Cd70 binding molecules and methods of use thereof

Abstract

This disclosure provides anti-CD70 antibodies comprising antigen-binding molecules that specifically bind to CD70, their antigen-binding fragments, chimeric antigen receptors (CARs) and engineered T-cell receptors (TCRs), polynucleotides encoding them, and in vitro cells comprising them. The polynucleotides, peptides, and in vitro cells described herein can be used in engineered TCRs and / or CAR T-cell therapies for the treatment of patients with cancer. In one specific embodiment, the polynucleotides, peptides, and in vitro cells described herein can be used to treat multiple myeloma.

Description

Technical Field

[0001] This disclosure relates to chimeric antigen receptors (CARs) comprising antigen-binding molecules that bind to CD70 and engineered T-cell receptors (TCRs), the polynucleotides encoding them, and methods for treating cancer in patients using them. Cross-reference materials for related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 458,879, filed February 14, 2017, the entire of which is incorporated herein by reference. sequence list

[0003] This application contains a sequence list that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy created on February 8, 2018, is named K-1034_02_SL.txt and is 183,361 kilobytes in size. Prior Technology

[0004] Human cancers are essentially normal cells that have undergone genetic or epigenetic transformation into abnormal cancer cells. When this transformation occurs, cancer cells begin to express proteins and other antigens that differ from those expressed by normal cells. These abnormal tumor antigens can be used by the body's innate immune system to specifically target and kill cancer cells. However, cancer cells employ various mechanisms to prevent immune cells (such as T and B lymphocytes) from successfully targeting them.

[0005] Human T-cell therapy relies on enriched or modified human T cells to target and kill a patient's cancer cells. To enhance the ability of T cells to target and kill specific cancer cells, methods have been developed to engineer T cells to express constructs that guide T cells to target specific cancer cells. Chimeric antigen receptors (CARs) containing binding domains that interact with specific tumor antigens and engineered T-cell receptors (TCRs) allow T cells to target and kill cancer cells expressing those specific tumor antigens.

[0006] Current therapies for hematologic malignancies have demonstrated varying degrees of effectiveness. Therefore, there is a need to identify novel and improved therapies for treating diseases and disorders related to CD70. Summary of the Invention

[0007] In one state, a single polynucleotide encoding a chimeric antigen receptor (CAR) or T-cell receptor (TCR) contains an antigen-binding molecule that specifically binds to CD70.In one specific embodiment, the antigen-binding molecule comprises: (a) a heavy chain variable region (VH) complementarity-determining region (CDR) 1, which comprises, is composed of, or is substantially composed of amino acid sequences selected from the group consisting of: GFTFSSY (SEQ ID NO: 71), GDSIISGGY (SEQ ID NO: 73), and GYTFTSY (SEQ ID NO: 75); (b) a heavy chain variable region (VH) complementarity-determining region (CDR) 2, which comprises, is composed of, or is substantially composed of amino acid sequences selected from the group consisting of: WYDGSN (SEQ ID NO: 72), FYSGS (SEQ ID NO: 74), and DPSGGS (SEQ ID NO: 75). NO:76); (c) Heavy chain variable region (VH) complementarity-determining region (CDR) 3, comprising, consisting of, or substantially consisting of amino acid sequences selected from the following groups: DLLRGVKGYAMDV (SEQ ID NO:64), SGYSYALFDH (SEQ ID NO:67), and DYGDYVFDY (SEQ ID NO:76); (d) Light chain variable region (VL) complementarity-determining region (CDR) 1, comprising, consisting of, or substantially consisting of amino acid sequences selected from the following groups: RASQSLRRIYLA (SEQ ID NO:53), RASQFIGRYFN (SEQ ID NO:56), and SGSSSNIGTNTVN (SEQ ID NO:76). NO:59); (e) Light chain variable region (VL) complementarity-determining region (CDR) 2, which comprises an amino acid sequence selected from the following groups, is composed of an amino acid sequence selected from the following groups, or is substantially composed of an amino acid sequence selected from the following groups: DVFDRAT (SEQ ID NO:54), AESSLQS (SEQ ID NO:57), and INNQRPS (SEQ ID NO:60); (f) Light chain variable region (VL) complementarity-determining region (CDR) 3, which comprises an amino acid sequence selected from the following groups, is composed of an amino acid sequence selected from the following groups, or is substantially composed of an amino acid sequence selected from the following groups: QQYSDSPFT (SEQ ID NO:55), QQSYSTPFT (SEQ ID NO:58), and ATWDDSLNGPVV (SEQ ID NO:61).In one specific embodiment, VH CDR3 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 64, 67, and 70.

[0008] In another embodiment, a single polynucleotide encoding a chimeric antigen receptor (CAR) or T-cell receptor (TCR) is disclosed to contain an antigen-binding molecule that specifically binds to CD70. In one specific embodiment, the antigen-binding molecule comprises VH CDR3, which contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 64, 67, and 70. In one specific embodiment, VH CDR1 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 71, 73, and 75.

[0009] In another specific embodiment, VH CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 72, 74 and 76.

[0010] In another embodiment, VL CDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 53, 56, and 59. In another embodiment, VL CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 54, 57, and 60. In another embodiment, VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 55, 58, and 61.

[0011] In another specific embodiment, VH CDR1, VH CDR2, and VH CDR3 each contain the amino acid sequences of the variable light chains VH CDR1, VH CDR2, and VH CDR3 shown in Figures 7A, 7C, or 7E.

[0012] In another specific embodiment, VL CDR1, VL CDR2, and VL CDR3 each comprise the amino acid sequences of the antibodies VL CDR1, VL CDR2, and VL CDR3 shown in Figures 7B, 7D, or 7F. In another specific embodiment, the antigen-binding molecule comprises a heavy chain variable region sequence containing an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 7, and 11.

[0013] In another specific embodiment, the antigen-binding molecule includes a light chain variable region sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 9 and 13.

[0014] In another specific embodiment, the antigen-binding molecule comprises one of the following: (a) a VH CDR1 region containing the amino acid sequence of SEQ ID NO:71; a VH CDR2 region containing the amino acid sequence of SEQ ID NO:72; a VH CDR3 region containing the amino acid sequence of SEQ ID NO:64; a VL CDR1 region containing the amino acid sequence of SEQ ID NO:53; a VL CDR2 region containing the amino acid sequence of SEQ ID NO:54; and a VL CDR3 region containing the amino acid sequence of SEQ ID NO:55; (b) a VH CDR1 region containing the amino acid sequence of SEQ ID NO:73; a VH CDR2 region containing the amino acid sequence of SEQ ID NO:74; a VH CDR3 region containing the amino acid sequence of SEQ ID NO:67; a VL CDR1 region containing the amino acid sequence of SEQ ID NO:56; a VL CDR2 region containing the amino acid sequence of SEQ ID NO:74; a VH CDR3 region containing the amino acid sequence of SEQ ID NO:67; a VL CDR1 region containing the amino acid sequence of SEQ ID NO:56; a VL CDR2 region containing the amino acid sequence of SEQ ID NO:74; and a VL CDR3 region containing the amino acid sequence of SEQ ID NO:67. The amino acid sequence NO:57; and the VL CDR3 region, which contains the amino acid sequence SEQ ID NO:58; or (c) the VH CDR1 region, which contains the amino acid sequence SEQ ID NO:75; the VH CDR2 region, which contains the amino acid sequence SEQ ID NO:76; the VH CDR3 region, which contains the amino acid sequence SEQ ID NO:70; the VL CDR1 region, which contains the amino acid sequence SEQ ID NO:59; the VL CDR2 region, which contains the amino acid sequence SEQ ID NO:60; and the VL CDR3 region, which contains the amino acid sequence SEQ ID NO:61. In another specific embodiment, the antigen-binding molecule includes one of the following: (a) VH, which contains the amino acid sequence of SEQ ID NO:3; and VL, which contains the amino acid sequence of SEQ ID NO:5; (b) VH, which contains the amino acid sequence of SEQ ID NO:7; and VL, which contains the amino acid sequence of SEQ ID NO:9; or (c) VH, which contains the amino acid sequence of SEQ ID NO:11; and VL, which contains the amino acid sequence of SEQ ID NO:13.

[0015] In another specific embodiment, the nucleotide sequence is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the nucleotide sequences selected from the group consisting of SEQ ID NO:2 and 4.

[0016] In another specific embodiment, the nucleotide sequence is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the nucleotide sequences selected from the group consisting of SEQ ID NO: 6 and 8.

[0017] In another specific embodiment, the nucleotide sequence is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the nucleotide sequences selected from the group consisting of SEQ ID NO: 10 and 12.

[0018] In another specific embodiment, the antigen-binding molecule is a single chain.

[0019] In various specific embodiments, the antigen-binding molecule is selected from the group consisting of: scFv, Fab, Fab', Fv, F(ab')2, dAb, and any combination thereof, and in a particular specific embodiment, the antigen-binding molecule comprises scFv.

