., humanized mouse antibodies) and heteroconjugate antibodies (e.g., bispecific antibodies). See also Pierce Catalog and Handbook, 1994–1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rd edition, WH Freeman & Co., New York, 1997.

[0226] Antibodies not found in nature can be constructed using solid-phase peptide synthesis, recombinantly produced, or obtained by screening combinatorial libraries consisting of variable heavy and variable light chains, as described, for example, in Huse et al., Science Vol. 246: pp. 1275-1281 (1989), incorporated herein by reference. These and other methods for producing chimeric, humanized, CDR-grafted, single-chain, and bifunctional antibodies are well known to those skilled in the art (Winter and Harris, Immunol. Today Vol. 14: pp. 243-246 (1993); Ward et al., Nature). Volume 341: pp. 544-546 (1989); Harlow and Lane, above, 1988; Hilyard et al., Protein Engineering: A practical approach (IRL Press 1992); Borrabeck, Antibody Engineering, 2nd edition (Oxford University Press 1995); each of these is incorporated herein by reference).

[0227] A reference antibody and an antibody that "binds to the same epitope" refer to an antibody that blocks 50% or more of the binding of the reference antibody to its antigen in a competitive assay, and conversely, a reference antibody blocks 50% or more of the binding of its antibody to its antigen in a competitive assay. Antibody competitive assays are well known, and exemplary competitive assays are provided herein.

[0228] The "humanized" antibody or antigen-binding fragment is derived from a human framework region and one or more CDRs derived from a non-human (e.g., mouse, rat, or synthetic) antibody or antigen-binding fragment. This includes the non-human antibody or antigen-binding fragment that provides the CDR, which is called the “donor,” and the human antibody or antigen-binding fragment that provides the framework, which is called the “acceptor.” In one embodiment, all CDRs are derived from the donor immunoglobulin in the humanized immunoglobulin. A constant region is not required to be present, but if present, it may be substantially identical to the human immunoglobulin constant region, e.g., at least about 85–90%, e.g., about 95% or more identical. Thus, all parts of the humanized antibody or antigen-binding fragment are substantially identical to the corresponding parts of the natural human antibody sequence, except perhaps the CDR.

[0229] A "chimeric antibody" is an antibody that contains sequences derived from two different antibodies, typically from different species. In some cases, a chimeric antibody contains one or more CDRs and / or framework regions from one human antibody as well as CDRs and / or framework regions from another human antibody.

[0230] A “fully human antibody” or “human antibody” is an antibody that contains sequences derived from (or originating from) the human genome but does not contain sequences from another species. In some embodiments, a human antibody contains CDRs, framework regions, and (if present) Fc regions derived from (or originating from) the human genome. Human antibodies can be identified and isolated, for example, by phage display, using technologies for generating sequences based on sequences derived from the human genome, or by using transgenic animals (see, e.g., Barbas et al. Phage display: A Laboratory Manuel. 1st ed. New York: Cold Spring Harbor Laboratory Press, 2004 Print.; Lonberg, Nat. Biotech., vol. 23: pp. 1117-1125, 2005; Lonberg, Curr. Opin. Immunol., vol. 20: pp. 450-459, 2008).

[0231] Antibodies may have one or more binding sites. If there are more than one binding sites, these sites may be identical or different. For example, naturally occurring immunoglobulins have two identical binding sites, single-chain antibodies or Fab fragments have one binding site, while bispecific or bifunctional antibodies have two different binding sites.

[0232] Methods for testing antibodies for their ability to bind to any functional portion of a CAR are known in the art and include any antibody-antigen binding assays, such as radioimmunoassays (RIA), ELISA, Western blotting, immunoprecipitation, and competitive inhibition assays (see, for example, Janeway et al., U.S. Patent Application Publication No. 2002 / 0197266Al, and U.S. Patent No. 7,338,929).

[0233] Furthermore, CARs, CAR-expressing T cells, antibodies, or their antigen-binding moieties may 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).

[0234] C. Conjugate Monoclonal antibodies or antigen-binding fragments thereof that are specific to CARs, CAR-expressing T cells, or one or more of the antigens disclosed herein may be conjugated to drugs such as effector molecules or detectable markers by any means known to those skilled in the art. Both covalent and non-covalent means may be used. The conjugate may include antibodies that specifically bind to one or more of the antigens disclosed herein. or molecules having covalent linkage of an effector molecule or a detectable marker to an antigen-binding fragment, etc. Those skilled in the art will know of chemotherapeutic agents, anti-angiogenic agents, toxins, radioactive agents, for example, 125 I, 32 P, 14 C, 3 H and 35 It is understood that various effector molecules and detectable markers, including (but not limited to) S, other labels, target moieties, and ligands, may be used.

[0235] The selection of a specific effector molecule or detectable marker depends on the specific target molecule or cell and the desired biological effect. For example, an effector molecule could be a cytotoxic substance used to induce the death of a specific target cell (e.g., tumor cells).

[0236] The procedure for conjugating an effector molecule or detectable marker to an antibody or antigen-binding fragment varies depending on the chemical structure of the effector. Polypeptides typically contain various functional groups; for example, carboxylic acid (COOH), free amine (-NH2), or sulfhydryl (-SH) groups, which are available for reaction with suitable functional groups on the antibody to result in the binding of the effector molecule or detectable marker. Alternatively, the antibody or antigen-binding fragment is derivatized to expose or bind further reactive functional groups. Derivatization may involve the binding of one of several known linker molecules, such as those available from Pierce Chemical Company, Rockford, IL. The linker can be any molecule used to conjugate the antibody or antigen-binding fragment to the effector molecule or detectable marker. The linker can form a covalent bond to both the antibody or antigen-binding fragment and the effector molecule or detectable marker. Suitable linkers are well known to those skilled in the art and include, but are not limited to, linear 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 may be attached to the constituent amino acids via their side groups (e.g., via disulfide linkage to cysteine) or to the amino and carboxyl groups of the alpha carbon of the terminal amino acids.

[0237] In some embodiments, the linker may include a spacer element, if present, which increases the size of the linker, thereby increasing the distance between the effector molecule or detectable marker and the antibody or antigen-binding fragment. Exemplary spacers are known to those skilled in the art and include U.S. Patents 7,964,566, 7,498,298, 6,884,869, 6,323,315, 6,239,104, 6,034,065, 5,780,588, 5,665,860, 5,663,149, 5,635,483, 5,599,902, 5,554,725, 5,530,097, and 5,521 This includes U.S. Patent Nos. 284, 5,504,191, 5,410,024, 5,138,036, 5,076,973, 4,986,988, 4,978,744, 4,879,278, 4,816,444 and 4,486,414, as well as those listed in U.S. Patent Application Publication Nos. 20110212088 and U.S. Patent Application Publication Nos. 20110070248, each of which is incorporated herein by reference in its entirety.

[0238] In some embodiments, the linker is cleavable under intracellular conditions, and as a result, cleavage of the linker causes the release of effector molecules or detectable markers from the antibody or antigen-binding fragment into the intracellular environment. In yet other embodiments, the linker is incleavable, and the effector molecules or detectable markers are released, for example, by antibody degradation. In some embodiments, the linker is released into the intracellular environment (e.g., lysosomes or endosows). It can be cleaved by cleavage agents present in the cell or caveolae. The linker may be a peptide linker cleaved by an intracellular peptidase or protease enzyme, including but not limited to lysosome or endosomal proteases. In some embodiments, the peptide linker is at least 2 amino acid long or at least 3 amino acid long. However, the linker may be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid long, e.g., 1-2, 1-3, 2-5, 3-10, 3-15, 1-5, 1-10, 1-15 amino acid long. Proteases may include cathepsins B and D, as well as plasmin, all of which are known to hydrolyze dipeptide drug derivatives, resulting in the release of the active drug inside the target cell (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics vol. 83: pp. 67-123). For example, peptide linkers cleavable by cathepsin B, a thiol-dependent protease, may be used (e.g., phenylalanine-leucine or glycine-phenylalanine-leucine-glycine linkers). Other examples of such linkers are described, for example, in U.S. Patent No. 6,214,345, incorporated herein by reference. In specific embodiments, the peptide linker cleavable by an intracellular protease is a valine-citrulline linker or a phenylalanine-lysine linker (see, for example, U.S. Patent No. 6,214,345, which describes the synthesis of doxorubicin using a valine-citrulline linker).

[0239] In other embodiments, the cleavable linker is pH-sensitive, i.e., susceptible to hydrolysis at a specific pH value. Typically, pH-sensitive linkers are hydrolyzable under acidic conditions. For example, acid-unstable linkers hydrolyzable in lysosomes (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amides, orthoesters, acetals, ketals, etc.) may be used (see, e.g., U.S. Patent No. 5,122,368; U.S. Patent No. 5,824,805; U.S. Patent No. 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics, Vol. 83: pp. 67-123; Neville et al., 1989, Biol. Chem., Vol. 264: pp. 14653-14661). Such linkers are relatively stable under neutral pH conditions, such as in blood, but are unstable at pH levels below 5.5 or 5.0, which is the approximate pH of lysosomes. In certain embodiments, the hydrolyzable linker is a thioether linker (e.g., a thioether conjugated to the therapeutic agent via an acylhydrazone linkage) (see, for example, U.S. Patent No. 5,622,929).

[0240] In other embodiments, the linker is cleavable under reducing conditions (e.g., a disulfide linker). Various disulfide linkers are known in the field, including, for example, SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyldithio)toluene), which can be formed using SPDB and SMPT (e.g., Thorpe et al., 1987, Cancer Res. Vol. 47: pp. 5924-5931; Wawrzynczak et al., Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (CWVogel, ed., Oxford U. Press, 1987); Phillips et al., Cancer See Res. 68: pp. 9280-9290, 2008. Also see U.S. No. 4,880,935.

[0241] In yet another specific embodiment, the linker is a malonate linker (Johnson et al., 1995, Anticancer Res. Vol. 15: pp. 1387-1393), Murray These are midobenzoyl linkers (Lau et al., 1995, Bioorg-Med-Chem. Vol. 3 (No. 10): pp. 1299-1304) or 3'-N-amide analogs (Lau et al., 1995, Bioorg-Med-Chem. Vol. 3 (No. 10): pp. 1305-12).

[0242] In yet another embodiment, the linker is incapable of cleavage, and the effector molecule or detectable marker is released by antibody degradation (see U.S. Patent Application Publication 2005 / 0238649, the full contents of which are incorporated herein by reference).

[0243] In some embodiments, the linker is resistant to cleavage in the extracellular environment. For example, when the conjugate is present in the extracellular environment (e.g., plasma), approximately 20%, 15%, 10%, 5%, 3%, or 1% of the linker in the conjugate sample is cleaved. Whether the linker is resistant to cleavage in the extracellular environment can be determined, for example, by incubating the conjugate containing the linker of interest with plasma for a predetermined period (e.g., 2, 4, 8, 16, or 24 hours) and then quantifying the amount of free effector molecules or detectable markers present in the plasma. Various exemplary linkers that may be used in a conjugate are described in WO2004-010957, U.S. Patent Application Publication 2006 / 0074008, U.S. Patent Application Publication 20050238649, and U.S. Patent Application Publication 2006 / 0024317, each of which is incorporated herein by reference in its entirety.

[0244] In some embodiments, CAR conjugates, CAR-expressing T cells, antibodies or their antigen-binding moieties, and one or more small molecule toxins, such as calitiamycin, maytansinoid, drastatin, auristatin, trichothecin, and CC1065, as well as derivatives of these toxins having toxic activity.

[0245] Maytansine compounds suitable for use as the toxin portion of mitansinoids are well known in the art and can be isolated from natural sources according to known methods, produced using genetic engineering techniques (see Yu et al. (2002) PNAS Vol. 99: pp. 7968-7973), or synthesized using known methods to produce maytansinol and maytansinol analogs. Mitansinoids are mitotic inhibitors that act by inhibiting tubulin polymerization. Maytansine was first isolated from the East African shrub Maytenus serrata (U.S. Patent No. 3,896,111). Subsequently, it was discovered that certain microorganisms also produce maytansinoids such as maytansinol and C-3 maytansinol ester (U.S. Patent No. 4,151,042). Synthetic mytansinol and its derivatives and analogs are, for example, U.S. Patent Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,31 U.S. Patent No. 3,946; U.S. Patent No. 4,315,929; U.S. Patent No. 4,317,821; U.S. Patent No. 4,322,348; U.S. Patent No. 4,331,598; U.S. Patent No. 4,361,650; U.S. Patent No. 4,364,866; U.S. Patent No. 4,424,219; U.S. Patent No. 4,450,254; U.S. Patent No. 4,362,663; and U.S. Patent No. 4,371,533, each of which is incorporated herein by reference. Conjugates containing mytansinoids, methods for preparing them, and their therapeutic uses are disclosed, for example, U.S. Patent No. 5,208,020; U.S. Patent No. 5,416,064; U.S. Patent No. 6,441,16 Disclosed in Patent No. 3 and European Patent EP0 425 235 B1, the disclosures of which are expressly incorporated herein by reference.

[0246] Further toxins may be used in conjunction with CARs, CAR-expressing T cells, antibodies, or their antigen-binding moieties. Exemplary toxins include Pseudomonas exotoxin (PE), hematopoxin, abrin, diphtheria toxin and its subunits, ribotoxin, ribonuclease, saporin and calitiamycin, and botulinum toxins A–F. These toxins are well known in the art and many are readily available from commercial sources (e.g., Sigma Chemical Company, St. Louis, MO). The toxins intended may also include variants of these toxins (see, for example, U.S. Patent Nos. 5,079,163 and 4,689,401).

[0247] Saporins are toxins derived from Saponaria officinalis that disrupt protein synthesis by inactivating the 60S portion of ribosome complexes (Stirpe et al., Bio / Technology, Vol. 10: pp. 405-412, 1992). However, these toxins lack a mechanism for specific entry into cells and therefore require conjugation to antibodies or antigen-binding fragments that recognize internalized cell surface proteins in order to be efficiently taken up by cells.

[0248] Diphtheria toxin is isolated from Corynebacterium diphtheriae. Typically, diphtheria toxin for use in immunotoxins is mutated to reduce or eliminate nonspecific toxicity. A variant known as CRM107, which has full enzymatic activity but significantly reduced nonspecific toxicity, has been known since the 1970s (Laird and Groman, J. Virol. 19:220, 1976) and has been used in human clinical trials. See U.S. Patents No. 5,792,458 and 5,208,021.

[0249] Castor bean toxin is lectin RCA60 derived from Ricinus communis (castor bean). For examples of castor bean toxin, see U.S. Patent Nos. 5,079,163 and 4,689,401. Ricinus communis aglutinin (RCA) has molecular weights of approximately 65 kD and 120 kD, respectively. 60 and RCA 120 It exists in two forms, known as (Nicholson and Blaustein, J. Biochim. Biophys. Acta vol. 266: p. 543, 1972). Chain A is responsible for inactivating protein synthesis and cell death. Chain B binds hematin to galactose residues on the cell surface and promotes the transport of chain A into the cytosol (Olsnes et al., Nature vol. 249: pp. 627-631, 1974 and US Patent No. 3,060,165).

[0250] Ribonucleases have also been conjugated into targeted molecules for use as immunotoxins (see Suzuki et al., Nat. Biotech. Vol. 17: pp. 265-267, 1999). Exemplary ribotoxins, such as α-sarcin and restrictocin, are discussed, for example, in Rathore et al., Gene Vol. 190: pp. 31-35, 1997; and in Goyal and Batra, Biochem. Vol. 345 Part 2: pp. 247-244, 2000. Kalithiamycin, first isolated from Micromonospora echinospora, is a member of the enediin antitumor antibiotic family, inducing double-strand breaks in DNA that lead to apoptosis (see, for example, Lee et al., J. Antibiot. Vol. 42: pp. 1070-1087, 1989). This drug is the toxic portion of an immunotoxin in clinical trials (e.g., Gillespie et al., Ann. Oncol). (See Volume 11: pp. 735-7341, 2000).

[0251] Abrin contains toxic lectins derived from Abrus precatorius. The toxic elements abrins a, b, c, and d have molecular weights of approximately 63 kD and 67 kD, and are composed of two disulfide-linked polypeptide chains A and B. Chain A inhibits protein synthesis; chain B (abrin-b) binds to D-galactose residues (see Funatsu et al., Agr. Biol. Chem. Vol. 52: p. 1095, 1988; and Olsnes, Methods Enzymol. Vol. 50: pp. 330-335, 1978).

