Compositions and methods for treating cancer with anti-CD33 immunotherapy

Abstract

To provide a method for providing anti-tumor immunity in a human subject and a method of treating cancer in the human subject.SOLUTION: A method for providing anti-tumor immunity in a human subject includes a step which brings the human subject the anti-tumor immunity by administering an effective amount of isolated T cells or isolated natural killer (NK) cells. The isolated T cells or the isolated NK cells include vectors including nucleic acid molecules encoding chimeric antigen receptors (CAR). The CAR includes from N terminal to C terminal (i) a heavy chain variable region having one of specific two kinds of amino acid sequences or a single-chain segment variable (ScFv) domain selected from a group comprising other specific four kinds of amino acid sequences and includes at least one extracellular antigen binding domain bound to CD33.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 62 / 620,139, filed January 22, 2018, and U.S. Provisional Patent Application No. 62 / 476,438, filed March 24, 2017, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on March 20, 2018, is named Sequence_Listing.txt and is 124 kilobytes in size.

[0003] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made in the implementation of a Cooperative Research and Development Agreement with the National Institutes of Health, an agency of the U.S. Department of Health and Human Services. The U.S. Government has certain rights in this invention.

[0004] Field of the Disclosure The present application relates to the field of cancer, in particular to CD33 antigen-binding domains and chimeric antigen receptors (CARs) comprising such CD33 antigen-binding domains, and methods of use thereof. [Background technology]

[0005] background Cancer is one of the most deadly threats to human health. In the United States alone, cancer affects nearly 1.3 million new patients each year, making it the second leading cause of death after cardiovascular disease, accounting for approximately one-quarter of all deaths. Solid tumors are responsible for most of these deaths. Although significant advances have been made in the medical treatment of certain cancers, the overall 5-year survival rate for all cancers has improved by only about 10% over the past 20 years. Cancer, or malignant tumors, metastasize and grow rapidly and uncontrolled, making treatment extremely difficult.

[0006] CD33 is a 67 kDa transmembrane cell surface glycoprotein receptor. CD33 is a member of the sialic acid-binding immunoglobulin-like lectin (SIGLEC) family. Proteins in this family mediate adhesion of leukocytes to endothelial cells by binding to sialylated glycans (Kelm S, Schauer R, Crocker PR). Glycoconj J. 1996;13:913-926). Furthermore, CD33 functions as an inhibitory receptor via immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Activation of the CD33 receptor leads to phosphorylation of two tyrosines (Y340 and Y358) in the CD33 cytoplasmic tail, which serves as a docking site for SHP phosphatases and participates in inhibitory signaling cascades, including downregulation of calcium mobilization (Paul SP1, Taylor LS, Stansbury EK, McVicar DW Blood. 2000 July 15;96(2):483-90).

[0007] CD33 is a myeloid lineage differentiation antigen and is highly expressed on myeloid progenitor cells (Andrews RG, Torok-Storb B, Bernstein ID. Blood CD33 is expressed at low levels on differentiated myeloid cells, namely macrophages and granulocytes (Simmons D, Seed BJ Immunol. 1983; 141: 2797-2800). Meanwhile, CD33 has been reported to be expressed in 87.8%-99% of acute myeloblastic leukemias (AML) (A Ehninger et al. Blood Cancer Journal (2014) 4: e218; Christina Krupka et al. Blood 2014 123: 356-365). AML is a severe disease with a 5-year survival rate of approximately 26% (available on the World Wide Web at cancer.net / cancer-types / leukemia-acute-myeloid-aml / statistics). The current standard of care for AML consists of induction treatment with high-dose chemotherapy or high-dose radiation, followed by consolidation, if necessary, consisting of allogeneic stem cell transplantation and further courses of chemotherapy (available on the World Wide Web at cancer.org / cancer / acute-myeloid-leukemia / treating / typical-treatment-of-aml.html). The high toxicity associated with this treatment and the risk of complications such as myelosuppression or GVHD have driven the search for better therapeutic alternatives.

[0008] Several novel approaches for treating AML are currently under investigation, including antibody-drug conjugates (SGN-CD33A, vadastuximab butarilin, Stein AS et al. (2015). Blood, 126(23):324; Phase I-II clinical trial NCT02706899), bispecific T cell-engaging antibodies (AMG330, Laszlo GS et al. Blood 2013:123(4):554-561, NCT02520427), and CART-33 cells (Wang QS et al. Mol Ther. 2015 Jan;23(1):184-91, NCT01864902). However, some of the novel approaches have been hindered by clinical toxicity. A Seattle Genetics Phase I clinical trial testing an SGN-CD33 drug was recently put on hold due to the risk of liver toxicity (available on the World Wide Web at businesswire.com / news / home / 20161227005087 / en / Seattle-Genetics-Announces-Clinical-Hold-Phase-1). Gemtuzumab ozogamicin (Mylotarg, Pfizer / Wyeth) was voluntarily withdrawn from the market by its manufacturer in 2010 after the development of potentially fatal veno-occlusive liver disease observed in a post-marketing clinical trial (Jacob M. Rowe and Bob Lowenberg Blood 2013 121:4838-4841). The FDA has + Despite the recent reintroduction of Mylotarg for adult AML and relapsed / refractory pediatric AML, new, more conservative, smaller dosages and new regimens are prescribed for the drug (FDA press release, September 2017, available on the worldwide web at fda.gov). The efficacy of this treatment, the durability of patient responses to Mylotarg, instances of tumor antigen escape, and its safety profile under the new regimens remain to be determined. Thus, there remains a pressing need for safe, effective, and durable AML treatments.

[0009] Chimeric antigen receptors (CARs) are hybrid molecules that contain three essential units: (1) an extracellular antigen-binding motif, (2) a linking / transmembrane motif, and (3) an intracellular T cell signaling motif (Long AH, Haso WM, Orentas RJ. Lessons learned from a highly-active CD2-specific chimeric antigen receptor. Oncoimmunology. 2013;2(4):e23621). The antigen-binding motif of CARs is generally based on the single-chain fragment variable (ScFv), the smallest binding domain of an immunoglobulin (Ig) molecule. Alternative antigen-binding motifs include, for example, receptor ligands (i.e., IL-13 binds to tumor-expressed IL-13 receptors). CAR vectors have been engineered (e.g., intact immune receptors, library-derived peptides, and innate immune system effector molecules (e.g., NKG2D). Alternative cellular targets for CAR expression (e.g., NK or gamma-delta T cells) are also under development (Brown CE et al. Clin Cancer Res. 2012;18(8):2199-209; Lehner M et al. PLoS One. 2012;7(2):e31210). Considerable effort remains to be undertaken in defining the most active T cell populations for transduction with CAR vectors, determining optimal culture and expansion techniques, and defining the molecular details of the CAR protein structure itself.

[0010] The linking motif of the CAR can be a relatively stable structural domain, such as the constant domain of IgG, or can be designed to be an extended, flexible linker. Structural motifs, such as those derived from the constant domain of IgG, can be used to extend the ScFv-binding domain away from the T cell plasma membrane surface. This may be important for some tumor targets, where the binding domain is particularly close to the tumor cell surface membrane (e.g., for disialoganglioside GD2; Orentas et al., unpublished observation). To date, the signaling motif used in CARs has always included the CD3-ζ chain, because this core motif is an important signal for T cell activation. The first reported second-generation CARs featured the CD28 signaling domain and CD28 transmembrane sequence. This motif was also used in third-generation CARs containing the CD137 (4-1BB) signaling motif (Zhao Y et al., J Immunol. 2009; 183(9):5563-74). With the advancement of new technologies, activation of T cells by beads linked to anti-CD3 and anti-CD28 antibodies, and the presence of the canonical "signal 2" derived from CD28, no longer needs to be encoded by the CAR itself. Using bead activation, third-generation vectors were found to be no superior to second-generation vectors in in vitro assays and offered no clear benefit over second-generation vectors in mouse models of leukemia (Haso W, Lee DW, Shah NN, Stetler-Stevenson M, Yuan CM, Pastan IH, Dimitrov DS, Morgan RA, FitzGerald DJ, Barrett DM, Wayne AS, Mackall CL, Orentas RJ. Anti-CD22-chimeric antigen receptors targeting B cell precursor acute lymphoblastic leukemia. Blood. 2013;121(7):1165-74; Kochenderfer JN et al. Blood. 2012;119(12):2709-20).This is due to the second generation CD28 / CD3-ζ (Lee DW et al. American Society of Hematology Annual Meeting. New Orleans, LA; December 7-10, 2013) and CD137 / CD3-ζ signaling formats (Porter. This is supported by the clinical success of CD19-specific CARs (DL et al. N Engl J Med. 2011;365(8):725-33). In addition to CD137, other tumor necrosis factor receptor superfamily members, such as OX40, can also provide important sustained signals in CAR-transduced T cells (Yvon E et al. Clin Cancer Res. 2009;15(18):5852-60). The culture conditions under which the CAR T cell population is cultured are equally important.

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

[0012] T cell-based immunotherapy is an emerging field in synthetic biology; multiple promoters and gene products are envisioned to direct these highly potent cells to the tumor microenvironment, where they can escape negative regulatory signals and mediate effective tumor killing. Elimination of unwanted T cells via drug-induced dimerization of an inducible caspase-9 construct with AP1903 demonstrates one way in which a powerful switch can be pharmacologically initiated to control T cell populations (Di Stasi A et al. N Engl J Med. 2011;365(18):1673-83). Creation of an effector T cell population immune to the negative regulatory effects of transforming growth factor-β by expression of a decoy receptor further demonstrates the extent to which effector T cells can be engineered for optimal antitumor activity (Foster AE et al. J Immunother. 2008;31(5):500-5). Thus, CARs appear to be able to induce T cell activation in a manner similar to endogenous T cell receptors; however, to date, the major obstacles to the clinical application of this technology have been the limited in vivo expansion of CAR T cells, the rapid loss of cells after infusion, and disappointing clinical activity. Several antibody-based modalities for targeting CD33-positive tumors are currently under development, including anti-CD33 antibody-drug conjugates (Stein AS et al., Blood, 2015, 126(23):324), bispecific T cell inducers (BiTEs) (Laszlo GS et al., Blood, 2013, 123(4):554-561), and CAR T cells (Wang QS et al., Mol. Ther., 2015, January; 23(1):184-91).Recent studies in preclinical models of AML have shown that lysis of CD33-positive AML blasts and tumor cell lines by CD33-targeting modalities is achievable in vitro and in vivo, but treatment-related toxicity (Rowe JM and Lowenberg B, Blood 2013;121:4838-4841; Wang QS et al. Mol Ther. 2015;23(1):184-91; available at: NCT01864902) and suboptimal efficacy (Walter RB et al. Blood. 2012;119(26):6198-6208; Cowan AJ et al. Biosci. Several challenges of this approach have become apparent in clinical settings, including the need for high-density CD33 antigen expression and additional T cell costimulation / checkpoint inhibition for optimal BiTE function (Laszlo GS et al. Blood. 2014; 123(4):554-56; Laszlo GS et al. Blood Cancer Journal (2015) 5, e340). Therefore, novel compositions for the treatment of AML using approaches that can exhibit specific and effective antitumor effects without the above-mentioned drawbacks are needed. There is an urgent and long-felt need in the art to discover methods and methods for treating cancer. [Means for solving the problem]

[0013] The present invention addresses these needs by providing CAR compositions and therapeutic methods that can be used to treat cancer and other diseases and / or conditions. In particular, the invention disclosed and described herein provides CARs that can be used to treat diseases, disorders, or conditions associated with dysregulation of CD33 expression, wherein the CAR comprises a CD33 antigen-binding domain that exhibits high surface expression on transduced T cells and exhibits high levels of cytolysis and in vivo proliferation and persistence of the transduced T cells.

[0014] overview Provided herein are novel anti-CD33 antibodies or their antigen-binding domains, chimeric antigen receptors (CARs) containing such CD33 antigen-binding domains, host cells (e.g., T cells) expressing the receptors, and nucleic acid molecules encoding the receptors. CARs may consist of a single molecule expressed on the surface of effector cells, or may consist of a signaling module and a soluble targeting module expressed in effector cells; for example, a complete functional CAR is formed when the soluble targeting module binds to the signaling module expressed in the cells. CARs exhibit high surface expression on transduced T cells, high levels of cytolysis, and in vivo proliferation and persistence of transduced T cells. Also provided are methods using the disclosed CARs, host cells, and nucleic acid molecules, for example, to treat cancer in a subject.

[0015] Thus, in one aspect, an isolated polynucleotide encoding a human anti-CD33 antibody or fragment thereof is provided, comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, and 11.

[0016] In one embodiment, an isolated polynucleotide encoding a fully human anti-CD33 antibody or fragment thereof is provided, wherein the antibody or fragment thereof comprises a fragment selected from the group consisting of a Fab fragment, a F(ab')2 fragment, an Fv fragment, and a single-chain Fv (ScFv).

[0017] In one embodiment, an isolated polynucleotide encoding a fully human anti-CD33 antibody or fragment thereof is provided, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, and 12.

[0018] In one embodiment, an isolated nucleic acid molecule is provided that encodes a chimeric antigen receptor (CAR) comprising, from N-terminus to C-terminus, at least one CD33 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, and 11.

[0019] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded extracellular CD33 antigen-binding domain comprises at least one single-chain variable fragment of an antibody that binds to CD33.

[0020] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded extracellular CD33 antigen-binding domain comprises at least one heavy chain variable region of an antibody that binds to CD33.

[0021] In one embodiment, the targeting domain of the CAR is expressed separately in the form of a monoclonal antibody, ScFv Fab, Fab'2, and is linked to an additional binding tag or epitope. and an antigen targeting domain comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, and 11, while the CAR component expressed in the effector cell comprises a binding domain that is specifically directed to bind to a tag or epitope expressed on the soluble CAR module, e.g., when the soluble component of the CAR specifically binds to the cell-bound CAR component, a complete functional CAR structure is formed.

[0022] In another embodiment, the targeting domain of the CAR is separately expressed in the form of a monoclonal antibody, an ScFv Fab, Fab'2, and comprises an antigen targeting domain comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, and 11, and an additional scFv, while the CAR component expressed in the effector cell comprises a tag or epitope that specifically reacts with the additional scFv expressed in the soluble CAR module, e.g., when the soluble component of the CAR specifically binds to the cell-bound CAR component, a complete functional CAR structure is formed.

[0023] In yet another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded CAR extracellular CD33 antigen-binding domain further comprises at least one lipocalin-based antigen-binding antigen (anticalin) that binds to CD33.

[0024] In one embodiment, an isolated nucleic acid molecule is provided in which the encoded extracellular CD33 antigen-binding domain is connected to the transmembrane domain by a linker domain.

[0025] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded CD33 extracellular antigen-binding domain is preceded by a sequence encoding a leader or signal peptide.

[0026] In yet another embodiment, an isolated nucleic acid molecule is provided encoding a CAR comprising at least one CD33 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, and 11, wherein the CAR further encodes an extracellular antigen-binding domain that targets an antigen including, but not limited to, CD19, CD20, CD22, ROR1, mesothelin, CD33, CD38, CD123 (IL3RA), CD138, BCMA (CD269), GPC2, GPC3, FGFR4, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, NY-ESO-1 TCR, MAGE A3 TCR, or any combination thereof.

[0027] In certain embodiments, the further encoded extracellular antigen-binding domain is an anti-CD19 ScFv antigen-binding domain, an anti-CD20 ScFv antigen-binding domain, an anti-CD22 ScFv antigen-binding domain, an anti-ROR1 ScFv antigen-binding domain, an anti-mesothelin ScFv antigen-binding domain, an anti-CD33 ScFv antigen-binding domain, an anti-CD38 ScFv antigen-binding domain, an anti-CD123 (IL3RA) ScFv antigen-binding domain, an anti-CD138 ScFv antigen-binding domain, an anti-BCMA (CD269) ScFv antigen-binding domain, an anti-GPC2 ScFv antigen-binding domain, an anti-GPC3 ScFv antigen-binding domain, an anti-FGFR4 ScFv antigen-binding domain, an anti-c-Met ScFv antigen-binding domain, an anti-PMSA ScFv antigen-binding domain, an anti-glycolipid F77 ScFv antigen-binding domain, or an anti-EGFRvIII ScFv. Provided is an isolated nucleic acid molecule encoding a CAR comprising an ScFv antigen-binding domain, an anti-GD-2 ScFv antigen-binding domain, an anti-NY-ESo-1 TCR ScFv antigen-binding domain, an anti-MAGE A3 TCR ScFv antigen-binding domain, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or any combination thereof.

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

[0029] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, in which an extracellular CD33 antigen-binding domain, an intracellular signaling domain, or both, is connected to a transmembrane domain by a linker or spacer domain.

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

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

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

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

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

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

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

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

[0038] In one aspect, provided herein is a chimeric antigen receptor (CAR) comprising, from N-terminus to C-terminus, at least one CD33 antigen-binding domain, at least one transmembrane domain, and at least one intracellular signaling domain.

[0039] In one embodiment, the extracellular CD33 antigen-binding domain comprises at least one single chain variable fragment of an antibody that binds to the antigen, or at least one heavy chain variable region of an antibody that binds to the antigen, or a combination thereof.

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

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

[0042] In one embodiment, the extracellular antigen-binding domain is an anti-CD19 ScFv antigen-binding domain, an anti-CD20 ScFv antigen-binding domain, an anti-CD22 ScFv antigen-binding domain, an anti-ROR1 ScFv antigen-binding domain, an anti-mesothelin ScFv antigen-binding domain, an anti-CD33 ScFv antigen-binding domain, an anti-CD38 ScFv antigen-binding domain, an anti-CD123 (IL3RA) ScFv antigen-binding domain, an anti-CD138 ScFv antigen-binding domain, an anti-BCMA (CD269) ScFv antigen-binding domain, an anti-GPC2 ScFv antigen-binding domain, an anti-GPC3 ScFv antigen-binding domain, an anti-FGFR4 ScFv antigen-binding domain, an anti-c-Met ScFv antigen-binding domain, an anti-PMSA ScFv antigen-binding domain, an anti-glycolipid F77 ScFv antigen-binding domain, or an anti-EGFRvIII ScFv. CARs are provided that comprise an ScFv antigen-binding domain, an anti-GD-2 ScFv antigen-binding domain, an anti-NY-ESo-1 TCR ScFv antigen-binding domain, an anti-MAGE A3 TCR ScFv antigen-binding domain, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or any combination thereof.

[0043] In another embodiment, the extracellular antigen-binding domain is an immunoglobulin variable heavy chain alone (VH) anti-CD19 antigen-binding domain, anti-CD20 VH antigen-binding domain, anti-CD22 VH antigen-binding domain, anti-ROR1 VH antigen-binding domain, anti-mesothelin VH antigen-binding domain, anti-CD33 VH antigen-binding domain, anti-CD38 VH antigen-binding domain, anti-CD123 (IL3RA) VH antigen-binding domain, anti-CD138 VH antigen-binding domain, anti-BCMA (CD269) VH antigen-binding domain, anti-GPC2 VH antigen-binding domain, anti-GPC3 VH antigen-binding domain, anti-FGFR4 VH antigen-binding domain, anti-c-Met VH antigen-binding domain, anti-PMSA VH antigen-binding domain, anti-glycolipid F77 VH antigen-binding domain, anti-EGFRvIII VH antigen-binding domain, anti-GD-2 VH antigen-binding domain, anti-NY-ESO-1 TCR CARs are provided that comprise 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 thereto, or any combination thereof.

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

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

[0046] In one embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 15 (LTG 1905 EF1a VH-2 CD33 -CD8 TM-41BB-CD3 zeta nucleic acid sequence (Figure 2A)). In one embodiment, the nucleic acid sequence comprises SEQ ID NO: 16 (LTG 1905 EF1a VH-2 CD33 -CD8 TM-41BB-CD3 zeta amino acid sequence (Figure 2A) encodes a CAR.

[0047] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 17 (LTG 1906 Ef1a- VH-4 CD33 -CD8 TM-41BB-CD3 zeta nucleic acid sequence (Figure 2B)). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 18 (LTG 1906 Ef1a- VH-4 CD33 -CD8 TM-41BB-CD3 zeta amino acid sequence (Figure 2B)).

[0048] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 19 (LTG1936 Ef1a ScFv9 CD33 CD8 TM-41BB-CD3 Zeta CAR nucleotide sequence (Figure 2C)). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 20 (LTG1936 Ef1a ScFv9 CD33 CD8 TM-41BB-CD3 Zeta CAR amino acid sequence (Figure 2C)).

[0049] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 21 (LTG1937 Ef1a ScFv10 CD33 CD8 TM-41BB-CD3 zeta nucleic acid sequence (Figure 2D)). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 22 (LTG1937 Ef1a ScFv10 CD33 CD8 TM-41BB-CD3 zeta amino acid sequence (Figure 2D)).

[0050] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 23 (LTG1938 EF1a ScFv12 CD33 CD8 TM-41BB-CD3 zeta nucleic acid sequence (Figure 2E)). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 24 (LTG1938 EF1a ScFv12 CD33 CD8 TM-41BB-CD3 zeta amino acid sequence (Figure 2E)).

[0051] In another embodiment, the nucleic acid sequence encoding the CAR is the nucleic acid sequence of SEQ ID NO: 25 (LTG1939 EF1a_ScFv15 CD33 CD8 TM-41BB-CD3 enzyme In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 26 (LTG1939 EF1a ScFv15 CD33 CD8 TM-41BB-CD3 zeta amino acid sequence (Figure 2F)).

[0052] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 69 (LTG1927 EF1a-CD33_4 CD8 TM-CD28-CD3zeta nucleic acid sequence (Figure 12A)). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 70 (LTG1927 EF1a-CD33_4 CD8 TM-CD28-CDzeta amino acid sequence (Figure 12A)).

[0053] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 71 (LTG_D0033 Ef1a-CD33_4 VH TNFRSF19 H_TM_CD28z nucleic acid sequence (Figure 12B)). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 72 (LTG_D0033 (Ef1a-CD33_4 VH TNFRSF19 H_TM_CD28z) amino acid sequence (Figure 12B)).

[0054] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 73 (LTG_D0034 Ef1a-CD33_4 VH TNFRSF19 H_TM_4-1BBz nucleic acid sequence (Figure 12C)). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 74 (LTG_D0034 Ef1a-CD33_4 VH TNFRSF19 H_TM_4-1BBz amino acid sequence (Figure 12C)).

[0055] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 87 (LTG_D0035 Ef1a_CD33_4 VH H CH2 CH3 IgG4_CD8TM_CD28z nucleic acid sequence (Figure 12F)). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 88 (LTG_D0035 Ef1a_CD33_4 VH H CH2 CH3 IgG4_CD8TM_CD28z amino acid sequence (Figure 12F)).

[0056] In one embodiment, the CARs disclosed herein are modified to express or contain a detectable marker for use in diagnosis, monitoring and / or prediction of treatment outcome, such as progression-free survival in cancer patients, or to monitor the progress of such treatment.

[0057] In one embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 75 (LTG_D0015 Ef1a-CD33_4 VH CD8 BBz T2A tEGFR nucleic acid sequence (Figure 12D)). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 76 (LTG_D0015 Ef1a-CD33_4 VH CD8 BBz T2A tEGFR amino acid sequence (Figure 12D)).

[0058] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 77 (LTG_D0016 Ef1a-CD33_4 VH CD8 28z T2A tEGFR nucleic acid sequence (Figure 12E)). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 78 (LTG_D0015 Ef1a-CD33_4 VH CD8 28z T2A tEGFR amino acid sequence (Figure 12E)).

[0059] In one embodiment, the nucleic acid molecules encoding the disclosed CARs can be contained in a vector, such as a viral vector, such as a DNA vector, an RNA vector, a plasmid vector, a cosmid vector, a herpes virus vector, a measles virus vector, a lentivirus vector, an adenovirus vector, or a retrovirus vector, or is a combination of them.

[0060] In certain embodiments, the vector further comprises a promoter that is an inducible promoter, a tissue-specific promoter, a constitutive promoter, a suicide promoter, or any combination thereof.

[0061] In yet another embodiment, the CAR-expressing vector can be further modified to include one or more operable elements to control the expression of CAR T cells or to eliminate CAR-T cells by a suicide switch. The suicide switch can include, for example, an apoptosis-inducing signaling cascade or a drug that induces cell death. In a preferred embodiment, the CAR-expressing vector can be further modified to express an enzyme such as thymidine kinase (TK) or cytosine deaminase (CD).

[0062] In another aspect, a host cell comprising a nucleic acid molecule encoding a CAR is also provided. In some embodiments, the host cell is a T cell, for example, a primary T cell obtained from a subject. In one embodiment, the host cell is a CD8 + T cells.

[0063] In yet another aspect, a pharmaceutical composition is provided comprising an antitumor effective amount of a human T cell population, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), the CAR comprising at least one extracellular antigen-binding domain, at least one linker domain, at least one transmembrane domain, and at least one intracellular signaling domain, the extracellular antigen-binding domain comprising a CD33 antigen-binding domain comprising the amino acid sequence of SEQ ID NOs: 2, 4, 6, 8, 10, and 12. The T cells are from a human with cancer. The cancer includes, inter alia, hematological cancers, such as leukemia (e.g., chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), or chronic myelogenous leukemia (CML)), lymphoma (e.g., mantle cell lymphoma, non-Hodgkin's lymphoma, or Hodgkin's lymphoma), or multiple myeloma, or a combination thereof.

[0064] In one embodiment, a pharmaceutical composition is provided, wherein at least one transmembrane domain of the CAR comprises a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, mesothelin, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154, or a combination thereof.

[0065] In another embodiment, pharmaceutical compositions are provided wherein the human cancer comprises oral and pharyngeal cancer (tongue, mouth, pharynx, head and neck), digestive system cancer (esophagus, stomach, small intestine, colon, rectum, anus, liver, intrahepatic bile duct, gallbladder, pancreas), respiratory system cancer (larynx, lung and bronchus), bone and joint cancer, soft tissue cancer, skin cancer (melanoma, basal cell carcinoma and squamous cell carcinoma), childhood tumors (neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma), tumors of the central nervous system (brain tumor, astrocytoma, glioblastoma, glioma), and adult cancers including cancer of the breast, reproductive system (cervix, uterus, ovary, vulva, vagina, prostate, testicle, penis, endometrium), urinary system (bladder, kidney and renal pelvis, ureter), eye and orbit, endocrine system (thyroid), and brain and other nervous system, or any combination thereof.

[0066] In yet another embodiment, a pharmaceutical composition is provided comprising an anti-tumor effective amount of a population of human T cells from a human with cancer, wherein the cancer is a refractory cancer that is unresponsive to one or more chemotherapeutic agents. The cancer may be hematopoietic cancer, myelodysplastic syndrome, pancreatic cancer, head and neck cancer, skin tumor, acute lymphoblastic leukemia (ALL), minimal residual disease (MRD) in acute myeloid leukemia (AML), CLL (chronic lymphocytic leukemia), CML (chronic myeloid leukemia), or other cancers. and / or other hematological cancers and solid tumors, or any combination thereof.

[0067] In another aspect, a method of generating CAR-containing T cells (hereinafter "CAR T cells") is provided, which method comprises transducing T cells with a vector or nucleic acid molecule encoding the disclosed CAR that specifically binds to CD33, thereby generating the CAR T cells.

[0068] In yet another aspect, a method of generating a population of RNA engineered cells is provided, comprising introducing in vitro transcribed or synthetic RNA of a nucleic acid molecule encoding a disclosed CAR into cells of a subject to generate CAR cells.

[0069] In yet another aspect, a method is provided for diagnosing a disease, disorder, or condition associated with CD33 expression in a cell, the method comprising the steps of: (a) contacting the cell with a human anti-CD33 antibody or fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, and 12; and (b) detecting the presence of CD33, wherein the presence of CD33 diagnoses the disease, disorder, or condition associated with CD33 expression.