[0020] In another embodiment, VH and VL are connected by a connector. In one particular embodiment, VH is located at the N end of the connector and VL is located at the C end of the connector; and in another particular embodiment, VL is located at the N end of the connector and VH is located at the N end of the connector.

[0021] In another specific embodiment, the linker comprises at least about 5, at least about 8, at least about 10, at least about 13, at least about 15, at least about 18, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, or at least about 100 amino acids, and in another specific embodiment, the linker comprises an amino acid sequence that is at least 75%, at least 85%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to one of the amino acid sequences in SEQ ID NO: 80 and 81.

[0022] In another specific embodiment, the antigen-binding molecule binds CD70 with a KD of less than about 1×10⁻⁶ M, less than about 1×10⁻⁷ M, less than about 1×10⁻⁸ M, or less than about 1×10⁻⁹ M.

[0023] In another specific embodiment, the CAR or TCR further includes a constant region, and in yet another specific embodiment, the CAR or TCR further includes a transmembrane domain, and in yet another specific embodiment, the transmembrane domain is one of the following transmembrane domains: CD28, 4-1BB / CD137, CD8α, CD4, CD19, CD3ε, CD45, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, the α chain of the T cell receptor, the β chain of the T cell receptor, the ζ chain of the T cell receptor, or any combination thereof. In a particular specific embodiment, the transmembrane domain is the CD28T transmembrane domain. In another specific embodiment, the CD28T transmembrane domain contains an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO:15 or 19.

[0024] In another specific embodiment, the CD28T transmembrane domain is encoded by a nucleotide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 16 or 18.

[0025] In another specific embodiment, the transmembrane domain is a CD8 α transmembrane domain. In another specific embodiment, the CD8 α transmembrane domain contains an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 17, 21, or 94.

[0026] In another specific embodiment, the CD8 α transmembrane domain is encoded by a nucleotide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO:16 or 20.

[0027] In another specific embodiment, the CAR or TCR further includes a hinge region between the transmembrane domain and the antigen-binding molecule. In another specific embodiment, the hinge region includes all or fragments of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, CD28, or CD8α. In another specific embodiment, the hinge region is the hinge region of CD28T. In another specific embodiment, the hinge region includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence selected from the group consisting of SEQ ID NO: 15 and 83. In another specific embodiment, the hinge region is encoded by a nucleotide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence selected from the group consisting of SEQ ID NO: 14 and 82.

[0028] In another embodiment, the hinge region is the hinge region of CD8 α. In another embodiment, the hinge region comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence selected from the group consisting of SEQ ID NO: 17 and 85, and in another embodiment, the hinge region is encoded by a nucleotide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence selected from the group consisting of SEQ ID NO: 16 and 84.

[0029] In another specific embodiment, the CAR or TCR further includes a co-stimulatory region. In another specific embodiment, the co-stimulatory region is CD2, CD3δ, CD3ε, CD3γ, CD4, CD7, CD8α, CD8β, CD11a (ITGAL), CD11b (ITGAM), CD11c (ITGAX), CD11d (ITGAD), CD18 (ITGB2), CD19 (B4), CD27 (TNFRSF7), CD28, CD28T, CD29 (ITGB1), CD30 (TNFRSF8), CD40 (TNFRSF5), CD48 (SLAMF2), CD49a (ITGA1), CD49d (ITGA4), CD49f (ITGA6), CD66a (CEACAM1), CD66b (CEACAM8), CD66c (CEACAM6), CD66d (CEACAM3), CD66e (CEACAM5), CD69 (CLEC2), CD79A... (β-chain of B-cell antigen receptor complex-associated), CD79B (β-chain of B-cell antigen receptor complex-associated), CD84 (SLAMF5), CD96 (Tactile), CD100 (SEMA4D), CD103 (ITGAE), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD158A (KIR2DL1), CD158B1 (KIR2DL2), CD158B2 (KIR2DL3), CD158C (KIR3DP1), CD158D (KIRDL4), CD158F1 (KIR2DL5A), CD158F2 (KIR2DL5B), CD158K (KIR3DL2), CD160 (BY55), CD162 (SELPLG), CD226 (DNAM1), CD229 (SLAMF3), CD244 (SLAMF4), CD247 (CD3-ζ), CD258 (LIGHT), CD268 (BAFFR), CD270 (TNFSF14), CD272 (BTLA), CD276 (B7-H3), CD279 (PD-1), CD314 (NKG2D), CD319 (SLAMF7), CD335 (NK-p46), CD336 (NK-p44), CD337 (NK-p30), CD352 (SLAMF6), CD353 (SLAMF8), CD355 (CRTAM), CD357 (TNFRSF18), Inducible T cell costimulators (ICOS), LFA-1(CD11a / CD18), NKG2C, DAP-10, ICAM-1, NKp80 (KLRF1), IL-2Rβ, IL-2Rγ, IL-7Rα, LFA-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1 / CBP, CD83 ligand, Fcγ receptor, MHC class 1 molecules, MHC class 2 molecules, TNF receptor protein, immunoglobulin protein, cell mediator receptor, integrin, activated NK cell receptor, Toll ligand receptor, and signal transduction regions of fragments or combinations thereof.

[0030] In another specific embodiment, the co-stimulatory region is a CD28 co-stimulatory region. In another specific embodiment, the co-stimulatory region comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 23, and in another specific embodiment, the co-stimulatory region is encoded by a nucleotide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 22.

[0031] In another specific embodiment, the co-stimulatory region is a CD137 (4-1BB) co-stimulatory region. In another specific embodiment, the co-stimulatory region comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 25. In yet another specific embodiment, the co-stimulatory region is encoded by a nucleotide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 24.

[0032] In another specific embodiment, the CAR or TCR further includes an activation domain. And, in another specific embodiment, the activation domain is a CD3ζ domain. In another specific embodiment, the activation domain includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 27 or 92, and in another specific embodiment, the activation domain is encoded by a nucleotide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 26.

[0033] In another specific embodiment, the CAR or TCR further comprises a leader peptide. In another specific embodiment, the leader peptide comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 28, and in another specific embodiment, the leader peptide is encoded by a nucleotide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 95.

[0034] In another embodiment, a polynucleotide encoding a CAR or TCR is provided. In a particular embodiment, the polynucleotide is selected from the group consisting of SEQ ID NO: 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, and 51. In another embodiment, a vector comprising any of the polynucleotides disclosed herein is provided. In another particular embodiment, the vector is a retroviral vector, a DNA vector, a plastid, an RNA vector, an adenovirus vector, an adenovirus-associated vector, a lentiviral vector, or any combination thereof.

[0035] In another embodiment, the CAR or TCR is encoded by the polynucleotide or vector disclosed herein. In yet another embodiment, cells comprising the polynucleotide disclosed herein, the vector disclosed herein, the CAR or TCR disclosed herein, or any combination thereof are provided. In another specific embodiment, the cells comprise immune cells, and in yet another specific embodiment, the cells are T cells. In yet another specific embodiment, the T cells are tumor-infiltrating lymphocytes (TILs), autologous T cells, engineered autologous T cells (eACT), allogeneic T cells, or any combination thereof, and in yet another specific embodiment, the cells are in vitro cells.

[0036] In another embodiment, the composition comprises the polynucleotides disclosed herein, the vectors disclosed herein, the CARs or TCRs disclosed herein, or the cells disclosed herein. In another specific embodiment, the composition is formulated for delivery to an individual.

[0037] In another embodiment, the method for producing cells expressing CAR or TCR includes, under appropriate conditions, using the polynucleotide transduced cells disclosed herein. In yet another specific embodiment, the method further includes isolated cells.

[0038] In another embodiment, a method for inducing tumor-targeting immunity is provided, and in one specific embodiment, this includes administering to an individual an effective amount of cells containing the polynucleotides disclosed herein, the vectors disclosed herein, or the CARs or TCRs disclosed herein.

[0039] In another embodiment, a method for treating cancer in an individual with a need is provided, and in one specific embodiment, it includes delivery to the individual of the polynucleotide disclosed herein, the vector disclosed herein, the CAR or TCR disclosed herein, the cell disclosed herein, or the composition disclosed herein. In one specific embodiment, the cancer is a blood cancer. In another specific embodiment, the cancer is a cancer of white blood cells. In a further specific embodiment, the cancer is a cancer of plasma cells. In another specific embodiment, the cancer is leukemia, lymphoma, or myeloma. In a particular specific embodiment, the cancer is multiple myeloma, Hodgkin's disease, non-Hodgkin's lymphoma (NHL), primary mediastinal large B-cell lymphoma (PMBC), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), transformed follicular lymphoma, splenic marginal zone lymphoma (SMZL), chronic or acute leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia (ALL). (Including non-T-cell ALL), chronic lymphocytic leukemia (CLL), T-cell lymphoma, one or more B-cell acute lymphoblastic leukemias (“BALL”), T-cell acute lymphoblastic leukemia (“TALL”), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell tumor, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, blastic leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative condition, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, spinal cord The following are considered cancers: dysplasia and myelodysplastic syndromes, plasmablastic lymphoma, plasmacytoid dendritic cell tumor, Waldenstrom macroglobulinemia, plasmacytic dysplasia (e.g., asymptomatic myeloma (depressive multiple myeloma or painless myeloma)), monoclonal gamma globulinosis of undetermined significance (MGUS), plasmacytomas (e.g., plasmacytic cachexia, solitary myeloma, solitary plasmacytoma, extramedullary plasmacytoma, and multiple plasmacytomas), systemic amyloid light chain amyloidosis, POEMS syndrome (also known as Crow-Fukase syndrome, Takatsuki disease, and PEP syndrome), or combinations thereof. In a specific embodiment, the cancer is multiple myeloma. It is also disclosed as a CAR or TCR, wherein the CAR or TCR comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, and 52. In various specific embodiments, the CAR or TCR comprises a leader sequence that does not exist in SEQ ID NO: 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, and 52. Simple Explanation of the Diagram

[0040] [Figures 1A-1H] are sketches illustrating examples of CAR structures provided in this paper; showing representative components and their preferred configurations.