[0252] CARs, CAR-expressing T cells, monoclonal antibodies specific to one or more of the antigens disclosed herein, and their antigen-binding fragments may also be conjugated with detectable markers; for example, detectable markers detectable by ELISA, spectrophotometry, flow cytometry, microscopy or imaging techniques (e.g., computed tomography (CT), computed axial tomography (CAT) scans, magnetic resonance imaging (MRI), nuclear magnetic resonance imaging (NMRI), magnetic resonance imaging (MTR), ultrasound, fiber optic testing and laparoscopic testing). Specific non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzymatic linkages, radioactive isotopes and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI). For example, useful detectable markers include fluorescent compounds such as fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, and lanthanidrin photomeridians. Bioluminescent markers such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP) are also used. CARs, CAR-expressing T cells, antibodies, or their antigen-binding moieties can also be conjugated to enzymes useful for detection, such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, and glucose oxidase. When CARs, CAR-expressing T cells, antibodies, or their antigen-binding moieties are conjugated with a detectable enzyme, they can be detected by adding further reagents used by the enzyme to produce a identifiable reaction product. For example, in the presence of the drug horseradish peroxidase, the addition of hydrogen peroxide and diaminobenzidine results in a visually detectable colored reaction product. CARs, CAR-expressing T cells, antibodies, or their antigen-binding moieties can also be conjugated with biotin and detected via indirect measurement of avidin or streptavidin binding. It should be noted that avidin itself can be conjugated with enzymes or fluorescent labels.

[0253] A CAR, a T cell expressing the CAR, an antibody or an antigen-binding portion thereof can be conjugated with a paramagnetic agent such as gadolinium. Paramagnetic agents such as superparamagnetic iron oxide are also used as labels. Antibodies can also be conjugated with lanthanides (e.g., europium and dysprosium) and manganese. An antibody or antigen-binding fragment can also be labeled with a predetermined polypeptide epitope (e.g., leucine zipper pairing sequence, binding site for a secondary antibody, metal-binding domain, epitope tag) recognized by a secondary reporter.

[0254] A CAR, a T cell expressing the CAR, an antibody or an antigen-binding portion thereof can also be conjugated with radiolabeled amino acids. The radiolabel can be used for both diagnostic and therapeutic purposes. For example, the radiolabel can be used to detect one or more of the antigens and antigen-expressing cells disclosed herein by x-ray, luminescence spectroscopy or other diagnostic techniques. Further, the radiolabel can be used therapeutically as a toxin for the treatment of tumors in a subject, e.g., for the treatment of neuroblastoma. Examples of labels for polypeptides include, but are not limited to, the following radioisotopes or radionucleotides: 3 H, 14 C, 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I.

[0255] Means for detecting such detectable markers are well known to those skilled in the art. For example, radioactive labels can be detected using photographic film or a scintillation counter, and fluorescent markers can be detected using a photodetector to detect the emitted illumination. Enzymatic labels are typically detected by providing a substrate to an enzyme and detecting the reaction product produced by the enzyme's action on the substrate, and chromogenic labels are detected by simply visualizing the colored label.

[0256] D. Nucleotides, expression, vectors, and host cells A nucleic acid comprising a nucleotide sequence encoding any of the CARs, antibodies or their antigen-binding portions (including their functional portions and functional variants) described herein is further provided by one embodiment of the present invention. The nucleic acid of the present invention may comprise a nucleotide sequence encoding any of the leader sequences, antigen-binding domains, transmembrane domains and / or intracellular T cell signaling domains described herein.

[0257] In some embodiments, nucleotide sequences may be codon-modified. While not bound by any particular theory, codon optimization of nucleotide sequences is thought to increase the translation efficiency of mRNA transcripts. Codon optimization of nucleotide sequences may involve replacing native codons with other codons that encode the same amino acid but can be translated by tRNA that is more readily available in the cell, thereby increasing translation efficiency. Optimization of nucleotide sequences may also reduce secondary mRNA structures that interfere with translation, thereby increasing translation efficiency.

[0258] In embodiments of the present invention, the nucleic acid may comprise a codon-modified nucleotide sequence encoding the antigen-binding domain of the CAR of the present invention. In another embodiment of the present invention, the nucleic acid may comprise a codon-modified nucleotide sequence encoding any of the CARs (including their functional portions and functional variants) described herein.

[0259] As used herein, “nucleic acid” includes “polynucleotide,” “oligonucleotide,” and “nucleic acid molecule,” and generally refers to a polymer of DNA or RNA that may be single-stranded or double-stranded, synthetic or derived from natural sources (e.g., isolated and / or purified), may contain natural, unnatural or modified nucleotides, and may contain natural, unnatural or modified internucleotide linkages, such as phosphoramidate linkages or phosphorothioate linkages, instead of phosphodiesters found between nucleotides in unmodified oligonucleotides. In some embodiments, the nucleic acid does not contain any insertions, deletions, inversions, and / or substitutions. However, in some cases, as discussed herein, the nucleic acid may appropriately contain one or more insertions, deletions, inversions, and / or substitutions.

[0260] Recombinant nucleic acids may have sequences that do not exist in nature, or sequences that are created by artificially combining two or otherwise separated segments of a sequence. This artificial combination is often achieved by chemical synthesis, or more generally, by artificially manipulating isolated segments of nucleic acids using genetic engineering techniques, such as those described above by Sambrook et al. Nucleic acids can be constructed based on chemical synthesis and / or enzymatic ligation reactions using procedures known in the art. See, for example, Sambrook et al., above, and Ausubel et al., above. For example, nucleic acids may be naturally occurring nucleotides, or molecules designed to increase the biological stability of a molecule. Alternatively, nucleic acids can be chemically synthesized using various modified nucleotides (e.g., phosphorothioate derivatives and acridine-substituted nucleotides) designed to increase the physical stability of the double helix formed during hybridization. Examples of modified nucleotides that can be used to produce nucleic acids include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-substituted adenine, 7-methylguanine This includes, but is not limited to, nin, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid(v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleic acids of the present invention can be purchased from companies such as Integrated DNA Technologies (Coralville, IA, USA).

[0261] Nucleic acids may include any isolated or purified nucleotide sequences encoding either a CAR or a functional portion or functional variant thereof. Alternatively, a nucleotide sequence may include a nucleotide sequence that is degenerate with any of the sequences, or a combination of degenerate sequences.

[0262] One embodiment also provides isolated or purified nucleic acids comprising a nucleotide sequence complementary to any of the nucleotide sequences of the nucleic acids described herein, or a nucleotide sequence that hybridizes with any of the nucleotide sequences of the nucleic acids described herein under stringent conditions.

[0263] Nucleotide sequences that hybridize under stringent conditions may also hybridize under high stringency conditions. “High stringency conditions” means that the nucleotide sequence hybridizes specifically to a target sequence (a nucleotide sequence of any of the nucleic acids described herein) in a detectably strong amount compared to nonspecific hybridization. High stringency conditions include conditions that allow for the identification of polynucleotides with precisely complementary sequences, or polynucleotides containing only a few scattered mismatches, from random sequences that coincidentally have several subregions (e.g., 3–10 bases) that match the nucleotide sequence. These complementary subregions are more readily melted than full-length complements of 14–17 or more bases, and high stringency hybridization makes them readily distinguishable. Relatively high stringency conditions include low-salt and / or high-temperature conditions, such as those provided at a temperature of about 50–70°C with about 0.02–0.1 M NaCl or equivalent. Such highly stringent conditions tolerate little to no mismatch between the nucleotide sequence and the template or target strand, if any, and are particularly suitable for detecting the expression of any of the CARs of the present invention. Generally, it is understood that the conditions can be made more stringent by the addition of gradually increasing amounts of formamide.

[0264] Also provided are nucleic acids having nucleotide sequences that are identical to any of the nucleic acids described herein by at least about 70% or more, for example, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%.

[0265] In one embodiment, nucleic acids may be incorporated into a recombinant expression vector. In this regard, one embodiment provides a recombinant expression vector comprising any of the nucleic acids. For the purposes of this specification, the term “recombinant expression vector” means a genetically modified oligonucleotide or polynucleotide construct in which the construct comprises a nucleotide sequence encoding mRNA, protein, polypeptide, or peptide, and the vector enables the expression of mRNA, protein, polypeptide, or peptide by a host cell when the vector is brought into contact with the cell under conditions sufficient for the mRNA, protein, polypeptide, or peptide to be expressed in the cell. The vector as a whole does not exist in nature.

[0266] However, some vector components may be naturally occurring. Recombinant expression vectors may be single-stranded or double-stranded, synthetic or partially derived from natural sources, and may contain any type of nucleotide, including but not limited to DNA and RNA, which may contain natural, non-natural, or modified nucleotides. Recombinant expression vectors may contain naturally occurring or non-natural nucleotide linkages, or both. Preferably, non-natural or modified nucleotides or nucleotide linkages do not interfere with the transcription or replication of the vector.

[0267] In one embodiment, the recombinant expression vector can be any suitable recombinant expression vector and can be used to transform or transfect any suitable host cell. Suitable vectors include those designed for propagation and growth or for expression, or both, such as plasmids and viruses. The vector can be selected from the group consisting of the pUC series (Fermentas Life Sciences, Glen Burnie, MD), the pBluescript series (Stratagene, LaJolla, CA), the pET series (Novagen, Madison, WI), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, CA).

[0268] Bacteriophage vectors, such as, for example, λUTI 0, λUTI 1, λZapII (Stratagene), EMBL4, and λNMI 149, can also be used. Examples of plant expression vectors include pBIOl, pBI101.2, pBHOl.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). The recombinant expression vector can be a viral vector, such as, for example, a retroviral vector or a lentiviral vector. Lentiviral vectors are vectors derived from at least a portion of the lentiviral genome, including, in particular, self-inactivating lentiviral vectors such as those provided by Milone et al., Mol. Ther. 17(8):1453-1464 (2009). Other examples of lentiviral vectors that can be used in the clinic include, for example, but not limited to, the LENTIVECTOR® gene delivery technology of Oxford BioMedica plc, the LENTIMAX™ vector system of Lentigen, and the like. Non-clinical lentiviral vectors are also available and are known to those skilled in the art.

[0269] Several transfection techniques are generally known in the art (see, e.g., Graham et al., Virology, 52:456-467 (1973); Sambr ook et al., supra; Davis et al., Basic Methods in Molecular Biology, Elsevier (1986); and Chu et al., Gene, 13:97 (1981)).

[0270] Transfection methods include calcium phosphate co-precipitation (see, e.g., Graham et al., supra), direct microinjection into cultured cells (see, e.g., Capecchi, Cell, 22:479-488 (1980)), electroporation (see, e.g., Shigekawa et al., BioTechniques, 6:742-751 (1988)), liposome-mediated gene transfer (see, e.g., Mannino et al., BioTechniques, 6:682-690 (1988)), lipid-mediated transfection (see, e.g., Feigner et al., Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)) and nucleic acid delivery using high velocity microprojectiles (see, e.g., Klein et al., Nature, 327:70-73 (1987)).

[0271] In one embodiment, the recombinant expression vector can be prepared using standard recombinant DNA techniques described, e.g., in Sambrook et al., supra and Ausubel et al., supra. Expression vector constructs, which are circular or linear, can be prepared to contain a replication system functional in prokaryotic or eukaryotic host cells. The replication system can be derived from, e.g., ColE1, 2μ plasmid, λ, SV40, bovine papilloma virus, etc.

[0272] Recombinant expression vectors, taking into account whether the vector is DNA-based or RNA-based, may, if necessary, include regulatory sequences specific to the type of host cell into which the vector is introduced (e.g., bacteria, fungi, plants, or animals), such as transcription and translation start and termination codons. Recombinant expression vectors may also include restriction sites to facilitate cloning.

[0273] Recombinant expression vectors may contain one or more marker genes that allow for the selection of transformed or transfected host cells. Marker genes may include those for biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., and nutritional complementation in the host to provide protrophotrophy. Suitable marker genes for the 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.

[0274] Recombinant expression vectors may include native or non-native promoters operably ligated to nucleotide sequences encoding CARs (including their functional portions and functional variants), or to nucleotide sequences complementary to or hybridizing to CAR-encoding nucleotide sequences. The choice of promoter, e.g., strong, weak, inducible, tissue-specific, and developmentally specific, is within the scope of the art. Similarly, combining nucleotide sequences with promoters is also within the scope of the art. Promoter may be a non-viral promoter or a viral promoter, e.g., a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, or a promoter found in the terminal repeat sequences of mouse stem cell viruses.

[0275] Recombinant expression vectors can be designed for transient expression, stable expression, or both. Furthermore, recombinant expression vectors can be constructed for constitutive or inducible expression.

[0276] Furthermore, recombinant expression vectors can be constructed to contain suicide genes. When used, the term "suicide gene" refers to a gene that causes the death of cells that express it. A suicide gene may be a gene that confers sensitivity to drugs or other agents to cells that express it, or a gene that causes cells to die when they come into contact with or are exposed to a drug. Suicide genes are well known in the field (see, for example, Suicide Gene Therapy: Methods and Reviews, Springer, Caroline J. (Cancer Research UK Centre for Cancer Therapeutics at the Institute of Cancer Research, Sutton, Surrey, UK), Humana Press, 2004), and include, for example, the herpes simplex virus (HSV) thymidine kinase (TK) gene, cytosine deaminase (daminase), purine nucleoside phosphorylase, and nitroreductase.

[0277] One embodiment further provides a host cell containing any of the recombinant expression vectors described herein. As used herein, the term “host cell” means any type of cell that may contain the recombinant expression vectors of the present invention. The host cell may be a eukaryotic cell, e.g., a plant, animal, fungus, or algae, or a prokaryotic cell, e.g., a bacterium or protist. The host cell may be a cultured cell or a primary cell, i.e., it may be isolated directly from an organism such as a human. The host cell may be an adherent cell or a suspension cell, i.e., a cell that grows in a suspension. Suitable host cells are known in the art and include, for example, DH5a E. coli cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, etc. If the purpose is to amplify or replicate the recombinant expression vector, the host cell may be a prokaryotic cell, e.g., a DH5a cell. If the purpose is to produce recombinant CAR, the host cell may be a mammalian cell. The host cell may be a human cell. The host cell can be any cell type, may originate from any type of tissue, and may be at any developmental stage, but the host cell may be a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC). The host cell may be a T cell.

[0278] For the purposes of this specification, T cells may be any T cells, e.g., cultured T cells, e.g., primary T cells, or cultured T cell lines, e.g., T cells derived from Jurkat, SupTl, etc., or T cells obtained from mammals. When obtained from mammals, T cells may be obtained from a number of sources, including but not limited to blood, bone marrow, lymph nodes, thymus, or other tissues or body fluids. T cells may also be enriched or purified. T cells may be human T cells. T cells may be T cells isolated from humans. T cells may be any type of T cell, including but not limited to CD4+ / CD8+ double-positive T cells, CD4+ helper T cells, e.g., Th1 and Th2 cells, CD8+ T cells (e.g., cytotoxic T cells), tumor-infiltrating cells, memory T cells, memory stem cells, i.e., Tscm naive T cells, etc., and may be of any developmental stage. T cells may be CD8+ T cells or CD4+ T cells.

[0279] In one embodiment, the CAR described herein may be used in appropriate non-T cells. Such cells are cells with immune effector function, such as NK cells and T-like cells generated from pluripotent stem cells.

[0280] A population of cells comprising at least one host cell described herein is also provided by one embodiment. The population of cells may be a heterogeneous population comprising at least one other cell, e.g., a host cell (e.g., a T cell), or a cell other than a T cell, e.g., a B cell, macrophage, neutrophil, erythrocyte, hepatocyte, endothelial cell, epithelial cell, muscle cell, brain cell, etc., in addition to a host cell comprising any of the described recombinant expression vectors. Alternatively, the population of cells may be a substantially homogeneous population, where The population mainly comprises host cells containing (e.g., essentially derived from) recombinant expression vectors. The population can also be a clonal population of cells, where all cells in the population are clones of a single host cell containing a recombinant expression vector, and consequently all cells in the population contain that recombinant expression vector. In one embodiment of the present invention, the population of cells is a clonal population comprising host cells containing the recombinant expression vectors described herein.

[0281] CARs (including their functional parts and variants), nucleic acids, recombinant expression vectors, host cells (including their populations), and antibodies (including their antigen-binding parts) can be isolated and / or purified. For example, a purified (or isolated) host cell preparation is a preparation in which the host cells are purer than those in their natural environment within the body. Such host cells can be produced, for example, by standard purification techniques. In some embodiments, the host cell preparation is purified so that the host cells exhibit at least about 50% of the total cell content of the preparation, for example, at least about 70%. For example, purity can be at least about 50%, and can be above about 60%, about 70%, or about 80%, or it can be about 100%.

[0282] E. Treatment Method The CARs disclosed herein are intended to be used in methods for treating or preventing disease in mammals. In this regard, one embodiment provides a method for treating or preventing cancer in a mammal, comprising the step of administering to a mammal an effective amount for treating or preventing cancer in the mammal, a CAR, a nucleic acid, a recombinant expression vector, a host cell, a population of cells, an antibody and / or its antigen-binding moiety, and / or a pharmaceutical composition.