[0070] In one embodiment, the disease, disorder or condition associated with CD33 expression is cancer, including hematopoietic cancer, myelodysplastic syndrome, pancreatic cancer, head and neck cancer, skin tumor, adult B-cell malignancies including acute lymphoblastic leukemia (ALL), minimal residual disease (MRD) in acute myeloid leukemia (AML), CLL (chronic lymphocytic leukemia), CML (chronic myeloid leukemia), non-Hodgkin's lymphoma (NHL), pediatric B-cell malignancies (including B-cell lineage ALL (acute lymphocytic leukemia)), multiple myeloma, lung cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, melanoma or other hematological cancers and solid tumors, or any combination thereof.

[0071] In another embodiment, a method is provided for diagnosing, prognosing, or determining the risk of a disease associated with CD33 in a mammal, the method comprising detecting CD33 expression in a sample from a mammal, the method comprising the steps of: (a) contacting the sample with a human anti-CD33 antibody or fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, and 12; and (b) detecting the presence of CD33, wherein the presence of CD33 is diagnostic of the disease associated with CD33 in the mammal.

[0072] In another embodiment, a method of inhibiting CD33-dependent T cell inhibition is provided, comprising contacting a cell with a human anti-CD33 antibody or fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, and 12. In one embodiment, the cell is selected from the group consisting of a CD33-expressing tumor cell, a tumor-associated macrophage, and any combination thereof.

[0073] In another embodiment, a method is provided for altering the tumor microenvironment to block T cell inhibition mediated by cells expressing CD33 and inhibit tumor growth in a mammal, the method comprising administering to the mammal an effective amount of a composition comprising an isolated anti-CD33 antibody or fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, and 12. In one embodiment, the cells are tumor cells, tumor-associated macrophages, and CD33-expressing cells. Any combination thereof is selected from the group consisting of:

[0074] In another embodiment, a method is provided for inhibiting, suppressing, or preventing immunosuppression of an anti-tumor or anti-cancer immune response in a mammal, comprising the step of administering to the mammal an effective amount of a composition comprising an isolated anti-CD33 antibody or fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, and 12. In one embodiment, the antibody or fragment thereof inhibits an interaction between a first cell and a T cell, wherein the first cell is selected from the group consisting of a CD33-expressing tumor cell, a tumor-associated macrophage, and any combination thereof.

[0075] In another aspect, provided is a method of inducing anti-tumor immunity in a mammal, the method comprising administering to the mammal a therapeutically effective amount of T cells transduced with a vector or nucleic acid molecule encoding a disclosed CAR.

[0076] In another embodiment, a method of treating or preventing cancer in a mammal is provided, comprising administering to the mammal one or more of the disclosed CARs in an amount effective to treat or prevent cancer in the mammal. The method comprises administering to the subject a therapeutically effective amount of host cells expressing the disclosed CARs that specifically bind to CD33 and / or one or more of the above-mentioned antigens under conditions sufficient to form an immune complex between the antigen-binding domain of the CAR and the extracellular domain of CD33 and / or one or more of the above-mentioned antigens in the subject.

[0077] In yet another embodiment, a method is provided for treating a mammal having a disease, disorder, or condition associated with elevated expression of a tumor antigen, comprising administering to the subject an anti-tumor effective amount of a pharmaceutical composition comprising a population of T cells, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises at least one extracellular CD33 antigen-binding domain, at least one linker or spacer domain, at least one transmembrane domain, at least one intracellular signaling domain comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, or 12, or any combination thereof, and wherein the T cells are from a subject with cancer.

[0078] In yet another embodiment, a method of treating cancer in a subject in need thereof is provided, comprising administering to the subject an anti-tumor effective amount of a pharmaceutical composition comprising a population of T cells, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises at least one CD33 antigen-binding domain comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, or 12, or any combination thereof, at least one linker or spacer domain, at least one transmembrane domain, and at least one intracellular signaling domain, and the T cells are from a subject with cancer. In some embodiments of the above-described methods, the at least one transmembrane domain comprises the transmembrane alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, mesothelin, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154, or a combination thereof.

[0079] In yet another embodiment, a method is provided for generating a persistent population of engineered T cells in a human diagnosed with cancer. In one embodiment, the method comprises administering to the human T cells engineered to express a CAR, wherein the CAR comprises at least one CD33 antigen-binding domain, at least one transmembrane domain, and at least one intracellular signaling domain comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, or 12, or any combination thereof; and administering to the human T cells engineered to express a CAR. The persistent population, or population of progeny of T cells, persists in the human for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, or 3 years after administration.

[0080] In one embodiment, the progeny T cells in the human comprise memory T cells. In another embodiment, the T cells are autologous T cells.

[0081] In all of the aspects and embodiments of the methods described herein, any of the above-mentioned cancers, diseases, disorders, or conditions associated with elevated expression of tumor antigens can be treated or prevented or ameliorated using one or more of the CARs disclosed herein.

[0082] In yet another aspect, there is provided a kit for generating the chimeric antigen receptor T cells as described above, or for preventing, treating, or ameliorating any of the cancers, diseases, disorders, or conditions associated with elevated expression of a tumor antigen in the subject as described above, the kit comprising a container containing any one of the nucleic acid molecules, vectors, host cells, or compositions disclosed above, or any combination thereof, and instructions for using the kit.

[0083] It is understood that the CARs, host cells, nucleic acids, and methods are useful beyond the specific aspects and embodiments described in detail herein. The foregoing features and advantages of the present disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying drawings. [Brief explanation of the drawings]

[0084]

Figure 1

Figure 2A

[0023] Figure 2A shows several chimeric antigen receptors (CARs) containing novel extracellular CD33 antigen-binding domain sequences. The general schematic for a CAR includes, from N- to C-terminus, a signal peptide, an anti-CD33 binder variable heavy chain fragment or a linked single-chain fragment variable (ScFv), an extracellular linker, a transmembrane domain, 4-1BB, and CD3 zeta. Figure 2A shows a lentiviral vector expressing a CAR containing the LTG 1905 EF1a VH-2 CD33-CD8 TM-41BB-CD3 zeta nucleic acid sequence and the encoded amino acid sequence.

Figure 2B

Figure 2C

[0023] Figure 2C shows several chimeric antigen receptors (CARs) containing novel extracellular CD33 antigen-binding domain sequences. The general schematic for a CAR includes, from N- to C-terminus, a signal peptide, an anti-CD33 binder variable heavy chain fragment or a linked single-chain fragment variable (ScFv), an extracellular linker, a transmembrane domain, 4-1BB, and CD3 zeta. Figure 2C shows a lentiviral vector expressing a CAR containing the LTG1936 EF1a ScFv9 CD33 CD8 TM-41BB-CD3 zeta nucleotide sequence and the encoded amino acid sequence.

Figure 2D

[0023] Figure 2D shows several chimeric antigen receptors (CARs) containing novel extracellular CD33 antigen-binding domain sequences. The general schematic for a CAR includes, from N- to C-terminus, a signal peptide, an anti-CD33 binder variable heavy chain fragment or a linked single-chain fragment variable (ScFv), an extracellular linker, a transmembrane domain, 4-1BB, and CD3 zeta. Figure 2D shows a lentiviral vector expressing a CAR containing the LTG1937 EF1a ScFv10 CD33 CD8 TM-41BB-CD3 zeta nucleic acid sequence and the encoded amino acid sequence.

Figure 2E

[0023] Figure 2B shows several chimeric antigen receptors (CARs) containing novel extracellular CD33 antigen-binding domain sequences. The general schematic for a CAR includes, from N- to C-terminus, a signal peptide, an anti-CD33 binder variable heavy chain fragment or a linked single-chain fragment variable (ScFv), an extracellular linker, a transmembrane domain, 4-1BB, and CD3 zeta. Figure 2E shows a lentiviral vector expressing a CAR containing the LTG1938 EF1a ScFv12 CD33 CD8 TM-41BB-CD3 zeta nucleic acid sequence and the encoded amino acid sequence.

Figure 2F

[0023] Figure 2B shows several chimeric antigen receptors (CARs) containing novel extracellular CD33 antigen-binding domain sequences. The general schematic for a CAR includes, from N- to C-terminus, a signal peptide, an anti-CD33 binder variable heavy chain fragment or a linked single-chain fragment variable (ScFv), an extracellular linker, a transmembrane domain, 4-1BB, and CD3 zeta. Figure 2F shows a lentiviral vector expressing a CAR containing the LTG1939 EF1a ScFv15 CD33 CD8 TM-41BB-CD3 zeta nucleic acid sequence and the encoded amino acid sequence.

Figure 3

Figure 4

[0023] Figure 1 shows that anti-CD33 CAR T cells incorporating immunoglobulin heavy chain variable domain binders demonstrate cytolysis of CD33-positive tumors in vitro. CAR T cells expressing anti-CD33 constructs were incubated overnight with CD33-high (HL-60), CD33-intermediate (K562), and CD33-low (Reh) targets stably transduced with firefly luciferase at effector-to-target ratios of 5, 10, and 20. The cytotoxic activity of the CAR T cells was then assessed by luciferase activity measurement, as described in Materials and Methods. N=3 + / - SEM.

Figure 5

Figure 6

Figure 7

[0023] Figure 1 shows that anti-CD33 CAR T cells incorporating immunoglobulin heavy chain variable domain binders demonstrate cytolysis of CD33-positive tumors in vitro. CAR T cells expressing anti-CD33 constructs were incubated overnight with CD33-high (HL-60, MOLM-14), CD33-intermediate (K562), and CD33-low (Reh) targets stably transduced with firefly luciferase at effector-to-target ratios of 5, 10, and 20. The cytotoxic activity of the CAR T cells was then assessed by luciferase activity measurement, as described in Materials and Methods. N=3 + / - SEM.

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12A

Figure 12B

Figure 12C

Figure 12D

[0033] Figure 12B shows several chimeric antigen receptors (CARs) containing the novel extracellular VH CD33_4 antigen-binding domain sequence in the context of different CAR configurations. The general scheme for a CAR includes, from N- to C-terminus, a signal peptide, an anti-CD33 binder variable heavy chain fragment, an extracellular linker, a transmembrane domain, a costimulatory domain, and a CD3 zeta activation domain. Some sequences contain a tEGFR tag peptide downstream from the CAR sequence, separated by a 2A ribosomal skip sequence. Figure 12C shows a lentiviral vector expressing a CAR containing the LTG_D0015 CD33_4VH CD8 BB CD3 zeta T2A tEGFR nucleic acid sequence and the encoded amino acid sequence.

Figure 12E

[0033] Figure 12B shows several chimeric antigen receptors (CARs) containing the novel extracellular VH CD33_4 antigen-binding domain sequence in the context of different CAR configurations. The general scheme for a CAR includes, from N- to C-terminus, a signal peptide, an anti-CD33 binder variable heavy chain fragment, an extracellular linker, a transmembrane domain, a costimulatory domain, and a CD3 zeta activation domain. Some sequences contain a tEGFR tag peptide downstream from the CAR sequence, separated by a 2A ribosomal skip sequence. Figure 12E shows a lentiviral vector expressing a CAR containing the LTG_D0016 CD33_4VH CD8 28 CD3 zeta T2A tEGFR nucleic acid sequence and the encoded amino acid sequence.

Figure 12F

[0085] Detailed Description definition As used herein, the singular forms "a," "an," and "the" refer to both the singular and the plural unless the context clearly indicates otherwise. For example, the term "an antigen" includes one or more antigens and can be considered equivalent to the phrase "at least one antigen." As used herein, the term "comprises" means "includes." Thus, "comprising an antigen" means "including one antigen," without excluding other elements. The phrase "and / or" means "and" or "or." It should be further understood that any and all base or amino acid sizes and all molecular weight or molecular mass values ​​given for nucleic acids or polypeptides are approximate and provided for convenience, unless otherwise specified. Although many methods and materials similar or equivalent to those described herein can be used, particularly suitable methods and materials are described below. In case of conflict, the present specification, including explanations of terms, will control. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting. The following explanations of terms are provided to facilitate review of the various embodiments.

[0086] The term "about," when referring to a measurable value, e.g., amount, temporal duration, etc., is meant to encompass variations of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% from the specified value, where such variations are appropriate for practicing the disclosed methods.

[0087] Unless otherwise specified, technical terms herein are used according to conventional usage. Definitions of common terms in molecular biology are found in Benjamin Lewin, Genes VII, 1999, published by Oxford University Press; Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, 1994, published by Blackwell Science Ltd.; and Robert J. A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, 1995; and other similar references.

[0088] The present disclosure provides a CD33 antibody or a fragment thereof, and a chimeric antigen receptor (CAR) having such a CD33 antigen-binding domain. The enhanced functional activity of the CAR is directly related to the enhanced functional activity of T cells expressing the CAR. As a result of one or more of these modifications, the CAR exhibits both high cytokine-induced cytolysis and cell surface expression on transduced T cells, along with increased in vivo T cell proliferation and persistence levels of transduced CAR-expressing T cells.

[0089] The unique ability to combine functional moieties from different protein domains is a key and innovative feature of chimeric antigen receptors (CARs). The selection of each of these protein domains, as well as the specific combinations they employ, is a key design feature. Each design domain is an essential component that can be used to manipulate lymphocyte function in various CAR platforms. For example, the selection of an extracellular binding domain can enable an otherwise ineffective CAR.

[0090] The non-variable framework components of immunoglobulin-derived protein sequences used to create the extracellular antigen-binding domain of a CAR can be completely neutral or self-associate, rendering the T cell metabolically exhausted and therefore the therapeutic T cells expressing that CAR extremely ineffective. This occurs regardless of the antigen-binding function of that CAR domain. Furthermore, the choice of intracellular signaling domain(s) can also determine the activity and durability of the therapeutic lymphocyte population used in immunotherapy. While the ability of these extracellular and intracellular domains to bind target antigens and deliver activation signals to T cells, respectively, are critical aspects of CAR design, the delivery of extracellular antigen-binding fragments is crucial. It has also become clear that the choice of source can have a significant effect on the efficacy of CARs and therefore play a crucial role in the function and clinical utility of CARs.

[0091] Surprisingly and unexpectedly, it has now been discovered that the functional activity of CAR-expressing T cells can also be determined by using a fully human antigen-binding domain in the CAR, rather than using a mouse-derived antigen-binding fragment (see UPenn-funded clinical trial, NCT02159716, using a mouse-derived SS1 ScFv sequence), which tends to induce anti-mouse immune responses and CAR T elimination in the host.

[0092] The CARs disclosed herein are expressed at high levels in cells. Cells expressing CARs have a high proliferation rate in vivo, produce large amounts of cytokines, and have high cytotoxicity against cells bearing the CD33 antigen to which the CAR binds. The use of a human extracellular CD33 antigen-binding domain results in the generation of CARs that function better in vivo, while avoiding the induction of anti-CAR immunity in the host immune response and the death of the CAR T cell population. CARs expressing a fully human extracellular CD33 ScFv antigen-binding domain exhibit superior activity / characteristics, including: i) prevention of the lack of CAR T persistence and function observed with mouse-derived binding sequences; ii) lack of effective local (i.e., intrapleural) delivery of CARs; and iii) the ability to generate CAR T cell designs based on both high-affinity and low-affinity binders for CD33. This last property allows researchers to better tune the efficacy and / or tissue specificity of CAR T products to toxicity, as tumors express more CD33 than normal tissues, allowing lower affinity binders to have greater specificity for tumors over normal tissues, thereby preventing non-on-target tumor toxicity and bystander cell killing.

[0093] Below is a detailed description of the CARs of the present invention, including a description of their extracellular CD33 antigen-binding domain, transmembrane domain, and intracellular domain, along with further description of CARs, antibodies and antigen-binding fragments thereof, conjugates, nucleotides, expression, vectors, and host cells, methods of treatment, compositions, and kits using the disclosed CARs.

[0094] A. Chimeric Antigen Receptor (CAR) The CARs disclosed herein comprise at least one CD33 antigen-binding domain capable of binding to CD33, at least one transmembrane domain, and at least one intracellular domain.

[0095] Chimeric antigen receptors (CARs) are artificially constructed hybrid proteins or polypeptides containing an antibody antigen-binding domain (e.g., a single-chain variable fragment (ScFv)) linked to a T cell signaling domain via a transmembrane domain. Characteristics of CARs include their ability to redirect T cell specificity and reactivity toward selected targets, leveraging the antigen-binding properties of monoclonal antibodies in a non-MHC-restricted manner. Non-MHC-restricted antigen recognition confers on CAR-expressing T cells the ability to recognize antigens independently of antigen processing, thus bypassing a major mechanism of tumor escape. Furthermore, when expressed in T cells, CARs advantageously do not dimerize with the alpha and beta chains of endogenous T cell receptors (TCRs).

[0096] As disclosed herein, the intracellular T cell signaling domain of a CAR can include, for example, a T cell receptor signaling domain, a T cell costimulatory signaling domain, or both. A T cell receptor signaling domain refers to a portion of a CAR that includes the intracellular domain of a T cell receptor, such as, but not limited to, the intracellular portion of the CD3 zeta protein. A costimulatory signaling domain is a domain that regulates the efficiency of lymphocytes against antigens. The term CAR refers to a portion of a CAR that contains the intracellular domain of a costimulatory molecule, a cell surface molecule other than an antigen receptor or their ligand, that is required for an effective response.

[0097] 1. Extracellular domain In one embodiment, CAR comprises target-specific binding element, otherwise called antigen-binding domain or part.The selection of domain depends on the type and number of ligands that define the surface of target cell.For example, antigen-binding domain can be selected to recognize the ligand that acts as a cell surface marker on target cells that is associated with specific disease state.Therefore, the examples of cell surface markers that can act as ligands for the antigen-binding domain in CAR include those associated with virus, bacteria and parasite infection, autoimmune disease and cancer cell.

[0098] In one embodiment, CAR can be engineered to target the tumor antigen of interest by engineering the desired antigen binding domain that specifically binds to the antigen on tumor cell.Tumor antigen is the protein produced by tumor cell that induces immune response, especially T cell-mediated immune response.The choice of antigen binding domain depends on the specific type of cancer to be treated. Tumor antigens include, for example, glioma-associated antigen, carcinoembryonic antigen (CEA), beta-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, Her2 / neu, survivin, and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, and CD33. The tumor antigens disclosed herein are included by way of example only; this list is not intended to be exhaustive, and further examples will be readily apparent to those of skill in the art.

[0099] In one embodiment, the tumor antigen comprises one or more antigenic cancer epitopes associated with malignant tumors. Malignant tumors express several proteins that can serve as target antigens for immune attack. These molecules include, but are not limited to, tissue-specific antigens, such as MART-1, tyrosinase, and GP100 in melanoma, and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation-related molecules, such as the oncogene HER-2 / Neu / ErbB-2. Yet another group of target antigens is oncofetal antigens, such as carcinoembryonic antigen (CEA). In B-cell lymphomas, tumor-specific idiotypic immunoglobulins constitute truly tumor-specific immunoglobulin antigens unique to individual tumors. B-cell differentiation antigens, such as CD19, CD20, and CD37, are other candidate target antigens in B-cell lymphomas. Some of these antigens (CEA, HER-2, CD19, CD20, idiotype) have been used as targets for passive immunotherapy with monoclonal antibodies with limited success.

[0100] In one preferred embodiment, the tumor antigen is CD33, and tumors associated with CD33 expression include lung mesothelioma, ovarian and pancreatic cancer, or any combination thereof, which express high levels of the extracellular protein CD33.

[0101] A type of tumor antigen can also be a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). TSAs are unique to tumor cells and are not present on other cells in the body. TAAs are not unique to tumor cells and instead cannot induce a state of immunological tolerance to the antigen. Under certain conditions, it is also expressed on normal cells.The expression of antigen on tumor can occur under the condition that allows immune system to respond to antigen.TAA can be the antigen that is expressed on normal cells during fetal development, when immune system is immature and unable to respond, or it can be the antigen that is usually present at a very low level on normal cells, but is expressed at a fairly high level on tumor cells.

[0102] Non-limiting examples of TSAs or TAAs include differentiation antigens such as MART-1 / MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, HER-2 / neu; unique tumor antigens resulting from chromosomal translocations such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3 / CA 27.29 / BCAA, CA 195, CA These include 242, CA-50, CAM43, CD68 / P1, CO-029, FGF-5, G250, Ga733 / EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 / Mac-2 binding protein / cyclophilin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0103] In one embodiment, the antigen binding domain portion of the CAR targets an antigen including, but not limited to, CD19, CD20, CD22, ROR1, CD33, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, MY-ESO-1 TCR, MAGE A3 TCR, etc.

[0104] In a preferred embodiment, the antigen-binding domain portion of the CAR targets the extracellular CD33 antigen.

[0105] In a preferred embodiment, an isolated nucleic acid molecule encoding the extracellular CD33 VH-2 antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 1, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD33 VH-2 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0106] In a preferred embodiment, an isolated nucleic acid molecule encoding the extracellular CD33 VH-4 antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 3, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD33 VH-4 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 thereto.

[0107] In one preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD33 ScFv9 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, wherein the encoded extracellular CD33 ScFv9 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 thereto.

[0108] In one preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD33 ScFv10 antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 7, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD33 ScFv10 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0109] In one preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD33 ScFv12 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, wherein the encoded extracellular CD33 ScFv12 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 thereto.

[0110] In one preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD33 ScFv15 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, wherein the encoded extracellular CD33 ScFv15 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 thereto.

[0111] The generation and binding characteristics of the specific CD33 variable heavy chain alone and ScFv antigen-binding fragments or antigen binders described herein are shown in Example 1.

[0112] In various embodiments of the CD33-specific CAR disclosed herein, a general schematic is shown in Figure 1, which includes, from N-terminus to C-terminus, a signal or leader peptide, an anti-CD33 ScFv, an extracellular linker, a CD8 transmembrane segment, 4-1BB, and CD3 zeta, with bold text indicating the cloning site for the linking domain.

[0113] In one embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 15, which encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 16 [LTG 1905:EF1a VH-2 CD33-CD8 TM-41BB-CD3 zeta amino acid sequence (shown in Figure 2A)].

[0114] In one embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 15, or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and the amino acid sequence set forth in SEQ ID NO: 16, or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto [LTG 1905 EF1a VH-2 CD33 -CD8 TM-41BB-CD3 Zeta The CAR encodes a CAR comprising the amino acid sequence (shown in Figure 2A )].

[0115] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 17, which encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 18 [LTG 1906 Ef1a- VH-4 CD33 -CD8 TM-41BB-CD3 zeta amino acid sequence (shown in Figure 2B)].

[0116] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 17, or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 18, or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto [LTG1906 Ef1a- VH-4 CD33 -CD8 TM-41BB-CD3zeta amino acid sequence (shown in Figure 2B)].

[0117] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 19, which encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 20 [LTG1936 Ef1a ScFv9 CD33 CD8 TM-41BB-CD3 Zeta CAR amino acid sequence (shown in Figure 2C)].

[0118] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 19, or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 20, or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto [LTG1936 Ef1a ScFv9 CD33 CD8 TM-41BB-CD3 Zeta CAR amino acid sequence (shown in Figure 2C)].

[0119] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 21 and is selected from the group consisting of SEQ ID NO: 22 [LTG1937 Ef1a ScFv10 CD33 CD8 TM-41BB-CD3 amino acid sequence (shown in Figure 2D)].

[0120] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 21, or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 22, or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto [LTG1937 Ef1a ScFv10 CD33 CD8 TM-41BB-CD3 amino acid sequence (shown in Figure 2D)].

[0121] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 23, which encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 24 [LTG1938 EF1a ScFv12 CD33 CD8 TM-41BB-CD3 zeta amino acid sequence (shown in Figure 2E)].

[0122] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 23, or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 24, or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto [LTG1938 EF1a ScFv12 CD33 CD8 TM-41BB-CD3 zeta amino acid sequence (shown in Figure 2E)].

[0123] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 25 and the amino acid sequence shown in SEQ ID NO: 26 [(LTG1939 EF1a ScFv15 CD33 CD8 TM-41BB-CD3 zeta amino acid sequence (shown in Figure 2F)].

[0124] In yet another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 25, or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and encodes a CAR comprising the amino acid sequence set forth in SEQ ID NO: 26, or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto [(LTG1939 EF1a ScFv15 CD33 CD8 TM-41BB-CD3 Zeta amino acid sequence (shown in Figure 2F)].

[0125] The surface expression of anti-CD33 CARs incorporating immunoglobulin heavy chain variable domain (VH) and single-chain fragment variable (ScFv) sequences reactive with the CD33 antigen is shown in Example 2 below and summarized in Table 2. Expression levels for each CAR containing ScFv or VH were determined by flow cytometry analysis of LV-transduced T cells from healthy donors using recombinant CD33-Fc peptide followed by anti-human FcF(ab')2 fragments conjugated with AF647, detected in the APC channel (see Example 2, Figures 3 and 6). VH-based anti-CD33 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, no CAR expression was detected on negative control, non-transduced T cells (gray line) or GFP controls (not shown), thus demonstrating the specificity of the detection method used (see Example 2, Figure 3 and Table 2). Similarly, ScFv-based anti-CD33 CAR constructs 1936, 1937, 1938, and 1939 were highly expressed in human primary T cells (black lines) compared to non-transduced T cell controls (gray lines). Representative results from one donor are shown.

[0126] As shown in Example 2, Figures 4 and 7, lentiviral vectors (LVs) expressing the following CARs were created and tested for anti-leukemia activity, demonstrating the high cytolytic activity of the CD33 CARs. Each experimental CAR contains a 4-1BB / CD3-zeta chain signaling motif and a specific anti-CD33 binding motif / domain as specified therein. Four leukemia target lines with different CD33 surface expression were used: HL-60 and MOLM-14 (high), Reh and K562 (low). The VH domain-based CAR-T constructs LTG1905 and LTG1906 lysed CD33-low K562 cells, with LTG1906 exhibiting superior cytolytic function at the effector-to-target (E:T) ratios indicated on the x-axis (see Figure 4, LTG1905 and LTG1906, filled diamonds and filled circles, respectively). When combined with the CD33-high HL-60 tumor line, LTG1906, but not LTG1905, demonstrated potent killing function, highlighting the potency of the construct LTG1906. On the other hand, the negative control groups: NT (non-transduced T cells) and 1398 (GFP control-transduced T cells) both failed to exert specific cytolytic activity. Therefore, the anti-CD33 CAR activity observed by the inventors against CD33-expressing tumor lines is not significant. The cytolytic activity of LTG1906 and LTG1905 is target-specific and CART-dependent.

[0127] In comparison, the ScFv-based anti-CD33 CAR constructs LTG1936 and LTG1939 were able to efficiently lyse the CD33-high tumor lines HL-60 and MOLM-14, but only partially lyse the CD33-low Reh tumor lines and had no specific lytic activity against K562 (see Figure 7, LTG1398 and LTG1936 open squares and open inverted triangles, respectively). This finding demonstrates the efficiency and specificity of the generated CAR constructs. Unexpectedly, additional CAR constructs tested in this set, LTG1937 and LTG1938, were inefficient at lysing the CD33-high tumor lines. Thus, it was again demonstrated that CART design is not trivial and that soluble antibody characteristics do not directly translate to CAR functionality.

[0128] The cytokine secretion capacity of anti-CD33 CAR T cells was then evaluated. Tumor cells were co-incubated with CAR T cells or control T cells at an effector-to-target ratio of 10:1 overnight, and culture supernatants were analyzed for IFN-gamma, TNF-alpha, and IL-2 by ELISA (see Figure 5 and Table 2). Notably, CAR T-expressing cells LTG1905 and LTG1906 produced high levels of IFN-gamma, TNF-alpha, and IL-2, while the negative control NT and 1398 groups produced little cytokine induction. Surprisingly, CD33 CAR LTG1905 tended to produce greater levels of induced cytokines in all tumor lines tested compared to construct LTG1906. This result contrasts with the in vitro cytolytic function of LTG1905, which was less than that of LTG1906 (see Figure 4), suggesting that multiple CAR T functional endpoints should be tested on a construct-by-construct basis.