[0041] [Figure 2] A series of graphs to demonstrate CD70 expression in various target cell lines, each corresponding to a range of blood cancers; Top row, from left to right: Human lymphoblastic cell line CEM (also known as CCRF-CEM), human eosinophilic leukocyte cell line EoL-1, human myeloid cell line HL-60, human myeloid cell line KG-1a, and human myeloid cell line MV4-11; Bottom row, from left to right: Human B cell line Namalwa, B lymphocyte cell line Raji, human B lymphocyte cell line Toledo, and human myeloid cell line U-937.

[0042] [Figures 3A and 3B] are a series of graphs demonstrating various CAR expressions against CD70 in lentivirally transduced T cells (isolated from the first healthy human T cell donor (Figure 3A) and the second healthy human T cell donor (Figure 3B)); transparent histograms represent simulated transduction, while gray-filled histograms represent cells expressing transduced CARs, and the numbers in the cells show the percentage of cells found to be CAR-positive.

[0043] [Figures 4A-4F] are a series of bar graphs depicting the production of IFNγ, TNFα, and IL-2 in lentivirus-transduced T cells from two healthy donors expressing four different CARs (28T-28z, 28T-4Bz, 8K-28z, and 8K-4Bz), each containing different scFvs (8G1, 1C8, and 6E9) (after 16 hours of co-culture with KG-1a, Raji, or Namalwa target cell lines). Figures 4A and 4B show the production of IFNγ (pg / ml; y-axis) in lentivirus-transduced CAR T cells (from donor 1 (Figure 4A) and from donor 2 (Figure 4B)), Figures 4C and 4D show the production of TNFα (pg / ml; y-axis) in lentivirus-transduced CAR T cells (from donor 1 (Figure 4C) and donor 2 (Figure 4D)), and Figures 4E and 4F show the production of TNFα in lentivirus-transduced CAR T cells. IL-2 production (pg / ml; y-axis) from T cells (derived from the first donor (Fig. 4E) and the second donor (Fig. 4F)).

[0044] [Figures 5A-5D] show the lentiviral transduced T cells of CAR (obtained from healthy donor 1 (Figures 5A and 5C) or healthy donor 2 (Figures 5B and 5D)) co-cultured with KG-1a (CD70-) (Figures 5A and 5B), Raji (CD70+) (Figure 5C), or Namalwa (CD70+) (Figure 5D) target cells for 112 hours, and the cell lysis activity over time (as a percentage of remaining viable target cells; y-axis).

[0045] [Figure 6] is a table showing the complementarity-determining regions (CDRs) of the antigen-binding molecules disclosed in this paper, encoded using the Kabat, Chothia, and IGMT systems.

[0046] [Figures 7A-7F] are a series of tables showing the variable weight (VH) and variable lightness (VL) sequences of each of the CARs or TCRs provided in this paper; CDRs are also presented and displayed as encoded using the Kabat, Chothia and IGMT systems.

[0047] [Figures 8A-8L] depict the nucleic acid sequences encoding the various CARs disclosed herein; the corresponding amino acid sequences are also provided.

[0048] [Figure 9] is a bar graph that confirms the expression of CD70 CAR C3L against CD70 in lentivirally transduced T cells.

[0049] [Figures 10A-10D] show the lentiviral transduced T cells of CAR (obtained from healthy donor 3 (Figures 10A and 10B) or healthy donor 4 (Figures 10C and 10D)) co-cultured with KG-1a or Raji at an E:T ratio of 4:1 (Figures 10A and 10C) or 1:1 (Figures 10B and 10D) for 112 hours, and the cell lysis activity over time (as a percentage of remaining viable target cells; y-axis).

[0050] [Figures 11A-11D] are a series of bar graphs depicting IFNγ production by lentivirus-transduced T cells (from two healthy donors expressing CD70 CAR C3L) (28T-28z, containing 8G1 scFv) after 16 hours of co-culture with KG-1a or Raji target cell lines; Figures 11A and 11B show IFNγ production (pg / ml; y-axis) in lentivirus-transduced CAR T cells (from donor 3) at E:T ratios of 4:1 and 1:1, respectively; Figures 11C and 11D show IFNγ production (pg / ml; y-axis) in lentivirus-transduced CAR T cells (from donor 4) at E:T ratios of 4:1 and 1:1, respectively.

[0051] [Figures 12A-12D] are a series of bar graphs depicting IL-2 production in lentivirus-transduced T cells (from two healthy donors expressing CD70 CAR C3L) (28T-28z, containing 8G1 scFv) after 16 hours of co-culture with KG-1a or Raji target cell lines; Figures 12A and 12B show IL-2 production (pg / ml; y-axis) in lentivirus-transduced CAR T cells (from donor 3) at E:T ratios of 4:1 and 1:1, respectively; Figures 12C and 12D show IL-2 production (pg / ml; y-axis) in lentivirus-transduced CAR T cells (from donor 4) at E:T ratios of 4:1 and 1:1, respectively.

[0052] [Figures 13A-13D] are a series of bar graphs depicting TNFα production by lentivirus-transduced T cells (from two healthy donors expressing CD70 CAR C3L) (28T-28z, containing 8G1 scFv) after 16 hours of co-culture with KG-1a or Raji target cell lines; Figures 13A and 13B show IL2 (pg / ml; y-axis) production in lentivirus-transduced CAR T cells (from donor 3) at E:T ratios of 4:1 and 1:1, respectively; Figures 13C and 13D show TNFα (pg / ml; y-axis) production in lentivirus-transduced CAR T cells (from donor 4) at E:T ratios of 4:1 and 1:1, respectively. Implementation

[0053] This disclosure relates to antibodies binding to CD70, their antigen-binding molecules, chimeric antigen receptors (CARs) and engineered T-cell receptors (TCRs), the polynucleotides encoding them, and in vitro cells containing them. The polynucleotides, peptides, and in vitro cells described herein can be used in engineered CAR T-cell therapies (e.g., autologous cell therapy (eACT™)) for the treatment of patients with cancer. In particular, the polynucleotides, peptides, and in vitro cells described herein can be used to treat multiple myeloma. definition

[0054] To facilitate understanding of this disclosure, some terms are first defined. Unless otherwise expressly provided herein, the following terms as used in this application shall have the meanings set forth below. Further definitions are set forth throughout this application. The headings provided herein are not intended to limit the various forms of this disclosure; such forms can be understood by referring to the entire specification.

[0055] It should be understood that whenever a state is described in this text by the language “contains”, it is also provided that other similar states are described by “composed of” and / or “substantially composed of”.

[0056] The use of substitutions (e.g., "or") should be understood to refer to one, both, or any combination thereof. As used herein, the indefinite article "a" or "an" should be understood to refer to "one or more" of any claimed or enumerated components.

[0057] The term "and / or" as used herein is to be regarded as specifically revealing each of the two specified characteristics or components (with or without the presence of the other). Therefore, the term "and / or" as used in phrases (such as "A and / or B") herein is intended to include "A and B," "A or B," "A (alone)," and "B (alone)." Similarly, the term "and / or" as used in phrases (such as "A, B, and / or C") herein is intended to cover the following states: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0058] The units, prefixes, and symbols used in this document are provided in their International System of Units (SI) accepted form. Numerical ranges include the numbers that define the range.

[0059] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art related to this invention. For example, Juo, [The Concise Dictionary of Biomedicine and Molecular Biology], 2nded., (2001), CRC Press; [The Dictionary of Cell & Molecular Biology], 5 thed., (2013), Academic Press; and [The Oxford Dictionary of Biochemistry and Molecular Biology], Cammack et al., eds., 2nd ed., (2006), Oxford University Press, provides a general dictionary of many terms used in this disclosure for those skilled in the art to which the invention pertains.