[0283] One embodiment further includes a step of lymphodepleting the mammal prior to the step of administering the CAR disclosed herein. Examples of lymphodepletion may include, but are not limited to, non-myeloablative lymphodepletion chemotherapy, myeloablative lymphodepletion chemotherapy, and total body irradiation.

[0284] For methods of administering host cells or populations of cells, the cells may be homogeneous or autologous to the mammal. Preferably, the cells are autologous to the mammal. As used herein, homogeneous means any material originating from different animals of the same species as the individual into which the material is introduced. Two or more individuals are said to be homogeneous if their genes are not identical at one or more loci. In some embodiments, homogeneous material from individuals of the same species may be genetically distinct enough to interact antigenically. As used herein, autologous means any material originating from the same individual that is subsequently reintroduced into the individual.

[0285] Mammals referred to herein may be any mammal. As used herein, the term “mammal” means any mammal, including but not limited to rodents, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits. Mammals may be of Carnivora origin, including felines (cats) and canines (dogs). Mammals may be of Artiodactyla origin, including bovines (cows) and swines (pigs), or Perissodactyla origin, including equines (horses). Mammals may be of Primates, New World Cebioids or Simoids (monkeys) or Anthropoids (humans and apes). Preferably, the mammal is human.

[0286] Regarding these methods, cancer includes acute lymphoblastic carcinoma, acute myeloid leukemia, and alveolar striated muscle cancer. Sarcoma, bladder cancer (e.g., bladder cancer), bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, cancer of the anus, anal canal or anorectum, eye cancer, intrahepatic bile duct cancer, joint cancer, cancer of the neck, gallbladder or pleura, cancer of the nose, nasal cavity or middle ear, oral cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid cancer It may be any cancer, including any of the following: cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, head and neck cancer (e.g., head and neck squamous cell carcinoma), Hodgkin lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, leukemia, humoral neoplasm, 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 lymphoma, B-chronic lymphocytic leukemia, hairy cell leukemia, acute lymphoblastic leukemia (ALL) and Burkitt lymphoma, ovarian cancer, pancreatic cancer, cancer of the peritoneum, retinoplasm and mesentery, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumor, synovial sarcoma, gastric cancer, testicular cancer, thyroid cancer and ureteral cancer.

[0287] The terms “treat” and “prevent,” and words derived therefrom, as used herein, do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention that a person skilled in the art would recognize as having a potentially beneficial or therapeutic effect. In this regard, this method may provide treatment or prevention of cancer in mammals of any amount or level.

[0288] Furthermore, the treatment or prevention provided by this method may include the treatment or prevention of one or more conditions or symptoms of a disease, such as cancer, being treated or prevented. Also, for the purposes of this specification, “prevention” may include delaying the onset of the disease, or its symptoms or conditions.

[0289] Another embodiment is a method for detecting the presence of cancer in a mammal, comprising: (a) contacting a sample containing one or more cells derived from a mammal with a CAR, nucleic acid, recombinant expression vector, host cell, population of cells, antibody and / or its antigen-binding portion, or pharmaceutical composition, thereby forming a complex; and (b) detecting the complex, wherein detection of the complex indicates the presence of cancer in the mammal.

[0290] The sample can be obtained by any suitable method, such as biopsy or autopsy. A biopsy is the removal of tissue and / or cells from an individual. Such removal can be the collection of tissue and / or cells from an individual for performing experiments on the removed tissue and / or cells. These experiments can include experiments for determining whether an individual has a particular condition or disease state and / or is suffering from a particular condition or disease state. The condition or disease can be, for example, cancer.

[0291] Regarding embodiments of a method for detecting the presence of a growth disorder, such as cancer, in a mammal, a sample containing mammalian cells can be a sample containing whole cells, their lysates, or fractions of whole cell lysates, such as nuclear or cytoplasmic fractions, whole protein fractions, or nucleic acid fractions. When the sample contains whole cells, these cells can be cells of any organ or tissue containing any mammalian cells, such as blood cells or endothelial cells.

[0292] The contacting step can occur in vitro or in vivo with respect to the mammal. Preferably, the contacting step is in vitro.

[0293] Also, detection of the complex can be performed by many methods known in the art. For example, the CAR, polypeptide, protein, nucleic acid, recombinant expression vector, host cell, population of cells, or antibody and / or its antigen-binding portion disclosed herein in this specification They may be labeled with detectable labels, such as the radioactive isotopes disclosed above, fluorophores (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), enzymes (e.g., alkaline phosphatase, horseradish peroxidase), and elemental particles (e.g., gold particles).

[0294] Methods for testing CARs for their ability to recognize target cells and antigen specificity are well known in the field. For example, Clay et al., J.Immunol, Vol. 163: pp. 507-513 (1999) teach a method for measuring the release of cytokines (e.g., interferon-γ, granulocyte / monocyte colony stimulating factor (GM-CSF), tumor necrosis factor α (TNF-α), or interleukin-2 (IL-2)). Furthermore, CAR function can be evaluated by measuring cytotoxicity, as described by Zhao et al., J.Immunol, Vol. 174: pp. 4415-4423 (2005).

[0295] Another embodiment provides the use of the CARs, nucleic acids, recombinant expression vectors, host cells, cell populations, antibodies or their antigen-binding moieties, and / or pharmaceutical compositions of the present invention to treat or prevent proliferative disorders such as cancer in mammals. Cancer may be any of the cancers described herein.

[0296] Any method of administration, including topical and systemic administration, may be used for the disclosed therapeutic agent. For example, topical, oral, intravascular, intramuscular, intraperitoneal, intranasal, intradermal, subarachnoid, and subcutaneous administration may be used. The specific mode of administration and drug regimen will be selected by the attending clinician, taking into account the characteristics of the case (e.g., the subject, the disease, the disease state involved, and whether the treatment is prophylactic). If more than one drug or composition is administered, one or more routes of administration may be used; for example, chemotherapeutic agents may be administered orally, and antibodies or antigen-binding fragments or conjugates or compositions may be administered intravenously. Methods of administration include injections in which CARs, CAR T cells, conjugates, antibodies, antigen-binding fragments or compositions are delivered in a non-toxic, pharmaceutically acceptable carrier, such as water, saline, Ringer's solution, dextrose solution, 5% human serum albumin, solid oil, ethyl oleate, or liposomes. In some embodiments, topical administration of the disclosed compound may be used, for example, by applying an antibody or antigen-binding fragment to an area of ​​tissue from which a tumor has been removed, or to an area suspected of being prone to tumor development. In some embodiments, sustained intratumoral (or near-tumor) release of a pharmaceutical preparation containing a therapeutically effective amount of antibody or antigen-binding fragment may be beneficial. In other examples, the conjugate may be applied topically as eye drops to the cornea, or intravitreally to the eye.

[0297] The disclosed therapeutic agents can be formulated in unit dosage forms appropriate for individual administrations of precise dosages. Furthermore, the disclosed therapeutic agents can be administered in single doses or in a multi-dose schedule. A multi-dose schedule is one in which the main course of treatment may consist of more than one separate dose, e.g., 1 to 10 doses, followed by other doses given at subsequent time intervals as needed to maintain or enhance the effect of the composition. Treatment may involve a daily dose or multiple daily doses of the compound over a period of several days to several months or even several years. Thus, the dosing regimen is also determined at least in part based on the specific requirements of the subject being treated and depends on the judgment of the practitioner administering it.

[0298] Typical dosages of antibodies or conjugates can range from approximately 0.01 to approximately 30 mg / kg, for example, from approximately 0.1 to approximately 10 mg / kg.

[0299] In certain cases, the subjects may be given multiple daily medication schedules, for example, at least two consecutive days, ten consecutive days, etc., over a period of several weeks, months, or years, such as conjugation. A therapeutic composition comprising one or more of the following is administered: a conjugate, antibody, composition, CAR, CAR T cells, or further agents. In one example, the subject is administered the conjugate, antibody, composition, or further agent for a period of 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.

[0300] In some embodiments, the disclosed methods include providing surgical, radiotherapy, and / or chemotherapeutic agents to a target (e.g., sequentially, substantially simultaneously, or concurrently) in combination with disclosed antibodies, antigen-binding fragments, conjugates, CARs, or CAR-expressing T cells. Such agents and methods of treatment and therapeutic dosages are known to those skilled in the art and can be determined by a skilled clinician. Preparation and dosing schedules for further agents may be used according to the manufacturer's instructions or may be determined experimentally by those skilled in the art. Preparation and dosing schedules for such chemotherapeutic agents are also described in Chemotherapy Service, (1992), edited by MCPerry, Williams & Wilkins, Baltimore, Md.

[0301] In some embodiments, combination therapy may involve administering a therapeutically effective dose of an additional cancer inhibitor to the subject. Non-limiting examples of additional therapeutic agents that may be used in combination therapy include microtubule-binding agents, DNA intercalators or crosslinkers, DNA synthesis inhibitors, DNA and RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, gene regulators, and angiogenesis inhibitors. These agents (administered in therapeutically effective doses) and treatments may be used alone or in combination. For example, any suitable anticancer or anti-angiogenic agent may be administered in combination with CARs, CAR-T cells, antibodies, antigen-binding fragments, or conjugates disclosed herein. Methods and therapeutic doses of such agents are known to those skilled in the art and may be determined by a skilled clinician.

[0302] Further chemotherapeutic agents include alkylating agents, e.g., nitrogen mustards (e.g., chlorambucil, chlormethine, cyclophosphamide, ifosfamide, and melphalan), nitrosoureas (e.g., carmustine, fotemustine, lomustine, and streptozocin), platinum compounds (e.g., carboplatin, cisplatin, oxaliplatin, and BBR3464), busulfan, dacarbazine, mechloretamine, procarbazine, temozolomide, thiotepa, and uramustine; antimetabolites Drugs, e.g., folic acid (e.g., methotrexate, pemetrexed, and larcitrexed), purines (e.g., cladribine, clofarabine, fludarabine, mercaptopurine, and thioguanine), pyrimidines (e.g., capecitabine), cytarabine, fluorouracil, and gemcitabine); plant alkaloids, e.g., podophyllum (e.g., etoposide and teniposide), taxanes (e.g., docetaxel and paclitaxel), vinca (e.g., vinblastine, vincristine, vindesine, and vinorelbine); Cytotoxic / antitumor antibiotics, e.g., members of the anthracycline family (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, and barurubicin), bleomycin, rifampicin, hydroxyurea, and mitomycin; topoisomerase inhibitors, e.g., topotecan and irinotecan; monoclonal antibodies, e.g., alemtuzumab, bevacizumab, cetuximab, gemtuzumab, rituximab, panitumumab, pertuzumab, and trastuzumab; tumor affinity photosensitive dyes, For example, aminolevulinic acid, methyl aminolevulinic acid, porfimer sodium and verteporfin; as well as other drugs, for example, alitretinoin, altretamine, amsacrin, anagrelide, arsenic trioxide, asparaginase, axitinib, bexarotene, bevacizumab, bortezomib, celecoxib, denileukin difutitox, erlotinib, estramustine, gefitinib, hydroxycarbamide, imatinib, lapatinib, pazopanib, pentostatin, masopropyl alcohol, mitotane, pegaspargase, tammon This includes, but is not limited to, xifene, sorafenib, sunitinib, vemurafenib, vandetanib, and tretinoin. The selection and therapeutic dosage of such drugs are known to those skilled in the art and can be determined by a skilled clinician.

[0303] Combination therapy can provide and prove to be synergistic; that is, the effect achieved when active ingredients are used together is greater than the sum of the effects that could result from using those compounds separately. Synergistic effects can be achieved when active ingredients are (1) co-formulated and administered or delivered simultaneously with the unit dose formulations used in combination; (2) delivered alternately or in parallel as separate formulations; or (3) in part with other regimens. When delivered alternately, synergistic effects can be achieved when the compounds are administered or delivered sequentially, for example, by different injections in separate syringes. Generally, during alternation, effective doses of each active ingredient are administered sequentially, i.e., in order; however, in combination therapy, effective doses of two or more active ingredients are administered together.

[0304] In one embodiment, an effective amount of an antibody or antigen-binding fragment or conjugate that specifically binds to one or more of the antigens disclosed herein is administered to a subject with a tumor after anticancer treatment. After a sufficient amount of time has elapsed for the administered antibody or antigen-binding fragment or conjugate to form immune complexes with the antigens expressed on each cancer cell, the immune complexes are detected. The presence (or absence) of immune complexes indicates the effectiveness of the treatment. For example, an increase in immune complexes compared to a control taken before the treatment indicates that the treatment is ineffective, while a decrease in immune complexes compared to a control taken before the treatment indicates that the treatment is effective.

[0305] F. Biopharmaceutical Compositions Provided herein are biopharmaceutical compositions or biologic compositions (hereinafter referred to as "Compositions") for use in gene therapy, immunotherapy and / or cell therapy, comprising, in a carrier (e.g., a pharmaceutically acceptable carrier), one or more of the disclosed CARs, or CAR-expressing T cells, antibodies, antigen-binding fragments, conjugates, or CAR-expressing T cells that specifically bind to one or more antigens disclosed herein. These compositions may be prepared in unit dose forms for administration to a subject. The amount and timing of administration to achieve the desired outcome is at the discretion of the clinician performing the treatment. These compositions may be formulated for systemic (e.g., intravenous) or topical (e.g., intratumoral) administration. In one example, the disclosed CARs, or CAR-expressing T cells, antibodies, antigen-binding fragments, conjugates, etc., may be formulated for parenteral administration, such as intravenous administration. Compositions comprising the CARs, or CAR-expressing T cells, conjugates, antibodies, or antigen-binding fragments disclosed herein may be used for the treatment and detection of tumors, such as, but not limited to, neuroblastoma. In some cases, these compositions are useful for treating or detecting cancer. Compositions comprising CARs, or CAR-expressing T cells, conjugates, antibodies, or antigen-binding fragments disclosed herein, can also be used, for example, for the detection of pathological angiogenesis.

[0306] The compositions for administration may include a solution of CAR, or CAR-expressing T cells, conjugates, antibodies, or antigen-binding fragments dissolved in a pharmaceutically acceptable carrier such as an aqueous carrier. Various aqueous carriers, such as buffered saline, may be used. These solutions are sterile and generally do not contain undesirable substances. The compositions can be sterilized by conventional, well-known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusters and buffers, toxicity modifiers, and adjuvants, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate. The concentrations of CAR, or CAR-expressing T cells, antibodies, or antigen-binding fragments or conjugates in these formulations can vary widely and are selected for specific characteristics. The dosage form is selected primarily based on volume, viscosity, body weight, etc., according to the requirements of the prescribed administration method and the target population. Practical methods for preparing such dosage forms for use in gene therapy, immunotherapy and / or cell therapy are known or will become apparent to those skilled in the art.

[0307] Typical compositions for intravenous administration contain approximately 0.01 to 30 mg / kg of antibody or antigen-binding fragment or conjugate per subject per day (or a corresponding dose of CAR, or a conjugate containing CAR-expressing T cells, antibody or antigen-binding fragment). Practical methods for preparing administerable compositions are known or obvious to those skilled in the art and are described in more detail in publications such as Remington's Pharmaceutical Science, 19th edition, Mack Publishing Company, Easton, PA (1995).

[0308] CARs, or CAR-expressing T cells, antibodies, antigen-binding fragments, or conjugates, may be supplied in lyophilized form and rehydrated with sterile water before administration, or they may also be supplied in sterile solutions of known concentrations. The solution of CARs, or CAR-expressing T cells, antibodies, or antigen-binding fragments or conjugates, is then added to an infusion bag containing 0.9% sodium chloride, USP, and administered in some cases at doses of 0.5–15 mg / kg body weight. Considerable experience in the administration of antibody or antigen-binding fragments and conjugate drugs is available in this field; for example, antibody drugs have been commercially available in the United States since the approval of Rituxan® in 1997. CARs, or CAR-expressing T cells, antibodies, antigen-binding fragments, and their conjugates may be administered by slow infusion rather than by intravenous injection or intravenous bolus. In one example, a higher loading dose is administered along with a subsequent maintenance dose administered at a lower level. For example, an initial loading dose of 4 mg / kg of antibody or antigen-binding fragment (or a conjugate containing the antibody or antigen-binding fragment at a corresponding dose) may be infused over a period of approximately 90 minutes, followed by a weekly maintenance dose of 2 mg / kg over 30 minutes for 4 to 8 weeks, provided that the previous dose was well tolerated.