[0129] Without intending to be limited to any particular mechanism of action, possible reasons for the enhanced therapeutic function associated with exemplary CARs of the present invention are believed to include, for example, but not by way of limitation, a) improved lateral movement within the plasma membrane, allowing for more efficient signaling; b) superior location within plasma membrane microdomains, such as lipid rafts, and a greater ability to interact with transmembrane signaling cascades associated with T cell activation; c) superior location within the plasma membrane due to preferential movement away from inhibitory or down-regulatory interactions, e.g., less proximity to or fewer interactions with phosphatases such as CD45; and d) superior assembly into the T cell receptor signaling complex (i.e., the immune synapse), or any combination thereof.

[0130] Although the present disclosure is exemplified using exemplary extracellular CD33 variable heavy chain alone and ScFv antigen-binding domains, other nucleotide and / or amino acid variants within the CD33 variable heavy chain alone and ScFv antigen-binding domains may be used to obtain CD33 antigen-binding domains for use in the CARs described herein.

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

[0132] In one exemplary embodiment, the antigen-binding domain portion of the CAR further targets CD19. Preferably, the antigen-binding domain in the CAR is an anti-CD19 ScFv, wherein the nucleic acid sequence of the anti-CD19 ScFv comprises the sequence set forth in SEQ ID NO: 37. In one embodiment, the anti-CD19 ScFv comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 30. In another embodiment, the anti-CD19 ScFv portion of the CAR comprises the amino acid sequence set forth in SEQ ID NO: 38.

[0133] In one embodiment of the present invention, the present invention provides a method for the treatment of infectious diseases, including, but not limited to, viral infections, including but not limited to, viral infections from Retroviridae (e.g., human immunodeficiency viruses, e.g., HIV-1 and HIV-LP), Picornaviridae (e.g., poliovirus, hepatitis A virus, enterovirus, human coxsackievirus, rhinovirus, and echovirus), rubella virus, coronavirus, vesicular stomatitis virus, rabies virus, Ebola virus, parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus, influenza virus, hepatitis B virus, parvovirus, Adenoviridae, Herpesviridae (e.g., type 1 and and herpes simplex virus type 2 (HSV), varicella-zoster virus, cytomegalovirus (CMV), and herpesviruses, poxviruses (e.g., smallpox virus, vaccinia virus, and poxviruses), or hepatitis C virus, or any combination thereof.

[0134] In another aspect of the present invention, a CAR capable of binding to an antigen derived from a bacterial strain of Staphylococci, Streptococcus, Escherichia coli, Pseudomonas, or Salmonella is provided. In particular, a CAR capable of binding to an antigen derived from an infectious bacterium, such as Helicobacter pyloris, Legionella pneumophilia, a bacterial strain of Mycobacteria sp. (e.g., M. tuberculosis, M. avium, M. intracellulare, M. kansaii, or M. gordonea), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitides, Listeria monocytogenes, Streptococcus pyogenes, Group A Streptococcus, Group B Streptococcus (Streptococcus agalactiae), Streptococcus pneumoniae, or Clostridium tetani, or a combination thereof, is provided.

[0135] 2. Transmembrane domain With respect to the transmembrane domain, the CAR comprises one or more transmembrane domains fused to the extracellular CD33 antigen-binding domain of the CAR.

[0136] Transmembrane domains can be derived from either natural or synthetic sources. If the source is natural, the domain can be derived from any membrane-bound or transmembrane protein.

[0137] The transmembrane region particularly used in the CAR described herein can be derived from (i.e., can include at least one of) the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, mesothelin, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154. Alternatively, the transmembrane domain can be synthetic, in which case it predominantly contains hydrophobic residues such as leucine and valine. Preferably, a phenylalanine, tryptophan, and valine triplet is found at each end of the synthetic transmembrane domain. Optionally, a short oligopeptide or polypeptide linker, preferably between 2 and 10 amino acids in length, can form the link between the transmembrane domain and the cytoplasmic signaling domain of the CAR. A glycine-serine duo provides a particularly suitable linker.

[0138] In one embodiment, a transmembrane domain naturally associated with one of the domains in the CAR is used in addition to the transmembrane domain.

[0139] In some cases, transmembrane domains may be selected by amino acid substitution to avoid binding of such domains to transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

[0140] In one embodiment, the transmembrane domain in the CAR of the invention is a CD8 transmembrane domain. In one embodiment, the CD8 transmembrane domain comprises the nucleic acid sequence of SEQ ID NO: 27. In one embodiment, the CD8 transmembrane domain comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 28. In another embodiment, the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 28. .

[0141] In one embodiment, the encoded transmembrane domain comprises an amino acid sequence having at least one, two, or three alterations (e.g., substitutions) but not more than 20, 10, or 5 alterations (e.g., substitutions) of the amino acid sequence of SEQ ID NO:28, or a sequence having 95-99% identity to the amino acid sequence of SEQ ID NO:28.

[0142] In some instances, the transmembrane domain of the CAR comprises a CD8 alpha hinge domain. In one embodiment, the CD8 hinge domain comprises the nucleic acid sequence of SEQ ID NO: 29. In one embodiment, the CD8 hinge domain comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 30. In another embodiment, the CD8 hinge domain comprises the amino acid sequence of SEQ ID NO: 30, or a sequence having 95-99% identity thereto.

[0143] In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded linker domain is derived from the extracellular domain of CD8 and is linked to a transmembrane CD8 domain, a transmembrane CD28 domain, or a combination thereof.

[0144] In one embodiment, the transmembrane domain in the CAR of the invention is a TNFRSF19 transmembrane domain. In one embodiment, the TNFRSF19 transmembrane domain comprises the nucleic acid sequence of SEQ ID NO: 51. In one embodiment, the TNFRSF19 transmembrane domain comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 52. In another embodiment, the TNFRSF19 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 52.

[0145] In one embodiment, the encoded transmembrane domain comprises an amino acid sequence having at least one, two, or three alterations (e.g., substitutions) but not more than 20, 10, or 5 alterations (e.g., substitutions) of the amino acid sequence of SEQ ID NO: 52, or a sequence having 95-99% identity to the amino acid sequence of SEQ ID NO: 52.

[0146] 3. Spacer domain In CARs, a spacer domain, also called a hinge domain, can be located between the extracellular domain and the transmembrane domain or between the intracellular domain and the transmembrane domain. A spacer domain refers to any oligopeptide or polypeptide that functions to link the transmembrane domain to the extracellular domain and / or the transmembrane domain to the intracellular domain. A spacer domain contains up to 300 amino acids, preferably 10-100 amino acids, and most preferably 25-50 amino acids.

[0147] In some embodiments, the linker may include a spacer element, which, if present, increases the size of the linker, thereby increasing the distance between the effector molecule or detectable marker and the antibody or antigen-binding fragment. Exemplary spacers are known to those skilled in the art, including those described in U.S. Patent Nos. 7,964,566, 7,498,298, 6,884,869, 6,323,315, 6,239,104, 6,034,065, 5,780,588, 5,665,860, 5,663,149, 5,635,483, 5,599,902, 5,554,725, 5, Nos. 5,530,097, 5,521,284, 5,504,191, 5,410,024, 5,138,036, 5,076,973, 4,986,988, 4,978,744, 4,879,278, 4,816,444 and 4,486,414, as well as U.S. Patent Application Publication Nos. 20110212088 and 20110070248. and others, each of which is incorporated herein by reference in its entirety.

[0148] The spacer domain preferably has a sequence that promotes the binding of CAR to the antigen and enhances signal transduction into the cell. Examples of amino acids that are predicted to promote binding include cysteine, charged amino acids, and serine and threonine in potential glycosylation sites, and these amino acids can be used as amino acids that constitute the spacer domain.

[0149] The spacer domain may be the entire or a portion 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 315-396 of CD4 (NCBI RefSeq: NP_000607.1), or amino acids 137-152 of CD28 (NCBI RefSeq: NP_006130.1). Alternatively, the spacer domain may be a portion of the constant region of an antibody heavy or light chain (e.g., the CH1 region or CL region, e.g., a peptide having the amino acid sequence set forth in SEQ ID NO: 32). Furthermore, the spacer domain may be an artificially synthesized sequence.

[0150] Furthermore, all or part of the amino acids comprising the constant region of human IgG4 (UniProt ID: P01861), including CH1 (amino acid numbers 1 to 98), hinge, SEQ ID NO: 80, and corresponding nucleotide SEQ ID NO: 79 (amino acid numbers 99 to 110), CH2, amino acid number 81 and corresponding nucleotide SEQ ID NO: 80 (amino acid numbers 111 to 220), and CH3, SEQ ID NO: 84 and corresponding nucleotide SEQ ID NO: 83 (amino acid numbers 221 to 327), or a combination thereof, can be used, for example, the IgG4 hinge CH2 CH3 domain, SEQ ID NO: 86, and corresponding nucleotide SEQ ID NO: 85.

[0151] In one embodiment, the spacer domain of the CAR comprises a TNFRSF19 hinge domain comprising the nucleic acid sequence of SEQ ID NO: 53. In one embodiment, the TNFRSF19 hinge domain comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 54. In another embodiment, the TNFRSF19 hinge domain comprises the amino acid sequence of SEQ ID NO: 54, or a sequence having 95-99% identity thereto.

[0152] In one embodiment, the spacer domain of the CAR comprises a truncated hinge domain of TNFRSF19 comprising the nucleic acid sequence of SEQ ID NO: 55. In one embodiment, the truncated hinge domain of TNFRSF19 comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 56. In another embodiment, the truncated hinge domain of TNFRSF19 comprises the amino acid sequence of SEQ ID NO: 56, or a sequence having 95-99% identity thereto.

[0153] In one embodiment, the TNFRSF19 hinge and transmembrane domain comprises the nucleic acid sequence of SEQ ID NO: 49. In one embodiment, the TNFRSF19 hinge and transmembrane domain comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 50. In another embodiment, the TNFRSF19 hinge and transmembrane domain comprises the amino acid sequence of SEQ ID NO: 50, or a sequence having 95-99% identity thereto.

[0154] In one embodiment, the CD8a hinge domain is fused to the TNFRSF19 transmembrane domain comprising the nucleic acid sequence of SEQ ID NO: 57. In one embodiment, the CD8a hinge domain fused to the TNFRSF19 transmembrane domain comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 58. In another embodiment, the CD8a hinge domain fused to the TNFRSF19 transmembrane domain comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 58. The fused CD8a hinge domain comprises the amino acid sequence of SEQ ID NO: 58, or a sequence having 95-99% identity thereto.

[0155] Furthermore, a signal peptide sequence, also called a leader peptide, can be linked to the N-terminus of a CAR. Signal peptide sequences are present at the N-terminus of many secretory proteins and membrane proteins and are 15 to 30 amino acids in length. Many of the protein molecules referred to above as intracellular domains have signal peptide sequences, and these signal peptides can be used as signal peptides for a CAR. In one embodiment, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 14.

[0156] In one embodiment, the CD8 alpha leader peptide comprises the nucleic acid sequence of SEQ ID NO: 43. In one embodiment, the CD8 alpha leader peptide comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 44. In another embodiment, the CD8a hinge domain fused to the TNFRSF19 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 44, or a sequence having 95-99% identity thereto.

[0157] In another embodiment, the GMCSF leader peptide comprises the nucleic acid sequence of SEQ ID NO: 39. In one embodiment, the GMCSF leader peptide comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 40. In another embodiment, the CD8a hinge domain fused to the TNFRSF19 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 40, or a sequence having 95-99% identity thereto.

[0158] In another embodiment, the TNFRSF19 leader peptide comprises the nucleic acid sequence of SEQ ID NO: 41. In one embodiment, the TNFRSF19 leader peptide and the CD8 alpha leader peptide comprise a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 42. In another embodiment, the CD8a hinge domain fused to the TNFRSF19 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 42, or a sequence having 95-99% identity thereto.

[0159] In one embodiment, the tag sequence encoding a truncated sequence of epidermal growth factor receptor (tEGFR) comprises the nucleic acid sequence of SEQ ID NO: 67. In one embodiment, the tEGFR comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 68. In another embodiment, the tEGFR tag comprises the amino acid sequence of SEQ ID NO: 68, or a sequence having 95-99% identity thereto.

[0160] In one embodiment, the furin recognition site and downstream T2A self-cleaving peptide sequence designed for bicistronic co-expression of the tag sequence and CAR sequence comprise the nucleic acid sequence of SEQ ID NO: 65. In one embodiment, the furin and T2A sequences comprise a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 66. In another embodiment, the tEGFR tag comprises the amino acid sequence of SEQ ID NO: 66, or a sequence having 95-99% identity thereto.

[0161] In one embodiment, the upstream furin recognition site, T2A self-cleaving peptide sequence, and downstream furin recognition site designed for bicistronic co-expression of the tag sequence and CAR sequence comprise the nucleic acid sequence of SEQ ID NO: 67. In one embodiment, the furin and T2A sequences comprise a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 68. In another embodiment, the tEGFR tag comprises the amino acid sequence of SEQ ID NO: 68, or a sequence having 95-99% identity thereto.

[0162] In one embodiment, the targeting domain of the CAR is a monoclonal antibody, ScFv Fa b, expressed separately in the form of Fab'2 and contains a binding tag or epitope, while the component of the CAR expressed on the effector cell contains a binding domain that is specifically directed to bind to the tag or epitope expressed on the soluble CAR module, e.g., when the soluble component of the CAR specifically binds to the cell-bound component, a complete functional CAR structure is formed.

[0163] 4. Intracellular domain The cytoplasmic domain or other intracellular signaling domain of a CAR is responsible for activating at least one of the normal effector functions of the immune cell in which the CAR is placed. The term "effector function" refers to a specialized function of a cell. For example, the effector function of a T cell can be cytolytic activity or helper activity, including cytokine secretion. Thus, the term "intracellular signaling domain" refers to the portion of a protein that transmits an effector function signal and instructs the cell to perform a specialized function. While the entire intracellular signaling domain can usually be used, it is often not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the intact chain, as long as it transmits the effector function signal. Thus, the term intracellular signaling domain is meant to include any truncated portion of the intracellular signaling domain sufficient to transmit the effector function signal.

[0164] Preferred examples of intracellular signaling domains for use in CARs include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivatives or variants of these sequences, and any synthetic sequences with the same functional capability.

[0165] It is known that signals generated through the TCR alone are insufficient for full activation of T cells, and that secondary or costimulatory signals are also required. Thus, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences) and those that act antigen-dependently to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences).

[0166] Primary cytoplasmic signaling sequences regulate the primary activation of the TCR complex in either a stimulatory or inhibitory manner. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs, or ITAMs.

[0167] Examples of ITAMs containing primary cytoplasmic signaling sequences of particular use in the CARs disclosed herein include those derived from TCR zeta (CD3 zeta), FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. Specific, non-limiting examples of ITAMs include amino acids 51-164 of CD3 zeta (NCBI RefSeq: NP_932170.1), amino acids 45-86 of Fc epsilon RI gamma (NCBI RefSeq: NP_004097.1), amino acids 201-244 of Fc epsilon RI beta (NCBI RefSeq: NP_000130.1), amino acids 139-182 of CD3 gamma (NCBI RefSeq: NP_000064.1), amino acids 128-171 of CD3 delta (NCBI RefSeq: NP_000723.1), amino acids 153-207 of CD3 epsilon (NCBI RefSeq: NP_000724.1), and CD5 (NCBI RefSeq: NP_000724.1). RefSeq:NP_055022.2) and amino acids 402–495 of 0022 (NCBI RefSeq:NP_001762). 2), amino acids 707 to 847 of CD79a (NCBI RefSeq: NP_001774.1), amino acids 166 to 226 of CD79b (NCBI RefSeq: NP_000617.1), and amino acids 182 to 229 of CD66d (NCBI Included herein are peptides having a sequence of amino acid numbers 177 to 252 of NCBI RefSeq:NP_001806.2), as well as variants thereof having the same functions as these peptides. The amino acid numbers based on the amino acid sequence information in NCBI RefSeq ID or GenBank described herein are numbered based on the full length of the precursor of each protein (including signal peptide sequences, etc.). In one embodiment, the cytoplasmic signaling molecule in the CAR comprises a cytoplasmic signaling sequence derived from CD3 zeta.

[0168] In a preferred embodiment, the intracellular domain of the CAR can be designed to include a CD3-zeta signaling domain, either alone or in combination with any other desired cytoplasmic domain(s) useful in the context of the CAR. For example, the intracellular domain of the CAR can include a CD3 zeta chain portion and a costimulatory signaling region. The costimulatory signaling region refers to a portion of the CAR that includes the intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for efficient lymphocyte response to antigens. Examples of such costimulatory molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83. Specific, non-limiting examples of such costimulatory molecules include amino acids 236-351 of CD2 (NCBI RefSeq: NP_001758.2), amino acids 421-458 of CD4 (NCBI RefSeq: NP_000607.1), amino acids 402-495 of CD5 (NCBI RefSeq: NP_055022.2), amino acids 207-235 of CD8 alpha (NCBI RefSeq: NP_001759.3), amino acids 196-210 of CD83 (GenBank: AAA35664.1), amino acids 181-220 of CD28 (NCBI RefSeq: NP_006130.1), and CD137 (4-1BB, NCBI The present disclosure primarily exemplifies 4-1BB as a costimulatory signaling element, but other costimulatory elements are within the scope of the present disclosure.

[0169] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR can be linked to each other randomly or in a specific order. Optionally, a short oligopeptide or polypeptide linker, preferably between 2 and 10 amino acids in length, can form the linkage. Glycine-serine duplexes provide particularly suitable linkers.

[0170] In one embodiment, the intracellular domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of CD28. In another embodiment, the intracellular domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of 4-1BB. In yet another embodiment, the intracellular domain is designed to comprise the signaling domain of CD3-zeta and the signaling domains of CD28 and 4-1BB.

[0171] In one embodiment, the intracellular domain in the CAR is designed to comprise the signaling domain of 4-1BB and the signaling domain of CD3-zeta, wherein the signaling domain of 4-1BB is set forth in SEQ ID NO: 33, SEQ ID NO: 45, or SEQ ID NO: 59, respectively. and the signaling domain of CD3-zeta comprises the nucleic acid sequence set forth in SEQ ID NO:35, SEQ ID NO:47, or SEQ ID NO:61, respectively.

[0172] In one embodiment, the intracellular domain in the CAR is designed to comprise the signaling domain of 4-1BB and the signaling domain of CD3-zeta, wherein the signaling domain of 4-1BB comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:34, SEQ ID NO:46, or SEQ ID NO:60, respectively, and the signaling domain of CD3-zeta comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:36, or SEQ ID NO:48, or SEQ ID NO:62.

[0173] In one embodiment, the intracellular domain in the CAR is designed to comprise the signaling domain of 4-1BB and the signaling domain of CD3-zeta, wherein the signaling domain of 4-1BB comprises the amino acid sequence set forth in SEQ ID NO:34, SEQ ID NO:46, or SEQ ID NO:60, respectively, and the signaling domain of CD3-zeta comprises the amino acid sequence set forth in SEQ ID NO:36, SEQ ID NO:48, or SEQ ID NO:62, respectively.

[0174] In one embodiment, the intracellular domain of the CAR is designed to comprise the signaling domain of CD28 and the signaling domain of CD3 zeta, wherein the signaling domain of CD28 comprises the nucleic acid sequence set forth in SEQ ID NO:45 or SEQ ID NO:59, respectively, and the signaling domain of CD3 zeta comprises the nucleic acid sequence set forth in SEQ ID NO:35, SEQ ID NO:47, or SEQ ID NO:61, respectively.

[0175] In one embodiment, the intracellular domain of the CAR is designed to comprise the signaling domain of CD28 and the signaling domain of CD3 zeta, wherein the signaling domain of CD28 comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 46 or SEQ ID NO: 60, respectively, and the signaling domain of CD3 zeta comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 36, or SEQ ID NO: 48, or SEQ ID NO: 62.

[0176] In one embodiment, the intracellular domain of the CAR is designed to comprise the signaling domain of CD28 and the signaling domain of CD3 zeta, wherein the signaling domain of CD28 comprises the amino acid sequence set forth in SEQ ID NO: 46 or SEQ ID NO: 60, respectively, and the signaling domain of CD3 zeta comprises the amino acid sequence set forth in SEQ ID NO: 36, SEQ ID NO: 48, or SEQ ID NO: 62, respectively.

[0177] 5. Further description of CAR The functional portion of the CAR disclosed herein is also expressly included within the scope of the present invention. The term "functional portion", when used in reference to a CAR, refers to any one or more parts or fragments of the CAR disclosed herein, which part or fragment retains the biological activity of the CAR (parent CAR) of which it is a part. A functional portion includes, for example, a part of a CAR that retains the ability to recognize target cells or detect, treat or prevent disease to a similar degree, the same degree, or a higher degree than the parent CAR. With respect to a parent CAR, a functional portion can, for example, comprise about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95% or more of the parent CAR.

[0178] The functional portion can comprise additional amino acids at the amino or carboxy end of the portion, or at both ends, which additional amino acids are not found in the amino acid sequence of the parent CAR. Preferably, the additional amino acids do not interfere with the biological function of the functional portion, such as, for example, recognizing target cells, detecting cancer, treating or preventing cancer, etc. More preferably, the additional amino acids enhance the biological activity of the functional portion compared to the biological activity of the parent CAR.

[0179] The functional variants of the CAR disclosed herein are included within the scope of this disclosure.The term "functional variant" as used herein refers to a CAR, polypeptide or protein that has substantial or significant sequence identity or similarity with the parent CAR, and this functional variant retains the biological activity of the CAR it is a variant of.Functional variants include, for example, variants of the CAR (parent CAR) described herein that retain the ability to recognize target cells to a similar degree, the same degree, or a higher degree than the parent CAR.With respect to the parent CAR, functional variants can be, for example, at least about 30%, 50%, 75%, 80%, 90%, 98% or more identical in amino acid sequence to the parent CAR.

[0180] A functional variant can, for example, comprise the amino acid sequence of a parent CAR with at least one conservative amino acid substitution. Alternatively, or in addition, a functional variant can comprise the amino acid sequence of a parent CAR with at least one non-conservative amino acid substitution. In this case, it is preferred that the non-conservative amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. The non-conservative amino acid substitution can enhance the biological activity of the functional variant, so that the biological activity of the functional variant is increased compared to the parent CAR.

[0181] The amino acid substitutions in the CAR are preferably conservative amino acid substitutions. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid with certain physical and / or chemical properties is replaced with another amino acid with the same or similar chemical or physical properties. For example, a conservative amino acid substitution can be an acidic / negatively charged polar amino acid substituting another acidic / negatively charged polar amino acid (e.g., Asp or Glu), an amino acid having a nonpolar side chain substituting another amino acid having a nonpolar side chain (e.g., Ala, Gly, Val, He, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), a basic / positively charged polar amino acid substituting another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.), an uncharged amino acid with a polar side chain substituting another uncharged amino acid with a polar side chain (e.g., Asn, Gin, Ser, Thr, Tyr, etc.), an amino acid with a beta-branched side chain substituting another amino acid with a beta-branched side chain (e.g., He, Thr, and Val), an amino acid with an aromatic side chain substituting another amino acid with an aromatic side chain (e.g., His, Phe, Trp, and Tyr), etc.

[0182] A CAR can consist essentially of the specified amino acid sequence(s) described herein, such that other components, e.g., other amino acids, do not significantly alter the biological activity of the functional variant.

[0183] CARs (including functional portions and functional variants) can be of any length, i.e., comprise any number of amino acids, provided that the CAR (or functional portion or variant thereof) retains its biological activity, e.g., the ability to specifically bind to an antigen, detect diseased cells in a mammal, or treat or prevent a disease in a mammal, etc. For example, a CAR can be about 50 to about 5,000 amino acids in length, e.g., 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1,000 or more amino acids in length.

[0184] CARs (including functional portions and functional variants of the invention) can contain synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, -amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3-hydroxyproline and trans-4-hydroxyproline, 4-aminophen ... N-aminoalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, 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.

[0185] CARs (including functional portions and functional variants) can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized, e.g., via disulfide bridges, or converted into acid addition salts and / or optionally dimerized or polymerized, or conjugated.

[0186] CAR (including its functional part and functional variant) can be obtained by methods known in the art.CAR can be produced by any suitable method of producing polypeptide or protein.Suitable methods for de novo synthesis of polypeptide and protein are described in references such as Chan et al., Fmoc Solid Phase Peptide Synthesis, Oxford University Press, Oxford, United Kingdom, 2000; Peptide and Protein Drug Analysis, Reid, R. (ed.), Marcel Dekker, Inc., 2000; Epitope Mapping, Westwood et al. (ed.), Oxford University Press, Oxford, United Kingdom, 2001; and U.S. Patent No. 5,449,752.In addition, polypeptide and protein can be produced recombinantly using the nucleic acid described herein by using standard recombinant methods. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. Furthermore, some CARs (including functional portions and functional variants thereof) can be isolated and / or purified from sources such as plants, bacteria, insects, mammals, e.g., rats, humans, etc. Isolation and purification methods are well known in the art. Alternatively, the CARs described herein (including functional portions and functional variants thereof) can be commercially synthesized by companies. In this regard, CARs can be synthetic, recombinant, isolated, and / or purified.

[0187] B. Antibodies and Antigen-Binding Fragments One embodiment further provides a CAR, a T cell expressing a CAR, an antibody that specifically binds to one or more of the antigens disclosed herein, or an antigen-binding domain or portion thereof. As used herein, "a T cell expressing a CAR" or "CAR T cell" refers to a T cell that expresses a CAR, e.g., has antigen specificity determined by the antibody-derived targeting domain of the CAR.

[0188] As used herein, an "antigen-binding domain" may include antibodies and antigen-binding fragments thereof. The term "antibody" is used in the broadest sense herein and includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., antibodies) as long as they exhibit the desired antigen-binding activity. The present invention encompasses various antibody structures, including, but not limited to, antibodies (e.g., bispecific antibodies), and antigen-binding fragments thereof. Non-limiting examples of antibodies include, for example, intact immunoglobulins known in the art, as well as variants and fragments thereof that retain binding affinity for an antigen.

[0189] A "monoclonal antibody" is an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and directed against a single antigenic epitope. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. In some examples, a monoclonal antibody is an antibody produced by a single clone of B lymphocytes, or an antibody produced by a cell transfected with nucleic acid encoding the antibody light and heavy chain variable regions of a single antibody (or antigen-binding fragment thereof), or its progeny. In some examples, a monoclonal antibody is isolated from a subject. A monoclonal antibody may have conservative amino acid substitutions that have substantially no effect on antigen binding or other immunoglobulin functions. Exemplary methods for producing monoclonal antibodies are known, see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual, 2nd ed. Cold Spring Harbor Publications, New York (2013).

[0190] Typically, immunoglobulins have heavy (H) chains and light (L) chains interconnected by disulfide bonds. Immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable domain genes. There are two types of light chains, lambda (λ) and kappa (κ). There are five major heavy chain classes (or isotypes): IgM, IgD, IgG, IgA, and IgE, which determine the functional activity of antibody molecules.

[0191] Each heavy and light chain contains a constant region (or constant domain) and a variable region (or variable domain; see, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p. 91 (2007)). In some embodiments, the variable regions of the heavy and light chains combine to specifically bind to an antigen. In further embodiments, only the heavy chain variable region is required. For example, naturally occurring camelid antibodies consisting only of heavy chains are functional and stable in the absence of light chains. (See, e.g., Hamers-Casterman et al., Nature, 363:446-448, 1993; Sheriff et al., Nat. Struct. Biol., 3:733-736, 1996). References to "VH" or "VH" refer to the variable region of an antibody heavy chain, including antigen-binding fragments such as Fv, ScFv, dsFv, or Fab. References to "VL" or "VL" refer to the variable domain of an antibody light chain, including those of Fv, ScFv, dsFv, or Fab.