[0060] As used herein, the twenty known (e.g., naturally occurring) amino acids and their abbreviations follow conventional usage. See, for example... [Immunology-A Synthesis] (2nd Edition), Golub and Green, eds., Sinauer Assoc., Sunderland, Mass. (1991), which is incorporated herein by reference for any purpose. Stereoisomers of twenty known amino acids (e.g., D-amino acids), non-natural amino acids such as α-, α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other uncommon amino acids may also be suitable components of the polypeptides disclosed herein. Examples of non-common amino acids include: 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, eN-acetylsine, O-phosphoserine, N-acetylsine, N-methionylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and other similar amino acids and imines (e.g., 4-hydroxyproline). In the polypeptide representation used herein, according to standard usage and convention, the left-hand direction represents the amino terminus and the right-hand direction represents the carboxyl terminus.

[0061] As used herein, the term "about" refers to a value or composition within an acceptable margin of error for a particular value or composition as determined by someone skilled in the art to which the invention pertains, which will depend in part on how the value or composition was measured or determined, i.e., the limitations of the measurement system. For example, "about" or "substantially includes" may mean within one or more standard deviations as performed in the art. Alternatively, "about" or "substantially includes" may mean within a range of up to 10% (i.e., ±10%). For example, about 5 mg may include any value between 4.5 mg and 5.5 mg. Furthermore, especially when relating to biological systems or processes, the term may refer to at most one order of magnitude or at most five times the value. Unless otherwise stated, when a particular value or composition is provided in this invention, the meaning of "about" or "substantially includes" should be assumed to be within an acceptable margin of error for that particular value or composition.

[0062] Unless otherwise specified, any concentration range, percentage range, proportion range or integer range described herein shall be understood to be a numerical value of any integer included in the enumerated range, and, where appropriate, a fraction of that integer (such as one-tenth and one-hundredth of an integer).

[0063] "Administration" refers to the physical introduction of a reagent into an individual using any of the various methods and delivery systems known to those skilled in the art to which this invention pertains. Exemplary administration routes for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral administration routes, such as by injection or infusion. As used herein, the phrase "parenteral administration" means a mode of administration other than enteral and local administration, typically by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrasheath, intralymphatic, intralesional, intracapsular, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions, as well as intracorporeal electroporation. In some specific embodiments, the formulation is administered via a non-parenteral route, such as oral administration. Other non-gastrointestinal routes include local, epidermal, or mucosal administration, such as intranasal, vaginal, rectal, sublingual, or other local administration routes. Administration may also be performed, for example, once, multiple times, and / or over one or more extended periods.

[0064] The term "antibody" (Ab) includes, but is not limited to, glycoprotein immunoglobulins that specifically bind to antigens. Generally, an antibody may comprise at least two heavy (H) chains and two light (L) chains linked by disulfide bonds, or its antigen-binding molecule. Each H chain contains a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region contains three constant domains CH1, CH2, and CH3. Each light chain contains a light chain variable region (VL) and a light chain constant region. The light chain constant region contains one constant domain, CL. The VH and VL regions may be further refined... The immunoglobulin is divided into highly variable regions, called complementarity-determining regions (CDRs), interspersed with more preserved regions, called framework regions (FRs). Each VH and VL contains three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the immunoglobulin mediate the binding of immunoglobulins to host tissues or factors, including various immune system cells (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0065] Antibodies may include, for example, both naturally occurring and non-naturally occurring (recombinant) antibodies, human and non-human antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabody (see, for example, Stocks, (2004) Drug Discovery Today 9(22): 960-66), antibody fusions (this term encompasses antibody-drug conjugates) and is sometimes referred to herein as “antibody conjugates”), heteroconjugated antibodies, single-domain antibodies, monovalent antibodies, single-chain antibodies or single-chain Fv (scFv), camelified antibodies, affybody, Fab fragment, F(ab') 2. Fragments, disulfide-linked Fvs (sdFv), anti-atopic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), microantibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimics") and their antigen-binding fragments. In some specific embodiments, the antibody system described herein refers to a group of multiple antibodies.

[0066] Immunoglobulins are tetrameric molecules, normally composed of two pairs of identical polypeptide chains, each pair having a light (approximately 25 kDa) and a heavy (approximately 50-70 kDa) chain. The amino-terminal portion of each chain includes a variable region of approximately 100 to 130 or more amino acids primarily responsible for antigen recognition. The carboxyl-terminal portion of each chain defines a constant region primarily responsible for effector functions. Human light chains are classified as κ and λ light chains. Heavy chains are classified as μ, δ, γ, α, or ε, and their antibody isotypes are defined as IgM, IgD, IgG, IgA, or IgE, respectively. In both light and heavy chains, the variable and constant regions are linked by a J-region of approximately 12 or more amino acids, and the heavy chain also includes a D-region of approximately 10 or more amino acids. See generally Berzofsky & Berkower, Ch. 7. [Fundamental Immunology] (Paul, W., ed., Lippincott Williams & Wilkins (2012); for all purposes, its chapters and volumes are incorporated herein by reference in their entirety). The variable regions of each light chain / heavy chain pair form antibody binding sites, giving intact immunoglobulins two major binding sites.

[0067] Immunoglobulins can be derived from any generally known isotype, including, but not limited to, IgA, secretory IgA, IgG, and IgM. IgG subclasses are also well known to those skilled in the art to which this invention pertains, and include, but are not limited to, human IgG1, IgG2, IgG3, and IgG4. "Isotype" refers to an antibody class or subclass (e.g., IgM or IgG1) encoded by a heavy chain constant region gene. For example, the term "antibody" includes naturally occurring and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human or non-human antibodies; fully synthetic antibodies; and single-chain antibodies. Non-human antibodies can be humanized by recombinant methods to reduce their immunogenicity in humans. Unless the context otherwise requires, the term "antibody" also includes any antigen-binding fragment or antigen-binding molecule of any of the above-described immunoglobulins, and includes monovalent and bivalent fragments or partial and single-chain antibodies (i.e., scFv).

[0068] As used herein, the terms “single-chain antibody” and “single-chain fragment variable (scFv)” are used interchangeably and refer to antigen-binding molecules in which the VL and VH regions are linked by a linker to form a continuous protein chain, wherein the linker is long enough that the protein chain folds back to form a monovalent antigen-binding site (see, for example, Bird et al., (1988) Science 242: 423-26 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85: 5879-83 (1988).

[0069] As used herein, the term "diabody" or "dAB" refers to a bivalent antibody comprising two polypeptide chains, each containing VH and VL domains linked by a linker that is too short to pair between the two domains on the same chain, thus allowing each domain to pair with a complementary domain on the other polypeptide chain (see, for example, Holliger et al., (1993) Proc Natl Acad Sci USA90: 6444-48, Poljak et al., (1994) Structure2: 1121-23, and Perisic et al., (1994) Structure2(12): (1217-26). If the two polypeptide chains of a bisomic antibody are identical, then the bisomic antibodies derived from their pairing will have two identical antigen-binding sites. Polypeptide chains with different sequences can be used to manufacture bisomic antibodies with two different antigen-binding sites. Similarly, tribody and tetrabody antibodies are antibodies containing three and four polypeptide chains, respectively, and forming three and four antigen-binding sites, which can be identical or different.

[0070] As used herein, the terms "Fab fragment" refer to a monovalent fragment having VL, VH, CL, and CH domains; "F(ab')2 fragment" refer to a bivalent fragment having two Fab fragments (connected by disulfide bridges in the hinge region); "Fv fragment" refer to a single arm of an antibody with VH and VL domains; and "dAb fragment" refer to a VH domain, VL domain, or antigen-binding fragment having either a VH or VL domain.

[0071] "Antigen-binding molecule," "antigen-binding moiety," or "antibody fragment" refers to any molecule containing an antigen-binding moiety (e.g., a CDR) of an antibody derived from that molecule. An antigen-binding molecule may include an antigen complementarity-determining region (CDR). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, dAb, linear antibodies, scFv antibodies, and multispecific antibodies formed from an antigen-binding molecule. Peptide antibodies (i.e., Fc fusion molecules containing a peptide-binding domain) are another example of suitable antigen-binding molecules. In some specific embodiments, the antigen-binding molecule binds to an antigen on tumor cells. Antigen binding. In some specific embodiments, the antigen-binding molecule binds to antigens, viral antigens, or bacterial antigens on cells involved in hyperproliferative diseases. In some specific embodiments, the antigen-binding molecule binds to CD70. In a further specific embodiment, the antigen-binding molecule is an antibody containing a fragment thereof (including one or more of its complementarity-determining regions (CDRs)). In a further specific embodiment, the antigen-binding molecule is a single-chain variable fragment (scFv). In some specific embodiments, the antigen-binding molecule comprises or is composed of high-affinity polymers (avimers).

[0072] Antigen-binding molecules may comprise, for example, alternative protein scaffolds or artificial scaffolds having a transplanted complementarity-determining region (CDR) or a CDR derivative. These scaffolds include, but are not limited to, antibody-derived scaffolds containing introduced mutations to, for example, stabilize the three-dimensional structure of the antigen-binding molecule and fully synthetic scaffolds containing, for example, biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1): 121-129 (2003); Roque et al., Biotechnol. Prog.20: 639-654 (2004). Furthermore, peptide antibody mimics (“PAM”) and antibody mimic-based scaffolds (which utilize various components, such as fibronectin, as scaffolds) can be used. Antigen-binding molecules may have, for example, the structure of naturally occurring immunoglobulins.