[0309] Controlled-release parenteral formulations can be prepared as implants, oily injections, or granular 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). Granular systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain a therapeutic protein, such as a cytotoxin or drug, as a central core. In microspheres, the therapeutic agent is dispersed throughout the particle. Particles smaller than about 1 μm, microspheres and microcapsules, are generally called nanoparticles, nanospheres, and nanocapsules, respectively. Capillaries have a diameter of about 5 μm so that only nanoparticles are administered intravenously. Microparticles are typically about 100 μm in diameter and are administered subcutaneously or intramuscularly. See, for example, Kreuter, J., Colloidal Drug Delivery Systems, edited by J. Kreuter, Marcel Dekker, Inc., New York, NY, pp. 219–342 (1994); and Tice and Tabibi, Treatise on Controlled Drug Delivery, edited by A. Kydonieus, Marcel Dekker, Inc., New York, NY, pp. 315–339 (1992).

[0310] The polymers may be used for the ion-controlled release of CARs, or CAR-expressing T cells, antibodies, or antigen-binding fragments or conjugate compositions disclosed herein. Various degradable and non-degradable polymer matrices are used for controlled drug delivery. The hydroxyapatite is well known in this field (Langer, Accounts Chem. Res. Vol. 26: pp. 537-542, 1993). For example, the block copolymer polaxamer 407 exists as a viscous but mobile liquid at low temperatures, but forms a semi-fluid gel at body temperature. It has been shown to be an effective vehicle for the formulation and sustained delivery of recombinant interleukin-2 and urease (Johnston et al., Pharm. Res. Vol. 9: pp. 425-434, 1992; and Pec et al., J.Parent. Sci. Tech. Vol. 44 (No. 2): pp. 58-65, 1990). Alternatively, hydroxyapatite has been used as a microcarrier for the controlled release of proteins (Ijntema et al., Int. J. Pharm. Vol. 112: pp. 215-224, 1994). In yet another embodiment, liposomes are used for the controlled release and drug targeting of lipid-encapsulated drugs (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA (1993)). Numerous further systems for the controlled delivery of therapeutic proteins are known (see U.S. Patent Nos. 5,055,303; 5,188,837; 4,235,871; 4,501,728; 4,837,028; 4,957,735; 5,019,369; 5,055,303; 5,514,670; 5,413,797; 5,268,164; 5,004,697; 4,902,505; 5,506,206; 5,271,961; 5,254,342 and 5,534,496).

[0311] G. Kitt In one embodiment, kits using the CARs disclosed herein are also provided. For example, a kit for treating a tumor in a subject or a kit for producing CAR T cells expressing one or more of the CARs disclosed herein. The kit typically includes, as disclosed herein, a disclosed antibody, antigen-binding fragment, conjugate, nucleic acid molecule, CAR, or CAR-expressing T cells. More than one of the disclosed antibodies, antigen-binding fragment, conjugate, nucleic acid molecule, CAR, or CAR-expressing T cells may be included in the kit.

[0312] The kit may include a container and labels or accompanying documents on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. Containers may be formed from a variety of materials, such as glass or plastic. Typically, a container holds a composition comprising one or more of the disclosed antibodies, antigen-binding fragments, conjugates, nucleic acid molecules, CARs, or CAR-expressing T cells. In some embodiments, the container may have a sterile access port (for example, the container may be an intravenous solution bag or vial with a stopper that can be pierced by a subcutaneous needle). Labels or accompanying documents indicate that the composition is used to treat a particular condition.

[0313] Labels or accompanying documents typically further include instructions for the use of disclosed antibodies, antigen-binding fragments, conjugates, nucleic acid molecules, CARs, or CAR-expressing T cells in, for example, methods for treating or preventing tumors or methods for producing CAR T cells. Accompanying documents typically include instructions habitually included in the market packaging of a therapeutic product, including information on indications, usage, dosage, administration, contraindications, and / or warnings relating to the use of the therapeutic product. Teaching materials may be written in electronic form (e.g., computer diskette or compact disk) or visual (e.g., video files). Kits may also include further components to facilitate the specific application for which the kit is designed. Thus, for example, a kit may include means for detecting a label (e.g., yeast The kit may further include an enzyme substrate for primary labeling, a filter set for detecting the fluorescent label, and appropriate secondary labels such as a secondary antibody. The kit may further include buffers and other reagents conventionally used for carrying out a particular method. Such kits and suitable contents are well known to those skilled in the art. [Examples]

[0314] The present invention is further illustrated by the following embodiments, which should not be construed as limiting the scope of the invention. On the contrary, it will be readily apparent that various other embodiments, modifications, and equivalents can be conceived after reading this specification without departing from the spirit of the invention and / or the appended claims, and these may come to the mind of those skilled in the art.

[0315] Example 1 Isolation of human CD123-specific antibodies from a fully human yeast display ScFv library. material and method: The anti-human CD123 antibodies described herein were isolated using a large yeast display human naive single-chain variable fragment (ScFv) antibody library. The library was constructed using a collection of human antibody gene repertoire from more than 60 individuals. Three rounds of magnetically activated cell sorting (MACS) were performed to enrich the human ScFv binder to recombinant human CD123-Fc. In the first round of yeast library panning, the yeast display ScFv library (5x10) was performed. 10 The cells were incubated with 5 μg / mL CD123-Fc in 15 ml PBSA (consisting of 0.1% bovine serum albumin (BSA) in Dulbecco's phosphate-buffered saline (PBS) buffer) on a rotator at room temperature for 1.5 hours. After washing twice with 25 ml PBSA, the yeast library mix was incubated with 100 μL of protein G microbeads (Miltenyi Biotec) on a rotator at room temperature for 30 minutes. After washing once, the library mix was resuspended in 50 ml PBSA and loaded onto a MACS cell isolation column (LS column). After washing three times with 10 ml PBSA, the yeast presented on the column with the ScFv binder was then eluted twice with 2 ml PBSA. These eluted yeast cells were combined and resuspended in 50 ml of SDCAA medium (1 L water, 20 g D-glucose, 6.7 g amino acid-free BD Difco™ yeast nitrogen base, 5 g Bacto™ casamino acid, 5.4 g Na2.HPO4, 8.56 g NaH2PO4.H2O) and amplified at 225 rpm, 30°C for 20 hours with shaking. The amplified pool was then induced in SGCAA medium (same composition as SDCAA medium, but with galactose instead of glucose) and shaken for a further 16 hours at 225 rpm, 30°C before being used for panning in the next round. The same process was repeated two more times to enrich the CD123-Fc-specific binder.

[0316] To further enrich the binder with higher affinity and better specificity, the strongest binder was isolated from the pool using FACS-based sorting. The induced pool was incubated with 1 μg / ml CD123-Fc at room temperature for 1 hour, then stained with Anti-c-Myc-Alexa 488 and goat anti-Hu-Fc PE conjugate, and the top 1% pool with the best PE vs. FITC signal was gated and sorted. The sorted pool was amplified in SDCAA medium, yeast plasmid DNA was extracted, and the cells were transformed into bacteria for single-clonal DNA sequencing. Forty random clones were sequenced to identify 36 unique sequences. Fifteen CD123 ScFv clones were created as M12301, M12303, M12304, M12305, M12306, M12308, M12309, M12310, M12311, M12313, M12314, M12315, M12316, M12317, and M12318, respectively. As specified, and as shown in Example 2 and Table 1, the CAR constructs were cloned for CAR-T functional screening.

[0317] Example 2 Generation of a CD123-targeting CAR T construct incorporating a fully human binder ScFv sequence derived from a yeast display library. There are few treatment options for AML, and treatment-related toxicity and disease relapse after treatment are common. Furthermore, immunotherapies using non-human sequences, such as mouse-derived antibodies, can lead to treatment refusal or side effects in patients. To develop a novel CAR T treatment method for AML, we designed 15 CD123-targeting CAR T constructs incorporating a fully human ScFv-targeting domain and evaluated their antitumor activity.

[0318] material and method: (a) Cell line AML cell lines MOLM-14 and Kg-1a are each classified as DSMZ (Leibniz). The cells were purchased from the Institute DSMZ (Braunschwieg, Germany) and the American Tissue Culture Collection (ATCC, Manassass, VA). The human fetal kidney line 293T was purchased from ATCC (Gibco / Thermo Fisher Scientific, Grand Island, NY). Single-cell clones of luciferase-expressing cell lines were generated by stably transducing wild-type tumor lines with a lentiviral vector encoding firefly luciferase (Lentigen). Whole blood was collected from healthy volunteers at the Oklahoma Blood Institute (OBI) with the written consent of the donors. Processed buffy coat was purchased from OBI (Oklahoma City, OK). CD4-positive and CD8-positive human T cells were purified from the buffy coat by positive selection using a 1:1 mixture of CD4 and CD8-MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's protocol.

[0319] (b) Creation of chimeric antigen receptors (CARs) - expression vectors The antigen-binding domain, scFv, and sequence of the CAR were derived from human anti-CD123 ScFv. The CAR T construct was generated by ligating the binder sequence in frame to the CD8a ligation and transmembrane domain (aa123-191, Ref sequence ID NP001759.3), followed by the 4-1BB (CD137, aa214-255, UniProt sequence ID Q07011) signaling domain and the CD3 zeta signaling domain (CD247, aa52-163, Ref sequence ID: NP_000725.1). The CAR construct sequence was cloned into a third-generation lentiviral plasmid backbone (Lentigen Technology Inc., Gaithersburg, MD). Transient transfection of HEK293 T cells generated supernatant containing the lentiviral vector (LV), and the supernatant containing the lentiviral vector was centrifugated to pellet the vector, which was stored at -80°C.

[0320] (c) Purification and transduction of primary T cells Primary human T cells from healthy volunteers were purified from whole blood or buffy coat (purchased from a commercial provider with the donor's written consent) using immunomagnetic bead selection of CD4+ and CD8+ cells, according to the manufacturer's protocol (Miltenyi Biotec, Bergisch Gladbach, Germany). The T cells were then selected at a density of 0.3–2 x 10⁶. 6 Cells were cultured at a concentration of 1 / ml in TexMACS® medium supplemented with 200 IU / ml IL-2, activated with CD3 / CD28MACS® GMP T cell TransAct reagent (Miltenyi Biotec), and 1 on day 2. Lentiviral vectors encoding the CAR construct were transduced overnight in the presence of 0 μg / ml protamine sulfate (Sigma-Aldrich, St. Louis, MO), and the culture medium was changed on day 3. The cultures were grown in TexMACS® medium supplemented with 200 IU / ml IL-2 until harvesting on days 8-13.

[0321] (d) Immunoeffector assays (CTLs and cytokines) To determine cell-mediated cytotoxicity (CTL assay), 5,000 target cells stably transduced with firefly luciferase were combined with CAR T cells in various effector-to-target ratios and incubated overnight. SteadyGlo reagent (Promega, Madison WI) was added to each well, and the resulting luminescence was quantified as counts per second (sample CPS). The assay range was determined using wells containing only targets (maximum CPS) and wells containing only targets with 1% Tween-20 added (minimum CPS). The percentage of specific lysis was calculated as (1 - (sample CPS - minimum CPS) / (maximum CPS - minimum CPS)). The supernatant from co-cultures with an E:T ratio of 10:1 was removed, and IFNγ and TNFα concentrations were analyzed by ELISA (eBioscience, San Diego, CA).

[0322] (e) Flow cytometry analysis of CAR surface expression For cell staining, 500,000 CAR T transduced cells were isolated from the culture and washed twice with cold AutoMACS® buffer supplemented with 0.5% bovine serum albumin (Miltenyi Biotec). CAR surface expression was detected by staining with protein L-biotin, followed by streptavidin-PE conjugate (Jackson ImmunoResearch, West Grove, PA). Anti-CD4 antibody conjugated with VioBlue® fluorophore (Miltenyi Biotec) was used as needed, according to the vendor's protocol. Non-transduced cells were used as a negative control. In all studies, dead cells were excluded by 7AAD staining (BD Biosciences, San Jose, CA). Cells were washed twice and resuspended in 200 μl of staining buffer before quantitative analysis by flow cytometry. Flow cytometry analysis was performed using MACSQuant® 10 Analyzer (Miltenyi Biotec), and data plots were generated using FlowJo software (Ashland, OR).

[0323] result: This example describes the creation of CAR T cells that target the tumor antigen CD123 for the treatment of AML and other CD123+ malignancies.

[0324] Each CAR consisted of a human ScFv binder, a CD8 hinge and transmembrane domain, a 4-1BB costimulatory domain, and a CD3z activating domain (Figure 1).

[0325] Table 1 below details the CAR123 constructs that were developed and designated by their LTG numbers, and the ScFv used in each construct.

[0326] [Table 1]

[0327] Figure 1 shows an overview of the CD123 CAR design. A fully human ScFv binder targeting CD123 was ligated in-frame to the CD8 hinge and transmembrane domain, the 4-1BB costimulatory domain, and the CD3 zeta-activating domain. The CAR sequence was incorporated into a third-generation lentiviral vector and applied to primary human T cells for transduction.

[0328] The surface expression of anti-CD123 CARs incorporating single-chain fragment variable (ScFv) sequences is shown in Figure 2. Expression levels for each CAR containing ScFv were determined by flow cytometry analysis of healthy donor-derived LV transducible T cells using protein L-biotin followed by streptavidin-PE. A subset of ScFv-based anti-CD123 CAR constructs were detected by protein L and were highly expressed in human primary T cells compared to untransduced T cell controls (UTD) (Figure 2). These constructs included LTG##2074, 2075, 2076, 2078, 2079, 2084, and 2088.

[0329] As shown in Figure 3, the high cytolytic activity of CD123 CAR was demonstrated for a subset of the constructs analyzed. Untransduced T cells (UTDs) and GFP-transduced T cells (LTG1398) were used as negative controls for CAR cytolytic function.

[0330] Human primary T cells were transduced with LV encoding CAR constructs (see Methods) and then incubated for 18 hours with either the CD123+ tumor cell line MOLM-14 or the CD123-negative control cell line HEK293. For a luminescence-based in vitro cell death assay, firefly luciferase was stably transduced into each target cell line. MOLM-14 cells were effectively lysed by constructs LTG2075, 2076, 2078, 2079, 2082, 2083, 2085, 2087, and 2088. However, CAR LTG##2074, 2080, 2081, 2084, and 2086 were not effective. Ultimately, CD123+MOLM-14 tumor cells could not be lysed, which is consistent with the relatively low expression of CARs LTG2074, 2084, and 2087. The expression levels of LTG#2080 and 2081 were undetectable. In particular, no cell death function was detected in the negative control group, UTD, and GFP-transduced T cells LTG1398. These results demonstrate that the intensity of CAR lytic activity is dependent on the CAR expression level and is therefore CAR-specific (Figure 3). Subsequently, CAR constructs targeting CD123 were tested against the CD123-negative cell line, 293T. Although dramatic cell lysis was observed in CD123-positive cell lines for some CAR123 constructs, none of the CD123 CAR constructs tested showed cell lytic activity exceeding the negative control level (T cells only - UTD, or GFP-transduced T cells, LTG1398). This observation demonstrates that the cytolytic activity of CAR T cells is target-dependent (Figure 3).

[0331] Next, we evaluated the ability of anti-CD123 CAR T cells to produce cytokines in response to antigen-expressing target cells (Figure 4). We incorporated CAR123 constructs LTG##2074~2088 into Cd123-positive tumor lines MOLM-14 and KG-1a, or the CD123-negative tumor line 293T, in an effector-to-target ratio of 10:1. T cells, or negative non-transduced T cells (UTDs), or GFP-transduced T cells (LTG1398), were co-incubated overnight, and the culture supernatant was analyzed by ELISA for IFN gamma and TNF-alpha. CAR123 constructs LTG##2076, 2078, and 2088 strongly induced cytokines in response to tumor cells, while the remaining CAR123 constructs tested, and the negative control (non-transduced, UTD; GFP, LTG1398), did not produce any noticeable cytokine induction. Importantly, CAR123 constructs LTG##2076, 2078, and 2088 did not generate cytokine secretion in the absence of tumor cells (CAR-ART-only group), which further confirms the specificity of CAR and indicates a lack of sustained signaling by tandem CARs.

[0332] While not intended to limit to any particular mechanism of action, possible reasons for the enhanced therapeutic function associated with exemplary CD123 targeting the CARs of the present invention include, for example, but not as an limitation, a) improved lateral movement within the plasma membrane, enabling more efficient signaling; b) superior location within plasma membrane microdomains such as lipid rafts and a higher ability to interact with transmembrane signaling cascades associated with T cell activation; c) superior location within the plasma membrane, such as less proximity to or less interaction with phosphatases like CD45, due to preferential movement away from repressive or downregulatory interactions; and d) superior assembly to T cell receptor signaling complexes (i.e., immune synapses); or e) superior ability to engage with tumor antigens due to two distinct targeting domains present in each CAR molecule, or any combination thereof.

[0333] Each application and patent cited herein, and each document or reference cited in each application and patent (including each granted patent in litigation, “Application Reference Documents”), and each PCT and foreign application or patent corresponding to and / or claiming priority to any of these applications and patents, and each document cited or referenced in each Application Reference Document, are incorporated herein by express reference and can be used in practicing the present invention. More generally, documents or reference documents are cited in the text, in the list of references preceding the claims, or in the text itself, and each of these documents or reference documents (“Cited Reference Documents”), and each document or reference cited in each Cited Reference Document (including any manufacturer’s specifications, instructions, etc.) are incorporated herein by express reference. .