[0192] The variable regions of the light and heavy chains contain a "framework" region interrupted by three hypervariable regions, also called "complementarity-determining regions" or "CDRs" (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, USDapartment of Health and Human Services, 1991). The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, which is the combined framework regions of the constituent light and heavy chains, functions to position and align the CDRs in three-dimensional space.

[0193] CDRs are primarily responsible for binding to an epitope of an antigen. The amino acid sequence boundaries of a given CDR are , Kabat et al. ("Sequences of Proteins of Immunological Interest", 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991; "Kabat" numbering scheme), Al-Lazikani et al. (JMB Vol. 273, pp. 927-948, 1997; "Chothia" numbering scheme), and Lefranc et al. ("IMGT unique numbering for immunoglobulin and T cell receptor The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3 (N- to C-terminus) and are also typically identified by the chain in which a particular CDR is located. Thus, a VH CDR3 is the CDR3 from the variable domain of the heavy chain of the antibody in which it is found, while a VL CDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found. Light chain CDRs are sometimes referred to as LCDR1, LCDR2, and LCDR3. Heavy chain CDRs are sometimes referred to as HCDR1, HCDR2, and HCDR3.

[0194] "Antigen-binding fragments" are portions of full-length antibodies that retain the ability to specifically recognize their cognate antigen, as well as various combinations of such portions. Non-limiting examples of antigen-binding fragments include Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., ScFv); and multispecific antibodies formed from antibody fragments. Antibody fragments include antigen-binding fragments produced by engineering whole antibodies or those synthesized de novo using recombinant DNA methodologies (see, e.g., Kontermann and Dubel (eds.), Antibody Engineering, Vols. 1-2, 2nd Edition, Springer Press, 2010).

[0195] Single-chain antibodies (ScFvs) are genetically engineered molecules containing the VH and VL domains of one or more antibodies (or antibodies) linked by a suitable polypeptide linker as a genetically fused single-chain molecule (see, e.g., Bird et al., Science, 242:423-426, 1988; Huston et al., Proc. Natl. Acad. Sci., 85:5879-5883, 1988; Ahmad et al., Clin. Dev. Immunol., 2012, doi:10.1155 / 2012 / 980250; Marbry, IDrugs, 13:543-549, 2010). The intramolecular orientation of the VH and VL domains in ScFvs is typically not critical for ScFvs. Thus, ScFvs with both possible configurations (VH domain-linker domain-VL domain; VL domain-linker domain-VH domain) can be used.

[0196] In dsFv, the heavy and light variable chains are mutated to introduce disulfide bonds to stabilize the association of the chains. Also included are diabodies, which are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain but use a linker that is too short to allow pairing between the two domains on the same chain, thereby pairing the domains with complementary domains on another chain to create two antigen-binding sites (see, for example, Holliger et al., Proc. Natl. Acad. Sci., 90:6444-6448, 1993; Poljak et al., Structure, 2:1121-1123, 1994).

[0197] Antibodies include chimeric antibodies (e.g., humanized murine antibodies) and heteroconjugate antibodies (e.g., Also included are genetically engineered forms, such as bispecific antibodies. See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rd ed., W.H. Freeman & Co., New York, 1997.

[0198] Non-naturally occurring antibodies can be constructed using solid-phase peptide synthesis, can be produced recombinantly, or can be obtained by screening combinatorial libraries consisting of variable heavy and variable light chains, as described, for example, in Huse et al., Science 246:1275-1281 (1989), which is incorporated herein by reference. These and other methods of generating, for example, chimeric, humanized, CDR-grafted, single-chain, and bifunctional antibodies are well known to those of skill in the art (Winter and Harris, Immunol. Today 14:243-246 (1993); Ward et al., Nature 14:243-246 (1993)). 341:544-546 (1989); Harlow and Lane, supra, 1988; Hilyard et al., Protein Engineering: A practical approach (IRL Press 1992); Borrabeck, Antibody Engineering, 2nd Edition (Oxford University Press 1995); each of which is incorporated herein by reference.

[0199] An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competition assay; conversely, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competition assay. Antibody competition assays are known, and exemplary competition assays are provided herein.

[0200] A "humanized" antibody or antigen-binding fragment comprises a human framework region and one or more CDRs derived from a non-human (e.g., mouse, rat, or synthetic) antibody or antigen-binding fragment. The non-human antibody or antigen-binding fragment providing the CDRs is referred to as the "donor," and the human antibody or antigen-binding fragment providing the framework is referred to as the "acceptor." In one embodiment, all CDRs are derived from the donor immunoglobulin in the humanized immunoglobulin. Constant regions need not be present, but if present, can be substantially identical to human immunoglobulin constant regions, e.g., at least about 85-90%, e.g., about 95% or more identical. Thus, all parts of a humanized antibody or antigen-binding fragment, except possibly for the CDRs, are substantially identical to the corresponding parts of a natural human antibody sequence.

[0201] A "chimeric antibody" is an antibody that contains sequences from two different antibodies, typically from different species. In some instances, a chimeric antibody contains one or more CDRs and / or framework regions from one human antibody and CDRs and / or framework regions from another human antibody.

[0202] A "fully human antibody" or "human antibody" is an antibody that contains sequences derived from the human genome (or derived from the human genome) but does not contain sequences derived from another species. In some embodiments, a human antibody contains CDRs, framework regions, and (if present) an Fc region derived from the human genome (or derived from the human genome). Human antibodies can be identified and isolated, for example, by phage display, using technology to create sequences based on sequences derived from the human genome, or using transgenic animals (see, e.g., Barbas et al., Phage display: A Laboratory Manuel. 1st ed. New York: Cold Spring Harbor Laboratory Press, 2004. Print.; Lonberg, Nat. Biotech., 23:1117-1125, 2005; Lonenberg, Cu (See rr. Opin. Immunol., 20:450-459, 2008).

[0203] An antibody can have one or more binding sites. If more than one binding site is present, these binding sites may be identical to one another or different. For example, naturally occurring immunoglobulins have two identical binding sites, single-chain antibodies or Fab fragments have one binding site, while bispecific or bifunctional antibodies have two different binding sites.

[0204] Methods for testing antibodies for the ability to bind to any functional portion of a CAR are known in the art and include any antibody-antigen binding assay, such as radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation, and competitive inhibition assays (see, e.g., Janeway et al., infra, U.S. Patent Application Publication No. 2002 / 0197266 A1, and U.S. Patent No. 7,338,929).

[0205] In addition, the CAR, CAR-expressing T cells, antibodies, or antigen-binding portions thereof, can be modified to contain 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).

[0206] C. Conjugate CARs, T cells expressing CARs, or monoclonal antibodies or antigen-binding fragments thereof specific for one or more of the antigens disclosed herein can be conjugated to agents such as effector molecules or detectable markers using several means known to those skilled in the art. Both covalent and non-covalent binding means can be used. Conjugates include, but are not limited to, molecules in which an effector molecule or detectable marker is covalently linked to an antibody or antigen-binding fragment that specifically binds to one or more of the antigens disclosed herein. Those skilled in the art will recognize that the conjugation of an effector molecule or detectable marker to an antibody or antigen-binding fragment that specifically binds to one or more of the antigens disclosed herein can be used to conjugate an effector molecule or detectable marker to an antibody or antigen-binding fragment that specifically binds to one or more of the antigens disclosed herein. 125 I, 32 P, 14 C. 3 H and 35 It will be understood that a variety of effector molecules and detectable markers may be used, including, but not limited to, S, as well as other labels, targeting moieties, ligands, and the like.

[0207] The choice of a particular effector molecule or detectable marker will depend on the particular target molecule or cell and the desired biological effect. Thus, for example, the effector molecule can be a cytotoxin used to bring about the death of a particular target cell (e.g., a tumor cell).

[0208] The procedure for attaching an effector molecule or detectable marker to an antibody or antigen-binding fragment varies according to the chemical structure of the effector. Polypeptides typically contain various functional groups, such as carboxylic acid (COOH), free amine (-NH2), or sulfhydryl (-SH) groups, which are available for reaction with appropriate functional groups on an antibody to result in attachment of an effector molecule or detectable marker. Alternatively, the antibody or antigen-binding fragment is derivatized to expose or attach additional reactive functional groups. Derivatization can include attachment of any of several known linker molecules, such as those available from Pierce Chemical Company, Rockford, IL. The linker can be any molecule used to attach an antibody or antigen-binding fragment to an effector molecule or detectable marker. The linker is capable of forming covalent bonds to both the antibody or antigen-binding fragment and the effector molecule or detectable marker. Suitable linkers are well known to those skilled in the art and may be used in conjunction with other suitable linkers. include, but are not limited to, straight or branched chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. When the antibody or antigen-binding fragment and effector molecule or detectable marker are polypeptides, the linkers can be attached to the constituent amino acids through their side groups (e.g., via a disulfide linkage to cysteine) or to the amino and carboxyl groups of the alpha carbon of the terminal amino acid.

[0209] In some embodiments, the linker may include a spacer element, which, if present, increases the size of the linker, thereby increasing the distance between the effector molecule or detectable marker and the antibody or antigen-binding fragment. Exemplary spacers are known to those of skill in the art, including those described in U.S. Patent Nos. 7,964,566, 7,498,298, 6,884,869, 6,323,315, 6,239,104, 6,034,065, 5,780,588, 5,665,860, 5,663,149, 5,635,483, 5,599,902, 5,554,725, 5,530,097, 5,521 Nos. 5,284, 5,504,191, 5,410,024, 5,138,036, 5,076,973, 4,986,988, 4,978,744, 4,879,278, 4,816,444 and 4,486,414, as well as those listed in U.S. Patent Application Publication Nos. 20110212088 and 20110070248, each of which is incorporated herein by reference in its entirety.

[0210] In some embodiments, the linker is cleavable under intracellular conditions, such that cleavage of the linker releases the effector molecule or detectable marker from the antibody or antigen-binding fragment in the intracellular environment. In yet other embodiments, the linker is non-cleavable, and the effector molecule or detectable marker is released, for example, by antibody degradation. In some embodiments, the linker is cleavable by a cleaving agent present in the intracellular environment (e.g., within a lysosome, endosome, or caveolae). The linker can be, for example, a peptide linker that is cleaved by an intracellular peptidase or protease enzyme, including, but not limited to, a lysosomal or endosomal protease. In some embodiments, the peptide linker is at least two amino acids long or at least three amino acids long. However, the linker can also be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids long, e.g., 1-2, 1-3, 2-5, 3-10, 3-15, 1-5, 1-10, or 1-15 amino acids long. Proteases can include cathepsins B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives, resulting in the release of active drugs inside target cells (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). For example, a peptide linker cleavable by the thiol-dependent protease cathepsin B can be used (e.g., a phenylalanine-leucine or glycine-phenylalanine-leucine-glycine linker). Other examples of such linkers are described, for example, in U.S. Pat. No. 6,214,345, which is incorporated herein by reference. In a specific embodiment, the peptide linker cleavable by intracellular proteases is a valine-citrulline linker or a phenylalanine-lysine linker (see, e.g., U.S. Pat. No. 6,214,345, which describes the synthesis of doxorubicin using a valine-citrulline linker).

[0211] In other embodiments, the cleavable linker is pH-sensitive, i.e., sensitive to hydrolysis at a certain pH value. Typically, a pH-sensitive linker is hydrolyzable under acidic conditions. For example, an acid-labile linker (e.g., hydrazone, semicarbazone, thiosemicarbazone, cis-aconitamide ( Linkers such as cis-aconitic amides, orthoesters, acetals, ketals, etc., can be used (see, e.g., U.S. Pat. No. 5,122,368; U.S. Pat. No. 5,824,805; U.S. Pat. No. 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661). Such linkers are relatively stable under neutral pH conditions, such as in blood, but are unstable at pHs below 5.5 or 5.0, the approximate pH of lysosomes. In certain embodiments, the hydrolyzable linker is a thioether linker (e.g., a thioether attached to a therapeutic agent via an acylhydrazone bond) (see, e.g., U.S. Pat. No. 5,622,929).

[0212] In other embodiments, the linker is cleavable under reducing conditions (eg, a disulfide linker). A variety of disulfide linkers are known in the art, including, for example, those that can be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene)-, SPDB, and SMPT (see, e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (C.W. Vogel, ed., Oxford University Press, 1987); Phillips et al., Cancer Res. 68:9280-9290, 2008). See also U.S. Patent No. 4,880,935.

[0213] In yet other specific embodiments, the linker is a malonate linker (Johnson et al., 1995, Anticancer Res. 15:1387-93), a maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1299-1304), or a 3'-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1305-12).

[0214] In still other embodiments, the linker is non-cleavable and the effector molecule or detectable marker is released by antibody degradation (see U.S. Patent Application Publication No. 2005 / 0238649, the entire contents of which are incorporated herein by reference).

[0215] In some embodiments, the linker is resistant to cleavage in an extracellular environment. For example, when the conjugate is present in an extracellular environment (e.g., in plasma), about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 3% or less, or about 1% or less of the linkers in the sample of the conjugate are cleaved. Whether a linker is resistant to cleavage in an extracellular environment can be determined, for example, by incubating the conjugate containing the desired linker with plasma for a predetermined period (e.g., 2, 4, 8, 16, or 24 hours), and then quantifying the amount of free effector molecule or detectable marker present in the plasma. Various exemplary linkers that can be used in the conjugates are described in WO2004-010957, U.S. Patent Application Publication No. 2006 / 0074008, U.S. Patent Application Publication No. 20050238649, and U.S. Patent Application Publication No. 2006 / 0024317, each of which is incorporated herein by reference in its entirety.

[0216] In some embodiments, a conjugate of a CAR, a T cell expressing a CAR, an antibody or or an antigen-binding portion thereof, and one or more small molecule toxins, such as calicheamicin, maytansinoids, dolastatins, auristatins, trichothecines, and CC1065, and derivatives of these toxins that have toxin activity, are provided.

[0217] Maytansine compounds suitable for use as maytansinoid toxin moieties are well known in the art and can be isolated from natural sources according to known methods, produced using genetic engineering techniques (see Yu et al. (2002) PNAS 99:7968-7973), or synthetically prepared maytansinol and maytansinol analogs according to known methods. Maytansinoids are mitotic inhibitors that act by inhibiting tubulin polymerization. Maytansine was first isolated from the East African shrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it was discovered that certain microorganisms also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042). Synthetic maytansinol and its derivatives and analogs are described, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428; and 4,311,140. Nos. 3,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533, each of which is incorporated herein by reference. Maytansinoid-containing conjugates, methods for making same, and their therapeutic uses are disclosed, for example, in U.S. Patent Nos. 5,208,020; 5,416,064; 6,441,163 and European Patent EP 0 425 235 B1, the disclosures of which are expressly incorporated herein by reference.

[0218] Additional toxins can be used with CARs, CAR-expressing T cells, antibodies, or antigen-binding portions thereof. Exemplary toxins include Pseudomonas exotoxin (PE), ricinus toxin, abrin, diphtheria toxin and its subunits, ribotoxin, ribonuclease, saporin and calicheamicin, and botulinum toxins A-F. These toxins are well known in the art, and many are readily available from commercial sources (e.g., Sigma Chemical Company, St. Louis, MO). Contemplated toxins also include variants of these toxins (see, e.g., U.S. Patent Nos. 5,079,163 and 4,689,401).

[0219] Saporin is a toxin derived from Saponaria officinalis that disrupts protein synthesis by inactivating the 60S portion of the ribosomal complex (Stirpe et al., Bio / Technology, 10:405-412, 1992). However, this toxin does not have a mechanism for specific entry into cells and therefore requires conjugation to an antibody or antigen-binding fragment that recognizes an internalized cell surface protein in order to be efficiently taken up by cells.

[0220] Diphtheria toxin is isolated from Corynebacterium diphtheriae. Typically, diphtheria toxin for use in immunotoxins is mutated to reduce or eliminate nonspecific toxicity. Diphtheria toxin has full enzymatic activity but exhibits significant nonspecific toxicity. A mutant known as CRM107, which has a reduced ribonucleotide count, has been known since the 1970s (Laird and Groman, J. Virol. 19:220, 1976) and has been used in human clinical trials. See U.S. Patent Nos. 5,792,458 and 5,208,021.

[0221] Castor toxin is the lectin RCA60 from Ricinus communis (castor bean). For examples of castor toxins, see U.S. Patent Nos. 5,079,163 and 4,689,401. Ricinus communis agglutinin (RCA) is a lectin derived from the plant Ricinus communis (castor bean), according to its molecular weight of approximately 65 kD and 120 kD, respectively. 60 and RCA 120 The toxin exists in two forms, termed the A chain and the B chain (Nicholson and Blaustein, J. Biochim. Biophys. Acta 266:543, 1972). The A chain is responsible for inactivating protein synthesis and killing the cell. The B chain binds the toxin to cell surface galactose residues and facilitates transport of the A chain into the cytosol (Olsnes et al., Nature 249:627-631, 1974 and U.S. Pat. No. 3,060,165).

[0222] Ribonucleases have also been conjugated to targeting molecules for use as immunotoxins (see Suzuki et al., Nat. Biotech. 17:265-70, 1999). Exemplary ribotoxins, such as α-sarcin and restrictocin, are discussed, for example, in Rathore et al., Gene 190:31-5, 1997; and Goyal and Batra, Biochem. 345(2):247-54, 2000. Calicheamicin, originally isolated from Micromonospora echinospora, is a member of the enediyne antitumor antibiotic family, generating double-strand breaks in DNA that lead to apoptosis (see, for example, Lee et al., J. Antibiot. 42:1070-87, 1989). This drug is the toxic portion of an immunotoxin in clinical trials (see, eg, Gillespie et al., Ann. Oncol. 11:735-41, 2000).

[0223] Abrin includes toxic lectins from Abrus precatorius. The toxicants, abrins a, b, c, and d, have molecular weights of approximately 63 kD and 67 kD and are composed of two disulfide-linked polypeptide chains, A and B. The A chain inhibits protein synthesis; the B chain (abrin-b) binds to D-galactose residues (see Funatsu et al., Agr. Biol. Chem. 52:1095, 1988; and Olsnes, Methods Enzymol. 50:330-335, 1978).

[0224] CARs, CAR-expressing T cells, monoclonal antibodies specific for one or more of the antigens disclosed herein, and antigen-binding fragments thereof can also be conjugated to a detectable marker; for example, a detectable marker detectable by ELISA, spectrophotometry, flow cytometry, microscopy, or imaging techniques (e.g., computed tomography (CT), computed axial tomography (CAT) scan, magnetic resonance imaging (MRI), nuclear magnetic resonance imaging (NMRI), magnetic resonance tomography (MTR), ultrasound, fiber optic testing, and laparoscopic testing). Specific non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzymatic linkages, radioactive isotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI). For example, useful detectable markers include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, and the like. Bioluminescent markers such as green fluorescent protein (GFP) and yellow fluorescent protein (YFP) are also used. CARs, CAR-expressing T cells, antibodies, or antigen-binding portions thereof, can also be conjugated to enzymes useful for detection, such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, glucose oxidase, etc. When CARs, CAR-expressing T cells, antibodies, or antigen-binding portions thereof are conjugated to a detectable enzyme, they can be detected by adding an additional reagent that the enzyme uses to produce a discernible reaction product. For example, in the presence of the agent horseradish peroxidase, the addition of hydrogen peroxide and diaminobenzidine results in a visually detectable colored reaction product. CARs, CAR-expressing T cells, antibodies, or antigen-binding portions thereof can also be conjugated to biotin and detected through indirect measurement of avidin or streptavidin binding. It should be noted that avidin itself can be conjugated to an enzyme or fluorescent label.

[0225] CAR, CAR-expressing T cells, antibodies, or their antigen-binding portions can be conjugated with paramagnetic agents such as gadolinium. Paramagnetic agents such as superparamagnetic iron oxide can also be used as labels. Antibodies can also be conjugated with lanthanides (e.g., europium and dysprosium) and manganese. Antibodies or antigen-binding fragments can also be labeled with a predetermined polypeptide epitope recognized by a secondary reporter (e.g., leucine zipper pair sequence, binding site for secondary antibody, metal binding domain, epitope tag).

[0226] CAR, CAR-expressing T cells, antibodies, or antigen-binding portions thereof can also be conjugated with radiolabeled amino acids.Radiolabels can be used for both diagnostic and therapeutic purposes.For example, radiolabels can be used to detect one or more of the antigens and antigen-expressing cells disclosed herein by x-ray, emission spectroscopy, or other diagnostic techniques.In addition, radiolabels can be used therapeutically as toxins for treating tumors in subjects, for example, for treating neuroblastoma.Examples of labels for polypeptides include, but are not limited to, the following radioisotopes or radionucleotides: 3 H, 14 C. 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I.

[0227] Means for detecting such detectable markers are well known to those skilled in the art. Thus, for example, radiolabels can be detected using photographic film or scintillation counters, fluorescent markers can be detected using a photodetector to detect emitted illumination, enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and chromogenic labels are detected by simply visualizing the colored label.

[0228] D. Nucleotides, Expression, Vectors and Host Cells Further provided by one embodiment of the present invention is a nucleic acid comprising a nucleotide sequence encoding any of the CARs, antibodies, or antigen-binding portions thereof described herein (including functional portions and functional variants thereof). The nucleic acids of the present invention may comprise a nucleotide sequence encoding any of the leader sequences, antigen-binding domains, transmembrane domains, and / or intracellular T cell signaling domains described herein.

[0229] In some embodiments, nucleotide sequences can be codon-modified.Without being bound by any particular theory, it is believed that codon optimization of nucleotide sequences can increase the translation efficiency of mRNA transcripts.The codon optimization of nucleotide sequences can include replacing native codons with other codons that code for the same amino acid but can be translated by tRNAs that are more readily available in cells, thereby increasing translation efficiency.Optimization of nucleotide sequences It may also reduce secondary mRNA structures that interfere with translation, thus increasing translation efficiency.

[0230] In embodiments of the invention, a nucleic acid can comprise a codon-modified nucleotide sequence encoding the antigen-binding domain of a CAR of the invention. In another embodiment of the invention, a nucleic acid can comprise a codon-modified nucleotide sequence encoding any of the CARs described herein (including functional portions and functional variants thereof).

[0231] "Nucleic acid," as used herein, includes "polynucleotides," "oligonucleotides," and "nucleic acid molecules," and generally refers to a polymer of DNA or RNA that can be single- or double-stranded, synthetic or obtained from natural sources (e.g., isolated and / or purified), and can contain natural, non-natural, or modified nucleotides, and can contain natural, non-natural, or modified internucleotide linkages, e.g., phosphoramidate or phosphorothioate linkages, in place of the phosphodiesters found between nucleotides in unmodified oligonucleotides. In some embodiments, a nucleic acid does not contain any insertions, deletions, inversions, and / or substitutions. However, in some cases, as discussed herein, it may be appropriate for a nucleic acid to contain one or more insertions, deletions, inversions, and / or substitutions.

[0232] Recombinant nucleic acids can have sequences that do not occur in nature or that are created by the artificial combination of two otherwise separate segments of sequence. This artificial combination is often achieved by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acid, for example, by genetic engineering techniques such as those described in Sambrook et al., supra. Nucleic acids can be constructed based on chemical synthesis and / or enzymatic ligation reactions using procedures known in the art. See, for example, Sambrook et al., supra and Ausubel et al., supra. For example, nucleic acids can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides (e.g., phosphorothioate derivatives and acridine-substituted nucleotides) designed to increase the biological stability of the molecule or to increase the physical stability of the duplex formed upon hybridization.Examples of modified nucleotides that can be used to generate nucleic acids include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-substituted adenines, 7-methylguanine, and the like. Examples of nucleic acids include, but are not limited to, uracil-5-oxyacetic acid (v), ubutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleic acids of the present invention can be purchased from a company such as Integrated DNA Technologies (Coralville, IA, USA).

[0233] The nucleic acid can comprise any isolated or purified nucleotide sequence encoding a CAR or any of its functional portions or variants. Alternatively, the nucleotide sequence can comprise a nucleotide sequence that is degenerate to any of the sequences, or a combination of degenerate sequences.

[0234] One embodiment also provides an isolated or purified nucleic acid comprising a nucleotide sequence that is complementary to or that hybridizes under stringent conditions to the nucleotide sequence of any of the nucleic acids described herein.

[0235] Nucleotide sequences that hybridize under stringent conditions may also hybridize under high stringency conditions. "High stringency conditions" means that a nucleotide sequence specifically hybridizes to a target sequence (any nucleotide sequence of a nucleic acid described herein) in an amount detectably stronger than nonspecific hybridization. High stringency conditions include conditions that distinguish polynucleotides with exact complementary sequences, or polynucleotides containing only a few scattered mismatches, from random sequences that happen to have several small regions (e.g., 3-10 bases) that match the nucleotide sequence. Such small regions of complementarity are more easily 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 by about 0.02-0.1 M NaCl or equivalent at temperatures of about 50-70°C. Such highly stringent conditions tolerate little, if any, mismatch between the nucleotide sequence and the template or target strand and are particularly suitable for detecting expression of any of the CARs of the present invention. It is generally understood that conditions can be made more stringent by the addition of increasing amounts of formamide.

[0236] Also provided are nucleic acids comprising a nucleotide sequence that is at least about 70% or more, e.g., about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to any of the nucleic acids described herein.

[0237] In one embodiment, the nucleic acid may be incorporated into a recombinant expression vector. In this regard, one embodiment provides a recombinant expression vector comprising any of the nucleic acids. For purposes of this specification, the term "recombinant expression vector" refers to a genetically engineered oligonucleotide or polynucleotide construct that allows a host cell to express an mRNA, protein, polypeptide, or peptide when the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide, and when the vector is contacted with a cell under conditions sufficient for the mRNA, protein, polypeptide, or peptide to be expressed in the cell. The vector as a whole does not exist in nature.

[0238] However, portions of the vector may be naturally occurring. Recombinant expression vectors may be single-stranded or double-stranded, synthetic or derived from partially natural sources, and may contain any type of nucleotide, including, but not limited to, DNA and RNA, which may contain natural, non-natural, or modified nucleotides. Recombinant expression vectors may contain naturally occurring or non-naturally occurring internucleotide linkages, or both types of linkages. Preferably, the non-naturally occurring or modified nucleotides or internucleotide linkages do not interfere with the transcription or replication of the vector.

[0239] In one embodiment, the recombinant expression vector may be any suitable recombinant expression vector, Any suitable host cell can be transformed or transfected using vectors. Suitable vectors include those designed for propagation and propagation 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).

[0240] Bacteriophage vectors, such as λυTΙO, λυTΙ 1, λZapII (Stratagene), EMBL4, and λΝΜΙ 149, can also be used. Examples of plant expression vectors include pBI01, pBI101.2, pBHO1.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). The recombinant expression vector can be a viral vector, such as a retroviral or lentiviral vector. Lentiviral vectors are vectors derived from at least a portion of the lentiviral genome, including, in particular, self-inactivating lentiviral vectors such as those provided in Milone et al., Mol. Ther. 17(8):1453-1464 (2009). Other examples of lentiviral vectors that may be used in the clinic include, for example, but not limited to, Oxford BioMedica plc's LENTIVECTOR® gene delivery technology, Lentigen's LENTIMAX™ vector system, etc. Non-clinical forms of lentiviral vectors are also available and known to those skilled in the art.

[0241] Several transfection techniques are generally known in the art (see, e.g., Graham et al., Virology 52:456-467 (1973); Sambrook et al., supra; Davis et al., Basic Methods in Molecular Biology, Elsevier (1986); and Chu et al., Gene 13:97 (1981)).