[0073] Antigen-binding molecules can form components of CARs or TCRs and can act to guide CARs or TCRs to recognize targets of interest (e.g., CD70). As used herein, in the context of the disclosed CAR or TCR, antigen-binding molecule means any component of a CAR or TCR that guides the CAR or TCR to and associates with the desired target. In a particular embodiment, the antigen-binding molecule component of the CAR or TCR includes scFv, which contains heavy chain and light chain variable regions linked by linkers. The heavy chain and light chain variable regions can be derived from the same antibody or two different antibodies. The antigen-binding molecule used in the CAR or TCR can be derived from a known antibody or an antibody suspected of binding to a target of interest. In a particular embodiment, the antigen-binding molecule used in the CAR or TCR includes sequence pairs comprising the amino acid sequences of SEQ ID NO: 3 and 5, SEQ ID NO: 7 and 9, and SEQ ID NO: 11 and 13, as shown in Figures 7A-7F.

[0074] As used herein, the terms "variable region" or "variable domain" are used interchangeably and refer to a portion of an antibody, generally a portion of the light or heavy chain, typically the amino terminus of the antibody, comprising approximately 100 to 130 amino acids in the heavy chain and approximately 90 to 115 amino acids in the light chain. This varies considerably in sequence between antibodies and is used to determine the binding and specificity of a particular antibody to its specific antigen. Sequence variability is concentrated in regions called complementarity-determining regions (CDRs), while regions within the variable domain that are more highly preserved are called framework regions (FRs). The CDRs of both the light and heavy chains are primarily responsible for antibody-antigen interactions and specificity.

[0075] In some embodiments, the variable region is a human variable region. In further embodiments, the variable region includes a rodent, human, or mouse CDR and a human frame region (FR). In further embodiments, the variable region is a primate (e.g., a non-human primate) variable region. In yet another embodiment, the variable region is a rabbit variable region. In other embodiments, the variable region includes a human CDR and a non-human (e.g., rabbit, mouse, rat, or non-human primate) frame region (FR). In other embodiments, the variable region includes a non-human (e.g., rabbit, mouse, rat, or non-human primate) CDR and a human frame region (FR).

[0076] The terms “VLˮ”, “VL chainˮ”, or “VL domainˮ” are used interchangeably and refer to the variable region of the light chain of an antigen-binding molecule, antibody, or its antigen-binding fragment.

[0077] The terms “VHˮ”, “VH chainˮ”, and “VH domainˮ” are used interchangeably and refer to antigen-binding molecules, antibodies, or the variable region of the heavy chain of their antigen-binding fragments.

[0078] As used herein, the term "complementarity-determining region" or "CDR" refers to an amino acid sequence that contributes to antigen-binding specificity and affinity. Frame regions help maintain the correct identification of CDRs to facilitate binding between antigen-binding molecules and antigens. Many definitions of CDRs are commonly used: Kabat number, Chothia number, contact number, AbM number, or IMGT number. The AbM definition is a compromise between two definitions used by the Oxford Molecular AbM antibody modeling software. The contact definition is based on the analysis of available complex crystal structures. Table 1 summarizes the definitions of these nomenclature systems.

[0079]

[0080] The term "Kabat number" and similar terms are recognized in the art and refer to a system of numbered amino acid residues in the variable regions of the heavy and light chains of antibodies or their antigen-binding molecules. In some states, the CDR of an antibody can be determined according to the Kabat numbering system (see, for example, Kabat et al., in Sequences of [Proteins of Immunological Interest], 5th Ed., NIH Publication 91-3242, Bethesda MD 1991). Using the Kabat numbering system, intramolecular CDRs in antibody heavy chain molecules are typically located at amino acid positions 31 to 35 (which may include one or two additional amino acids after 35, referred to as 35A and 35B in the Kabat numbering scheme) (CDR1), amino acid positions 50 to 65 (CDR2), and amino acid positions 95 to 102 (CDR3). Using the Kabat numbering system, intramolecular CDRs in antibody light chain molecules are typically located at amino acid positions 24 to 34 (CDR1), amino acid positions 50 to 56 (CDR2), and amino acid positions 89 to 97 (CDR3).

[0081] In specific embodiments, the CDRs of the antibodies described herein can be described according to the Kabat numbering scheme, as shown in Figure 6 (although they can be easily deduced using other numbering systems in Table 1 above). Figure 6 also provides CDRs using the Chothia and IMGT numbering schemes.

[0082] In some morphologies, the CDR of the antibody can be determined according to the Chothia numbering scheme, which refers to the position of the loop in the immunoglobulin structure (see, for example, Chothia C & Lesk AM, (1987), J Mol Biol 196: 901-917; Al-Lazikani B et al., (1997) J Mol Biol 273: 927-948; Chothia C et al., (1992) J Mol Biol 227: 799-817; Tramontano A et al., (1990) J Mol Biol 215(1): 175-82; and U.S. Patent No. 7,709,226). Typically, when using the Kabat numbering rules, the Chothia CDR-H1 ring is present in heavy chain amino acids 26 to 32, 33, or 34; the Chothia CDR-H2 ring is present in heavy chain amino acids 52 to 56; and the Chothia CDR-H3 ring is present in heavy chain amino acids 95 to 102. The Chothia CDR-L1 ring is present in light chain amino acids 24 to 34; the Chothia CDR-L2 ring is present in light chain amino acids 50 to 56; and the Chothia CDR-L3 ring is present in light chain amino acids 89 to 97. When using the Kabat numbering rules, the termination of the Chothia CDR-HI ring varies between H32 and H34 depending on the ring length (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35A nor 35B exists, the ring ends at 32; if only 35A exists, the ring ends at 33; if both 35A and 35B exist, the ring ends at 34). See Table 1.

[0083] In a particular embodiment, the CDR of the antibody described herein has been determined according to the Chothia numbering scheme, as shown in Figure 6.

[0084] As used herein, the terms “constant region” and “constant domain” are interchangeable and have the meanings commonly used in the art. A constant region is an antibody portion, such as the carboxyl-terminal portion of the light and / or heavy chains that does not directly participate in antibody-antigen binding but can exhibit various effector functions (such as interaction with Fc receptors). Compared to the variable domains of immunoglobulins, the constant regions of immunoglobulin molecules typically have a more preserved amino acid sequence.

[0085] As used herein, when relating to antibody use, the term “heavy chain” based on an amino acid sequence of constant domains can refer to any different type, such as α, δ, ε, γ, and μ, which result in antibody classes IgA, IgD, IgE, IgG, and IgM, including subclasses of IgG, such as IgG1, IgG2, IgG3, and IgG4.

[0086] As used herein, when relating to antibody use, the term "light chain" based on an amino acid sequence with a constant structural domain can refer to any different type, such as κ or λ. Light chain amino acid sequences are well known in the art. In a particular specific embodiment, the light chain is a human light chain.

[0087] As used herein, the term “binding affinity” refers to the sum of the strengths of non-covalent interactions between a single binding site of a molecule (e.g., an antigen-binding molecule, such as an antibody) and its binding partner (e.g., an antigen). Unless otherwise stated, “binding affinity” as used herein refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y is typically represented by the dissociation constant (KD). Affinity can be measured and / or represented in a variety of ways known in the art, including, but not limited to, the equilibrium dissociation constant (KD) and the equilibrium association constant (KA). KD is calculated from the quotient of kopen / kopen, while KA is calculated from the quotient of kopen / kclose. kopen refers, for example, the association rate constant between an antibody and an antigen, while krelationship refers, for example, the dissociation rate constant between an antibody and an antigen. The k-on and k-off can be determined by standard techniques known to those skilled in the art to which this invention pertains (such as BIAcore® or KinExA or surface plasma resonance).

[0088] As used herein, “conservative amino acid substitution” refers to an amino acid residue in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having side chains have been defined in the art. These families include amino acids having the following side chains: basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), and uncharged polar side chains (e.g., glycine, asparagine, glutamic acid, serine, threonine, tyrosine). The amino acid residues may include: cysteine, tryptophan; nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine); β-branched side chains (e.g., threonine, valine, isoleucine); and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histamine). In some specific embodiments, one or more amino acid residues within the CDR of the antibody or its antigen-binding fragment, or within the frame region, may be replaced by amino acid residues having similar side chains.

[0089] The conserved amino acid substitutions covered in this disclosure can include non-naturally occurring amino acid residues that are typically incorporated through chemical peptide synthesis rather than synthesis in biological systems. These include peptide mimics and other inverted or reversed forms of amino acid moieties. Naturally occurring residues can be categorized based on their common side-chain properties: Hydrophobic: Leucine, Met, Ala, Val, Leu, Ile; Neutral and hydrophilic compounds: Cys, Ser, Thr, Asn, Gln; Acidic: Asp, Glu; Alkaline: His, Lys, Arg; Residues affecting chain orientation: Gly, Pro; and Aromatic compounds: Trp, Tyr, Phe.