[0334] The aforementioned descriptions of some specific embodiments provide sufficient information to enable others to easily modify or adapt the invention to various applications, such as specific embodiments, without departing from the general concept, by applying the knowledge of the invention. Therefore, such adaptations and modifications should and are intended to be understood as being within the meaning and scope of equivalents of the disclosed embodiments. It is understood that the terms or predicates used herein are for descriptive purposes only, not limiting purposes. Exemplary embodiments are disclosed in the drawings and description, and specific terminology may be used, but unless otherwise noted, they are used only in a general and descriptive sense, not limiting purposes, and therefore the claims are not so limited. Furthermore, those skilled in the art will understand that certain steps of the methods discussed herein can be arranged in a different order or combined. Therefore, the appended claims are not intended to be limited to the detailed embodiments disclosed herein. Those skilled in the art can understand and grasp many equivalents of the embodiments of the invention described herein using commonplace experiments. Such equivalents are encompassed by the following claims.

[0335] Sequence List The nucleic acid and amino acid sequences listed below are shown using standard abbreviations for nucleotide bases and one- or three-letter codes for amino acids, as specified in 37C.FR1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included by referring to the shown strand. In the attached sequence listing: Nucleotide sequence of SEQ ID NO: 1 CD123 hScFv binder M12301 GAAGTGCAACTCGTCCAAAGCGGAGCTGAAGTGAAGAAACCAGGCGGATCCCTGAGACTGTCTTGCGCCGCATCGGGCTTCACCTTCTCCTCGTATTCCATGAACTGGGTCAGACAGGCCCCTGGAAAGGGTCTGGAATGGGTGTCCTCCATTTCCTCCTCGTCGAGCTACATCTACTACGC CGACTCCGTGAAGGGGCGCTTCACAATCTCCCGGGACAACGCGAAGAACTCCCTGTACCTCCAAATGAACTCCCTGAGGCCGAGGATACTGCCGTGTACTACTGCGCCATCGAGAGCTGGGGCTCCCTCGACTATTGGGCCAGGGAACCCTGGTCACCGTGTCATCCGGCGGTGGAGGAT CGGGTGGTGGCGGATCCGGAGGAGGGGGATCCCAGAGCGTGCTGACCCAACCCCCGTCAGTGTCAGCCGCGCCTGGACAGAAGGTCACCATCAGCTGTAGCGGCTCATCCTCCAATATCGGCGACGATTACGTGTCCTGGTACCAGCAGCTTCCTGGAACCGCTCCCAAGCTCCTGATCTAC GACAACCACAAGCGCCCGTCGGGAATTCCGGACCGGTTTAGCGGTTCAAAGTCCGGGACTAGCGCGACTCTGGGGATTACCGGACTGCAGACGGGCGACGAAGCCGATTACTACTGCGGGACTTGGGATGACTCGCTTAGCGGAGTGGTGTTCGGTGGCGGGACCAAGCTCACTGTGTTGGGA Amino acid sequence of Sequence ID No. 2 CD123 hScFv binder M12301 EVQLVQSGAEVKKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAIESWGSLDYWGQGTLVTVSSGGGG SGGGGSGGGGSQSVLTQPPSVSAAPGQKVTISCSGSSSNIGDDYVSWYQQLPGTAPKLLIYDNHKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDDSLSGVVFGGGTKLTVLG Nucleotide sequence of sequence number 3 CD123 hScFv binder M12303 GAAGTGCAACTCGTCGAAACTGGAGCCGAAGTGAAAAAGCCTGGAGCGTCCGTCAAAGTGTCGTGCAAGGCCTCCGGCTACACCTTCACGACCTACTACGTGCACTGGGTCAGACAGGCTCCGGGTCAAGGGCTGGAGTGGATGGGCATCATTAACCCCTCCGGTGGAAGCACCTCCTATGCGCAAAAGTTCCAGGGTCGCGTCACCATGACTCGCGATACCTCCACTTCCACTGTGTACATGGAACTGAGCTCCCTGAGGTCCGAGGACACCGCCGTGTACTACTGCGCACGGGATGGAGGCTTGGGCGGCTACGAGGCTTGGGGACAGGGCACCCTCGTGACTGTGTCAAGCGGAGGGGGTGGATCCGGAGGGGGAGGATCAGGCGGTGGTGGAAGCGATATCCAGCTTACCCAGTCGCCTTCCGCGCTGTCTGCATCGGCCGGCGACAGAGTGACAATTACCTGTCAAGCCAGCCAGGACATCTCCAACTATCTGAACTGGTACCAGCAGAAGCCCGGAAAGGCTCCGAAGCTGCTGATCTACGACGCCAGCAACCTGGAACGGGGCGTGCCATCACGGTTCTCGGGATCAGGGTCGGGCACTGAGTTCACCTTCACCATCTCCTCCCTCCAACCCGAGGACATTGCCACCTACTACTGCCAGCAGTACGACAACCTCCCGATCACCTTTGGACAGGGGACTCGCCTGGAAATCAA Amino acid sequence of SEQ ID NO: 4 CD123 hScFv binder M12303 EVQLVETGAEVKKPGASVKVSCKASGYTFTTYYVHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVY MELSSLRSEDTAVYYCARDGGLGGYEAWGQGTLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSALSASAGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLERGVPSRFSGSGSGTEFTFTISSLQPEDIATYYCQQYDNLPITFGQGTRLEIK sequence number 5 CD123 hScFvバインダーM12304のnucleotide sequence GAAGTGCAATTGGTCCAGAGCGGAGGAGGACTTGTGAAGCCAGGCGGATCCCTGAGATTGTCATGCGCCGCATCGGGGGTTCACCTTTTCCTCCTACTCCATGAACTGGGTCAGACAGGCGCCCGGAAAGGGACTTGAATGGGTGTCGTCCATTTCCTCCTCCTGTCTACATCTACTACGCCGACTCC GTGAAGGGCCGCTTCACCACTCCCGGGACAACGCCAAGAACAGCCTGTATCTCCAAATGAACTCCCTGCGGCCGAAGATACTGCTGTGTATTACTGCGCTCGGACTTCCCGTACGACTCATCGGGCTATTACTCGGACGCGTTCGATATCTGGGGCCAGGGAACTATGGTCACCGTCAGCTCTGGTG GCGGTGGTTCGGAGGGGGTGGATCCGGTGGCGGAGGATCAGAGATTGTGCTGACCCAGTCCCCGCTGTCACTGCCCGTGACTCCGGGAGAGCCTGCCTCGATCCGTGTCGGTCCAGCCAGTCCCTGCTGCACTCGAATGGGTACAACTACCTCGATTGGTACCTCCAAAAGCCTGGGCAGTCACCCCA ACTGCTGATCTACCTCGGGAGCAACAGAGCCAGCGGAGTGCCTGACGCTTTAGCGGTTCCGGATCCGGCACCGACTTCCACCCTGAAAATCAGCCGGGTGGAAGCCGAGGATGTCGGCGTGTACTACTGCATGCAGGCACTGCAGACTCTGGGGTACACCTTCGGCCAGGGCACGAAGCTCGAGATCAAG Amino acid sequence of SEQ ID NO: 6 CD123 hScFv binder M12304 EVQLVQSGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDFPYDSSGYYSDAFDIWGQGTMVTVSSG GGGSGGGGSGGGGSEIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYCMQALQTLGYTFGQGTKLEIK Nucleotide sequence of SEQ ID NO: 7 CD123 hScFv binder M12305 CAAGTGCAACTCGTCCAATCCGGTGCCGAAGTCAAGAAGCCTGGTTCCTCCGTGAAAGTGTCGTGCAAAGCCAGCGGCGGGACTTTTAGCTCCTACGCGATCAGCTGGGTCAGACAGGCCCCTGGACAAGGCCTCGAGTGGATGGGCGGCATCATTCCGATTTTCGGTACCGCCAACTACGCCCAGAAGTTCCAGGGACGCGTGACCATTACTACCGACGAGAGCACCTCAACCGCATACATGGAACTGTCCAGCCTGCGCTCCGAGGACACGGCTGTGTACTATTGCGCCAGACGGGGATGGGGAGGATTCTCCTCCGGCTCCGCATTCGACATCTGGGGACAGGGCACTATGGTCACTGTGTCATCCGGGGGAGGAGGATCAGGCGGTGGAGGATCCGGTGGTGGCGGATCCAACTTCATGCTGACCCAGCCCCACTCAGTGTCGGAATCGCCCGGCAACACCGTGACTATCAGCTGCACCGGATCCAGCGGGACCATCGGCTCTAATTTCGTGCAGTGGTACCAGCAGTCCCCAGGGAGAGCTCCGACCCTGTTGATCTACGAGGACACAAAGCGGCCAAGCGGAGTGCCGCCTAGATTCGCCGGCTCCGTGGATTCCTCGTCCAACTCGGCGTCGCTGACCATCAGCGGACTCAAGACTGAAGATGAAGCCGACTACTACTGTCAGTCCTACGACTCGAGCAACTGGGTGTTTGGGGGCGGGACTAAGCTGACCGTGCTTGGA Amino acid sequence of Sequence No. 8 CD123 hScFv binder M12305 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITTDESTSTAYMELSSLRSEDTAVYYCARRGWGGFSSGSAFDIWGQGTMVTVSSG GGGSGGGGSGGGGSNFMLTQPHSVSESPGNTVTISCTGSSGTIGSNFVQWYQQSPGRAPTLLIYEDTKRPSGVPPRFAGSVDSSSNSASLTISGLKTEDEADYYCQSYDSSNWVFGGGTKLTVLG Nucleotide sequence of sequence number 9 CD123 hScFv binder M12306 GAAGTCCAATTGGTGCAGAGCGGATCCGAACTTAAGAAACCTGGCGCGAGCGTGAAAGTGTCCTGCAAGGCCTCCGGAGGGACTTTCTCGTCGTACGCCATTAGCTGGGTCCGCCAAGCTCCTGGCCAAGGCCTGGAGTGGATGGGCGGGATTATCCCCATCTTCGGGACTGCGAACTACGCCCAGAAGTTTCAGGGCCGGGTCACTATCACCGCCGACGAATCAACCTCGACCGCCTACATGGAACTGTCCTCGCTTCGGTCCGAGGATACTGCCGTGTACTATTGTGCCTCAACGGCCAGACGCGGATGGGACACCGCTGGTCCGCTCGATTACTGGGGCCAGGGAACCCTCGTGACCGTCAGCTCCGGAGGAGGAGGCTCCGGTGGTGGAGGATCCGGGGGTGGTGGATCCGACATCCAAATGACCCAGTCCCCCTCGTCCCTGAGCGCCTCTGTGGGCGACAGAGTGACAATTGCATGCAGGGCCTCACAGACTATCTCCCGCTACCTGAACTGGTACCAGCAGAAGCCAGGAAAGGCCCCTAAGCTGCTCATCTACGCTGCGTCCTCGCTCCAATCCGGGGTGTCCTCACGGTTTTCCGGATCGGGTTCCGGCACCGAGTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCAACCTACTTCTGCCAGCAAACCTACTCCCCGCCGATTACGTTCGGACAGGGGACTCGGCTGGAAATCAA Amino acid sequence of SEQ ID NO: 10 CD123 hScFv binder M12306 EVQLVQSGSELKKPGASVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCASTARRGWDTAGPLDYWGQGTLVTVS SGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTIACRASQTISRYLNWYQQKPGKAPKLLIYAASSLQSGVSSRFSGSGSGTEFTLTISSLQPEDFATYFCQQTYSPPITFGQGTRLEIK Nucleotide sequence of SEQ ID NO: 11 CD123 hScFv binder M12308 GAAGTGCAACTCGTCGAAACAGGGGCAGAAGTGAAAAACCCAGGCTCAAGCGTGAAAGTGTCGTGCAAGGCTTCGGGCGGAACTCTGTCCAACTACGCCATCTCCTGGGTCCGCCAAGCTCCGGGAAAGGGCCTCGAGTGGATGGGCGGAATCATTCCCATTTTCGGGACCGCCAACTACGCGCAAAAGTTCCAGGGCCGGGTCACTATCACCGCGGACGAAAGCACCAGCACCGCCTACATGGAACTGTCCTCCCTGCGCTCCGAGGACACTGCCGTGTACTATTGCGCCCGGAGGTCATCGTGGTACCCCGAGGGCTGCTTCCAGCACTGGGGACAGGGCACTCTCGTGACCGTGTCGTCGGGTGGTGGTGGATCAGGAGGGGGAGGATCCGGAGGAGGCGGAAGCGATATTCAGCTGACCCAGTCACCGAGCTCCCTGTCCGCCTCCACCGGAGACAGAGTGACCATCACGTGTCGGGCCTCCCAAGGGATCTCCTCCTACCTGGCCTGGTACCAGCAGAAGCCTGGAAAGGCACCGAAGTTGCTGATCTACGCCGCGAGCACCCTTCAGTCCGGAGTGCCTAGCCGCTTCTCGGGTTCCGGCTCTGGCACTGACTTCACTCTGACCATTAGCTGCCTGCAGTCCGAGGATTTTGCCACCTACTACTGCCAGCAGTACTATAGCTACCCCCTGACCTTCGGGGGCGGAACCAAGCTCGACATCAAG Amino acid sequence of SEQ ID NO: 12 CD123 hScFv binder M12308 EVQLVETGAEVKNPGSSVKVSCKASGGTLSNYAISWVRQAPGKGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARRSSWYPEGCFQHWGQGTLVTVSS GGGGSGGGGSGGGGSDIQLTQSPSSLSASTGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISCLQSEDFATYYCQQYYSYPLTFGGGTKLDIK Sequence ID 13: Nucleotide sequence of the reader / signal peptide sequence atgctgctgctggtgaccagcctgctgctgtgcgaactgccgcatccggcgtttctgctgattccg SEQ ID NO: 14 Amino acid sequence of the reader / signal peptide sequence MLLLVTSLLLCELPHPAFLLIP Nucleotide sequence of SEQ ID NO: 15 CD123 hScFv binder M12309 GAAGTCCAATTGGTGCAGAGCGGAGCTGAAGTGAAGAAACCTGGCGCGACCGTGAAGATCTCGTGCAAAGTGTCCGGCTACACTTTCACCGACTACTATATGCACTGGGTGCAACAGGCGCCGGGAAAGGGACTGGAGTGGATTGGCCTTGTGGACCCCGAAGATGGCGAAACCATCTACGCCGAGAAGTTCCAGGGCCGGGTCACTATCACCGCGGACACTTCCACGGACACCGCCTACATGGAACTGAGCTCCCTGAGATCCGAGGACACCGCCGTGTACTACTGCGCCACTGCCCCACTGGGGGAAGTCGGCGCAGCAGTGGACTACTGGGGACAGGGAACTCTCGTCACTGTGTCCAGCGGTGGAGGAGGCAGCGGTGGTGGAGGCTCCGGTGGTGGTGGATCCCATGTCATTCTGACTCAGCCGCCGTCAGTGTCAGCCGCCCCTGGACAAGAGGTGTCCATCTCCTGTTCGGGGTCCGATGCCAACATTGGGACCAACTTGGTGTCGTGGTACCAGCACGTGCCTGGAACAGCCCCCAAGCTGCTCATCTACGAGAACTCGAAGAGGCCATCCGGAATTCCCGCCCGGTTTTCATCGAGCCAGTCAGGGACCTCCGCTACCCTGGCTATCAGCGGGCTCCAGTCTGGGGATGAAGCGATCTACTACTGCCTGACCTGGGATCGCACCCTCTCCGGAAAGATCTTCGGTGGCGGCACTCAGCTGACCGTGCTTGGA Amino acid sequence of SEQ ID NO: 16 CD123 hScFv binder M12309 EVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWIGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATAPLGEVGAAVDYWGQGTLVTVSSGG GGSGGGGSGGGGSHVILTQPPSVSAAPGQEVSISCSGSDANIGTNLVSWYQHVPGTAPKLLIYENSKRPSGIPARFSSSQSGTSATLAISGLQSGDEAIYYCLTWDRTLSGKIFGGGTQLTVLG Nucleotide sequence of SEQ ID NO: 17 CD123 hScFv binder M12310 GAAGTGCAACTTGTCCAAAGCGGAGCCGAAGTGAAGAAGCCAGGATCCTCCGTGAAAGTGTCTTGCAAAGCATCCGGCGGCACTTTCTCCTCCTACGCCATCTCCTGGGTCAGACAGGCGCCTGGACAGGGTCTGGAGTGGATGGGCATTATCAATCCTAGCGGTGGCTCCACTTCGTATGCCCAGAAGTTCCAGGGTCGGGTCACCATGACCCGGGATACTTCAACTAGCACCGTGTACATGGAACTCTCCTCGCTGCGCTCGGACGATACCGCCGTGTACTACTGTGCCCGCGAGCTGCTCTGGTTTGGAGAGCTGGACACCTACGGAATGGACGTCTGGGGACAGGGGACCACTGTGACGGTGTCGTCAGGAGGCGGAGGCTCAGGAGGGGGTGGTTCCGGAGGGGGAGGATCCCTCCCGGTGCTGACCCAGCCCCCAAGCGTCAGCGTGGCTCCGGGAAAGACCGCCCGCATCACATGCGGCGGGAACAACATCGGCTCCAAGTCCGTGCATTGGTACCAGCAGAAGCCTGGACAAGCGCCGGTGCTGGTCATCTACGACGACTCAGATCGGCCCTCCGGCATTCCCGAGCGGTTCAGCGGCTCCAACTCGGGCAACACTGCTACTCTGACCATCTCGAGGGTGGAAGCGGGGGACGAAGCAGATTACTACTGCCAAGTCTGGGACTCCAGCTCCGACCACGGGGTGTTCGGCGGAGGAACCCAGCTGACCGTGTTGGGA Amino acid sequence of SEQ ID NO: 18 CD123 hScFv binder M12310 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSDDTAVYYCARELLWFGELDTYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSLPVLTQPPSVSVAPGKTARIT CGGNNIGSKSVHWYQQKPGQAPVLVIYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHGVFGGGTQLTVLG Nucleotide sequence of SEQ ID NO: 19, CD123 hScFv binder M12311 GAAGTGCAACTCGTCCAATCTGGTGCCGAAGTCAAGAAGCCTGGCTCAAGCGTGAAAGTGTCCTGCAAAGCGTCGGGAGGGACCTTCAGCTCCTACGCCATTTCCTGGGTCCGCCAAGCACCAGGACAGGGCCTGGAGTGGATGGGCGGCATCATCCCGATCTTCGGGACTGCCAACTACGCCCAGAAGTTCCAGGGGAGAGTGACCATTACCGCCGACGAGTCGACCAGCACGGCCTACATGGAACTGTCCAGCCTGCGCTCCGAGGACACTGCCGTGTACTACTGCGCGAGGGCCAGACTCGGTGGAGCGTTCGACATCTGGGGACAGGGCACCATGGTCACCGTGTCATCCGGTGGCGGAGGATCCGGTGGTGGCGGATCAGGAGGGGGAGGATCCCAGTCCGTGCTGACTCAGCCTCCCTCCGTGAGCGCTGCACCGGGACAGAAGGTCACCATCTCATGCTCGGGGGGAAGCTCCAACATCGGGAACCACTACGTGTCCTGGTACCAACAGTTGCCTGGTGCCGCTCCAAAGCTGCTGATCTATGACGATAACAAGCGGCCGTCCGGAATCCCCGACCGGTTCTCGGGGTCTAGATCCGGAACCAGCGCAACTCTCGGCATTACCGGACTGCAGAGCGGCGATGAGGCCGACTACTACTGTGGCACATGGGACTCGTCGCTGGCTGCCCACGTGTTTGGCACTGGCACCAAGGTCACCGTGCTTGGA Amino acid sequence of SEQ ID NO: 20, CD123 hScFv binder M12311 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARARLGGAFDIWGQGTMVTVSSGGGG SGGGGSGGGGSQSVLTQPPSVSAAPGQKVTISSCSGGSSNIGNHYVSWYQQLPGAAPKLLIYDDNKRPSGIPDRFSGSRSGTSATLGITGLQSGDEADYYCGTWDSSLAAHVFGTGTKVTVLG Nucleotide sequence of SEQ ID NO: 21 CD123 hScFv binder M12313 GAAGTGCAACTTGTCCAGAGCGGAGCCGAAGTGAAGAAACCTGGATCCTCCGTCAAAGTGTCGTGCAAGGCTTCGGGCGGAACCTTCTCCTCGTACGCGATCTCATGGGTCAGACAGGCACCCGGACAGGGACTGGAGTGGATGGGCGGCATCATTCCCATCTTCGGCACCGCTAATTACGCCCAGAAGTTTCAGGGGAGAGTGACCATCACCGCCGACGAGTCCACCTCCACTGCCTACATGGAACTGTCCTCACTGAGGTCCGAGGATACTGCCGTGTACTACTGCGCGTCGCAAAAGGGGGGTGGATGGTCCATTGACGCCTTCGATATTTGGGGACAGGGGACGATGGTCACAGTGTCATCCGGCGGTGGTGGATCCGGTGGTGGCGGATCCGGAGGAGGAGGCAGCCAGTCCGTGCTGACCCAGCCGCCTAGCGTGTCGGCCGCATCTGGGCAGCGCGTGACCATTTCCTGTTCCGGGTCCTCGTCCAACATCGGCAACAACTACGCCTCCTGGTACCAACAGCTCCCGGGAATGGCCCCTAAGCTGCTGATCTACGAGGACAACAAGCGGCCATCCGGGATCTCAGACCGGTTCAGCGGATCCCAGTCCGGCACTTCCGCGAGCCTCGCCATCACCGGACTGCAGGCTGAGGACGAAGCCGACTACTACTGCCAATCATATGACAGCTCGCTCAGCGGCGATGTGGTGTTCGGCGGTGGCACTAAGCTGACCGTGTTGGGA Amino acid sequence of SEQ ID NO: 22 CD123 hScFv binder M12313 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCASQKGGGWSIDAFDIWGQGTMVTVSSGG GGSGGGGSGGGGSQSVLTQPPSVSAASGQRVTISCSGSSSNIGNNYASWYQQLPGMAPKLLIYEDNKRPSGISDRFSGSQSGTSASLAITGLQAEDEADYYCQSYDSSLSGDVVFGGGTKLTVLG Nucleotide sequence of SEQ ID NO: 23 CD123 hScFv binder M12314 