[0242] Transfection methods include calcium phosphate coprecipitation (see, e.g., Graham et al., supra), direct microinjection into cultured cells (see, e.g., Capecchi, Cell 22:479-488 (1980)), electroporation (see, e.g., Shigekawa et al., BioTechniques 6:742-751 (1988)), liposome-mediated gene transfer (see, e.g., Mannino et al., BioTechniques 6:682-690 (1988)), lipid-mediated transduction (see, e.g., Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987)), and high velocity micropropellants. Microprojectile delivery (see, eg, Klein et al., Nature, 327:70-73 (1987)) is also included.

[0243] In one embodiment, recombinant expression vectors can be prepared using standard recombinant DNA techniques, for example, as described in Sambrook et al., supra, and Ausubel et al., supra. Circular or linear expression vector constructs can be prepared to contain a replication system functional in prokaryotic or eukaryotic host cells. Replication systems can be derived from, for example, ColEl, 2μ plasmid, λ, SV40, bovine papilloma virus, etc.

[0244] The recombinant expression vector may be selected from a variety of vectors, taking into account whether the vector is DNA- or RNA-based, as appropriate, and depending on the type of host cell into which the vector will be introduced (e.g., bacterial, fungal, plant, etc.). The recombinant expression vector may contain regulatory sequences specific to the host (or animal), such as transcription and translation initiation and termination codons. The recombinant expression vector may contain restriction sites to facilitate cloning.

[0245] The recombinant expression vector may contain one or more marker genes to allow for the selection of transformed or transfected host cells. Marker genes include biocide resistance, resistance to, e.g., antibiotics, heavy metals, etc., complementation in auxotrophic hosts to provide prototrophy, etc. Suitable marker genes for the expression vectors of the present invention include, for example, the neomycin / G418 resistance gene, the hygromycin resistance gene, the histidinol resistance gene, the tetracycline resistance gene, and the ampicillin resistance gene.

[0246] The recombinant expression vector may comprise a native or non-native promoter operably linked to the nucleotide sequence encoding the CAR (including its functional portion and functional variant), or to a nucleotide sequence complementary to or hybridizing with the nucleotide sequence encoding the CAR. The selection of a promoter, for example, strong, weak, inducible, tissue-specific, and developmentally specific, is within the skill of those skilled in the art. Similarly, combining a nucleotide sequence with a promoter is also within the skill of those skilled in the art. The promoter may be a non-viral promoter or a viral promoter, such as a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, or a promoter found in the long terminal repeat of murine stem cell virus.

[0247] Recombinant expression vectors can be designed for transient expression, stable expression, or both, and can be made for constitutive or inducible expression.

[0248] Furthermore, recombinant expression vectors can be made to contain suicide genes. As used herein, the term "suicide gene" refers to a gene that causes cells that express the suicide gene to die. A suicide gene can be a gene that confers sensitivity to a drug or other agent on the cell in which the gene is expressed, or a gene that causes the cell to die when contacted with or exposed to a drug. Suicide genes are known in the art (see, for example, Suicide Gene Therapy: Methods and Reviews, Springer, Caroline J. (Cancer Research UK Centre for Cancer Therapeutics at the Institute of Cancer Research, Sutton, Surrey, UK), Humana Press, 2004), and include, for example, herpes simplex virus (HSV) thymidine kinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase, and nitroreductase.

[0249] One embodiment further provides a host cell comprising any of the recombinant expression vectors described herein. As used herein, the term "host cell" refers to any type of cell that can contain a recombinant expression vector of the present invention. The host cell can be a eukaryotic cell, such as a plant, animal, fungus, or algae, or a prokaryotic cell, such as a bacterium or protist. The host cell can be a cultured or primary cell, i.e., directly isolated from an organism such as a human. The host cell can be an adherent cell or a suspension cell, i.e., a cell that grows in suspension. Suitable host cells are known in the art and include, for example, DH5a E. coli cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, etc. For the purpose of amplifying or replicating a recombinant expression vector, the host cell can be a prokaryotic cell, such as a DH5a cell. For the purpose of producing a recombinant CAR, the host cell can be a mammalian cell. Host Cell The host cells can be human cells. The host cells can be of any cell type, originate from any type of tissue, and be at any stage of development, but the host cells can be peripheral blood lymphocytes (PBLs) or peripheral blood mononuclear cells (PBMCs). The host cells can be T cells.

[0250] For purposes herein, T cells can be any T cells, e.g., cultured T cells, e.g., primary T cells, or T cells from cultured T cell lines, e.g., Jurkat, SupTl, etc., or T cells obtained from a mammal. If obtained from a mammal, T cells can be obtained from a number of sources, including, but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. T cells can also be enriched or purified. T cells can be human T cells. T cells can be T cells isolated from a human. T cells can be CD4 + / CD8 + Double positive T cells, CD4 + Helper T cells, e.g., Th1 and Th2 cells, CD8 + The T cells can be of any type and at any stage of development, including, but not limited to, T cells (e.g., cytotoxic T cells), tumor-infiltrating cells, memory T cells, memory stem cells, i.e., Tscm, naive T cells, etc. T cells can be CD8 + T cells or CD4 + It may be a T cell.

[0251] In one embodiment, the CARs described herein can be used in suitable non-T cells, such as cells with immune effector function, such as NK cells and T-like cells generated from pluripotent stem cells.

[0252] Also provided by one embodiment is a population of cells comprising at least one host cell described herein. The population of cells can be a heterogeneous population comprising host cells comprising any of the described recombinant expression vectors in addition to at least one other cell, e.g., a host cell (e.g., a T cell), that does not comprise any recombinant expression vector, or a cell other than a T cell, e.g., a B cell, macrophage, neutrophil, erythrocyte, hepatocyte, endothelial cell, epithelial cell, muscle cell, brain cell, etc. Alternatively, the population of cells can be a substantially homogeneous population, wherein the population primarily comprises host cells comprising (e.g., consisting essentially of) the recombinant expression vector. The population can also be a clonal population of cells, wherein all cells in the population are clones of a single host cell comprising the recombinant expression vector, such that all cells in the population comprise that recombinant expression vector. In one embodiment of the present invention, the population of cells is a clonal population comprising host cells comprising the recombinant expression vector described herein.

[0253] CARs (including functional portions and variants thereof), nucleic acids, recombinant expression vectors, host cells (including populations thereof), and antibodies (including antigen-binding portions thereof) can be isolated and / or purified. For example, a purified (or isolated) host cell preparation is one in which the host cells are more pure than the cells in their natural environment in the body. Such host cells can be produced, for example, by standard purification techniques. In some embodiments, a host cell preparation is purified such that the host cells represent at least about 50%, e.g., at least about 70%, of the total cell content of the preparation. For example, the purity can be at least about 50%, greater than about 60%, about 70%, or about 80%, or can be about 100%.

[0254] E. Treatment Method It is contemplated that the CARs disclosed herein can be used in methods for treating or preventing disease in mammals. In this regard, one embodiment provides a method for treating or preventing cancer in a mammal, comprising administering to the mammal a CAR, a nucleic acid, a recombinant expression vector, a host cell, a population of cells, an antibody and / or an antigen-binding portion thereof, and / or a pharmaceutical composition in an amount effective to treat or prevent cancer in the mammal.

[0255] One embodiment involves administering a CAR disclosed herein to a mammal. Examples of lymphodepletion may include, but are not limited to, non-myeloablative lymphodepleting chemotherapy, myeloablative lymphodepleting chemotherapy, total body irradiation, etc.

[0256] For the purpose of the method of administering host cells or a group of cells, the cells can be allogeneic or autologous to the mammal.Preferably, the cells are autologous to the mammal.As used herein, allogeneic refers to any material derived from a different animal of the same species as the individual into which the material is introduced.Two or more individuals are said to be allogeneic to each other if the genes are not identical at one or more loci.In some embodiments, allogeneic materials from individuals of the same species can be genetically sufficiently different to interact antigenically.As used herein, "autologous" refers to any material derived from the same individual that is subsequently reintroduced into the individual.

[0257] The mammal referred to herein may be any mammal. As used herein, the term "mammal" refers to any mammal, including, but not limited to, rodent mammals, e.g., mice and hamsters, and logomorph mammals, e.g., rabbits. The mammal may be from the carnivora order, including felines (cats) and canines (dogs). The mammal may be from the artiodactyla order, including bovines (cows) and swines (pigs), or from the perissodactyla order, including equines (horses). The mammal may be from the primate order, ceboids or simoids (monkeys), or anthropoids (humans and apes). Preferably, the mammal is a human.

[0258] For these methods, the cancer may be acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer (e.g., bladder carcinoma), bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, cancer of the anus, anal canal, or anorectum, eye cancer, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid carcinoma The cancer may be any cancer, including any of the following: colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, head and neck cancer (e.g., head and neck squamous cell carcinoma), Hodgkin's lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, leukemia, liquid tumors, liver cancer, lung cancer (e.g., non-small cell lung cancer and lung adenocarcinoma), lymphoma, mesothelioma, mast cell tumor, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin's lymphoma, B-chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia (ALL) and Burkitt's lymphoma, ovarian cancer, pancreatic cancer, peritoneal, omental and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumor, synovial sarcoma, gastric cancer, testicular cancer, thyroid cancer, and ureteral cancer.

[0259] The terms "treat" and "prevent," and words derived therefrom, as used herein, do not necessarily mean 100% or complete treatment or prevention. Rather, there are various degrees of treatment or prevention that one of skill in the art would recognize as having a potential beneficial or therapeutic effect. In this regard, the method may provide any amount or level of treatment or prevention of cancer in a mammal.

[0260] Furthermore, the treatment or prevention provided by this method can include treatment or prevention of one or more conditions or symptoms of the disease, such as cancer, being treated or prevented. Also, for purposes herein, "prevention" can include delaying the onset of the disease, or its symptoms or conditions.

[0261] Another embodiment provides a method of detecting the presence of cancer in a mammal, the method comprising: (a) contacting a sample comprising one or more cells from the mammal with a CAR, a nucleic acid, a recombinant expression vector, a host cell, a population of cells, an antibody and / or antigen-binding portion thereof, or a pharmaceutical composition, thereby forming a complex; and (b) detecting the complex, wherein detection of the complex indicates the presence of cancer in the mammal.

[0262] The sample can be obtained by any suitable method, for example, biopsy or autopsy.Biopsy is the removal of tissue and / or cells from an individual.Such removal can be the collection of tissue and / or cells from an individual in order to perform experiments on the removed tissue and / or cells.This experiment can include experiments to determine whether the individual has a certain condition or disease state and / or whether they are suffering from a certain condition or disease state.The condition or disease can be, for example, cancer.

[0263] For embodiments of methods for detecting the presence of a proliferation disorder, e.g., cancer, in a mammal, the sample containing mammalian cells can be a sample containing whole cells, a lysate thereof, or a fraction of a whole cell lysate, e.g., a nuclear or cytoplasmic fraction, a whole protein fraction, or a nucleic acid fraction. When the sample contains whole cells, these cells can be any cells of a mammal, e.g., cells of any organ or tissue, including blood cells or endothelial cells.

[0264] The contacting step can occur in vitro or in vivo with respect to a mammal. Preferably, the contacting step is in vitro.

[0265] Also, detection of complexes can be carried out by many methods known in the art.For example, the CAR disclosed herein, polypeptide, protein, nucleic acid, recombinant expression vector, host cell, cell population, or antibody or its antigen-binding portion can be labeled with detectable label, such as the radioisotope disclosed above, fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), enzyme (e.g., alkaline phosphatase, horseradish peroxidase) and element particle (e.g., gold particle), etc.

[0266] The method of testing CAR for its ability to recognize target cells and antigen specificity is known in the art.For example, Clay et al., J.Immunol, vol. 163: 507-513 (1999) teaches a method for measuring the release of cytokines (e.g., interferon-γ, granulocyte / monocyte colony stimulating factor (GM-CSF), tumor necrosis factor α (TNF-α) or interleukin 2 (IL-2)).In addition, CAR function can be evaluated by measuring cytotoxicity, as described in Zhao et al., J.Immunol. vol. 174: 4415-4423 (2005).

[0267] Another embodiment provides the use of the CARs, nucleic acids, recombinant expression vectors, host cells, populations of cells, antibodies or antigen-binding portions thereof, and / or pharmaceutical compositions of the invention to treat or prevent a proliferative disorder, such as cancer, in a mammal. The cancer can be any of the cancers described herein.

[0268] Any administration method, including local and systemic administration, can be used for the disclosed therapeutic agent. For example, topical, oral, intravascular (e.g., intravenous), intramuscular, intraperitoneal, intranasal, intradermal, intrathecal, and subcutaneous administration can be used. The specific administration mode and dosing regimen will be selected by the attending clinician, taking into account the characteristics of the case (e.g., subject, disease, involved disease state, and whether the treatment is preventive). When more than one agent or composition is administered, One or more routes of administration may be used; for example, the chemotherapeutic agent may be administered orally, and the antibody or antigen-binding fragment or conjugate or composition may be administered intravenously. Administration methods include injection, in which the CAR, CAR T cell, conjugate, antibody, antigen-binding fragment, or composition is provided in a non-toxic pharmaceutically acceptable carrier, such as water, saline, Ringer's solution, dextrose solution, 5% human serum albumin, solid oil, ethyl oleate, or liposomes. In some embodiments, local administration of the disclosed compounds may be used, for example, by applying the antibody or antigen-binding fragment to an area of ​​tissue from which a tumor has been removed or to an area suspected of being prone to tumor development. In some embodiments, sustained intratumoral (or near-tumoral) release of a pharmaceutical preparation containing a therapeutically effective amount of the antibody or antigen-binding fragment may be beneficial. In other examples, the conjugate is applied topically to the cornea as eye drops or intravitreally to the eye.

[0269] The disclosed therapeutic agents can be formulated in unit dosage forms suitable for individual administration of precise dosage amounts. Furthermore, the disclosed therapeutic agents can be administered in a single dose or in a multiple-dose schedule. A multiple-dose schedule is one in which the main course of treatment may involve more than one discrete 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(s) over a period of several days to several months or even years. Thus, the dosing regime will also be determined, at least in part, based on the specific needs of the subject being treated and will be dependent on the judgment of the administering practitioner.

[0270] Typical dosages of antibodies or conjugates can range from about 0.01 to about 30 mg / kg, for example, from about 0.1 to about 10 mg / kg.

[0271] In particular examples, the subject is administered a therapeutic composition comprising one or more of the conjugate, antibody, composition, CAR, CAR T cell, or additional agent in a multiple daily dosing schedule, e.g., at least 2 consecutive days, 10 consecutive days, etc., for a period of, e.g., weeks, months, or years. In one example, the subject is administered the conjugate, antibody, composition, or additional agent for a period of at least 30 days, e.g., at least 2 months, at least 4 months, at least 6 months, at least 12 months, at least 24 months, or at least 36 months.

[0272] In some embodiments, the disclosed methods include providing a subject with surgery, radiation therapy, and / or chemotherapy in combination with the disclosed antibodies, antigen-binding fragments, conjugates, CARs, or CAR-expressing T cells (e.g., sequentially, substantially simultaneously, or simultaneously). Such agents and treatment methods and therapeutic dosages are known to those skilled in the art and can be determined by a skilled clinician. Preparation and dosing schedules for additional agents can be used according to manufacturer's instructions or can be as empirically determined by one skilled in the art. Preparation and dosing schedules for such chemotherapy are also described in Chemotherapy Service, (1992) MC Perry (ed.), Williams & Wilkins, Baltimore, Md.

[0273] In some embodiments, the combination therapy may include administering to the subject a therapeutically effective amount of an additional cancer inhibitor. Non-limiting examples of additional therapeutic agents that can be used in the combination therapy include microtubule binding agents, DNA intercalators or crosslinkers, DNA synthesis inhibitors, DNA and RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, gene regulators, and angiogenesis inhibitors. These agents (administered in therapeutically effective amounts) and treatments can be used alone or in combination. For example, any suitable anti-cancer or anti-angiogenic agent can be administered in combination with the CAR, CAR-T cells, antibodies, antigen-binding fragments, or conjugates disclosed herein. Methods and therapeutic dosages of such agents are known to those skilled in the art and can be readily determined. This can be determined by the clinician in charge.

[0274] Additional chemotherapeutic agents include alkylating agents such as nitrogen mustards (e.g., chlorambucil, chlormethine, cyclophosphamide, ifosfamide, and melphalan), nitrosoureas (e.g., carmustine, fotemustine, lomustine, and streptozocin), platinum compounds (e.g., carboplatin, cisplatin, oxaliplatin, and BBR3464), busulfan, dacarbazine, mechlorethamine, procarbazine, temozolomide, thiotepa, and uramustine; antimetabolites such as folates (e.g., methotrexate, pemetrexed, and raltitrexed), purines (e.g., cladribine, clofarabine, fludarabine, mercaptopurine, and thioguanine), pyrimidines (e.g., capecitabine), cytarabine, fluorouracil, and gemcitabine; plant alkaloids, such as For example, podophyllum (e.g., etoposide and teniposide), taxanes (e.g., docetaxel and paclitaxel), vincas (e.g., vinblastine, vincristine, vindesine, and vinorelbine); cytotoxic / antitumor antibiotics, such as members of the anthracycline family (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, and valrubicin), bleomycin, rifampicin, hydroxyurea, and mitomycin; topoisomerase inhibitors, such as topotecan and irinotecan; monoclonal antibodies, such as alemtuzumab, bevacizumab, cetuximab, gemtuzumab, rituximab, panitumumab, pertuzumab, and trastuzumab; tumor-affinity photosensitizers, such as aminolevulinic acid, methyl aminolevulinate, porfimer sodium, and verteporfin;and other drugs, such as alitretinoin, altretamine, amsacrine, anagrelide, arsenic trioxide, asparaginase, axitinib, bexarotene, bevacizumab, bortezomib, celecoxib, denileukin diftitox, erlotinib, estramustine, gefitinib, hydroxycarbamide, imatinib, lapatinib, pazopanib, pentostatin, masoprocol, mitotane, pegaspargase, tamoxifen, sorafenib, sunitinib, 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.

[0275] Combination therapy can provide synergistic effects and can be proven to be synergistic, that is, the effect achieved when active ingredients are used together is greater than the sum of the effects that can be obtained from using these compounds separately.Synergistic effects can be achieved when active ingredients are (1) co-formulated and administered or delivered simultaneously as a combined unit dosage formulation; (2) delivered alternately or in parallel as separate formulations; or (3) by some other regimen.When delivered alternately, synergistic effects can be achieved when these compounds are administered or delivered sequentially, for example, by different injections in separate syringes.Generally, during alternation, each active ingredient in an effective dosage is administered continuously, that is, sequentially, whereas in combination therapy, two or more active ingredients in an effective dosage are administered together.

[0276] In one embodiment, an effective amount of an antibody or antigen-binding fragment or conjugate thereof that specifically binds to one or more of the antigens disclosed herein is administered to a subject with a tumor after anti-cancer treatment. After a sufficient amount of time has passed for the administered antibody or antigen-binding fragment or conjugate to form an immune complex with the antigen expressed on each cancer cell, the immune complex is detected. The presence (or absence) of the immune complex indicates the effectiveness of the treatment. For example, an increase in immune complexes compared to a control taken before treatment indicates that the treatment is ineffective, and a decrease in immune complexes compared to a control taken before treatment indicates that the treatment is effective.

[0277] F. Biopharmaceutical Compositions Provided herein are biopharmaceutical or biologic compositions (hereinafter "compositions") for use in gene therapy, immunotherapy, and / or cell therapy, comprising one or more of the disclosed CARs, or CAR-expressing T cells, antibodies, antigen-binding fragments, conjugates, CARs, or T cells expressing CARs that specifically bind to one or more antigens disclosed herein, in a carrier (e.g., a pharmaceutically acceptable carrier). These compositions can be prepared in unit dosage forms for administration to a subject. The amount and timing of administration to achieve a desired outcome is at the discretion of the treating clinician. These compositions can be formulated for systemic (e.g., intravenous) or local (e.g., intratumoral) administration. In one example, the disclosed CARs, or CAR-expressing T cells, antibodies, antigen-binding fragments, conjugates, are formulated for parenteral administration, such as intravenous administration. Compositions comprising the disclosed CARs, or CAR-expressing T cells, conjugates, antibodies, or antigen-binding fragments, are used for, e.g., treatment and detection of tumors, such as, but not limited to, neuroblastoma. In some examples, these compositions are useful for treating or detecting cancer. Compositions comprising the CARs disclosed herein, or T cells, conjugates, antibodies, or antigen-binding fragments expressing the CARs, are also used, for example, to detect pathological angiogenesis.

[0278] Compositions for administration can 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. A variety of aqueous carriers, such as buffered saline, can be used. These solutions are sterile and generally free of undesirable substances. The compositions can be sterilized by conventional, well-known sterilization techniques. The compositions can contain pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, adjuvant drugs, etc., such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of CAR, or CAR-expressing T cells, antibodies, or antigen-binding fragments, or conjugates in these formulations can vary widely and is selected primarily based on fluid volume, viscosity, body weight, etc., according to the particular mode of administration selected and the needs of the subject. Actual methods for preparing such dosage forms for use in gene therapy, immunotherapy, and / or cell therapy are known or will become apparent to those skilled in the art.

[0279] A typical composition for intravenous administration contains about 0.01 to about 30 mg / kg of antibody or antigen-binding fragment or conjugate (or a corresponding dose of CAR, or T cells expressing a CAR, or a conjugate comprising the antibody or antigen-binding fragment) per subject per day. Actual methods for preparing administrable compositions will be known or apparent to those of skill in the art and are described in more detail in publications such as Remington's Pharmaceutical Science, 19th Edition, Mack Publishing Company, Easton, PA (1995).

[0280] CARs, or CAR-expressing T cells, antibodies, antigen-binding fragments, or conjugates, can be provided in lyophilized form and rehydrated with sterile water before administration, but they can also be provided in sterile solutions of known concentrations. The CAR, or CAR-expressing T cells, antibodies, or antigen-binding fragments, or conjugates, solution is then added to an infusion bag containing 0.9% sodium chloride, USP, and in some cases administered at a dosage of 0.5 to 15 mg / kg body weight. Considerable experience is available in the field for administering antibody or antigen-binding fragment and conjugate drugs; for example, antibody drugs have been commercially available in the United States since the approval of Rituxan® in 1997. CARs, or CAR-expressing T cells, antibodies, antigen-binding fragments, and their conjugates, can be administered by slow infusion rather than intravenous injection or intravenous bolus. In one example, a higher loading dose is administered with subsequent maintenance doses administered at lower levels. For example, an initial loading dose of 4 mg / kg of antibody or antigen-binding fragment (or a corresponding dose of A conjugate containing the original binding fragment may be infused over a period of approximately 90 minutes, followed by weekly maintenance doses of 2 mg / kg for 4-8 weeks infused over a period of 30 minutes if the previous dose was well tolerated.

[0281] Controlled release parenteral formulations can be made as implants, oily injections, or as granular systems. For a broad review of protein delivery systems, see Banga, AJ, Thera See, Pneutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Technomic Publishing Company, Inc., Lancaster, PA (1995). Particulate systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain a therapeutic protein, such as a cytotoxin or drug, as a central core. In microspheres, the therapeutic agent is dispersed throughout the particle. Particles smaller than about 1 μm, microspheres, and microcapsules, are commonly referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Capillaries have a diameter of approximately 5 μm, so that only nanoparticles are administered intravenously. Microparticles are typically approximately 100 μm in diameter and are administered subcutaneously or intramuscularly. See, e.g., Kreuter, J., Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, NY, pp. 219-342 (1994); and Tice and Tabibi, Treatise on Controlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc., New York, NY, pp. 315-339 (1992).

[0282] The polymers can be used for ion-controlled release of the CARs disclosed herein, or T cells, antibodies or antigen-binding fragments, or conjugate compositions expressing the CARs. Various degradable and non-degradable polymer matrices used for controlled drug delivery are known in the art (Langer, Accounts Chem. Res. 26:537-542, 1993). For example, the block copolymer poloxamer 407 exists as a viscous but still mobile liquid at low temperatures, but forms a semi-fluid gel at body temperature. It has been shown to be an effective vehicle for the formulation and sustained delivery of recombinant interleukin-2 and urease (Johnston et al., Pharm. Res. 9:425-434, 1992; and Pec et al., J. Parent. Sci. Tech. 44(2):58-65, 1990). Alternatively, hydroxyapatite has been used as a microcarrier for the controlled release of proteins (Ijntema et al., Int. J. Pharm. 112:215-224, 1994). In yet another embodiment, liposomes are used for the controlled release and drug targeting of lipid-encapsulated drugs (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA (1993)). Numerous additional systems for controlled delivery of therapeutic proteins are known (see U.S. Patent Nos. 5,055,303; 5,188,837; 4,235,871; 4,501,728; 4,837,028; 4,957,735; 5,019,369; 5,055,303; 5,514,670; 5,413,797; 5,268,164; 5,004,697; 4,902,505; 5,506,206; 5,271,961; 5,254,342 and 5,534,496).

[0283] G.Kit In one aspect, kits using the CARs disclosed herein are also provided. , a kit for treating a tumor in a subject, or a kit for generating CAR T cells expressing one or more of the CARs disclosed herein. The kit typically includes the disclosed antibodies, antigen-binding fragments, conjugates, nucleic acid molecules, CARs, or CAR-expressing T cells disclosed herein. More than one of the disclosed antibodies, antigen-binding fragments, conjugates, nucleic acid molecules, CARs, or CAR-expressing T cells can be included in the kit.

[0284] The kit may include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container may be formed from a variety of materials, such as glass or plastic. The container typically holds a composition comprising one or more of the disclosed antibodies, antigen-binding fragments, conjugates, nucleic acid molecules, CARs, or CAR-expressing T cells. In some embodiments, the container may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle). The label or package insert indicates that the composition is used to treat a specific condition.

[0285] The label or package insert typically further includes instructions for using the disclosed antibody, antigen-binding fragment, conjugate, nucleic acid molecule, CAR, or CAR-expressing T cell, for example, in a method for treating or preventing tumors or in a method for generating CAR T cells. The package insert typically includes instructions customarily included in the commercial packaging of a therapeutic product, including information about indications, usage, dosage, administration, contraindications, and / or warnings regarding the use of the therapeutic product. The instructional material can be written in electronic form (e.g., a computer diskette or compact disc) or visual (e.g., a video file). The kit can also include additional components to facilitate the specific application for which the kit is designed. Thus, for example, the kit can further include a means for detecting the label (e.g., an enzyme substrate for an enzymatic label, a filter set for detecting a fluorescent label, an appropriate secondary label such as a secondary antibody, etc.). The kit can further include buffers and other reagents routinely used for the implementation of a particular method. Such kits and their appropriate contents are well known to those skilled in the art. [Example]

[0286] The present invention is further illustrated by the following examples, which should not be construed as imposing limitations on the scope of the present invention in any way. On the contrary, it is readily understood that recourse must be had to various other embodiments, modifications, and equivalents thereof, which may occur to those skilled in the art after reading the description herein without departing from the spirit of the present invention and / or the scope of the appended claims.