[0090] Non-conservative substitutions may involve exchanging members from one class with members from another class. These substituted residues may be introduced, for example, into regions of human antibodies homologous to non-human antibodies, or into non-homologous regions of the molecule. Exemplary conserved amino acid substitutions are illustrated in Table 2 below.

[0091]

[0092] As used herein, “antigen determinant” is a term in the art and refers to a local region of an antigen that an antibody can specifically bind to. An antigen determinant can be, for example, a continuous amino acid of a polypeptide (linear or continuous antigen determinant), or an antigen determinant can be, for example, a collection of two or more discontinuous regions (configurational, nonlinear, discontinuous, or non-continuous antigen determinants) of a polypeptide or group of polypeptides. In some specific embodiments, the antigen determinant that binds to the antibody can be determined by, for example, NMR spectroscopy, X-ray diffraction crystallography, ELISA analysis, hydrogen / deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography, electrospray mass spectrometry), array-based oligopeptide scanning analysis, and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). In X-ray crystallography, crystallization can be performed using any method known in the art (e.g., Giege et al., (1994) Acta Crystallogr D Biol Crystallogr50 (Pt 4): 339-350; McPherson, (1990) Eur J Biochem 189: 1-23; Chayen, (1997) Structure 5: 1269-1274; McPherson, (1976) J Biol Chem 251: 6300-6303. Antibodies: Antigen crystals can be studied using well-known X-ray diffraction techniques and can be refined using computer software, such as using X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, for example, Meth Enzymol (1985) Vols 114 & 115, eds Wyckoff et al.) and BUSTER (Bricogne, (1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1): 37-60;Bricogne, (1997) Meth Enzymol 276A: 361-423, ed. Carter; Roversi et al., (2000) Acta Crystallogr D Biol Crystallogr 56 (Pt 10): 1316-1323. Mutagenesis mapping studies can be performed using any method known to a person of ordinary skill in the art.See, for example, Champe et al., (1995) J Biol Chem 270: 1388-94 and Cunningham & Wells, (1989) Science 244: 1081-85, for descriptions of mutagenesis techniques, including alanine and arginine scanning mutagenesis.

[0093] As used herein, the term "cross-competition" refers to a situation where the interaction between an antigen (e.g., CD70) and a first antigen-binding molecule or its binding fragment blocks, restricts, inhibits, or reduces the ability of a reference antigen-binding molecule or its binding fragment (such as the antigen-binding molecules, CARs, and TCRs provided herein) to interact with the antigen. Cross-competition can be complete, for example, the binding of the binding molecule to the antigen completely blocks the ability of the reference binding molecule to bind to the antigen, or cross-competition can be partial, for example, the binding of the binding molecule to the antigen reduces the ability of the reference binding molecule to bind to the antigen. In some specific embodiments, the antigen-binding molecule that cross-competes with the reference antigen-binding molecule binds to the same or overlapping antigenic determinants as the reference antigen-binding molecule. In other specific embodiments, the antigen-binding molecule that cross-competes with the reference antigen-binding molecule binds to a different antigenic determinant than the reference antigen-binding molecule. Various types of competitive binding assays can be used to determine whether an antigen-binding molecule competes with another molecule, such as: solid-phase direct or indirect radioimmunoassay (RIA); solid-phase direct or indirect enzyme immunoassay (EIA); sandwich competitive assay (Stahli et al., (1983)). Methods: Enzymol 9:242-53); Solid-phase direct biotin-avidin EIA (Kirkland et al., (1986) J Immunol 137:3614-19); Solid-phase direct labeling analysis, solid-phase direct labeling sandwich analysis (Harlow and Lane, 1988, [Antibodies, A Laboratory Manual], Cold Spring Harbor Press); solid-phase direct labeling of RIA using I 125 (Morel et al., (1988) Molec Immunol 25:7-15); solid-phase direct biotin-avidin EIA (Cheung et al., (1990) Virology 176:546-52); and directly labeled RIA (Moldenhauer et al., (1990) Scand J Immunol 32:77-82).

[0094] As used herein, in the context of antibodies, the terms "immunospecific binding," "immunospecific recognition," "specific binding," and "specific recognition" are similar terms, and as understood by one of ordinary skill in the art to which this invention pertains, such binding refers to a molecule that binds to an antigen (e.g., an antigenic determinant or immune complex). For example, a molecule that specifically binds to an antigen may, generally, bind to other peptides or polypeptides with low affinity, such affinity being determined by, for example, immunoassays, BIAcore®, KinExA 3000 instruments (Sapidyne Instruments, Boise, ID), or other analytical assays known in the art. In a particular embodiment, the molecule that specifically binds to an antigen binds to the antigen with a KA of at least 2 log, 2.5 log, 3 log, 4 log, or a larger KA than when the molecule binds to another antigen.

[0095] In another specific embodiment, the molecule that specifically binds to the antigen (e.g., CD70), and the molecule including this sequence and the cell presenting this molecule, bind with a dissociation constant (Kd) of about 1 × 10⁻⁷ M. In some specific embodiments, when Kd is from about 1 × 10⁻⁹ M to about 5 × 10⁻⁹ M, the antigen-binding molecule binds to the antigen (e.g., CD70) with “high affinity.” In some specific embodiments, when Kd is from 1 × 10⁻¹⁰ M to about 5 × 10⁻¹⁰ M, the antigen-binding molecule binds to the antigen (e.g., CD70) with “very high affinity.”

[0096] In another specific embodiment, the molecule that specifically binds to the antigen does not cross-react with other proteins under similar binding conditions. In another specific embodiment, the molecule that specifically binds to the antigen does not cross-react with other non-CD70 proteins. In a particular specific embodiment, an antibody or fragment thereof is provided herein that has a higher affinity for binding to CD70 than for binding to another unrelated antigen. In some specific embodiments, an antibody or fragment thereof is provided herein that has an affinity for binding to CD70 (e.g., human CD70) that is 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more higher than for binding to another unrelated antigen, the binding affinity being measured by, for example, radioimmunoassay, surface plasma resonance, or kinetic exclusion analysis. In a particular embodiment, the anti-CD70 antibody or its antigen-binding fragment described herein binds to unrelated non-CD70 proteins to a degree that is 10%, 15%, or 20% less than the degree to which the antibody binds to CD70 proteins (this is measured, for example, by radioimmunoassay).

[0097] In one particular embodiment, this document provides an antibody or fragment thereof that has a higher affinity for binding to human CD70 than for binding to CD70 of another species. In some embodiments, this document provides an antibody or fragment thereof that has an affinity for binding to human CD70 that is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or higher than for binding to CD70 of another species, wherein the binding affinity is measured by, for example, radioimmunoassay, surface plasma resonance, or kinetic exclusion analysis. In one particular embodiment, the antibody or fragment thereof described herein that binds to human CD70 will bind to CD70 protein of another species by 10%, 15%, or 20% less than the antibody or fragment thereof binds to human CD70 protein, wherein the binding is measured by, for example, radioimmunoassay, surface plasma resonance, or kinetic exclusion analysis.

[0098] "Antigen" refers to any molecule that can elicit an immune response or can be bound by an antibody or antigen-binding molecule. An immune response may involve antibody production or activation of specific immune cells, or both. Those skilled in the art to which this invention pertains will readily understand that any macromolecule (including almost all proteins or peptides) can serve as an antigen. Generally, antigens can be endogenously expressed, i.e., expressed by genomic DNA, or recombinantly expressed, or chemically synthesized. Antigens can be specific to certain tissues, such as cancer cells, or they can be broadly expressed. Furthermore, fragments of larger molecules can serve as antigens. In one specific embodiment, the antigen is a tumor antigen. In some specific embodiments, the antigen is CD70, which may be conjugated with an adjuvant (such as keyhole limpet hemocyanin (KLH)) as needed.

[0099] The term "neutralization" refers to an antigen-binding molecule, scFv, antibody, or fragment thereof that binds to a ligand (e.g., CD70) and prevents or reduces the biological effects of that ligand. In some specific embodiments, the antigen-binding molecule, scFv, antibody, or fragment thereof directly blocks the binding site on the ligand or alters the binding ability of the ligand through indirect means (such as structural or energy changes of the ligand). In some specific embodiments, the antigen-binding molecule, scFv, antibody, or fragment thereof prevents the protein to which it binds from performing its biological function.