GAAGTCCAACTCGTCCAAAGCGGTGCAGAAGTGAAAAAGCCAGGCTCCTCAGTGAAAGTGTCCTGCAAAGCCTCGGGGGGAACCTTCTCCTCCTACGCCATCTCCTGGGTCCGCCAAGCACCAGGACAGGGCCTGGAGTGGATGGGCGGGATCATTCCGATCTTCGGCACCGCCAACTACGCCCAGAAGTTTCAGGGCCGCGTGACTATCACCGCCGACGAGTCCACCTCCACTGCGTACATGGAACTGTCCAGCCTGCGGTCCGAGGACACTGCCGTGTATTACTGCGCGAGAGTCGGTTGCTCCGGGGGATCGTGTTATCCCGACTACTGGGGACAGGGGACCCTCGTGACCGTGTCGTCGGGTGGTGGTGGAAGCGGCGGTGGAGGATCCGGTGGAGGAGGCAGCGAAATCGTGCTGACTCAGTCGCCGTCCTCGCTTTCCGCCTCCGTGGGAGATCGCGTGACCATCACGTGTCAGGCTTCTCAAGACATTAGCAACTACCTGAATTGGTACCAGCAGAAGCCTGGAAAGGCTCCGAAGCTGCTCATCTACGACGCGTCCAACCTGGAGACAGGGGTGCCTTCACGGTTCTCGGGAAGCGGATCCGGCACCGATTTCACCTTCACCATTTCAAGCCTGCAACCCGAGGATATTGCCACCTACTACTGCCAGCAGTACGACAACCTCCCCCTGACTTTCGGGGGCGGCACTAAGTTGGACATCAAG Amino acid sequence of SEQ ID NO: 24 CD123 hScFv binder M12314 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARVGCSGGSCYPDYWGQGTLVTVSS GGGGSGGGGSGGGGSEIVLTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPLTFGGGTKLDIK Nucleotide sequence of SEQ ID NO: 25 CD123 hScFv binder M12315 CAAGTCCAGTTGCAACAATGGGGAGCAGGCCTTCTGAAACCGTCCGAGACACTGAGCCTGACGTGCGCCGTCTATGGCGGATCGTTCTCCGGATACTACTGGTCGTGGATCAGACAGCCTCCGGGAAAGGGTCTGGAATGGATCGGCGAAATCAACCACAGCGGCAGCACCAATTACAACCCGTCACTGAAGTCAAGGGTCACCATTAGCGTGGACACTTCCAAGAACCAGTTCTCCCTGAAACTGTCGAGCGTGACCGCTGCCGATACTGCCGTGTACTACTGTGCCCGCGGCCAAGTCAAGTATAGCTCAAGCCTCGGCTACTGGGGCCAGGGAACCCTCGTGACCGTGTCCTCGGGTGGAGGAGGCTCCGGTGGTGGAGGATCCGGTGGCGGAGGATCGCAGTCCGTGCTGACCCAGCCTCCCTCCGTGTCTGCTGCCCCTGGGCAAAAGGTCACCATTTCGTGCTCCGGCTCATCGTCCAACATCGGGAACAACTTTGTGTCCTGGTACCAGCAGCTGCCCGGTACTGCCCCAAAGCTGCTGATCTACGAGGACAACAAGCGCCCATCCGGGATTCCGGATCGGTTCAGCGGATCACGGTCCGGAACTAGCGCGACCCTGGGGATCACCGGGCTCCAGACTGGCGACGAAGCGGACTACTACTGCGGAACTTGGGACTCCTCCTTGGGGGCCTGGGTGTTCGGCGGAGGGACCAAGCTCACCGTGCTTGGA Amino acid sequence of SEQ ID NO: 26 CD123 hScFv binder M12315 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGQVKYSSSLGYWGQGTLVTVSSGGG GSGGGGSGGGGSQSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNFVSWYQQLPGTAPKLLIYEDNKRPSGIPDRFSGSRSGTSATLGITGLQTGDEADYYCGTWDSSLGAWVFGGGTKLTVLG Sequence ID 27: Nucleotide sequence of the DNA CD8 transmembrane domain atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc accctttact gc SEQ ID NO: 28 Amino acid sequence of the CD8 transmembrane domain Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Sequence ID 29: Nucleotide sequence of the DNA CD8 hinge domain accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg gacttcgcct gtgat Amino acid sequence of the CD8 hinge domain (SEQ ID NO: 30) Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Amino acid sequence of the hinge region (amino acid numbers 118-178) of sequence number 31 CD8.alpha (NCBI RefSeq: NP.sub.--001759.3) Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Sequence ID 32: Amino acid sequence of the human IgG CL sequence Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala Pro Thr Glu Cys Ser Nucleotide sequence of the DNA signaling domain of SEQ ID NO: 33 4-1BB aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggagaggtgt surfaceactg Amino acid sequence of the signaling domain of SEQ ID NO: 34 4-1BB Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Sequence ID 35: Nucleotide sequence of the DNA signaling domain of CD3-zeta agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc tacgacgccc ttcacatgca ggccctgccc cctcgc Amino acid sequence of SEQ ID NO: 36 CD3 zeta Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp With Leu His Met Gln Ala Leu Pro Pro Arg Characteristics of 37 ScFv CD19 gataccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcaccatcagttgca gggcaagtca ggacattagt aaatatttta attggtacca gcagaaacca gatgactg ttaaactcct gatctaccat acatcagat tacaccagtcag aggtcagg aggtc The shapes eat eat. ccattagcaa cctggagcaa gaagatattg ccacttactt ttgccaacag ggtaatacgc ttccgtacac gttcggaggg gggaccaagc tggagatcac aggtggcggt ggctcgggcg gtggtgggtc gggtggcggc ggatctgagg tgaaactgca ggagtcagga cctggcctgg tggcgccctc acagagcctg tccgtcacat gcactgtctc aggggtctca ttacccgact atggtgtaag ctggattcgc cagcctccac gaaagggtct ggagtggctg ggagtaatat ggggtagtga aaccacatac tataattcag ctctcaaatc cagactgacc atcatcaagg acaactccaa gagccaagtt ttcttaaaaa tgaacagtct gcaaactgat gacacagcca tttactactg tgccaaacat tattactacg gtggtagcta tgctatggac tactggggcc aaggaacctc agtcaccgtc tcctca Amino acid sequence of SEQ ID NO:38 ScFv CD19 Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Nucleotide sequence of SEQ ID NO: 39 GMCSF leader peptide ATGCTGCTGCTGGTGACCAGCCTGCTGCTGTGCGAACTGCCGCATCCGGCGTTTCTGCTGATTCCG Amino acid sequence of SEQ ID NO: 40 GMCSF leader peptide MLLLVTSLLLCELPHPAFLLIP Nucleotide sequence of the TNFRSF19 leader peptide (SEQ ID NO: 41) GGCTCTGAAAGTGCTGTTGGAACAAGAAAAGACCTTCTTCACCTTGCTCGTGTTGCTGGGGTACCTGTCCTGCAAAGTCACCTGT Amino acid sequence of SEQ ID NO: 42 TNFRSF19 leader peptide MALKVLLEQEKTFFTLLVLLGYLSCKVTC Nucleotide sequence of SEQ ID NO: 43 CD8 alpha-leader peptide atggcgctgccggtgaccgcgctgctgctgccgctggcgctgctgctgcatgcggcgcgc ccg Amino acid sequence of SEQ ID NO: 44 CD8 alpha-leader peptide MALPVTALLLPLALLLHAARP Nucleotide sequence of the CD28 costimulatory domain (SEQ ID NO: 45) CGGTCGAAGAGGTCCAGACTCTTGCACTCCGACTACATGAACATGACTCCTAGAAGGCCCGGACCCACTAGAAAGCACTACCAGCCGTACGCCCCTCCTCGGGATTTCGCCGCATACCGG TCC Amino acid sequence of the CD28 costimulatory domain (SEQ ID NO: 46) RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS Sequence ID 47: Nucleotide sequence of the CD3 zeta-activating domain AGAGTGAAGTTCAGCCGCTCAGCCGATGCACCGGCCTACCAGCAGGGACAGAACCAGCTCTACAACGAGCTCAACCTGGGTCGGCGGGAAGAATATGACGTGCTGGACAAACGGCGCGGCAGAGATCCGGAGATGGGGGGAAAGCCGAGGAGGAAGAACCCTCAAGAG GGCCTGTACAACGAACTGCAGAAGGACAAGATGGCGGAAGCCTACTCCGAGATCGGCATGAAGGGAGAACGCCGGAGAGGGAAGGGTCATGACGGACTGTACCAGGGCCTGTCAACTGCCACTAAGGACACTTACGATGCGCTCCATATGCAAGCTTTGCCCCCGCGG Amino acid sequence of the CD3 zeta-activating domain (SEQ ID NO: 48) RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Sequence ID 49: Nucleotide sequences of the hinge and transmembrane domain of TNFRSF19 (the transmembrane domain is underlined). GCGGCCGCGGTCGGATTCCAAGACATGGAATGCGTGCCCTGCGGCGACCCGCCACCTCCTTACGAGCCGCACTGCGCATCGAAGGTCAACCTCGTGAAGATCGCGAGCACCGCGTCCTCACCCCGGGATACTGCTCTG GCCGCCGTGATTTGTTCCGCCTTGGCCACCGTGCTTCTGGCCCTGCTGATCCTCTGTGTGATC Amino acid sequence of the hinge and transmembrane domain of sequence number 50 TNFRSF19 (the transmembrane domain is underlined). AAAVGFQDMECVPCGDPPPPY EPHCASKVNLVKIASTASS PRDTA L A A V I C S A L A T V L L A L L I L C V I Sequence ID 51: Nucleotide sequence of the TNFRSF19 transmembrane domain GCCGCCGTGATTTGTTCCGCCTTGGCCACCGTGCTTCTGGCCCTGCTGATCCTCTGTGTGATC Amino acid sequence of the TNFRSF19 transmembrane domain (SEQ ID NO: 52) AAVICSALATVLLALLILCVI Nucleotide sequence of the TNFRSF19 hinge domain (SEQ ID NO: 53) GCGGCCGCGGTCGGATTCCAAGACATGGAATGCGTGCCCTGCGGCGACCCGCCACCTCCTTACGAGCCGCACTGCGCATCGAAGGTCAACCTCGTGAAGATCGCGAGCACCGCGTCCTCACCCCGGGATACTGCTCTG Amino acid sequence of the hinge domain of sequence number 54 TNFRSF19 AAAVGFQDMECVPCGDPPPPY EPHCASKVNLVKIASTASSPR DTAL Sequence ID 55: Nucleotide sequence of the abbreviated TNFRSF19 hinge domain TACGAGCCTCACTGCGCCAGCAAAGTCAACTTGGTGAAGATCGCGAGCACTGCCTCGTCCCCTCGGGACACTGCTCTGGC SEQ ID NO: 56 Amino acid sequence of the abbreviated TNFRSF19 hinge domain YEPHCASKVNLVKIASTASSP RDTAL Sequence ID 57: Nucleotide sequence of the CD8a hinge domain fused to the TNFRSF19 transmembrane domain (transmembrane sequence is underlined). GCGGCCGCGCCCGCCCCTCGGCCCCCGACTCCTGCCCGACGATCGCTTCCCAACCTCTCTCGCTGCCCGGAAGCATGCCGGCCCGCCGCCGGTGGCGCTGTCCACACTCGCGGACTGGACTTTGATACCGCACTG GCGGCCGTGATCTGTAGCGCCCTGGCCACCGTGCTGCTGGCGCTGCTCATCCTTTGCGTGATCTACTGCAAGCGGCAGCCTAGG Amino acid sequence of the CD8a hinge domain fused to the TNFRSF19 transmembrane domain (sequence number 58) (transmembrane sequence is underlined). AAAPAPRPPTPAPTIASQPLS LRPEACRPAAGGAVHTRGLDF DTAL A A V I C S A L A T V L L A L L I L C V I Y C K R Q P R Nucleotide sequence of the CD28 costimulatory domain (SEQ ID NO: 59) CGGTCGAAGAGGTCCAGACTCTTGCACTCCGACTACATGAACATGACTCCTAGAAGGCCCGGACCCACTAGAAAGCACTACCAGCCGTACGCCCCTCCTCGGGATTTCGCCGCATACCGGTCC Amino acid sequence of the CD28 costimulatory domain (SEQ ID NO: 60) RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS Sequence ID 61: Nucleotide sequence of CD3 zeta version 2 cgcgtgaaatttagccgcagcgcggatgcgccggcgtatcagcagggccagaaccagctg tataacgaactgaacctgggccgccgcgaagaatatgatgtgctggataaacgccgcggc cgcgatccggaaatgggcggcaaaccgcgccgcaaaaacccgcaggaaggcctgtataac gaactgcagaaagataaaatggcggaagcgtatagcgaaattggcatgaaaggcgaacgc cgccgcggcaaaggccatgatggcctgtatcagggcctgagcaccgcgaccaaagatacc tatgatgcgctgcatatgcaggcgctgccgccgcgc Amino acid sequence of SEQ ID NO: 62 CD3 zeta version 2 RVKFSRSADAPAYQQGQNQLY NELNLG RREEYDVLDKRRGRDPEMGGK PRRKNP QEGLYNELQKDKMAEAYSEIG MKGERRRGKGHDGLYQGLSTA TKDTYDALHMQALPPR Sequence ID 63: Nucleotide sequence of furin P2A furin CGCGCGAAACGCAGCGGCAGCGGCGCGACCAACTTTAGCCTGCTGAAACAGGCGGGCGAT GTGGAAGAAAACCCGGGCCCGCGAGCAAAGAGG Amino acid sequence of sequence number 64, furin P2A furin (furin sequence is underlined). RAKR SGSGATNFSLLKQAGDVEENPGP RAKR Sequence ID 65: Nucleotide sequence of furin T2A AGAGCTAAACGCTCTGGGTCTGGTGAAGGACGAGGTAGCCTTCTTACGTGCGGAGACGTGGAGGAAAACCCAGGACCC Amino acid sequence of sequence number 66, furin T2A (furin sequence is underlined) RAKR SGSGEGRGSLLTCGDVEENPGP Sequence ID 67: Nucleotide sequence of the truncated EGFR (tEGFR) tag SEQ ID NO: 68 Amino acid sequence of the shortened EGFR (tEGFR) tag RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCK ATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM Nucleotide sequence of SEQ ID NO: 69 CD123 hScFv binder M12316 GAAGTGCAGTTGGTGGAGAGCGGTGGAGGACTTGTGCAACCTGGTGGATCCCTGAGATTGTCGTGCGCAGCTTCAGGGTT CACCTTCTCCTCCTACGCCATGCACTGGGTCCGCCAAGCACCAGGAAAGGGCCTGGAATGGGTCAGCTCCATCTCCTCGTCGTCCTCGTACATCTACTATGCCGACTCCGTGAAGGGCCGCTTCACCATTAGCCGGGACAACTCAAAGAACACTCTGTACCTTCAAATGAACTCCCTGCGGGCTGAAGATACCGCCGTGTACTACTGCGCGAGGGATTGGGATGACGCGTTCGACATTTGGGGCCAGGGGACTACCGTCACCGTGTCGTCGGGTGGAGGAGGATCCGGGGGTGGAGGATCGGGAGGGGGTGGAAGCGACATTCAGATGACTCAGAGCCCGTCCTCCCTGTCGGCCTCAGTGGGCGACAGAGTGACCATCACCTGTCAAGCCAGCCAGGACATCTCAAACTACCTGAACTGGTACCAGCAGAAGCCCGGAAAGGCCCCTAAGCTGCTCATCTACGACGCCTCCAACCTGGAGACTGGAGTGCCCTCACGGTTTTCCGGCTCTGGAAGCGGCACCGATTTCACCTTCACGATCTCCTCCCTGCAACCGGAAGATATCGCGACCTACTACTGCCAGCAGTATGACAATCTCCCGCTCACCTTCGGTGGCGGCACTAAGCTCGAGATCAAA<H Amino acid sequence of SEQ ID NO: 70, CD123 hScFv binder M12316<H EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDWDDAFDIWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPLTFGGGTKLEIK<H Nucleotide sequence of SEQ ID NO: 71, CD123 hScFv binder M12317<H CAAGTCCAACTCGTCGAAACTGGTGGTGGCCTCGTGAAGCCTGGAGGATCCCTGCGCCTTTCCTGTGCCGCTTCCGGCTTTACTTTCTCGTCGTACTCCATGAACTGGGTCAGACAGGCTCCCGGAAAGGGCCTGGAATGGGTGTCCTCCATCTCGTCCTCATCCTCCTACATCTATTACGCGGACTCCGTGAAGGGCAGATTCACCATTTCCCGGGACAACGCCAAGAACAGCTTGTACCTCCAAATGAACTCCCTGCGGGCAGAGGACACCGCCGTGTACTACTGCGCGAGGGATGGGGATTTCTGGAGCGGAGCCATCGACTACTGGGGCCAGGGAACTCTCGTGACCGTCAGCTCCGGTGGTGGTGGAAGCGGAGGCGGAGGTTCTGGGGGGGGAGGATCAGACATTCAGCTGACCCAGTCGCCATCCTCCCTGAGCGCCTCAGTGGGGGACCGCGTGACTATTACATGCCAGGCCTCCCAAGATATCTCGAACTACCTGAACTGGTATCAGCAGAAGCCTGGAAAGGCCCCGAAGCTGTTGATCTACGATGCCAGCAACCTGGAGACTGGGGTGCCTTCCCGGTTCTCGGGATCAGGCTCGGGCACCGATTTCACCTTCACGATCAGCAGCCTGCAGCCCGAGGACATTGCAACCTACTACTGCCAGCAGTACGACAATCTGCCGCTTTTTGGGGGAGGCACCAAGCTGGAAATCAAA Amino acid sequence of SEQ ID NO: 72, CD123 hScFv binder M12317 QVQLVETGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDGDFWSGAIDYWGQGTLVTVSS GGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPLFGGGTKLEIK Nucleotide sequence of SEQ ID NO: 73 CD123 hScFv binder M12318 GAGGTGCAATTGGTGCAGTCAGGTGGTGGAGTGGTGCAGCCAGGAAGATCCCTTAGACTCTCGTGTGCGGCGTCAGGCTTTACCTTCTCCTCGTACTCCATGAACTGGGTCAGACAGGCACCGGGAAAGGGACTGGAATGGGTGTCCTCCATCTCGTCCTCCTCCTCCTACATCTACTACGCCGATAGCGTGAAGGGCCGGTTCACCATTTCGCGCGACAACGCCAAGAACACCCTGTACCTCCAAATGAATTCGCTGCGGGCCGAAGATACCGCTGTCTATTACTGCGCCCGCGACAACTGGGGCTCGCTGGACTATTGGGGCCAGGGAACCCTCGTCACCGTGTCAAGCGGAGGGGGTGGATCCGGAGGCGGAGGATCCGGTGGAGGGGGAAGCGACATTCAGATGACTCAGAGCCCGTCCTCCCTGTCTGCCTCCGTGGGGGATCGCGTGACCATCACATGCCAGGCCTCACAAGACATCAGCAATTACCTGAACTGGTACCAGCAGAAGCCTGGAAAGGCCCCCAAGCTGCTGATCTACGATGCCAGCAACCTGGAGACTGGGGTGCCTTCAAGGTTCTCCGGTTCCGGAAGCGGCACTGACTTCACCTTCACTATCTCGAGCCTGCAACCCGAGGACATTGCCACCTACTACTGCCAGCAGTACGACAACCTTCCGCACATGTACACGTTCGGCCAGGGCACCAAGCTCGAAATCAAA Amino acid sequence of SEQ ID NO: 74 CD123 hScFv binder M12318 EVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARDNWGSLDYWGQGTLVTVSSGGG GSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPHMYTFGQGTKLEIK Nucleotide sequence of Sequence ID No. 75 LTG2074 (hScFv aCD123 CD8 TM 4-1BB CD3 zeta) ATGCTGCTGCTGGTGACCAGCCTGCTGCTGTGCGAACTGCCGCATCCGGCGTTTCTGCTGATTCCGGAAGTGCAACTCGTCCAAAGCGGAGCTGAAGTGAAGAAACCAGGCGGATCCCTGAGACTGTCTTGCGCCGCATCGGGCTTCACCTTCTCCTCGT ATTCCATGAACTGGGTCAGACAGGCCCCTGGAAAGGGTCTGGAATGGGTGTCCTCCATTTCCTCCTCGTCGAGCTACATCTACTACGCCGACTCCGTGAAGGGGCGCTTCACAATCTCCCGGGACAACGCGAAGAACTCCCTGTACCTCCAAATGAACTC Amino acid sequence of sequence number 76 LTG2074 (hScFv aCD123 CD8 TM 4-1BB CD3 zeta) MLLLVTSLLLCELPHPAFLLIPEVQLVQSGAEVKKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAIES WGSLDYWGQGTLVTVSSGGGGSGGGGSGGGSQSVLTQPPSVSAAPGQKVTISCSGSSSNIGDDYVSWYQQLPGTAPKLLIYDNHKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGT WDDSLSGVVFGGGTKLTVLGAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Nucleotide sequence of sequence number 77 LTG2075 (hScFv aCD123 CD8 TM 4-1BB CD3 zeta) Amino acid sequence of Sequence ID No. 78 LTG2075 (hScFv aCD123 CD8 TM 4-1BB CD3 zeta) MLLLVTSLLLCELPHPAFLLIPEVQLVETGAEVKKPGASVKVSCKASGYTFTTYYVHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR DGGLGGYEAWGQGTLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSALSASAGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLERGVPSRFSGSGSGTEFTFTISSLQPEDIAT YYCQQYDNLPITFGQGTRLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Sequence ID 79: Nucleotide sequence of human IgG4 hinge GAGAGCAAATACGGGCCGCCATGTCCCCCGTGTCCG SEQ ID NO: 80 Amino acid sequence of human IgG4 hinge ESKYGPPCPPCP Sequence ID 81: Nucleotide sequence of the human IgG4 CH2 domain GCACCACCAGTTGCTGGCCCTAGTGTCTTCTTGTTCCCTCCCAAGCCCAAAGACACCTTGATGATTTCCAGAACTCCTGAGGTTACCTGCGTTGTCGTAGATGTTTCTCAGGAGGACCCAGAGGTCCAATTTAACTGGTACGTTGATGGGGTGGAAGTTCACA ATGCGAAGACAAAGCCGCGGGAAGAACAATTTCAGTCCACTTACCGGGTTGTCAGCGTTCTGACGGTATTGCATCAAGACTGGCTTAATGGAAAGGAATATAAGTGTAAGGTGTCCAACAAAGGTTTGCCGAGCAGTATTGAGAAGACCATATCAAAGGCGAAG Sequence ID 82: Amino acid sequence of the human IgG4 CH2 domain APPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV DGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKA K Sequence ID 83: Nucleotide sequence of the human IgG4 CH3 domain GGGCAGCCGCGCGAGCCACAAGTTTACACTTTGCCGCCATCTCAAGAGGAAATGACTAAAAACCAGGTATCCTTGACATGCCTCGTAAAAGGATTTTATCCATCTGATATTGCTGTGGAATGGGAGTCTAACGGGCAGCCGGAAAATAATTACAAAACTA CACCACCTGTGCTCGATTCAGATGGAAGTTTCTTTCCTTTACAGTAGACTTACGGTGGACAAATCTAGGTGGCAGGAAGGGAATGTGTTTAGTTGTAGTGTAATGCACGAGGCACTTCATAACCACTATACACAGAAGTCACTGAGTTTGAGTCTTGGCAAA Sequence ID 84: Amino acid sequence of the human IgG4 CH3 domain GQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Sequence ID 85: Nucleotide sequence of the human IgG4 hinge CH2 CH3 domain. GAGAGCAAATACGGGCCGCCATGTCCCCCGTGTCCGGCACCACCAGTTGCTGGCCCTAGTGTCTTCTTGTTCCCTCCCAAGCCCAAAGACACCTTGATGATTTCCAGAACTCCTGAGGTTACCTGCGTTGTCGTAGATGTTTCTCAGGAGGACCCAGAGGTCCAATTTAAC TGGTACGTTGATGGGGTGGAAGTTCACAATGCGAAGACAAAGCCGCGGGAAGAACAATTTCAGTCCACTTACCGGGTTGTCAGCGTTCTGACGGTATTGCATCAAGACTGGCTTAATGGAAAGGAATATAAGTGTAAGGTGTCCAACAAAGGTTTGCCGAGCAGTATTGAG AAGACCATATCAAAGGCGAAGGGGCAGCCGCGCGAGCCACAAGTTTACACTTTGCCGCCATCTCAAGAGGAAATGACTAAAAACCAGGTATCCTTGACATGCCTCGTAAAAGGATTTTATCCATCTGATATTGCTGTGGAATGGGAGTCTAACGGGCAGCCGGAAAATAAT TACAAAACTACACCACCTGTGCTCGATTCAGATGGAAGTTTCTTCCTTTACAGTAGACTTACGGTGGACAAATCTAGGTGGCAGGAAGGGAATGTGTTTAGTTGTAGTGTAATGCACGAGGCACTTCATAACCACTATACACAGAAGTCACTGAGTTTGAGTCTTGGCAAA Sequence ID 86: Amino acid sequence of the human IgG4 hinge CH2 CH3 domain. ESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Nucleotide sequence of SEQ ID NO: 87, LTG - 2076 (hScFv - αCD123 - CD8 - TM - 4 - 1BB - CD3 zeta) ATGCTGCTGCTGGTGACCAGCCTGCTGCTGTGCGAACTGCCGCATCCGGCGTTTCTGCTGATTCCGGAAGTGCAATTGGTCCAGAGCGGAGGAGGACTTGTGAAGCCAGGCGGATCCCTGAGATTGTCATGCGCCGCATCGGGGTTCACCTTTTCCTCCTACTCCATGAACTGGGTCAGACAGGCGCCCGGAAAGGGACTTGAATGGGTGTCGTCCATTTCCTCCTCCTCGTCCTACATCTACTACGCCGACTCCGTGAAGGGCCGCTTCACCATCTCCCGGGACAACGCCAAGAACAGCCTGTATCTCCAAATGAACTCCCTGCGGGCCGAAGATACTGCTGTGTATTACTGCGCTCGGGACTTCCCGTACGACTCATCGGGCTATTACTCGGACGCGTTCGATATCTGGGGCCAGGGAACTATGGTCACCGTCAGCTCTGGTGGCGGTGGTTCCGGAGGGGGTGGATCCGGTGGCGGAGGATCAGAGATTGTGCTGACCCAGTCCCCGCTGTCACTGCCCGTGACTCCGGGAGAGCCTGCCTCGATCTCGTGTCGGTCCAGCCAGTCCCTGCTGCACTCGAATGGGTACAACTACCTCGATTGGTACCTCCAAAAGCCTGGGCAGTCACCCCAACTGC TGATCTACCTCGGGAGCAACAGAGCCAGCGGAGTGCCTGACCGCTTTAGCGGTTCCGGATCCGGCACCGACTTCACCCTGAAAATCAGCCGGGTGGAAGCCGAGGATGTCGGCGTGTACTACTGCATGCAGGCACTGCAGACTCTGGGGTACACCTTCGGCCAGGGCACGAAGCTCGAGATCAAGGCGGCCGCAACTACCACCCCTGCCCCTCGGCCGCCGACTCCGGCCCCAACCATCGCAAGCCAACCCCTCTCCTTGCGCCCCGAAGCTTGCCGCCCGGCCGCGGGTGGAGCCGTGCATACCCGGGGGCTGGACTTTGCCTGCGATATCTACATTTGGGCCCCGCTGGCCGGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACCCTTTACTGCAAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAGCCGTTCATGCGGCCCGTGCAGACGACTCAGGAAGAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGAGGGGGGATGCGAACTGCGCGTCAAGTTCTCACGGTCCGCCGACGCCCCCGCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGG Amino acid sequence of SEQ ID NO: 88, LTG2076 (hScFv aCD123 CD8 TM 4-1BB CD3 zeta) MLLLVTSLLLCELPHPAFLLIPEVQLVQSGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDFPYD SSGYYSDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSEIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDV GVYYCMQALQTLGYTFGQGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGC SCRFPEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Nucleotide sequence of Sequence ID No. 89 LTG2077 (hScFv aCD123 CD8 TM 4-1BB CD3 zeta) Amino acid sequence of Sequence ID No. 90 LTG2077 (hScFv aCD123 CD8 TM 4-1BB CD3 zeta) MLLLVTSLLLCELPHPAFLLIPQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITTDESTSTAYMELSSLRSEDTAVYYCARRGWG GFSSGSAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSNFMLTQPHSVSESPGNTVTISCTGSSGTIGSNFVQWYQQSPGRAPTLLIYEDTKRPSGVPPRFAGSVDSSSNSASLTISGLKTEDEAD YYCQSYDSSNWVFGGGTKLTVLGAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCS CRFPEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Nucleotide sequence of sequence number 91 LTG2078 (hScFv aCD123 CD8 TM 4-1BB CD3 zeta) Amino acid sequence of Sequence ID No. 92 LTG2078 (hScFv aCD123 CD8 TM 4-1BB CD3 zeta) MLLLVTSLLLCELPHPAFLLIPEVQLVQSGSELKKPGASVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCASTAR RGWDTAGPLDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTIACRASQTISRYLNWYQQKPGKAPKLLIYAASSLQSGVSSRFSGSGSGTEFTLTISSLQPEDFATY FCQQTYSPPITFGQGTRLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Nucleotide sequence of sequence number 93 LTG2079 (hScFv aCD123 CD8 TM 4-1BB CD3 zeta) GATGCCTTGCATATGCAAGCACTCCCACCCCGG Amino acid sequence of Sequence ID No. 94 LTG2079 (hScFv aCD123 CD8 TM 4-1BB CD3 zeta) MLLLVTSLLLCELPHPAFLLIPEVQLVETGAEVKNPGSSVKVSCKASGGTLSNYAISWVRQAPGKGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARRS SWYPEGCFQHWGQGTLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASTGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGGTDFTLTISCLQSEDFATYY CQQYYSYPLTFGGGTKLDIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Nucleotide sequence of sequence number 95 LTG2080 (hScFv aCD123 CD8 TM 4-1BB CD3 zeta) Amino acid sequence of SEQ ID NO: 96 LTG2080 (hScFv aCD123 CD8 TM 4-1BB CD3 zeta) MLLLVTSLLLCELPHPAFLLIPEVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWIGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATAPL GEVGAAVDYWGQGTLVTVSSGGGGSGGGGSGGGGSHVILTQPPSVSAAPGQEVSISCSGSDANIGTNLVSWYQHVPGTAPKLLIYENSKRPSGIPARFSSSQSGTSATLAISGLQSGDEAIYYC LTWDRTLSGKIFGGGTQLTVLGAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Nucleotide sequence of sequence number 97 LTG2081 (hScFv aCD123 CD8 TM 4-1BB CD3 zeta) ATGCTGCTGCTGGTGACCAGCCTGCTGCTGTGCGAACTGCCGCATCCGGCGTTTCTGCTGATTCCGGAAGTGCAACTTGTCCAAAGCGGAGCCGAAGTGAAGAAGCCAGGATCCTCCGTGAAAGTGTCTTGCAAAGCATCCGGCGGCACTTTCTCCTCCTACGCCATCTCCTGGGTCAGACAGGCGCCTGGACAGGGTCTGGAGTGGATGGGCATTATCAATCCTAGCGGTGGCTCCACTTCGTATGCCCAGAAGTTCCAGGGTCGGGTCACCATGACCCGGGATACTTCAACTAGCACCGTGTACATGGAACTCTCCTCGCTGCGCTCGGACGATACCGCCGTGTACTACTGTGCCCGCGAGCTGCTCTGGTTTGGAGAGCTGGACACCTACGGAATGGACGTCTGGGGACAGGGGACCACTGTGACGGTGTCGTCAGGAGGCGGAGGCTCAGGAGGGGGTGGTTCCGGAGGGGGAGGATCCCTCCCGGTGCTGACCCAGCCCCCAAGCGTCAGCGTGGCTCCGGGAAAGACCGCCCGCATCACATGCGGCGGGAACAACATCGGCTCCAAGTCCGTGCATTGGTACCAGCAGAAGCCTGGACAAGCGCCGGTGCTGGTCATCTACGACGACTCAGATC GGCCCTCCGGCATTCCCGAGCGGTTCAGCGGCTCCAACTCGGGCAACACTGCTACTCTGACCATCTCGAGGGTGGAAGCGGGGGACGAAGCAGATTACTACTGCCAAGTCTGGGACTCCAGCTCCGACCACGGGGTGTTCGGCGGAGGAACCCAGCTGACCGTGTTGGGAGCGGCCGCAACTACCACCCCTGCCCCTCGGCCGCCGACTCCGGCCCCAACCATCGCAAGCCAACCCCTCTCCTTGCGCCCCGAAGCTTGCCGCCCGGCCGCGGGTGGAGCCGTGCATACCCGGGGGCTGGACTTTGCCTGCGATATCTACATTTGGGCCCCGCTGGCCGGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACCCTTTACTGCAAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAGCCGTTCATGCGGCCCGTGCAGACGACTCAGGAAGAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGAGGGGGGATGCGAACTGCGCGTCAAGTTCTCACGGTCCGCCGACGCCCCCGCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGG Amino acid sequence of SEQ ID NO: 98, LTG2081 (hScFv aCD123 CD8 TM 4-1BB CD3 zeta) MLLLVTSLLLCELPHPAFLLIPEVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSDDTAVYYCARELLW FGELDTYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSLPVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVIYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYC QVWDSSSDHGVFGGGTQLTVLGAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Nucleotide sequence of sequence number 99 LTG2082 (hScFv aCD123 CD8 TM 4-1BB CD3 zeta) Amino acid sequence of Sequence ID No. 100 LTG2082 (hScFv aCD123 CD8 TM 4-1BB CD3 zeta) MLLLVTSLLLCELPHPAFLLIPEVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARARL GGAFDIWGQGTMVTVSSGGGGSGGGGSGGGSQSVLTQPPSVSAAPGQKVTISSCSGGSSNIGNHYVSWYQQLPGAAPKLLIYDDNKRPSGIPDRFSGSRSGTSATLGITGLQSGDEADYYCGT WDSSLAAHVFGTGTKVTVLGAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Nucleotide sequence of Sequence ID No. 101 LTG2083 (hScFv aCD123 CD8 TM 4-1BB CD3 zeta) Amino acid sequence of Sequence ID No. 102 LTG2083 (hScFv aCD123 CD8 TM 4-1BB CD3 zeta) MLLLVTSLLLCELPHPAFLLIPEVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCASQKGG GWSIDAFDIWGQGTMVTVSSGGGGSGGGGSGGGSQSVLTQPPSVSAASGQRVTISCSSGSSSNIGNNYASWYQQLPGMAPKLLIYEDNKRPSGISDRFSGSQSGTSASLAITGLQAEDEADYYC QSYDSSLSGDVVFGGGTKLTVLGAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCS CRFPEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Nucleotide sequence of sequence number 103 LTG2084 (hScFv aCD123 CD8 TM 4-1BB CD3 zeta) GATGCCTTGCATATGCAAGCACTCCCACCCCGG Amino acid sequence of Sequence ID No. 104 LTG2084 (hScFv aCD123 CD8 TM 4-1BB CD3 zeta) MLLLVTSLLLCELPHPAFLLIPEVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARVG CSGGSCYPDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYY CQQYDNLPLTFGGGTKLDIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Nucleotide sequence of Sequence ID No. 105 LTG2085 (hScFv aCD123 CD8 TM 4-1BB CD3 zeta) Amino acid sequence of Sequence ID No. 106 LTG2085 (hScFv aCD123 CD8 TM 4-1BB CD3 zeta) MLLLVTSLLLCELPHPAFLLIPQVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGQVK YSSSLGYWGQGTLVTVSSGGGGSGGGGSGGGSQSVLTQPPSVSAAPGQKVTISSCSGSSSNIGNNFVSWYQQLPGTAPKLLIYEDNKRPSGIPDRFSGSRSGTSATLGITGLQTGDEADYYCG TWDSSLGAWVFGGGTKLTVLGAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Nucleotide sequence of Sequence ID No. 107 LTG2086 (hScFv aCD123 CD8 TM 4-1BB CD3 zeta) ATGCTGCTGCTGGTGACCAGCCTGCTGCTGTGCGAACTGCCGCATCCGGCGTTTCTGCTGATTCCGGAAGTGCAGTTGGTGGAGAGCGGTGGAGGACTTGTGCAACCTGGTGGATCCCTGAGATTGTCGTGCGCAGCTTCAGGGTTCACCTTCTCCTCCTACGCCATGCACTGGGTCCGCCAAGCACCAGGAAAGGGCCTGGAATGGGTCAGCTCCATCTCCTCGTCGTCCTCGTACATCTACTATGCCGACTCCGTGAAGGGCCGCTTCACCATTAGCCGGGACAACTCAAAGAACACTCTGTACCTTCAAATGAACTCCCTGCGGGCTGAAGATACCGCCGTGTACTACTGCGCGAGGGATTGGGATGACGCGTTCGACATTTGGGGCCAGGGGACTACCGTCACCGTGTCGTCGGGTGGAGGAGGATCCGGGGGTGGAGGATCGGGAGGGGGTGGAAGCGACATTCAGATGACTCAGAGCCCGTCCTCCCTGTCGGCCTCAGTGGGCGACAGAGTGACCATCACCTGTCAAGCCAGCCAGGACATCTCAAACTACCTGAACTGGTACCAGCAGAAGCCCGGAAAGGCCCCTAAGCTGCTCATCTACGACGCCTCCAACCTGGAGACTGGAGTGCCCT CACGGTTTTCCGGCTCTGGAAGCGGCACCGATTTCACCTTCACGATCTCCTCCCTGCAACCGGAAGATATCGCGACCTACTACTGCCAGCAGTATGACAATCTCCCGCTCACCTTCGGTGGCGGCACTAAGCTCGAGATCAAAGCGGCCGCAACTACCACCCCTGCCCCTCGGCCGCCGACTCCGGCCCCAACCATCGCAAGCCAACCCCTCTCCTTGCGCCCCGAAGCTTGCCGCCCGGCCGCGGGTGGAGCCGTGCATACCCGGGGGCTGGACTTTGCCTGCGATATCTACATTTGGGCCCCGCTGGCCGGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACCCTTTACTGCAAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAGCCGTTCATGCGGCCCGTGCAGACGACTCAGGAAGAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGAGGGGGGATGCGAACTGCGCGTCAAGTTCTCACGGTCCGCCGACGCCCCCGCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGG Amino acid sequence of SEQ ID NO: 108, LTG2086 (hScFv aCD123 CD8 TM 4-1BB CD3 zeta) MLLLVTSLLLCELPHPAFLLIPEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARD WDDAFDIWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTFTFTISSLQPEDIATYYCQ QYDNLPLTFGGGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRF PEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Nucleotide sequence of Sequence ID No. 109 LTG2087 (hScFv aCD123 CD8 TM 4-1BB CD3 zeta) Amino acid sequence of Sequence ID No. 110 LTG2087 (hScFv aCD123 CD8 TM 4-1BB CD3 zeta) MLLLVTSLLLCELPHPAFLLIPQVQLVETGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDG DFWSGAIDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYY CQQYDNLPLFGGGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Nucleotide sequence of sequence number 111 LTG2088 (hScFv aCD123 CD8 TM 4-1BB CD3 zeta) Amino acid sequence of Sequence ID No. 112 LTG2088 (hScFv aCD123 CD8 TM 4-1BB CD3 zeta) MLLLVTSLLLCELPHPAFLLIPEVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARDN WGSLDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQ YDNLPHMYTFGQGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