[0287] Example 1 Isolation of CD33-specific antibodies from phage-displayed fully human ScFv and VH libraries material and method: a) Production of phage-displayed human ScFv and VH CD33-specific antibodies A naive human scFv (recombinant single-chain fragment of immunoglobulin variable) phage display library (approximate diversity, 10) constructed from peripheral blood B cells of 50 healthy donors was used.10 The unique specificity of (ZYZhu and DSDimitrov, unpublished data), and a human VH (immunoglobulin heavy chain variable domain) library were used to select scFv or VH specific for recombinant human CD33. 12 The amplified libraries of ScFv or VH were incubated with 5, 3, and 1 μg of coated CD33 in a volume of 5 × 100 μl, and the first, second, and third ScFv / VH libraries were incubated with 5, 3, and 1 μg of coated CD33 in a volume of 5 × 100 μl. During the first and third rounds of biopanning, the phage were distributed equally among five wells of a 96-well plate and incubated at room temperature for 2 hours. After each round of incubation, the wells were washed five times with phosphate-buffered saline (PBST) containing 0.05% Tween 20 (PBST) in the first round and ten times in subsequent rounds to remove nonspecifically bound phage. The bound phage were mixed with TG1 competent cells at 37°C for 1 hour, and phage were amplified from the infected cells and used in the next round of biopanning. After the third round of biopanning, 380 clones were randomly picked from the infected TG1 cells and each was inoculated into 150 μl of 2YT medium containing 100 μg / ml carbenicillin and 0.2% glucose in a 96-well plate using an automated BioRobotics BioPick colony picking system (Genomic Solutions, Ann Arbor, MI). After the bacterial culture reached an optical density at 600 nm (OD600) of 0.5, helper phage M13K07 at a multiplicity of infection (MOI) of 10 and 50 μg / ml (final concentration) kanamycin were added to the medium, and the plates were further incubated overnight at 30°C on a shaker at 250 rpm. The phage supernatant was mixed with a 3% nonfat milk solution in PBS at a volume ratio of 4:1 and used in enzyme-linked immunosorbent assay (ELISA) to identify phage clones displaying ScFv or VH with high CD33 binding affinity. The supernatant was incubated with 50 ng of coated recombinant human CD33 per well in a 96-well plate at room temperature for 2 hours, washed five times with PBST, and then incubated overnight at 4°C, blocked with 3% nonfat milk in PBS, and washed three times with PBS containing 0.05% Tween 20. Phage bound to CD33 was detected using a goat anti-M13 antibody conjugated with horseradish peroxidase. After incubation with the antibody, the wells were washed to remove nonspecifically bound antibody, and 3,3',5,5'-tetramethylbenzidine (TMB) substrate was added and the solution absorbance at 450 nm (A450) was measured. Clones that bound to CD33 and had an A450 greater than 1.0 were selected for further characterization.

[0288] b) Expression and purification of selected soluble ScFv or VH The VH and VL of selected clones, as well as the VH of the domain binder, were DNA sequenced, and scFvs or VHs encoded by clones with unique sequences were expressed and purified as described below. Plasmids extracted from these clones were used to transform HB2151 cells. Single colonies were picked from plates containing newly transformed cells and inoculated into 200 ml of 2YT medium containing 100 μg / ml ampicillin and 0.2% glucose, and incubated at 37°C with shaking at 250 rpm. When the culture OD at 600 nm reached 0.90, isopropyl-β-d-thiogalactopyranoside was added to a final concentration of 0.5 mM, and the culture was further incubated overnight at 30°C. After centrifugation at 8,000 × g for 20 minutes, the bacterial pellet was collected and resuspended in PBS buffer containing 0.5 mU polymyxin B (Sigma-Aldrich, St. Louis, MO). After incubation for 30 min at room temperature with rotation at 50 rpm, the resuspended pellet was centrifuged at 25,000 × g for 25 min at 4°C, and the supernatant was used for ScFv purification using Ni-NTA resin according to the supplier's protocol (Qiagen).

[0289] c) ELISA Binding Assay 50 μl of recombinant human CD33 diluted to 2 μg / ml in PBS was coated onto a 96-well plate overnight at 4°C. Serial dilutions of purified ScFv or VH (from above) bearing His and Flag tags were added to the target protein-coated wells. After washing, a 1:3000 dilution of HRP-conjugated anti-Flag antibody was added for 1 hour at RT. After washing, 3,3,5,5'-tetramethylbenzidine (TMB) substrate was added and incubated for 10 minutes at room temperature. The reaction was stopped by adding 1N H2SO4, and the OD was read at 450 nm to determine the binding of ScFv to CD33. The relative ability to bind was quantified.

[0290] result: Based on the results of ELISA binding assays, four distinct ScFs clones specific for recombinant human CD33 were identified and designated human anti-CD33 ScFv binders m1033-9 (ScFv9), m1033-10 (ScFv10), m1033-12 (ScFv12), and m1033-15 (ScFv15), respectively. Two unique VH domain binders, m1033-2 (VH-2) and m1033-4 (VH-4), were also identified from ELISA binding assays. The generation of chimeric antigen receptors expressing VH-2, VH-4, ScFv9, ScFv10, ScFv12, and ScFv15 human anti-CD33 binders is outlined in Example 2 below.

[0291] Example 2 CARs expressing anti-CD33 fully human heavy chain Ig-only or scFv-based binding sequences This example describes novel fully human immunoglobulin heavy chain-alone or single-chain fragment variable (scFv) binder sequence-derived anti-CD33 CAR T cells. The novel anti-CD33 CART constructs demonstrated high-level expression in primary human T cells and specific and potent cytotoxic and cytokine function against CD33-positive tumor cells.

[0292] Homo sapiens CD33 (sialic acid-binding Ig-like lectin 3, SIGLEC3, SIGLEC-3, gp67, p67) is a well-studied target for acute myeloid leukemia (AML). A CD33 humanized antibody (lintuzumab) and a CD33 antibody-drug conjugate (gemtuzumab ozogamicin, or GO, Pfizer) have shown some efficacy but have not demonstrated robust therapeutic benefit in clinical trials (1. Feldman EJ et al. J Clin Oncol 2005;23(18):4110-4116; 2. Petersdorf SH et al. Blood 2013;121(24):4854-4860). AMG330, a CD33-CD3 bispecific T cell inducer (BiTE), is also under investigation (Krupka C et al. Blood 2014 123:356–365). As of this year, GO has been reintroduced into the clinic at a modified, much smaller dose and with a revised regimen, but sufficient clinical data still need to be accumulated for this agent to be reevaluated. Another agent currently in development, the CD33-targeted antibody-drug conjugate vadastuximab-butarilin (SGN-CD33A), recently led to the clinical suspension of several Phase I / II trials due to hepatotoxicity (available on the World Wide Web at investor.seattlegenetics.com / phoenix.zhtml?c=124860&p=irol-newsArticle&ID=2232880), highlighting the urgent need to identify safe and effective CD33-targeting modalities.

[0293] CD33 CARs were designed using CD33-binding sequences derived from either immunoglobulin VH domains or full-length ScFvs under the control of the EF1a promoter and tested in vitro for transduction efficiency, killing function, and cytokine production.

[0294] material and method: (a) Cell line Human cell lines: promyelocytic leukemia HL-60, acute lymphoblastic leukemia Reh, monocytic leukemia THP-1, and myeloid leukemia K562 cell lines were purchased from the American Tissue Culture Collection (ATCC, Manassas, VA). Acute myeloid leukemia MOLM-14 was purchased from the German Collection of Cells were purchased from Microorganisms and Cell Lines (DSMZ, Braunschweig, Germany). Cell lines were cultured in RPMI-1640 medium (ATCC) supplemented with 10% heat-inactivated fetal bovine serum. THP-1 culture medium also contained 0.05% beta-mercaptoethanol. Wild-type leukemia lines were stably transduced with a lentiviral vector encoding firefly luciferase (Lentigen Technology, Inc., Gaithersburg, MD) with or without GFP, followed by limiting dilution to select for luciferase-positive clones to generate luciferase-expressing subclones.

[0295] (b) Creation of chimeric antigen receptor (CAR)-expression vector The antigen-binding domain sequence of the CAR was derived from a human anti-CD33 ScFv or heavy chain variable fragment. CAR T constructs were generated by linking the binder sequence in frame to the CD8a binding and transmembrane domain (UniProt sequence ID P01732, aa 138-206), followed by the 4-1BB (CD137, aa 214-255, UniProt sequence ID Q07011) signaling domain and the CD3 zeta signaling domain (CD247, aa 52-163, Ref sequence ID: NP_000725.1). In some constructs, the CD28 costimulatory sequence was used instead of the 4-1BB costimulatory sequence. In some constructs, the CD8 binding and / or transmembrane domain was replaced with a domain from the TNFRSF19 protein. For some sequences, a truncated epidermal growth factor receptor (tEGFR) tag was incorporated into the CAR construct via the 2A peptide to enable tagging of transduced cells in vitro and as a suicide switch for in vivo applications. The CAR construct sequences were cloned into a third-generation lentiviral plasmid backbone (Lentigen Technology Inc., Gaithersburg, MD). Supernatants containing lentiviral vectors (LVs) were generated by transient transfection of HEK293T cells, and the supernatants containing lentiviral vectors were centrifuged to pellet the vectors and stored at -80°C.

[0296] (c) Primary T cell purification and transduction Human primary T cells from normal donors were transfected with CD4 T cells according to the manufacturer's protocol (Miltenyi Biotec, Bergisch Gladbach, Germany). + and CD8 + Cells were purified from the buffy coat after immunomagnetic bead selection and grown at densities of 0.3–2 × 10 6cells / ml, cultured in TexMACS medium supplemented with 40 IU / ml IL-2, activated with CD3 / CD28MACS® GMP TransAct reagent (Miltenyi Biotec), and transduced with a lentiviral vector encoding a CAR construct overnight in the presence of 10 μg / ml protamine sulfate (Sigma-Aldrich, St. Louis, MO) on day 2, with the medium changed on day 4. On day 3, cultures were transferred to TexMACS medium supplemented with 200 IU / ml IL-2 and propagated until harvest on days 7–10.

[0297] (d) Immune effector assays (CTL and cytokines) To determine cell-mediated cytotoxicity (CTL assay), 5,000 target cells stably transduced with firefly luciferase were combined with CAR T cells at various effector-to-target ratios and incubated overnight. SteadyGlo reagent (Promega, Madison, WI) was added to each well, and the resulting luminescence was quantified as counts per second (sample CPS). Target-only wells (maximum CPS) and target-only wells plus 1% Tween-20 (minimum CPS) were used to determine the assay range. The percent specific lysis was calculated as (1 - (sample CPS - minimum CPS) / (maximum CPS - minimum CPS)). Supernatants were removed from 10:1 E:T cocultures and analyzed for IFNγ, TNFα, and IL-2 concentrations by ELISA (eBioscience, San Diego, CA).

[0298] (e) Flow cytometry analysis of CAR surface expression For cell staining, 500,000 CAR T-transduced cells were harvested from culture and washed twice with cold AutoMACS buffer (Miltenyi Biotec) supplemented with 0.5% bovine serum albumin. CAR surface expression was detected by staining with CD33-Fc peptide (R&D, Minneapolis, MN) followed by anti-Fc-AF647 conjugate (Jackson ImmunoResearch, West Grove, PA). 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 on a MACSQuant® 10 Analyzer (Miltenyi Biotec), and data plots were generated using FlowJo software (Ashland, OR).

[0299] (f) In vivo analysis of CAR T function The functionality of CD33-targeted CAR T cells was assessed in vivo. On day 0, 1.0 × 10 CD33-targeted CAR T cells were injected into 6- to 8-week-old NSG mice, six mice per group. 6 MOLM-14 CD33 + Tumor burden was determined by IVIS bioluminescence imaging on day 4, and mice were randomized into groups with equal mean tumor burden on study day 5, with 5.0 × 10 6 CAR T + Cells / mouse were administered. Tumor regression was determined by bioluminescence imaging on days 14, 21, 28, and 35. Mouse survival was recorded and analyzed at the end of the study. Blood was collected from all animals on study day 19 to determine the presence of CAR T and tumor cells. Absolute numbers of blood CAR T cells and MOLM-14 tumor cells were determined by flow cytometry, and inflammatory cytokine levels in plasma were measured using the MACSPlex Cytokine 12 Human Kit (Miltenyi Biotec) according to the manufacturer's protocol.

[0300] (g) Flow cytometry analysis of CAR T and tumor cells in mouse blood. For flow cytometry, 50 μl of blood was collected and analyzed for the number of CAR T and MOLM-14 tumor cells. First, red blood cells were lysed with Red Blood Cells Lysis Solution (Miltenyi Biotec) according to the manufacturer's instructions, and white blood cells were analyzed by human CD45 immunofluorescence. + , CD3 + The cells were stained with 7-AAD (BD Biosciences, San Jose, CA) and acquired using a MACSQiant10 flow cytometer (Miltenyi Biotec). MOLM-14 cells stably expressing the GFP reporter gene were detected in the B1 channel. 7-AAD-positive dead cells were excluded from the analysis. To facilitate direct quantification of human T cell and MOLM-14 numbers in blood, CountBright Absolute Counting Beads (ThermoFischer Scientific, Waltham, MA) were added to each sample before data acquisition, and the corresponding absolute cell counts were calculated according to the manufacturer's protocol.

[0301] (h) Long-term CAR T and tumor co-incubation assay CART cell lines expressing various anti-CD33 CAR constructs and controls were combined with tumor-targeting HL-60 cells at effector-to-target ratios ranging from 5:1 to 0.04:1 for 5 or 11 days. Negative control UTD (untransduced cells), T cells alone (E:T 1:0), and GFP-expressing T cells (1398) were included. At each time point, cells were stained with anti-human CD33 and CD3 antibodies and 7-AAD, and data were acquired on a MACSQuant10 flow cytometer. Forward and side scatter, singlets, and 7-AAD were analyzed to determine the percentage of viable CAR T cells and tumor cells in each condition. - , CD3 + or CD33 + Cells were gated for .

[0302] result: To evaluate the novel anti-CD33 fully human ScFv binding sequences, CAR constructs were designed incorporating the heavy chain-only binder sequences VH-2 or VH-4, or one of the ScFv sequences ScFv9, ScFv10, ScFv12, or ScFv15 as tumor antigen-binding domains. Each CAR design included a tumor-targeting domain followed by a linker and transmembrane domain from the human CD8 protein, a 4-1BB costimulatory domain, and a CD3 zeta signaling domain (Table 1 below). Construct LTG1940, which incorporated an ScFv binding domain derived from the My96 sequence, was used as a reference control or comparison.

[0303] [Table 1]

[0304] Anti-CD33 chimeric antigen receptor-transduced T cells demonstrate surface expression and cytolytic activity.

[0305] a) Surface expression of anti-CD33 CAR To evaluate the novel anti-CD33 CAR, lentiviral vectors (LV) encoding the CAR construct under the control of the human EF1a promoter were generated as described in Materials and Methods. Human primary T cells from two separate healthy donors were then transduced with the four lentiviral vectors encoding the CAR. Non-transduced cells (NT) from the same donor or GFP-transduced cells from the same donor served as negative controls.

[0306] On day 0 of culture, T cells were activated with TransAct T cell reagent (active binding of CD3 and CD28 antigens, Miltenyi Biotec, Inc.) in the presence of IL-2 as described in Materials and Methods. On day 10 of culture, expression of anti-CD33 CAR on the T cell surface was detected with CD33-Fc peptide followed by anti-Fc-AF647 and analyzed by flow cytometry. The anti-CD33 CAR construct demonstrated surface CAR expression.

[0307] b) Cytolytic assay of anti-CD33 CAR To demonstrate the cytolytic function of the generated CAR T cells, luciferase-based killing assays were performed using the HL-60-luc, MOLM-14 (CD33 high), Reh-luc, and K562-luc (CD33 low) leukemia lines stably expressing firefly luciferase. CAR T cells and target cells were combined at effector-to-target (E:T) ratios of 20, 10, and 5, co-incubated overnight, and cell killing was assessed by luminescence as described in Materials and Methods (Figures 3, 4; 6, and 7). When VH-based anti-CD33 CARs were tested, the CAR T construct LTG1906 demonstrated strong E:T ratio-dependent cytotoxicity against the CD33-high HL-60-luc line, moderate cytolysis against the K562 line, which expresses less CD33, and only weak cytolytic activity against the CD33-low Reh-luc line. Therefore, cytolytic activity was directly related to the CD33 expression level of each leukemia. Furthermore, the negative control GFP construct LTG1398, and NT (untransduced T cells from the same donor) were not cytolytic, demonstrating that cytotoxicity was CART-dependent. Notably, the LTG1905 CAR construct was not cytolytic in the HL-60luc line and only weakly cytolytic in the K562-luc line.

[0308] Similarly, construct LTG1906 produced high levels of IFNγ, TNFα, and IL-2 in response to the highly CD33-positive tumor lines THP-1 and HL-60, but when exposed to the leukemia lines K562 or Reh, which express low levels of CD33 antigen, the cytokines secreted from the CAR T remained low (Figure 5). Interestingly, construct LTG1905 produced very high levels of IFNγ, TNFα, and IL-2 as detected by ELISA, despite inefficient in vitro killing of CD33-positive HL-60 leukemia. Thus, CAR design and binder selection are not trivial, as some binders are active in soluble IgG or ScFv formats and suitable for expression on the T cell surface in a CAR T format, yet are inefficient at killing CD33-positive tumors.

[0309] In comparison, when ScFv anti-CD33 CAR T cells were tested, constructs LTG1936 and LTG1939 demonstrated potent killing activity against the CD33-high tumor lines HL-60 and MOLM-14, while activity was much less pronounced against the CD33-low Reh tumor line and virtually undetectable against the CD33-low K562 cells (Figure 7). Surprisingly and unexpectedly, CAR constructs LTG1937 and LTG1938 were inefficient at lysing CD33-positive tumor targets. This again demonstrates that designing CAR T constructs based on antibody fragments is not trivial, as the binding and / or solubility and / or multimerization properties of soluble antibodies do not directly translate to CAR functionality. Similar to the VH-only construct 1906, the scFv-based constructs 1936, 1939, and the My96 scFv-based comparison construct 1940 all exhibited highly CD33-positive tumor cell death. + When exposed to the tumor lines HL-60 and MOLM14, the cells produced high levels of IFN-gamma and TNF-alpha, but CD33 低When CAR cells were incubated alone in the presence of the Reh strain or in the absence of the target strain, they showed virtually no cytokine induction. CAR constructs 1937 and 1938, which exhibited poor in vitro killing function, were also inefficient in cytokine production in response to tumor cells (data not shown). Comparing cytokine induction by MOLM14 and HL60, MOLM14 exhibited a higher CD33 antigen density (30,000 sites per cell for MOLM14 vs. 25,000 sites per cell for HL-60; data not shown), corresponding to the higher induction of IFN-gamma and TNF-alpha elicited by MOLM14 for all anti-CD33 constructs tested. Again, this demonstrates the antigen-specific nature of anti-CD33 CAR activation. Unexpectedly, IL-2 induction was strong with CAR constructs 1906 and 1940, but moderate with CAR constructs 1936 and 1939.

[0310] CAR T cells incorporating the various constructs were then transfected with HL-60 CD33 +Long-term co-incubation assays were performed by combining CAR constructs with tumor cells at E:T ratios ranging from 5:1 to 0.04:1. UTD, untransduced T cells, 1398, GFP-transduced T cells, and T cells alone at E:T ratios of 0.1 were used as assay controls. Cells were co-cultured for either 5 days (data not shown) or 11 days, and similar trends in HL-60 elimination were demonstrated for each CAR construct at both times (Figure 9). Negative control groups, UTD and 1398, resulted in tumor cell proliferation and T cell elimination, demonstrating the need for CAR-mediated T cell stimulation for cytolytic activity and extended CAR T cell survival. CAR construct 1398, which did not perform well in overnight in vitro assays, was also ineffective at killing HL-60 cells in this long-term assay. At all E:T ratios below 1:1, CAR T cells were eliminated from the culture and tumors persisted, similar to the negative control group. In contrast, anti-CD33 CAR constructs 1906, 1936, and 1939 were equally potent in CTL function as comparator construct 1940, successfully eliminating HL-60 tumor cells at E:T ratios as low as 0.2:1 (Figure 9). Constructs 1906, 1936, 1939, and 1940 were therefore selected for further evaluation in an in vivo model of AML.

[0311] To facilitate in vivo comparison of anti-CD33 CAR constructs, we utilized a xenograft mouse model as described in Materials and Methods. Briefly, NSG mice were inoculated with MOLM-14 cells stably expressing firefly luciferase and GFP on day 0 and administered 5 million CAR T cells per mouse on study day 5. Tumor growth kinetics was measured by IVIS bioluminescence imaging on study days 14, 21, 28, and 35, and CAR T function was assessed in mouse blood on study day 19.

[0312] As shown in Figure 10A, mice implanted with MOLM-14 tumors and left untreated (TA) or administered an untransduced T cell control (UTD) succumbed to disease by study day 14. CAR constructs 1936 and 1939 demonstrated partial efficacy and delayed tumor growth and prolonged survival. Strikingly, CAR construct 1906 and comparator construct 1940 mediated MOLM-14 tumor rejection, with all animals in these groups surviving until the end of the study on day 39 (Figures 10A and 10B).

[0313] Blood was collected from each animal on study day 19 to assess blood CAR T cell levels, blood MOLM-14 tumor cell levels, and blood cytokine levels secreted by CAR T for each treatment group. Absolute numbers of CAR T and tumor cells were measured in blood samples by flow cytometry (Figure 11A, left panel). There were no significant differences in the number of CAR T cells in each group, although CAR T cells expressing construct 1906 tended to be higher than the other groups, followed by CAR comparator construct 1940. Interestingly, even the UTD control group, comprised of untransduced T cells, showed a significantly higher number of CAR T cells (8.0 x 10), likely due to the initially higher number of cells infused into this group (8.0 x 10). 6 The T cell levels were high (cells / mouse). Notably, we detected a statistically significant reduction in the number of circulating blood MOLM-14 tumor cells in all CAR T groups compared with the UTD control (Figure 11A, right panel). Furthermore, when comparing the CAR T groups with each other, CAR1906 and 1940 stimulated the strongest MOLM-14 cell reduction, significantly greater than CAR1936 and 1939. Thus, CAR1906 and 1940 were the most efficient in controlling MOLM-14 levels in the blood, followed by CAR1936 and CAR1939.

[0314] Measurable levels of the inflammatory cytokines GM-CSF, IFN-gamma, and IL-2 were detected in mice dosed with CAR T cells or the UTD control. The differences between the levels of these cytokines were not significant, but the plasma GM-CSF and IL-2 levels for construct 1906 were significantly higher. IL-2 levels tended to be higher with CAR1906 and CAR constructs 1940 and 1936 (Figure 11B). These results highlight the elevated secretion of inflammatory cytokines by activated CAR T cells. CAR1906 and 1940, which were most effective at tumor rejection, also tended to secrete greater levels of cytokines, although no significant differences were detected between the experimental groups, likely because the CAR T cells had already passed maximal activation on study day 19 (notably, differences in tumor burden were detected as early as study day 14, Figure 10A).

[0315] In summary, the high functionality of the novel fully human anti-CD33 CAR construct LTG1906 and the partial functionality of constructs LTG1936 and LTG1939 (Table 2 below) were demonstrated in vitro and in vivo. It is conceivable that the functionality of constructs LTG1936 and LTG1939 could be further improved by redesigning the CAR spacer, linker, or costimulatory domain to better target specific epitopes or to increase the level of CAR response to tumor epitope binding. The VH-based anti-CD33 CAR construct LTG1905 had low cytolytic efficacy despite detectable surface expression by flow cytometry and high cytokine secretion. The ScFv-based CAR T constructs LTG1937 and LTG1938 were also inefficient at lysing target cell lines in vitro despite being highly expressed.

[0316] Example 3 By altering the structure of anti-CD33 CARs expressing fully human heavy chains alone or ScFv-based binding sequences, improved functional properties of the CD33 CAR moiety can be achieved. This example describes various structural configurations of anti-CD33 CAR T cells derived from novel fully human immunoglobulin heavy chain alone or scFv binder sequences.

[0317] By incorporating a single CD28-derived versus CD137 / 4-1BB-derived costimulatory domain in-frame into the CAR structure (second-generation CARs), or by incorporating multiple costimulatory domains in tandem (third-generation CARs), along with an activation domain such as CD3 zeta lacking the costimulatory domain (first-generation CARs), CAR T cells are expected to secrete greater or lesser levels of inflammatory cytokines such as IL-2, IFN-gamma, and TNF-alpha upon exposure to antigen-expressing tumor cells.

[0318] In some constructs, incorporation of novel hinge and transmembrane domains, such as those derived from the human TNFRSF19 sequence, may confer enhanced efficacy on CARs in tumor cell killing and cytokine response.

[0319] Furthermore, altering the length and composition of the CAR hinge or linker domain, such as by replacing the CD8 alpha-derived linker domain with TNFRSSF19-derived domains of various lengths, or domains derived from immunoglobulin constant regions and / or hinges, such as IgG1- or IgG4-derived linker domains incorporating the CH2, and / or CH3, and / or hinge domains of immunoglobulin molecules, or modifications thereof, may allow better access of tumor antigens to the CAR binding domain, since appropriate length and flexibility of the CAR hinge / linker domain is required for optimal accessibility, tumor antigen binding, and CAR T-cell activation.

[0320] Furthermore, incorporating a tag molecule into the CAR construct sequence expressed on the surface of CAR T cells can 1) facilitate the detection of CAR T cells by flow cytometry during manufacturing and clinical applications; 1) for identification, 2) for sorting / isolation of CAR T cells during manufacturing, and 3) as a suicide tag to eliminate CAR T cells from the patient's body in the event of CAR-associated toxicity, such as B-cell aplasia, cytokine release syndrome, or CAR-associated neurotoxicity in response to anti-CD19 CARs. To this end, the CAR construct sequence may include a truncated ectodomain and transmembrane portion of a native transmembrane protein, such as HER1 / EGFR, HER2 / Neu / erbB-2, NGFR / LNGFR / CD271, CD19, CD20, or other protein. Mimotopes of these or other sequences can also be used. Removal of tagged CAR T cells from the patient's circulation should be achieved by administration of a tag-reactive clinical-grade antibody, such as an antibody targeting EGFR (cetuximab), HER2 (trastuzumab), CD20 (rituximab), or other protein.

[0321] Selected examples of the above-referenced CAR configurations are shown in Figures 12A-12F, respectively. The CD33-binding sequence from the immunoglobulin VH domain CD33_4 was used to design the anti-CD33 CAR constructs shown, although binder sequences in ScFv format can also be used.

[0322] material and method: (a) Creation of chimeric antigen receptor (CAR) - expression vector The antigen-binding domain sequence of the CAR was derived from a human anti-CD33 ScFv or heavy chain variable fragment. CAR T constructs were generated by linking the binder sequence in frame to the CD8a binding and transmembrane domain (UniProt sequence ID P01732, aa 138-206), followed by the 4-1BB (CD137, aa 214-255, UniProt sequence ID Q07011) signaling domain and the CD3 zeta signaling domain (CD247, aa 52-163, Ref sequence ID: NP_000725.1). In some constructs, the CD28 costimulatory sequence (UniProt ID: P10747, transmembrane domain, aa 153-179) was used instead of the 4-1BB costimulatory sequence. In some constructs, the CD8 binding and / or transmembrane domain was replaced with domains of various lengths from the TNFRSF19 protein (UniProt ID: Q9NS68). In some sequences, a truncated epidermal growth factor receptor (tEGFR) tag (UniProt ID: P00533, various sequences) was incorporated into the CAR construct via the 2A peptide to enable tagging of transduced cells in vitro and as a suicide switch for in vivo applications. CAR construct sequences were cloned into a third-generation lentiviral plasmid backbone (Lentigen Technology Inc., Gaithersburg, MD). Supernatants containing lentiviral vectors (LVs) were generated by transient transfection of HEK293T cells, and the vectors were pelleted by centrifugation and stored at -80°C.

[0323] [Table 2]

[0324] Each application and patent cited herein, and each document or reference cited therein (including each issued patent in litigation, the "Application Citations"), and each PCT and foreign application or patent corresponding to and / or claiming priority to any of these applications and patents, and each document cited or referenced in each Application Citation, are hereby expressly incorporated herein by reference and may be used in the practice of the invention. More generally, documents or references are cited either in the text, in a reference list before the claims, or in the text itself, and each such document or reference (the "In-Herein Cited References"), and each document or reference cited in each In-Herein Cited Reference (including any manufacturer's specifications, instructions, etc.), is hereby expressly incorporated herein by reference.