[0100] As used in this article, the term "CD70" refers to molecules belonging to the tumor necrosis factor (TNF) ligand family, and is also known as CD27LG and TNFSF7. CD70 is a ligand of CD27 (TNFRSF27). It is a surface antigen on activated, but not quiescent, T and B lymphocytes. It induces the proliferation of co-stimulated T cells, enhances the production of cytotoxic T cells, and contributes to T cell activation. CD70 also plays a role in regulating B-cell activation, the cytotoxic function of natural killer cells, and immunoglobulin synthesis. See, for example, Goodwin et al., (1993) Cell73(3): 447-56 and Bowman et al., (1994) J. Immunol. 152(4): 1756-61. The term CD70 may include, but is not limited to, native CD70, CD70 isoforms, or interspecies CD70 homologs. CD70 (also known as CD27LG, CD27L, and TNFSF7) is expressed on the surface of T and B cell lymphoma cells. The amino acid sequence of human CD70 (hCD70) is provided in NCBI Accession NP_001243.1 (GI:4507605) (SEQ ID NO:1) and has the following amino acid sequence:

[0101] Residues 1 to 17 of SEQ ID NO:1 correspond to the cytoplasmic region of hCD70, residues 18 to 38 correspond to the transmembrane region of hCD70, and residues 39 to 193 correspond to the extracellular region of hCD70. At least one other isoform of hCD70 is known and identified by UniProt identifier P32970-2.

[0102] As used herein, CD70 includes human CD70 and non-human CD70 homologs, as well as their variants, fragments, or translated modified forms, including but not limited to, N- and O-linked glycosylated forms of CD70. The CD70 protein may further include fragments comprising all or part of SEQ ID NO:1 (e.g., amino acids 39 to 193 of SEQ ID NO:1, corresponding to the extracellular component of hCD70, and amino acids 18 to 193, corresponding to the transmembrane and extracellular components of hCD70).

[0103] As used herein, the term "autologous" refers to any substance that is derived from the same individual as which it is later reintroduced. For example, the engineered autologous cell therapy (eACT™) method described herein involves collecting lymphocytes from a patient, then engineering them to represent constructs, such as CAR constructs, and then reintroducing them into the same patient.

[0104] The term "allogeneic" refers to any substance derived from another individual of the same species as the individual into which it is later introduced, such as allogeneic T-cell transplantation.

[0105] As used herein, the terms “transduction” and “transduced” refer to a process by which exogenous DNA is introduced into cells via a viral vector (see Hartl and Jones (1997)). [Genetics: Principles and Analysis], 4th thed, Jones & Bartlett). In some specific embodiments, the vector is a retroviral vector, DNA vector, RNA vector, adenovirus vector, baculovirus vector, Epstein-Barr viral vector, papovaviral vector, vaccinia virus vector, herpes simplex virus vector, adenovirus-associated vector, lentiviral vector, or any combination thereof.

[0106] As used herein, the term "cancer" refers to a large group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth in the body lead to the formation of malignant tumors that invade adjacent tissues and can also metastasize to distant parts of the body via the lymphatic system or bloodstream. "Cancer" or "cancer tissue" may include tumors. Examples of cancers that can be treated by the methods disclosed herein include, but are not limited to, cancers of the immune system, including lymphoma, leukemia, myeloma, and other malignant white blood cell tumors. In some specific embodiments, the methods disclosed herein can be used to shrink, for example, those derived from the following groups. Tumor size: Bone cancer, pancreatic cancer, skin cancer, head or neck cancer, malignant melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, multiple myeloma, Hodgkin's disease, non-Hodgkin's lymphoma (NHL), primary mediastinal large B-cell lymphoma (PMBC), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), transformed follicular lymphoma, splenic marginal zone lymphoma (SMZ). Cancers of the esophagus, small intestine, endocrine system, thyroid, parathyroid, adrenal glands, soft tissue sarcomas, urethra, penis, chronic or acute leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia (ALL) (including non-T-cell ALL), chronic lymphocytic leukemia (CLL), solid tumors in children, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumors, etc. Tumor angiogenesis, spinal tumors, brainstem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, including those induced by asbestos, other B-cell malignancies, and combinations thereof. In some specific embodiments, the cancer is multiple myeloma. The particular cancer may respond to chemotherapy or radiation therapy or may not respond to such treatments. The term "refractory cancer" refers to cancer that cannot be surgically corrected and that either initially did not respond to chemotherapy or radiation therapy or became unresponsive over time.

[0107] As used in this article, the term "anti-tumor effect" refers to the reduction in tumor volume, number of tumor cells, proliferation of tumor cells, number of metastases, increase in overall survival or progression-free survival, increase in life expectancy, or improvement in various tumor-related physiological symptoms. Anti-tumor effect can also refer to the prevention of tumor development, such as through vaccines.

[0108] As used herein, the term "cytokine" refers to a non-antibody protein released by a cell in response to a specific antigen, wherein the cytokine interacts with a second cell to mediate the response in that second cell. Cytokines can be expressed endogenously by cells or administered to an individual. Cytokines can be released by immune cells (including macrophages, B cells, T cells, and mast cells) to amplify the immune response. Cytokines can induce various responses in recipient cells. Cytokines can include homeostatic cytokines, chemokines, pro-inflammatory cytokines, effectors, and acute-phase proteins. For example, homeostatic cytokines (including interleukin-7 (IL-7) and interleukin-15 (IL-15)) promote immune cell survival and proliferation, while pro-inflammatory cytokines promote inflammatory responses. Examples of homeostatic cytokines include, but are not limited to, IL-2, IL-4, IL-5, IL-7, IL-10, and IL-12p40. Examples of pro-inflammatory cytokines include, but are not limited to, IL-12p70, IL-15, and interferon (IFN)γ. Examples of pro-inflammatory cytokines include, but are not limited to, IL-1a, IL-1b, IL-6, IL-13, IL-17a, tumor necrosis factor (TNF)-α, TNF-β, fibroblast growth factor (FGF)2, granulocyte-macrophage colony-stimulating factor (GM-CSF), soluble intercellular adhesion molecule-1 (sICAM-1), soluble vascular adhesion molecule-1 (sVCAM-1), vascular endothelial growth factor (VEGF), VEGF-C, VEGF-D, and placental growth factor (PLGF). Examples of effectors include, but are not limited to, granzyme A, granzyme B, soluble Fas ligand (sFasL), and perforin. Examples of acute-phase proteins include, but are not limited to, C-reactive protein (CRP) and serum amyloid A (SAA).

[0109] As used herein, the term "chemokine" refers to a type of cytokine that mediates cellular chemotaxis or directed movement. Examples of chemokines include, but are not limited to, IL-8, IL-16, eotaxin, eotaxin-3, macrophage-derived chemokines (MDC or CCL22), monocyte chemotactic protein 1 (MCP-1 or CCL2), MCP-4, macrophage inflammatory protein 1α (MIP-1α, MIP-1a), MIP-1β (MIP-1b), γ-induced protein 10 (IP-10), and thymus activation-regulated chemokines (TARC or CCL17).

[0110] As used herein, the terms "therapeutic effective amount," "effective dose," "effective amount," and "therapeutic effective dose" for a therapeutic agent (e.g., engineered CAR T cells or cells expressing TCRs including the desired α chain, β chain, or both α and β chains) are used interchangeably and refer to any amount that, when used alone or in combination with another therapeutic agent, protects an individual from disease onset or promotes disease remission, evidence of which includes a reduction in the severity of disease symptoms, an increase in the frequency and duration of symptom-free periods, or prevention of injury or disability due to disease suffering. The ability of a therapeutic agent to promote disease remission can be assessed using a variety of methods known to a skilled practitioner, such as assessment in human individuals during clinical trials, assessment in animal model systems used to predict efficacy in humans, or assessment via analysis of the agent's activity in in vitro assays.

[0111] As used herein, the term "lymphocyte" refers to the white blood cells found in the immune system of vertebrates. Lymphocytes include natural killer (NK) cells, T cells, and B cells. NK cells are a type of cytotoxic lymphocyte that represents a major component of the innate immune system. NK cells repel tumor cells and virus-infected cells through apoptosis or programmed cell death. Because they do not require activation to kill cells, they are called "natural killers."

[0112] T cells play a crucial role in cell-mediated immunity (without antibody involvement). T cell types include: 1) Helper T cells (e.g., CD4+ cells); 2) Cytotoxic T cells (also known as TC, cytotoxic T lymphocytes, CTL, T killer cells, cytolytic T cells, CD8+ T- cells, or killer T cells); 3) Memory T cells, including: (i) Stem memory T SCM cells, similar to naïve cells, are CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+, and IL-7Rα+, but also exhibit abundant CD95, IL-2Rβ, CXCR3, and LFA-1, and display many unique functional properties of memory cells. (ii) Central memory TCM cells, exhibiting L-selectin and CCR7, secrete IL-2 but not IFNγ or IL-4; and (iii) Effector memory TEM cells, however, do not express L-selectin or CCR7, but produce effector cell mediators such as IFNγ and IL-4. 4) Regulatory T cells (Tregs, suppressor T cells, or CD4+CD25+ regulatory T cells); 5) Natural killer T cells (NKT); 6) γδ T cells; and 7) Mucosa-associated invariant T cells (MAIT).

[0113] B cells play a major role in humoral immunity (in which antibodies are involved). B cells produce antibodies and antigens, act as antigen-presenting cells (APCs), and transform into memory B cells after being activated by antigen-antigen interactions.