Claims

1. A method for generating a population of RNA-modified cells, comprising the step of introducing in vitro transcribed RNA or synthetic RNA into cells in vitro, wherein the RNA comprises an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR) comprising at least one extracellular antigen-binding domain including a CD123 antigen-binding domain having the amino acid sequence of SEQ ID NO: 4, at least one transmembrane domain, at least one intracellular signaling domain, and at least one linker or at least one spacer domain.

2. Use of an isolated nucleic acid molecule for producing a pharmaceutical composition that induces antitumor immunity in mammals, wherein the isolated nucleic acid molecule comprises a vector containing an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR) comprising at least one extracellular antigen-binding domain including a CD123 antigen-binding domain containing the amino acid sequence of SEQ ID NO: 4, at least one transmembrane domain, at least one intracellular signaling domain, and at least one linker or at least one spacer domain.

3. Use of a chimeric antigen receptor (CAR) for producing a pharmaceutical composition for treating or preventing cancer in mammals, wherein the CAR comprises at least one extracellular antigen-binding domain comprising a CD123 antigen-binding domain comprising the amino acid sequence of SEQ ID NO: 4, at least one transmembrane domain, at least one intracellular signaling domain, and at least one linker or at least one spacer domain.

4. Use of a T cell population for producing a pharmaceutical composition for treating a mammal having a disease, disorder or condition associated with elevated expression of a tumor antigen, wherein the T cell population comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR), the CAR comprising at least one extracellular antigen-binding domain comprising a CD123 antigen-binding domain comprising the amino acid sequence of SEQ ID NO: 4, at least one linker or spacer domain, at least one transmembrane domain, and at least one intracellular signaling domain, and the T cell population is a T cell population of a subject having cancer.

5. A method for producing chimeric antigen receptor-expressing cells, comprising the step of introducing an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR) comprising at least one extracellular antigen-binding domain including a CD123 antigen-binding domain having the amino acid sequence of SEQ ID NO: 4, at least one transmembrane domain, at least one intracellular signaling domain, and at least one linker or at least one spacer domain into cells in vitro.

6. A method for producing chimeric antigen receptor-expressing cells according to claim 5, wherein the cells are T cells or a population of cells containing T cells.

7. The method according to claim 1, wherein at least one transmembrane domain comprises a transmembrane domain of a protein containing the alpha, beta, or zeta chain of a T cell receptor, CD8, CD28, CD3 epsilon, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD83, CD86, CD134, CD137, and CD154.

8. The method according to claim 1, wherein at least one extracellular antigen-binding domain containing a CD123 antigen-binding domain, at least one intracellular signaling domain, or both are connected to a transmembrane domain by a linker or spacer domain.

9. The method according to claim 1, wherein at least one linker or spacer domain is derived from the extracellular domain of CD8, TNFRSF19, or CD28 and is transmembrane-linked.

10. The method according to claim 1, wherein at least one extracellular antigen-binding domain containing an extracellular CD123 antigen-binding domain is preceded by a leader nucleotide sequence encoding a leader peptide.

11. At least one intracellular signaling domain further comprises a CD3 zeta intracellular domain, or At least one intracellular signaling domain includes a co-stimulatory domain, a primary signaling domain, or any combination thereof, The method according to claim 1, wherein 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, or 4-1BB (CD137).

12. The use according to claim 3, wherein at least one transmembrane domain comprises a transmembrane domain of a protein containing the alpha, beta, or zeta chain of a T cell receptor, CD8, CD28, CD3 epsilon, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD83, CD86, CD134, CD137, and CD154.

13. The use according to claim 3, wherein at least one extracellular antigen-binding domain containing a CD123 antigen-binding domain, at least one intracellular signaling domain, or both are connected to a transmembrane domain by a linker or spacer domain.

14. The use according to claim 3, wherein at least one linker or spacer domain is derived from the extracellular domain of CD8, TNFRSF19, or CD28 and is transmembrane-linked.

15. The use according to claim 3, wherein at least one extracellular antigen-binding domain containing an extracellular CD123 antigen-binding domain is preceded by a leader nucleotide sequence encoding a leader peptide.

16. At least one intracellular signaling domain further comprises a CD3 zeta intracellular domain, or At least one intracellular signaling domain includes a co-stimulatory domain, a primary signaling domain, or any combination thereof, The use according to claim 3, wherein 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, or 4-1BB (CD137).

17. The use according to claim 3, wherein the cancer is a hematological cancer.

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

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

20. The use according to claim 18, wherein the lymphoma is mantle cell lymphoma, non-Hodgkin lymphoma, or Hodgkin lymphoma.

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