[0325] The foregoing description of some specific embodiments provides sufficient information to enable others, by applying knowledge of the present invention, to easily modify or adapt the specific embodiments for various applications without departing from the general concept, and it is therefore intended that such adaptations and modifications be understood as being within the meaning and range of equivalents of the disclosed embodiments. It is understood that the scope of the appended claims is for the purpose of clarity. In the drawings and description, illustrative embodiments have been disclosed, and although specific terminology may have been employed, unless otherwise stated, it is used in a generic and descriptive sense only and not for purposes of limitation, and therefore the scope of the claims is not so limited. Moreover, those skilled in the art will recognize that certain steps of the methods discussed herein can be sequenced in an alternate order or steps can be combined. Accordingly, it is intended that the appended claims not be limited to the precise embodiments disclosed herein. Those skilled in the art will be able to recognize, or ascertain using no more than routine experimentation, many equivalents to the embodiments of the invention described herein. Such equivalents are encompassed by the scope of the following claims.

[0326] Reference to sequence listing This application contains a Sequence Listing that has been submitted electronically to the United States Patent and Trademark Office in a PDF file titled "Sequence Listing." The Sequence Listing is incorporated by reference.

[0327] Sequences of the present disclosure The nucleic acid and amino acid sequences listed below are shown using standard abbreviations for nucleotide bases and three-letter codes for amino acids as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the represented strand. In the accompanying sequence listing: SEQ ID NO: 1 Nucleotide sequence of CD33-reactive immunoglobulin heavy chain variable domain (VH-2) gaggtgcagctggtggagtctgggggaggcttggtacagcctggagggtccctgagactctcctgtgcagcctctggattcaccttcagtagctatggcatgagctgggtccgccaggctccaaggaagggcctggagtggattgggaaatcaatcatagtggaagcaccaactaca acccgtccctcaagagtcgagtcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacagccacgtattactgtgcgagacccctcaactactactactactacatggacgtctggggcaaagggaccacggtcaccgtctcctca SEQ ID NO: 2: Amino acid sequence of CD33-reactive immunoglobulin heavy chain variable domain (VH-2) EVQLVESGGGLVQPGGSLRLS CAASGFTFSSYGMSWVRQAPR KGLEWIGEINHSGSTNYNPSL KSRVTISRDNSKNTLYLQMNS LRAEDTATYYCARPLNYYYYY MDVWGKGTTVTVSS Gateway 3 CD33 is the core of the VH-4 (VH-4). gaggtgcagctggtggagtctgggggaggcttggtacagcctggaggggtccctgagactctcctgtgcagcctctggattcaccttcagtagctatggcatgagctgggtccgccaggctccaagacaagggcttgagtgggtggccaacataaagcaagatggaagtg agaaatactatgcggactcagtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacagccacgtattactgtgcgaaagaaatgtggactggggccagggcaccctggtcaccgtctcctca Gateway 4 CD33 insert mounting bracket of the VH-4 (VH-4). EVQLVESGGGLVQPGGSLRLS CAASGFTFSSYGMSWVRQAPR QGLEWVANIKQDGSEKYYADS VKGRFTISRDNSKNTLYLQMN SLRAEDTATYYCAKENVDWGQ GTLVTVSS Differential strain 5 CD33-induced ScFv9-induced mitochondrial fibroblasts caggtgcagctggtgcaatctggggcagaggtgaaaaagcccggggagtctcgaggatctcctgtaagggttctggattcagttttcccacctactggatcggctgggtgcgccagatg cccgggaaagcctggagtggatggggatcatctatcctgctctgataccagatacagcccgtcctccaaggccaggtcaccatctcagccgacaagtccatcagcaccgcctac ctgcagtggagcagcctgaaggcctcggacaccgccatgtattactgtcgagactagttggagatggctacaatacgggggctttgatatctggggccaagggacaatggtcaccgtc tcttcaggaggtggcgggtctggtgggcggtagcggtgcggatcgatattgtgatgacccactccactctctctctctctcgtcacccctggacagccggcctccatctctgc aagtctagtcagagcctcctgcatagtaatggaaagacctatttgtattggtacctgcagaagccagcctccagctcctgatctatggagcttccaaccggttcttggagtgccagacaggttca gtggcagcgggtcagggacagatttcacactgaaaatcagccgggtggaggctgaggatgttggggttactgcatgcaaagtatacagcttcctatcaccttcGgccaagggacacgactggagattaaa The 6-CD33-ScFv9-ScFv9-ScFv9-ScFv9-ScFv9 QVQLVQSGAEVKKPGESLRIS CKGSGFSFPTYWIGWVRQMPG KGLEWMGIIYPGDSDTRYSPS FQGQVTISADKSISTAYLQ WSSLKASDTAMYYCARLVGDG YNTGAF DIWGQGTMVTVSSGGGGSGGG GSGGGG SDIVMTHTPLSLSVTPGQPAS ISCKSS QSLLHSNGKTYLYWYLQKPGQ PPQLLI YGASNRFSGVPDRFSGSGSGT DFTLKI SRVEAEDVGVYYCMQSIQLPI TFGQGTRLEIK SEQ ID NO: 7 Nucleotide sequence of CD33-reactive ScFv10 binding domain caggtacagctgcagcagtcaggctggctggaagccctcgcagccctctagcacactgctgcttggaactggatcaggcagtccccatcgagaggccttgagtggctggaaggacatactacaggtccagctgtat aatgattatgcagtccctgtgaaaagtcgaataaccatcaacccagacacatccaagaaccagttctccctgcagctgaactctgtgactcccgaggacacggctgtgtactgtgcaagagaaacgtattacttatggttcg gggagttattgggatgcttttgatatctggggccaagggaccacggtcaccgtctctccaggaggtggcgggtctggtaggtagcggtgggcggatcccagtctgtcgtgacgcgcccctcagtgtctgcggccc caggacagaaggtcaccatctcctgctctggaagcagctccacattgggaataattatgtatcctggtaccagcagctcccaggcacggcccccaactcttcatctataaaaatcagggccctcagaggtccctgaccg attctctggctccaagtctggcacctcagccctccctggccatgggctccagtctgacgatgaggctgactactactgtgcagcatgggatgacaggctgaatggatatgtctcggaactgggaccaaggtcaccgtccta The ScFv10 ScFv10 CD33-800000000000000000020000000000000000000000000000000000800008000000001000000000000000000000000000000008 QVQLQQSGPGLVKPSQTLSLT CAISGDSVSSNTAAWNWIRQS PSRGLEWLGRTYYRSKWYNDY AVPVKSRITINPDTSKNQF S L Q L N S V T P E D T A V Y Y C A R E T Y Y Y G S G S Y W D A F D I W G Q G T T V T V S S G G G G S G S G G S G G G G S Q S V V T Q P P S V S A A P G Q K V T I S C S G S S S N I G N N Y V S W Y Q Q L P G T A P K L F I Y K N N Q R P S E V P D R F S G S K S G T S A S L A I S G L Q S D D E A D Y Y C A A W D D R L N G Y V F G T G T K V T V L Nucleotide sequence of the array number 9 CD33-reactive ScFv12-binding domain caggtacagctgcagcagtcaggctggtgaagccctcgcagaccctcctcactgctgctcgggacagtctctctagcaacgtgctgcttggaactggatcaggcagtccccatcgagaggccttgagtgctgggggcatactacaggtccagtggcctga ttatgcagtatctgtgaaaagtcgaataattatcaacgcagacacatcgaagaaccagttctccctgcagctgaactctgtgactcccgagacacggctgtattactgtgcgaggggatacttatgatagtaccgactggttcgacccctggccagggaaccctggtcacgtcctcct caggaggtggcgggtctggtggtgcggtagcgggtggcggatctcttctgagctgactcgacccaactgtgtctgtggccttgggacagacagtcacatgcaaggacagcctcagaagctattatgcaagctggtaccagcagcagacaggcggcctgtactctc atctatggtaaaaaccggccctcagggatcccagaccgattctctgctccagctcaggaaacacagctttgaccatcactggggctcaggcggaagatgactacttactgttcctcccggcggcagtggtcatcatctcttcggacctgggaccaggttcaccgt ScFv12 CD33 10000000000000000000000000000000000000000000 QVQLQQSGPGLVKPSQTLSLT CAISGD SVSSNSAAWNWIRQSPSRGLE WLGGTYRSKWYNDYAVSVKS RIIINADTSKNQF SLQLNSVTPEDTAVYYCARGY YYDSTD WFDPWGQGTLVTVSSGGGGSG GGGSGG GGSSSELTQDPTVSVALGQTV RITCQG DSLRSYYASWYQQKPGQAPVL VIYGKN NRPSGIPDRFSGSSSGNTASL TITGAQ AEDEADYYCSSRDGGSGHPYLF GPGTKVTVL SEQ ID NO: 11 Nucleotide sequence of CD33-reactive ScFv15 binding domain gaggtccagctggtgcagtctggagcagaggtgaaaaagcccggggagtctctgaagatctcctgtaagggttctggatacagctttaccagctactggatcggctgggtgcgccagatgcccgggaaaggcctggagtggatggggatcatctatcctggtgactctgataccagatacagcccgtccttccaaggccaggtcaccatctcagccgacaagtccatcagcaccgcctacctgcagtggagcagcctgaaggcctcggacaccgccatgtattactgtgcgagactgactacggctgggggtatggacgtctggggccaagggaccacggtcaccgtctcctcaggaggtggcgggtctggtggtggcggtagcggtggtggcggatccgaaattgtgctgactcagtctccactctccctgcccgtcacccttggacagccggcctccatctcctgcaggtctagtcaaagcctcgtacacagtgatggaaacacctacttgagttggcttcaccagaggccaggccagcctccaagactcctaatgtataagatttctaaccggttctctggggtcacagacagattcagtggcagcgggtcagggacagatttcacactgaaaatcagccgggtggaggctgaggatgttggggtttattactgcatgcaaggtatacacctaccgctcactttcggcggagggaccAagctggagatcaaa Amino acid sequence of the CD33-reactive ScFv15-binding domain, SEQ ID NO: 12 EVQLVQSGAEVKKPGESLKIS CKGSGYSFTSYWIGWVRQMPG KGLEWMGIIYPGDSDTRYSPS FQGQVTISADKSISTAYLQWS SLKASDTAMYYCARLTTAGGM DVWGQGTTVTVSSGGGGSGGG GSGGGGSEIVLTQSPLSLPVT LGQPASISCRSSQSLVHSDGN TYLSWLHQRPGQPPRLLMYKI SNRFSGVTDRFSGSGSGTDFT LKISRVEAEDVGVYYCMQGIH LPLTFGGGTKLEIK SEQ ID NO: 13 Nucleotide sequence of leader / signal peptide sequence atgctgctgctggtgaccagcctgctgctgtgcgaactgccgcatccggcgtttctgctgattccg SEQ ID NO: 14: Amino acid sequence of leader / signal peptide sequence MLLLVTSLLLCELPHPAFLLIP SEQ ID NO: 15 Nucleotide sequence of LTG1905_(EF1a- VH-2 CD33-CD8 TM-41BB-CD3 zeta) SEQ ID NO: 16: Amino acid sequence of LTG1905_(EF1a- VH-2 CD33 -CD8 TM-41BB-CD3 zeta) MLLLVTSLLLCELPHPAFLLIPEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPRKGLEWIGEINHSGSTNYNPSLKSRVTISRDNSKNTLYLQMNSLRAEDTATYYCARPLNYYYYYMDVWGKGTTVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRG LDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 17 Nucleotide sequence of LTG1906 (EF1a- VH-4 CD33 -CD8 TM-41BB-CD3 zeta) nucleic acid sequence SEQ ID NO: 18: Amino acid sequence of LTG1906 (EF1a-VH-4 CD33 -CD8 TM-41BB-CD3 zeta) MLLLVTSLLLCELPHPAFLLIPEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPRQGLEWVANIKQDGSEKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTATYYCAKENVDWGQGTLVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDF ACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 19 Nucleotide sequence of LTG1936_(EF1a_ScFv9 CD33 CD8 TM-41BB-CD3 Zeta CAR) SEQ ID NO: 20 LTG1936_(EF1a_ScFv9 CD33 CD8 TM- Amino acid sequence of 41BB-CD3 zeta MLLLVTSLLLCELPHPAFLLIPQVQLVQSGAEVKPGESLRISCKGSGFSFPTYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARLVGD GYNTGAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIVMTHTPLSLSVTPGQPASISCKSSQSLLHSNGKTYLYWYLQKPGQPPQLLIYGASNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVG VYYCMQSIQLPITFGQGTRLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCS CRFPEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 21 Nucleotide sequence of LTG1937_(EF1a_ScFv10 CD33 CD8 TM-41BB-CD3 Zeta CAR) SEQ ID NO: 22 Amino acid sequence of LTG1937_(EF1a_ScFv10 CD33 CD8 TM-41BB-CD3 zeta) MLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNTAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVPVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARET YYYGSGSYWDAFDIWGQGTTVTVSSGGGGGSGGSGGGGSQSVVTQPPSVSAAPGQKVTISSCSGSSSNIGNNYVSWYQQLPGTAPKLFIYKNNQRPSEVPDRFSGKSGTSASLAISGLQSDDEA DYYCAAWDDRLNGYVFGTGTKVTVLAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGC SCRFPEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 23 Nucleotide sequence of LTG1938_(EF1a_ScFv12 CD33 CD8 TM-41BB-CD3 Zeta) ATGCTGCTGCTGGTGACCAGCCTGCTGCTGTGCGAACTGCCGCATCCGGCGTTTCTGCTGATTCCGCAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAGGGACATACTACAGGTCCAAGTGGTATAATGATTATGCAGTATCTGTGAAAAGTCGAATAATTATCAACGCAGACACATCGAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTACTGTGCGAGGGGATATTACTATGATAGTACCGACTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGAGGTGGCGGGTCTGGTGGTGGCGGTAGCGGTGGTGGCGGATCCTCTTCTGAGCTGACTCAGGACCCAACTGTGTCTGTGGCCTTGGGACAGACAGTCAGGATCACATGCCAAGGAGACAGCCTCAGAAGCTATTATGCAAGCTGGTACCAGCAGAAGCCAGGACAGGCCCCTGTACTTGTCATCTATGGTAAAAACAACCGGCCCTCAGGGATCCCAGACCGATTCTCTGGCTCCAGCTCAGGAAACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGACTATTACTGTTCCTCCCGGGACGGCAGTGGTCATCCATATCTCTTCGGACCTGGGACCAAGGTCAC CGTTCTTGCGGCCGCAACTACCACCCCTGCCCCTCGGCCGCCGACTCCGGCCCCAACCATCGCAAGCCAACCCCTCTCCTTGCGCCCCGAAGCTTGCCGCCCGGCCGCGGGTGGAGCCGTGCATACCCGGGGGCTGGACTTTGCCTGCGATATCTACATTTGGGCCCCGCTGGCCGGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACCCTTTACTGCAAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAGCCGTTCATGCGGCCCGTGCAGACGACTCAGGAAGAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGAGGGGGGATGCGAACTGCGCGTCAAGTTCTCACGGTCCGCCGACGCCCCCGCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGG Amino acid sequence of SEQ ID NO: 24, LTG1938_ (EF1a_ScFv12 CD33 CD8 TM-41BB-CD3 zeta) MLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGGTYYRSKWYNDYAVSVKSRIIINADTSKNQFSLQLNSVTPEDTAVYYCAR GYYYDSTDWFDPWGQGTLVTVSSGGGGSGGGGSGGGGSSSELTQDPTVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYY CSSRDGSGHPYLFGPGTKVTVLAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 25 Nucleotide sequence of LTG1939_(EF1a_ScFv15 CD33 CD8 TM-41BB-CD3 Zeta) SEQ ID NO: 26: Amino acid sequence of LTG1939_(EF1a_ScFv15 CD33 CD8 TM-41BB-CD3 zeta) MLLLVTSLLLCELPHPAFLLIPEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARLTT AGGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPLSLPVTLGQPASISCRSSQSLVHSDGNTYLSWLHQRPGQPPRLLMYKISNRFSGVTDRFSGSGSGTDFTLKISRVEAEEDVGVY YCMQGIHLPLTFGGGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 27 DNA CD8 transmembrane domain nucleotide sequence atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc accctttact gc SEQ ID NO: 28: Amino acid sequence of CD8 transmembrane domain Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys SEQ ID NO: 29 DNA CD8 hinge domain nucleotide sequence accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg gacttcgcct gtgat SEQ ID NO: 30 Amino acid sequence of CD8 hinge domain Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr SEQ ID NO: 31: Amino acid sequence of amino acids 118 to 178 of the hinge region of 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 SEQ ID NO: 32 Amino acid sequence of human IgG CL sequence Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala Pro Thr Glu Cys Ser SEQ ID NO: 33 Nucleotide sequence of the DNA signaling domain of 4-1BB aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggagaggtgt gaactg SEQ ID NO: 34: Amino acid sequence of the signaling domain of 4-1BB Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu SEQ ID NO: 35 Nucleotide sequence of the DNA signaling domain of CD3-zeta agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat gaactgcaga aagatagat ggcggaggcc tacagtgaga ttgggatga aggcgagcgc cggaggggca aggggcacga tggctttac cagggtctca gtacagccac cggacacc tacgacgccc ttcacatgca ggccctgccc cctcgc CD3 Photo Information Photo Information Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp With Leu His Met Gln Ala Leu Pro Pro Arg 37 Scvf cd19's cover photo gataccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcaccatcagttgca gggcaagtca ggacattagt aaatatta attggtatca gcagaaacca gatggactg ttaaactcct gatctac cat acatcaagat tacactcagg agtcccatca aggttcagtg gcagtgggtc tggaacagat tattctctca 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 ScvF cd19 Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser SEQ ID NO: 39 Nucleotide sequence of GMCSF leader peptide ATGCTGCTGCTGGTGACCAGCCTGCTGCTGTGCGAACTGCCGCATCCGGCGTTTCTGCTG ATTCCG SEQ ID NO: 40 Amino acid sequence of GMCSF leader peptide MLLLVTSLLLCELPHPAFLLIP SEQ ID NO: 41 Nucleotide sequence of TNFRSF19 leader peptide GGCTCTGAAAGTGCTGTTGGAACAAGAAAAGACCTTCTTCACCTTGCTCGTGTGCTGGG GTACCTGTCCTGCAAAGTCACCTGT SEQ ID NO: 42 Amino acid sequence of TNFRSF19 leader peptide MALKVLLEQEKTFFTLLVLLGYLSCKVTC SEQ ID NO: 43 Nucleotide sequence of CD8 alpha leader peptide atggcgctgccggtgaccgcgctgctgctgccgctggcgctgctgctgcatgcggcgcgc ccg SEQ ID NO: 44: Amino acid sequence of CD8 alpha leader peptide MALPVTALLLPLALLLHAARP SEQ ID NO: 45 Nucleotide sequence of CD28 costimulatory domain CGGTCGAAGAGGTCCAGACTCTTGCACTCCGACTACATGAACATGACTCCTAGAAGGCCCGGACCCACTAGAAAGCACTACCAGCCGTACGCCCCTCCTCGGGATTTCGCCGCATACCGG TCC SEQ ID NO: 46: Amino acid sequence of CD28 costimulatory domain RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS SEQ ID NO: 47 Nucleotide sequence of CD3 zeta activation domain AGAGTGAAGTTCAGCCGCTCAGCCGATGCACCGGCCTACCAGCAGGGACAGAACCAGCTCTACAACGAGCTCAACCTGGGTCGGCGGGAAGAATATGACGTGCTGGACAAACGGCGCGGCAGAGATCCGGAGATGGGGGGAAAGCCGAGGAGGAAGAACCCTCAAGAG GGCCTGTACAACGAACTGCAGAAGGACAAGATGGCGGAAGCCTACTCCGAGATCGGCATGAAGGGAGAACGCCGGAGAGGGAAGGGTCATGACGGACTGTACCAGGGCCTGTCAACTGCCACTAAGGACACTTACGATGCGCTCCATATGCAAGCTTTGCCCCCGCGG SEQ ID NO: 48 Amino acid sequence of CD3 zeta activation domain RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGER RRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 49 Nucleotide sequence of TNFRSF19 hinge and transmembrane domain (transmembrane domain underlined) GCGGCCGCGGTCGGATTCCAAGACATGGAATGCGTGCCCTGCGGCGACCCGCCACCTCCT TACGAGCCGCACTGCGCATCGAAGGTCAACCTCGTGAAGATCGCGAGCACCGCGTCCTCA CCCCGGGATACTGCTCTG GCCGCCGTGATTTGTTCCGCCTTGGCCACCGTGCTTCTGGCC CTGCTGATCCTCTGTGTGATC SEQ ID NO: 50 Amino acid sequence of TNFRSF19 hinge and transmembrane domain (transmembrane domain 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 SEQ ID NO: 51 nucleotide sequence of TNFRSF19 transmembrane domain GCCGCCGTGATTTGTTCCGCCTTGGCCACCGTGCTTCTGGCCCTGCTGATCCTCTGTGTG ATC SEQ ID NO: 52 Amino acid sequence of TNFRSF19 transmembrane domain AAVICSALATVLLALLILCVI SEQ ID NO: 53 Nucleotide sequence of TNFRSF19 hinge domain GCGGCCGCGGTCGGATTCCAAGACATGGAATGCGTGCCCTGCGGCGACCCGCCACCTCCT TACGAGCCGCACTGCGCATCGAAGGTCAACCTCGTGAAGATCGCGAGCACCGCGTCCTCA CCCCGGGATACTGCTCTG SEQ ID NO: 54: Amino acid sequence of TNFRSF19 hinge domain AAAVGFQDMECVPCGDPPPPY EPHCASKVNLVKIASTASSPR DTAL SEQ ID NO: 55 Nucleotide sequence of truncated TNFRSF19 hinge domain TACGAGCCTCACTGCGCCAGCAAAGTCAACTTGGTGAAGATCGCGAGCACTGCCTCGTCC CCTCGGGACACTGCTCTGGC SEQ ID NO: 56: Amino acid sequence of truncated TNFRSF19 hinge domain YEPHCASKVNLVKIASTASSP RDTAL SEQ ID NO: 57 Nucleotide sequence of CD8a hinge domain fused to TNFRSF19 transmembrane domain (transmembrane sequence underlined) GCGGCCGCGCCCGCCCCTCGGCCCCCGACTCCTGCCCCGACGATGCTTCCCAACCTCTC TCGCTGCGCCCGGAAGCATGCCGGCCCGCCGCCGGTGGCGCTGTCCACACTCGCGGACTG GACTTTGATACCGCACTG GCGGCCGTGATCTGTAGCGCCCTGGCCACCGTGCTGCTGGCG CTGCTCATCCTTTGCGTGATCTACTGCAAGCGGCAGCCTAGG SEQ ID NO: 58 Amino acid sequence of the CD8a hinge domain fused to the TNFRSF19 transmembrane domain (transmembrane sequence 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 SEQ ID NO: 59 Nucleotide sequence of CD28 costimulatory domain CGGTCGAAGAGGTCCAGACTCTTGCACTCCGACTACATGAACATGACTCCTAGAAGGCCC GGACCCACTAGAAAGCACTACCAGCCGTACGCCCCTCCTCGGGATTTCGCCGCATACCGG TCC SEQ ID NO: 60 Amino acid sequence of CD28 costimulatory domain RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS SEQ ID NO: 61 Nucleotide sequence of CD3 zeta version 2 cgcgtgaaatttagccgcagcgcggatgcgccggcgtatcagcagggccagaaccagctg tataacgaactgaacctgggccgccgcgaagaatatgatgtgctggataaacgccgcggc cgcgatccggaaatgggcggcaaaccgcgccgcaaaaacccgcaggaaggcctgtataac gaactgcagaaagataaaatggcggaagcgtatagcgaaattggcatgaaaggcgaacgc cgccgcggcaaaggccatgatggcctgtatcagggcctgagcaccgcgaccaaagatacc tatgatgcgctgcatatgcaggcgctgccgccgcgc SEQ ID NO: 62 Amino acid sequence of CD3 zeta version 2 RVKFSRSADAPAYQQGQNQLY NELNLG RREEYDVLDKRRGRDPEMGGK PRRKNP QEGLYNELQKDKMAEAYSEIG MKGERRRGKGHDGLYQGLSTA TKDTYDALHMQALPPR SEQ ID NO: 63 Nucleotide sequence of furin P2A furin CGCGCGAAACGCAGCGGCAGCGGCGCGACCAACTTTAGCCTGCTGAAACAGGCGGGCGAT GTGGAAGAAAACCCGGGCCCGCGAGCAAAGAGG SEQ ID NO: 64 Amino acid sequence of furin P2A furin (furin sequence underlined) RAKR SGSGATNFSLLKQAGDVEENPGP RAKR SEQ ID NO: 65 Nucleotide sequence of furin T2A AGAGCTAAACGCTCTGGGTCTGGTGAAGGACGAGGTAGCCTTCTTACGTGCGGAGACGTGGAGGAAAACCCAGGACCC SEQ ID NO: 66 Amino acid sequence of furin T2A (furin sequence underlined) RAKR SGSGEGRGSLLTCGDVEENPGP Nucleotide sequence of the array number 67 shortened EGFR (tEGFR) tag AGGAAGGTTTGCAATGGAATCGGTATAGGGGAGTTTAAGGATTCACTTAGCATAAACGCT ACTAATATTAAACACTTCAAAAACTGTACGAGTATAAGTGGAGATCTTCACATTTTGCCG GTTGCATTCCGAGGCGATTCATTCACCCACACGCCACCGCTTGACCCACAAGAATTGGAT ATTCTTAAAACCGTTAAAGAAATAACGGGGTTTTTGCTCATTCAAGCGTGGCCAGAAAAT CGCACTGACCTCCATGCTTTCGAGAACCTGGAGATTATAAGAGGACGAACTAAGCAGCAT GGTCAATTCTCCCTTGCTGTGGTCAGCCTGAACATCACCAGTCTTGGTTTGCGGTCCCTC AAGGAAATTTCAGATGGAGATGTCATCATAAGCGGCAACAAGAATTTGTGCTATGCAAAT ACCATAAACTGGAAAAAACTGTTTGGCACTTCCGGCCAGAAAACCAAGATTATTTCAAAT CGGGGTGAGAACAGCTGCAAAGCCACCGGCCAGGTTTGTCATGCCTTGTGCTCTCCGGAA GGCTGTTGGGGGCCAGAACCCAGGGACTGCGTCAGTTGCAGAAACGTCTCAAGAGGCCGC GAATGCGTTGACAAGTGTAACCTCCTTGAGGGTGAGCCACGAGAGTTTGTTGAGAACAGC GAGTGTATACAATGTCACCCTGAATGTTTGCCCCAGGCTATGAATATAACCTGCACAGGC CGCGGGCCTGATAACTGCATCCAGTGTGCTCATTACATAGATGGACCTCACTGTGTGAAA ACCTGCCCGGCCGGAGTTATGGGAGAAAACAACACTCTGGTGTGGAAATACGCTGATGCA GGCCACGTGTGCCACCTTTGTCACCCGAATTGTACATATGGGTGTACCGGTCCTGGACTT GAAGGTTGCCCTACCAATGGCCCTAAAATACCCAGTATCGCAACTGGCATGGTAGGCGCT CTTCTCTTGCTCTTGGTAGTTGCTCTCGGCATAGGTCTTTTTATG SEQ ID NO: 68: Amino acid sequence of truncated EGFR (tEGFR) tag RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELD ILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSL KEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPE GCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTG RGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGL EGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM SEQ ID NO: 69 Nucleotide sequence of LTG1927 (EF1a-CD33_4-CD8 TM-CD28-CD3zeta-cfrag) ATGCTGCTGCTGGTGACCAGCCTGCTGCTGTGCGAACTGCCGCATCCGGCGTTTCTGCTG ATTCCGGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTG AGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGGCATGAGCTGGGTCCGC CAGGCTCCAAGACAAGGGCTTGAGTGGGTGGCCAACATAAAGCAAGATGGAAGTGAGAAA TACTATGCGGACTCAGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACG CTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACAGCCACGTATTACTGTGCGAAA GAAAATGTGGACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGCGGCCGCGACTACC ACTCCTGCACCACGGCCACCTACCCCAGCCCCCACCATTGCAAGCCAGCCACTTTCACTG CGCCCCGAAGCGTGTAGACCAGCTGCTGGAGGAGCCGTGCATACCCGAGGGCTGGACTTC GCCTGTGACATCTACATCTGGGCCCCATTGGCTGGAACTTGCGGCGTGCTGCTCTTGTCT CTGGTCATTACCCTGTACTGCCGGTCGAAGAGGTCCAGACTCTTGCACTCCGACTACATG AACATGACTCCTAGAAGGCCCGGACCCACTAGAAAGCACTACCAGCCGTACGCCCCTCCT CGGGATTTCGCCGCATACCGGTCCAGAGTGAAGTTCAGCCGCTCAGCCGATGCACCGGCC TACCAGCAGGGACAGAACCAGCTCTACAACGAGCTCAACCTGGGTCGGCGGGAAGAATAT GACGTGCTGGACAAACGGCGCGGCAGAGATCCGGAGATGGGGGGAAAGCCGAGGAGGAAG AACCCTCAAGAGGGCCTGTACAACGAACTGCAGAAGGACAAGATGGCGGAAGCCTACTCC GAGATCGGCATGAAGGGAGAACGCCGGAGAGGGAAGGGTCATGACGGACTGTACCAGGGC CTGTCAACTGCCACTAAGGACACTTACGATGCGCTCCATATGCAAGCTTTGCCCCCGCGG SEQ ID NO: 70 Amino acid sequence of LTG1927 (EF1a-CD33_4-CD8 TM-CD28-CD3zeta-cfrag) MLLLVTSLLLCELPHPAFLLIPEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVR QAPRQGLEWVANIKQDGSEKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTATYYCAK ENVDWGQGTLVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDF ACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPP RDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 71: Nucleotide sequence of LTG_D0033 (Ef1a_CD33_4 VH TNFRSF19 H_TM_CD28z) nucleotide sequence ATGCTGCTGCTGGTGACCAGCCTGCTGCTGTGCGAACTGCCGCATCCGGCGTTTCTGCTG ATTCCGGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTG AGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGGCATGAGCTGGGTCCGC CAGGCTCCAAGACAAGGGCTTGAGTGGGTGGCCAACATAAAGCAAGATGGAAGTGAGAAA TACTATGCGGACTCAGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACG CTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACAGCCACGTATTACTGTGCGAAA GAAAATGTGGACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGCGGCCGCAGTCGGA TTCCAAGACATGGAATGCGTGCCCTGCGGCGACCCGCCACCTCCTTACGAGCCGCACTGC GCATCGAAGGTCAACCTCGTGAAGATCGCGAGCACCGCGTCCTCACCCCGGGATACTGCT CTGGCCGCCGTGATTTGTTCCGCCTTGGCCACCGTGCTTCTGGCCCTGCTGATCCTCTGT GTGATCCGGTCGAAGAGGTCCAGACTCTTGCACTCCGACTACATGAACATGACTCCTAGA AGGCCCGGACCCACTAGAAAGCACTACCAGCCGTACGCCCCTCCTCGGGATTTCGCCGCA TACCGGTCCAGAGTGAAGTTCAGCCGCTCAGCCGATGCACCGGCCTACCAGCAGGGACAG AACCAGCTCTACAACGAGCTCAACCTGGGTCGGCGGGAAGAATATGACGTGCTGGACAAA CGGCGCGGCAGAGATCCGGAGATGGGGGGAAAGCCGAGGAGGAAGAACCCTCAAGAGGGC CTGTACAACGAACTGCAGAAGGACAAGATGGCGGAAGCCTACTCCGAGATCGGCATGAAG GGAGAACGCCGGAGAGGGAAGGGTCATGACGGACTGTACCAGGGCCTGTCAACTGCCACT AAGGACACTTACGATGCGCTCCATATGCAAGCTTTGCCCCCGCGG SEQ ID NO: 72 LTG_D0033 (Ef1a_CD33_4 VH TNFRSF19 H_TM_CD28z) nucleotide sequence amino acid sequence MLLLVTSLLLCELPHPAFLLIPEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVR QAPRQGLEWVANIKQDGSEKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTATYYCAK ENVDWGQGTLVTVSSAAAVGFQDMECVPCGDPPPPYEPHCASKVNLVKIASTASSPRDTA LAAVICSALATVLLALLILCVIRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAA YRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEG LYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 73 LTG_D0034 (Ef1a_CD33_4 VH TNFRSF19 H_TM_4-1BBz) nucleotide sequence ATGCTGCTGCTGGTGACCAGCCTGCTGCTGTGCGAACTGCCGCATCCGGCGTTTCTGCTG ATTCCGGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTG AGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGGCATGAGCTGGGTCCGC CAGGCTCCAAGACAAGGGCTTGAGTGGGTGGCCAACATAAAGCAAGATGGAAGTGAGAAA TACTATGCGGACTCAGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACG CTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACAGCCACGTATTACTGTGCGAAA GAAAATGTGGACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGCGGCCGCAGTCGGA TTCCAAGACATGGAATGCGTGCCCTGCGGCGACCCGCCACCTCCTTACGAGCCGCACTGC GCATCGAAGGTCAACCTCGTGAAGATCGCGAGCACCGCGTCCTCACCCCGGGATACTGCT CTGGCCGCCGTGATTTGTTCCGCCTTGGCCACCGTGCTTCTGGCCCTGCTGATCCTCTGT GTGATCAAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAGCCGTTCATGCGGCCC GTGCAGACGACTCAGGAAGAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGAGGGG GGATGCGAACTGAGAGTGAAGTTCAGCCGCTCAGCCGATGCACCGGCCTACCAGCAGGGA CAGAACCAGCTCTACAACGAGCTCAACCTGGGTCGGCGGGAAGAATATGACGTGCTGGAC AAACGGCGCGGCAGAGATCCGGAGATGGGGGGAAAGCCGAGGAGGAAGAACCCTCAAGAG GGCCTGTACAACGAACTGCAGAAGGACAAGATGGCGGAAGCCTACTCCGAGATCGGCATG AAGGGAAACGCCGGAGAGGGAAGGGTCATGACGGACTGTACCAGGGCCTGTCAACTGCC ACTAAGGACACTTACGATGCGCTCCATATGCAAGCTTTGCCCCCGCGG SEQ ID NO: 74: Amino acid sequence of LTG_D0034 (Ef1a_CD33_4 VH TNFRSF19 H_TM_4-1BBz) MLLLVTSLLLCELPHPAFLLIPEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVR QAPRQGLEWVANIKQDGSEKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTATYYCAK ENVDWGQGTLVTVSSAAAVGFQDMECVPCGDPPPPYEPHCASKVNLVKIASTASSPRDTA LAAVICSALATVLLALLILCVIKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEG GCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE GLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 75 LTG_D0015(Ef1a_CD33_4 VH CD8 BBz Nucleotide sequence of T2A tEGFR ATGCTGCTGCTGGTGACCAGCCTGCTGCTGTGCGAACTGCCGCATCCGGCGTTTCTGCTG ATTCCGGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTG AGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGGCATGAGCTGGGTCCGC CAGGCTCCAAGACAAGGGCTTGAGTGGGTGGCCAACATAAAGCAAGATGGAAGTGAGAAA TACTATGCGGACTCAGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACG CTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACAGCCACGTATTACTGTGCGAAA GAAAATGTGGACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGCGGCCGCAACTACC ACCCCTGCCCCTCGGCCGCCGACTCCGGCCCCAACCATCGCAAGCCAACCCCTCTCCTTG CGCCCCGAAGCTTGCCGCCCGGCCGCGGGTGGAGCCGTGCATACCCGGGGGCTGGACTTT GCCTGCGATATCTACATTTGGGCCCCGCTGGCCGGCACTTGCGGCGTGCTCCTGCTGTCG CTGGTCATCACCCTTTACTGCAAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAG CCGTTCATGCGGCCCGTGCAGACGACTCAGGAAGAGGACGGATGCTCGTGCAGATTCCCT GAGGAGGAAGAGGGGGGATGCGAACTGCGCGTCAAGTTCTCACGGTCCGCCGACGCCCCC GCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAG TACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGG AAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTAC TCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAG GGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCC CGGTCTAGAGCTAAACGCTCTGGGTCTGGTGAAGGACGAGGTAGCCTTCTTACGTGCGGA GACGTGGAGGAAAACCCAGGACCCCGAGCCAAACGAATGCTGCTGCTTGTTACAAGCCTT TTGCTCTGCGAACTCCCCCATCCAGCTTTTCTCCTGATTCCAAGGAAGGTTTGCAATGGA ATCGGTATAGGGGAGTTTAAGGATTCACTTAGCATAAACGCTACTAATATTAAACACTTC AAAAACTGTACGAGTATAAGTGGAGATCTTCACATTTTGCCGGTTGCATTCCGAGGCGAT TCATTCACCCACACGCCACCGCTTGACCCACAAGAATTGGATATTCTTAAAACCGTTAAA GAAATAACGGGGTTTTTGCTCATTCAAGCGTGGCCAGAAAATCGCACTGACCTCCATGCT TTCGAGAACCTGGAGATTATAAGAGGACGAACTAAGCAGCATGGTCAATTCTCCCTTGCT GTGGTCAGCCTGAACATCACCAGTCTTGGTTTGCGGTCCCTCAAGGAAATTTCAGATGGA GATGTCATCATAAGCGGCAACAAGAATTTGTGCTATGCAAATACCATAAACTGGAAAAAA CTGTTTGGCACTTCCGGCCAGAAAACCAAGATTATTTCAAATCGGGGTGAGAACAGCTGC AAAGCCACCGGCCAGGTTTGTCATGCCTTGTGCTCTCCGGAAGGCTGTTGGGGGCCAGAA CCCAGGGACTGCGTCAGTTGCAGAAACGTCTCAAGAGGCCGCGAATGCGTTGACAAGTGT AACCTCCTTGAGGGTGAGCCACGAGAGTTTGTTGAGAACAGCGAGTGTATACAATGTCAC CCTGAATGTTTGCCCCAGGCTATGAATATAACCTGCACAGGCCGGGGCCTGATAACTGC ATCCAGTGTGCTCATTACATAGATGGACCTCACTGTGTGAAAACCTGCCCGGCCGGAGTT ATGGGAGAAAACAACACTCTGGTGTGGAAATACGCTGATGCAGGCCACGTGTGCCACCTT TGTCACCCGAATTGTCATATGGGTGTACCGGTCCTGGACTTGAAGGTTGCCCTACCAAT GGCCCTAAATACCCCAGTATCGCAACTGGCATGGTAGGCGCTCTTTCTCTGCTCTTGGTA GTTGCTCTCGGCATAGGTCTTTTTATG SEQ ID NO:76 LTG_D0015(Ef1a_CD33_4 VH CD8 BBz T2A tEGFR)の amino acid sequence MLLLVTSLLLCELPHPAFLLIPEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVR QAPRQGLEWVANIKQDGSEKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTATYYCAK ENVDWGQGTLVTVSSAAATTTPPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDF ACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFP EEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRR KNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPP RSRAKRSGSGEGRGSLLTCGDVEENPGPRAKRMLLLVTSLLLCELPHPAFLLIPRKVCNG IGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVK EITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDG DVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPE PRDCVSCRNVVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNC IQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTN GPKIPSIATGMVGALLLLLVVALGIGLFM SEQ ID NO: 77 LTG_D0016 (Ef1a CD33_4 VH CD8 28z Nucleotide sequence of T2A tEGFR ATGCTGCTGCTGGTGACCAGCCTGCTGCTGTGCGAACTGCCGCATCCGGCGTTTCTGCTG ATTCCGGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTG AGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGGCATGAGCTGGGTCCGC CAGGCTCCAAGACAAGGGCTTGAGTGGGTGGCCAACATAAAGCAAGATGGAAGTGAGAAA TACTATGCGGACTCAGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACG CTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACAGCCACGTATTACTGTGCGAAA GAAAATGTGGACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGCGGCCGCGACTACC ACTCCTGCACCACGGCCACCTACCCCAGCCCCCACCATTGCAAGCCAGCCACTTTCACTG CGCCCCGAAGCGTGTAGACCAGCTGCTGGAGGAGCCGTGCATACCCGAGGGCTGGACTTC GCCTGTGACATCTACATCTGGGCCCCATTGGCTGGAACTTGCGGCGTGCTGCTCTTGTCT CTGGTCATTACCCTGTACTGCCGGTCGAAGAGGTCCAGACTCTTGCACTCCGACTACATG AACATGACTCCTAGAAGGCCCGGACCCACTAGAAAGCACTACCAGCCGTACGCCCCTCCT CGGGATTTCGCCGCATACCGGTCCAGAGTGAAGTTCAGCCGCTCAGCCGATGCACCGGCC TACCAGCAGGGACAGAACCAGCTCTACAACGAGCTCAACCTGGGTCGGCGGGAAGAATAT GACGTGCTGGACAAACGGCGCGGCAGAGATCCGGAGATGGGGGGAAAGCCGAGGAGGAAG AACCCTCAAGAGGGCCTGTACAACGAACTGCAGAAGGACAAGATGGCGGAAGCCTACTCC GAGATCGGCATGAAGGGAGAACGCCGGAGAGGGAAGGGTCATGACGGACTGTACCAGGGC CTGTCAACTGCCACTAAGGACACTTACGATGCGCTCCATATGCAAGCTTTGCCCCCGCGG AGAGCTAAACGCTCTGGGTCTGGTGAAGGACGAGGTAGCCTTCTTACGTGCGGAGACGTG GAGGAAAACCCAGGACCCCGAGCCAAACGAATGCTGCTGCTTGTTACAAGCCTTTTGCTC TGCGAACTCCCCCATCCAGCTTTTCTCCTGATTCCAAGGAAGGTTTGCAATGGAATCGGT ATAGGGGAGTTTAAGGATTCACTTAGCATAAACGCTACTAATATTAAACACTTCAAAAAC TGTACGAGTATAAGTGGAGATCTTCACATTTTGCCGGTTGCATTCCGAGGCGATTCATTC ACCCACACGCCACCGCTTGACCCACAAGAATTGGATATTCTTAAAACCGTTAAAGAAATA ACGGGGTTTTTGCTCATTCAAGCGTGGCCAGAAAATCGCACTGACCTCCATGCTTTCGAG AACCTGGAGATTATAAGAGGACGAACTAAGCAGCATGGTCAATTCTCCCTTGCTGTGGTC AGCCTGAACATCACCAGTCTTGGTTTGCGGTCCCTCAAGGAAATTTCAGATGGAGATGTC ATCATAAGCGGCAACAAGAATTTGTGCTATGCAAATACCATAAACTGGAAAAAACTGTTT GGCACTTCCGGCCAGAAAACCAAGATTATTTCAAATCGGGGTGAGAACAGCTGCAAAGCC ACCGGCCAGGTTTGTCATGCCTTGTGCTCTCCGGAAGGCTGTTGGGGGCCAGAACCCAGG GACTGCGTCAGTTGCAGAAACGTCTCAAGAGGCCGCGAATGCGTTGACAAGTGTAACCTC CTTGAGGGTGAGCCACGAGAGTTTGTTGAGAACAGCGAGTGTATACAATGTCACCCTGAA TGTTTGCCCCAGGCTATGAATATAACCTGCACAGGCCGCGGGGCCTGATAACTGCATCCAG TGTGCTCATTACATAGATGGACCTCACTGTGTGAAAACCTGCCCGGCCGGAGTTATGGGA GAAAAACAACACTCTGGTGTGGAAATACGCTGATGCAGGCCACGTGTGCCACCTTTGTCAC CCGAATTGTCATATGGGTGTACCGGTCCTGGACTTGAAGGTTGCCCTACCAATGGCCCT AAAATACCCAGTATCGCAACTGGCATGGTAGGCGCTCTTCTCTTGCTCTTGGTAGTTGCT CTCGGCATAGGTCTTTTTATG sequence number78 LTG_D0016(Ef1a CD33_4 VH CD8 28z T2A tEGFR)の amino acid sequence MLLLVTSLLLCELPHPAFLLIPEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVR QAPRQGLEWVANIKQDGSEKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTATYYCAK ENVDWGQGTLVTVSSAAATTTPPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDF ACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPP RDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR RAKRSGSGEGRGSLLTCGDVEENPGPRAKRMLLLVTSLLLCELPHPAFLLIPRKVCNGIG IGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEI TGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDV IISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPR DCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQ CAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGP KIPSIATGMVGALLLLLVVALGIGLFM SEQ ID NO: 79 Nucleotide sequence of human IgG4 hinge GAGAGCAAATACGGGCCGCCATGTCCCCCGTGTCCG SEQ ID NO: 80 Amino acid sequence of human IgG4 hinge ESKYGPPCPPCP SEQ ID NO: 81 Nucleotide sequence of human IgG4 CH2 domain GCACCACCAGTTGCTGGCCCTAGTGTCTTCTTGTTCCTCCCAAGCCCAAAGACACCTTG ATGATTTCCAGAACTCCTGAGGTTACCTGCGTTGTCGTAGATGTTTCTCAGGAGGACCCA GAGGTCCAATTTAACTGGTACGTTGATGGGGTGGAAGTTCACAATGCGAAGACAAAGCCG CGGGAAGAACAATTTCAGTCCACTTACCGGGTTGTCAGCGTTCTGACGGTATTGCATCAA GACTGGCTTAATGGAAAGGAATATAAGTGTAAGGTGTCCAACAAAGGTTTGCCGAGCAGT ATTGAGAAGACCATATCAAAGGCGAAG SEQ ID NO: 82 Amino acid sequence of human IgG4 CH2 domain APPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV DGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKA K SEQ ID NO: 83 Nucleotide sequence of human IgG4 CH3 domain GGGCAGCCGCGCGAGCCACAAGTTTACACTTTGCCGCCATCTCAAGAGGAAATGACTAAA AACCAGGTATCCTTGACATGCCTCGTAAAAGGATTTTATCCATCTGATATTGCTGTGGAA TGGGAGTCTAACGGGCAGCCGGAAAATAATTACAAAACTACACCACCTGTGCTCGATTCA GATGGAAGTTTCTTCCTTTACAGTAGACTTACGGTGGACAAATCTAGGTGGCAGGAAGGG AATGTGTTTAGTTGTAGTGTAATGCACGAGGCACTTCATAACCACTATACACAGAAGTCA CTGAGTTTGAGTCTTGGCAAA SEQ ID NO: 84 Amino acid sequence of human IgG4 CH3 domain GQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO: 85 Nucleotide sequence of human IgG4 hinge CH2 CH3 domain GAGAGCAAATACGGGCCGCCATGTCCCCCGTGTCCGGCACCACCAGTTGCTGGCCCTAGT GTCTTCTTGTTCCCTCCCCAAGCCCAAAGACACCTTGATGATTTCCAGAACTCCTGAGGTT ACCTGCGTTGTCGTAGATGTTTCTCAGGAGGACCCAGAGGTCCAATTTAACTGGTACGTT GATGGGGTGGAAGTTCACAATGCGAAGACAAAGCCGCGGGAAGAACAATTTCAGTCCACT TACCGGGTTGTCAGCGTTCTGACGGTATTGCATCAAGACTGGCTTAATGGAAAGGAATAT AAGTGTAAGGTGTCCAACAAAGGTTTGCCGAGCAGTATTGAGAAGACCATATCAAAGGCG AAGGGGCAGCCGCGCGAGCCACAAGTTTACACTTTGCCGCCATCTCAAGAGGAAATGACT AAAAACCAGGTATCCTTGACATGCCTCGTAAAAGGATTTTATCCATCTGATATTGCTGTG GAATGGGAGTCTAACGGGCAGCCGGAAAATAATTACAAAACTACACCACCTGTGCTCGAT TCAGATGGAAGTTTCTTCCTTTACAGTAGACTTACGGTGGACAAATCTAGGTGGCAGGAA GGGAATGTGTTTAGTTGTAGTGTAATGCACGAGGCACTTCATAACCACTATACACAGAAG TCACTGAGTTTGAGTCTTGGCAAA SEQ ID NO: 86 Amino acid sequence of human IgG4 hinge CH2 CH3 domain ESKYGPPCPPCPAPPVAGPSVFLFPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV DGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKA KGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Nucleotide sequence of sequence number 87 LTG_D0035(Ef1a_CD33_4 VH H CH2 CH3 IgG4_CD8TM_CD28z) ATGCTGCTGCTGGTGACCAGCCTGCTGCTGTGCGAACTGCCGCATCCGGCGTTTCTGCTG ATTCCGGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTG AGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGGCATGAGCTGGGTCCGC CAGGCTCCAAGACAAGGGCTTGAGTGGGGTGGCCAACATAAAGCAAGATGGAAGTGAGAAA TACTATGCGGACTCAGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACG CTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACAGCCACGTATTACTGTGCGAAA GAAAATGTGGACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGCGGCCGCAGAGAGC AAATACGGGCCGCCATGTCCCCCGTGTCCGGCACCACCAGTTGCTGGCCCTAGTGTCTTC TTGTTCCCTCCCAAGCCCAAAGACACCTTGATGATTTCCAGAACTCCTGAGGTTACCTGC GTTGTCGTAGATGTTTCTCAGGAGGACCCAGAGGTCCAATTTAACTGGTACGTTGATGGG GTGGAAGTTCACAATGCGAAGACAAAGCCGCGGGAAGAACAATTTCAGTCCACTTACCGG GTTGTCAGCGTTCTGACGGTATTGCATCAAGACTGGCTTAATGGAAAGGAATATAAGTGT AAGGTGTCCAACAAAGGTTTGCCGAGCAGTATTGAGAAGACCATATCAAAGGCGAAGGGG CAGCCGCGCGAGCCACAAGTTTACACTTTGCCGCCATCTCAAGAGGAAATGACTAAAAAC CAGGTATCCTTGACATGCCTCGTAAAAGGATTTTATCCATCTGATATTGCTGTGGAATGG GAGTCTAACGGGCAGCCGGAAAATAATTACAAAACTACACCACCTGTGCTCGATTCAGAT GGAAGTTTCTTCCTTTACAGTAGACTTACGGTGGACAAATCTAGGTGGCAGGAAGGGAAT GTGTTTAGTTGTAGTGTAATGCACGAGGCACTTCATAACCACTATACACAGAAGTCACTG AGTTTGAGTCTTGGCAAAATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTT CTCCTGTCACTGGTTATCACCCTTTACTGCCGGTCGAAGAGGTCCAGACTCTTGCACTCC GACTACATGAACATGACTCCTAGAAGGCCCGGACCCACTAGAAAGCACTACCAGCCGTAC GCCCCTCCTCGGGATTTCGCCGCATACCGGTCCAGAGTGAAGTTCAGCCGCTCAGCCGAT GCACCGGCCTACCAGCAGGGACAGAACCAGCTCTACAACGAGCTCAACCTGGGTCGGCGG GAAGAATATGACGTGCTGGACAAACGGCGCGGCAGAGATCCGGAGATGGGGGGAAAGCCG AGGAGGAAGAACCCTCAAGAGGGCCTGTACACGAACTGCAGAAGGGACAAGATGGCGGAA GCCTACTCCGAGATCGGCATGAAGGGAGAACGCCGGAGAGGGAAGGGTCATGACGGACTG TACCAGGGCCTGTCCAACTGCCACTAAGGACACTTACGATGCGCTCCATATGCAAGCTTTG CCCCCGCGGG sequence number 88 LTG_D0035(Ef1a_CD33_4 VH H CH2 CH3 IgG4_CD8TM_CD28z) MLLLVTSLLLCELPHPAFLLIPEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVR QAPRQGLEWVANIKQDGSEKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTATYYCAK ENVDWGQGTLVTVSSAAAESKYGPPCPPCPAPPVAGPSVFLFPKPKDTLMISRTPEVTC VVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKC KVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSL SLSLGKIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGPTRKHYQPY APPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR

Claims

1. 1. Use of isolated T cells or isolated natural killer (NK) cells for the manufacture of a pharmaceutical composition for generating anti-tumor immunity in a human subject, wherein the isolated T cells or the isolated NK cells comprise a vector comprising a nucleic acid molecule encoding a chimeric antigen receptor (CAR), the CAR comprising, from N-terminus to C-terminus: (i) at least one extracellular antigen-binding domain that binds to CD33, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, or a single-chain fragment variable (ScFv) domain selected from the group consisting of SEQ ID NOs: 6, 8, 10, and 12; (ii) a transmembrane domain comprising a transmembrane domain of a protein selected from the group consisting of the alpha chain of the T cell receptor (TCR), the beta chain of the TCR, the zeta chain of the TCR, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, or any combination thereof; (iii) at least one costimulatory domain comprising a functional signaling domain selected from the group consisting of OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP12, and 4-1BB (CD137), or any combination thereof; and (iv) Use comprising an intracellular signaling domain comprising a functional domain selected from the group consisting of 4-1BB (CD137), CD28 and CD3 zeta signaling domains or a combination thereof.

2. 1. Use of isolated T cells or isolated natural killer (NK) cells for the manufacture of a pharmaceutical composition for treating cancer in a human subject, wherein the isolated T cells or the isolated NK cells comprise a vector comprising a nucleic acid molecule encoding a CAR, the CAR comprising, from N-terminus to C-terminus: (i) at least one extracellular antigen-binding domain that binds to CD33, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, or a single-chain fragment variable (ScFv) domain selected from the group consisting of SEQ ID NOs: 6, 8, 10, and 12; (ii) a transmembrane domain comprising a transmembrane domain of a protein selected from the group consisting of the alpha chain of the T cell receptor (TCR), the beta chain of the TCR, the zeta chain of the TCR, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, or any combination thereof; (iii) at least one costimulatory domain comprising a functional signaling domain selected from the group consisting of OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP12, and 4-1BB (CD137), or any combination thereof; and (iv) Use comprising an intracellular signaling domain comprising a functional domain selected from the group consisting of 4-1BB (CD137), CD28 and CD3 zeta signaling domains or a combination thereof.

3. 2. The use of claim 1, wherein the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 27 or an amino acid sequence having 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:

28.

4. 2. The use of claim 1, wherein the at least one extracellular antigen-binding domain and the intracellular signaling domain, or both, are connected to the transmembrane domain by a linker or spacer domain.

5. The use of claim 4, wherein the linker or spacer domain is isolated from the extracellular domain of CD8, TNFRSF19, IgG4, or CD28 and is linked to the transmembrane domain.

6. 2. The use of claim 1, wherein the CAR comprises the amino acid sequence of SEQ ID NO: 16, 18, 20, 22, 24, 26, 70, 72, 74, 76, 78, or 88.

7. 3. The use of claim 2, wherein the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 27 or an amino acid sequence having 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:

28.

8. 3. The use of claim 2, wherein the at least one extracellular antigen-binding domain and the intracellular signaling domain, or both, are connected to the transmembrane domain by a linker or spacer domain.

9. 9. The use of claim 8, wherein the linker or spacer domain is isolated from the extracellular domain of CD8, TNFRSF19, IgG4, or CD28 and is linked to the transmembrane domain.

10. 3. The use of claim 2, wherein the CAR comprises the amino acid sequence of SEQ ID NO: 16, 18, 20, 22, 24, 26, 70, 72, 74, 76, 78, or 88.

11. The use according to claim 2, wherein the cancer is leukemia.

12. 12. The use of claim 11, 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).

Images