[0114] As used herein, the terms "genetically engineered" or "engineered" are used interchangeably and refer to methods of modifying the genome of a cell, including, but not limited to, deleting coding or non-coding regions or portions thereof, or inserting coding regions or portions thereof. In some specific embodiments, the modified cell is a lymphocyte (e.g., a T cell) that may be obtained from a patient or donor. The cell may be modified to express a foreign construct, such as, for example, a chimeric antigen receptor (CAR) or a T cell receptor (TCR) incorporated into the cell's genome.

[0115] As used herein, the term “immune response” refers to the action of cells of the immune system (such as T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, and neutrophils) and soluble macromolecules (including antibodies, cellular mediators, and complement) produced by any of these cells or the liver, which result in the selective targeting, binding, damage, destruction, and / or exclusion of invading pathogens, pathogen-infected cells or tissues, cancer cells or other abnormal cells, or normal human cells or tissues in cases of autoimmunity or pathological inflammation.

[0116] As used herein, the term "immunotherapy" refers to treatment of an individual who has or is at risk of developing a disease or a recurrence of a disease, which is performed by means of methods including inducing, enhancing, suppressing, or otherwise modifying an immune response. Examples of immunotherapy include, but are not limited to, T-cell therapy. T-cell therapy may include adoptive T-cell therapy, tumor-infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACT™), and allogeneic T-cell transplantation. However, those skilled in the art to which this invention pertains will recognize that the conditioning methods disclosed herein will enhance the effectiveness of any transplanted T-cell therapy. Examples of T-cell therapy are described in U.S. Patent Publication No. 2014 / 0154228, U.S. Patents Nos. 5,728,388 and 6,406,699, and International Publication No. WO 2008 / 081035.

[0117] T cells for immunotherapy can be derived from any source. For example, T cells can be differentiated from stem cell populations in vitro, or T cells can be obtained from an individual. T cells can also be obtained from, for example, peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors. In addition, T cells can be derived from one or more available T cell lines. T cells can also be obtained from blood units collected from an individual using any number of techniques known to those skilled in the art, such as FICOLL™ separation and / or hematologic separation. Additional methods for isolating T cells for T cell therapy are disclosed in U.S. Patent Publication No. 2013 / 0287748 (the entire contents of which are incorporated herein by reference).

[0118] The term "engineered autologous cell therapy" (abbreviated as "eACT™", also known as adoptive cell transfer) refers to the process of collecting a patient's own T cells and then genetically engineering them to recognize and target one or more antigens expressed on the surface of one or more specific tumor cells or malignant tumor cells. T cells may be engineered to express, for example, chimeric antigen receptors (CARs) or T-cell receptors (TCRs). CAR-positive (CAR+) T cell lines are engineered to express CARs. CARs may include, for example, extracellular single-stranded variable fragments (scFvs) specific to a particular tumor antigen, which are directly or indirectly linked to an intracellular signaling portion containing at least one co-stimulatory domain (which is directly or indirectly linked to at least one activation domain); the components may be arranged in any order. The co-stimulatory domain may be derived from, for example, CD28 or CD28T, while the activation domain may be derived from, for example, any form of CD3-ζ. In some specific embodiments, the CAR line is designed to have two, three, four, or more co-stimulatory domains. CAR scFv can be designed to target, for example, CD19, a transmembrane protein expressed by cells of B-cell lineages (including all normal B cells and B-cell malignancies such as NHL, CLL, and non-T-cell ALL). In some specific embodiments, the CAR lineage is engineered to express the co-stimulatory domains as individual polypeptide chains. Examples of CAR T-cell therapies and constructs are described in U.S. Patent Publications 2013 / 0287748, 2014 / 0227237, 2014 / 0099309, and 2014 / 0050708, the entire contents of which are incorporated herein by reference for any purpose.

[0119] As used herein, the term "in vitro cell" means any cell cultured in vitro. In vitro cells may include human cells, such as T cells or dendritic cells, or may include CHO, SP2 / O, rabbit, and other non-human cells. In some specific embodiments, in vitro cells may include T cells.

[0120] As used herein, the term "patient" means any person who is receiving treatment for an abnormal physiological condition (such as cancer) or has been formally diagnosed with a disorder, someone without a formally identified disorder, someone receiving medical care, someone at risk of developing a disorder, etc. The terms "individual" and "patient" are used interchangeably herein and include both human and non-human animal individuals. As used herein, the terms "patient" and "individual" are used interchangeably. In some specific embodiments, the term "individual" or "patient" means any person suffering from cancer (e.g., lymphoma or leukemia).

[0121] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to compounds comprising two or more amino acid residues linked together by peptide bonds. A protein or peptide must contain at least two amino acids, but there is no limit to the maximum number of amino acids that may comprise a protein or peptide sequence. A polypeptide includes any peptide or protein comprising two or more amino acids linked together by peptide bonds. As used herein, the term encompasses both short chains (also commonly referred to in the art as peptides, oligopeptides, and oligomers) and longer chains (commonly referred to in the art as proteins, which have many types). “Polypeptide” includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, etc. The term “polypeptide” includes native peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0122] In some forms, the polypeptide has one or more amino acids deleted, added, and / or substituted from the antigen-binding protein, and in some specific embodiments, preferably, the number of amino acid substitutions does not exceed 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Useful polypeptide fragments may include immunofunctional fragments of antigen-binding molecules, including, but not limited to, one or more CDR regions of the heavy chain and / or light chain, variable domains, portions of other parts of the antibody chain, etc. The portions that can be substituted by one or more amino acids of the antigen-binding molecule include, for example, amino acids of the D or L form, amino acids different from those commonly found at the same position in the antigen-binding molecule, deleted or non-naturally occurring amino acids, and chemical analogs of amino acids. Furthermore, polypeptide fragments of activating and / or co-stimulatory molecules are within the scope of this disclosure.

[0123] As used herein, the terms “activation,” “stimulation,” and “stimulatory signal” refer to a primary response induced by the binding of an activating molecule to its homologous ligand, wherein this binding mediates a signal transduction event. Examples of activating molecules are provided herein. In one example, “activating molecule” or “stimulatory molecule” refers to a molecule on a T cell (e.g., the TCR / CD3 complex) that specifically binds to a homologous stimulatory ligand present on an antigen-presenting cell. This type of signal transduction is sometimes referred to as “signal 1” in the context of T cell activation.

[0124] As used herein, "stimulatory ligand" refers to a ligand that, when present on antigen-presenting cells (e.g., APCs, dendritic cells, B cells, etc.), can specifically bind to stimulatory molecules on T cells, thereby mediating primary T cell responses (including, but not limited to, activation, initiation of immune responses, proliferation, etc.). Stimulatory ligands include, but are not limited to, class I MHC molecules carrying peptides, anti-CD3 antibodies, super-effector anti-CD28 antibodies, and super-effector anti-CD2 antibodies.

[0125] As used herein, the term "co-stimulatory signal" refers to a signal that, when combined with a primary signal (such as TCR / CD3 binding), elicits a T cell response (such as, but not limited to, proliferation and / or upregulation or downregulation of key molecules). In one instance, co-stimulatory signaling is induced when CD28 on a T cell associates with its homologous ligand B7 on an antigen-presenting cell. This type of signal transduction is sometimes referred to as "signal 2" in the context of T cell activation.

[0126] As used herein, the terms “co-stimulatory molecule” and “co-stimulatory ligand” refer to homologous binding partners on T cells that specifically bind to co-stimulatory ligands, thereby mediating co-stimulatory responses of T cells (such as, but not limited to, proliferation). Co-stimulatory molecules include, but are not limited to, CD2, CD3δ, CD3ε, CD3γ, CD4, CD7, CD8α, CD8β, CD11a (ITGAL), CD11b (ITGAM), CD11c (ITGAX), CD11d (ITGAD), CD18 (ITGB2), CD19 (B4), CD27 (TNFRSF7), CD28, CD28T, CD29 (ITGB1), CD30 (TNFRSF8), CD40 (TNFRSF5), CD48 (SLAMF2), CD49a (ITGA1), CD49d (ITGA4), CD49f (ITGA6), CD66a (CEACAM1), CD66b (CEACAM8), CD66c (CEACAM6), CD66d (CEACAM3), CD66e (CEACAM5), CD69 (CLEC2), and CD79A. (β-chain of B-cell antigen receptor complex-associated), CD79B (β-chain of B-cell antigen receptor complex-associated), CD84 (SLAMF5), CD96 (Tactile), CD100 (SEMA4D), CD103 (ITGAE), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD158A (KIR2DL1), CD158B1 (KIR2DL2), CD158B2 (KIR2DL3), CD158C (KIR3DP1), CD158D (KIRDL4), CD158F1 (KIR2DL5A), CD158F2 (KIR2DL5B), CD158K (KIR3DL2), CD160 (BY55), CD162 (SELPLG), CD226 (DNAM1), CD229 (SLAMF3), CD244 (SLAMF4), CD247 (CD3-ζ), CD258 (LIGHT), CD268 (BAFFR), CD270 (TNFSF14), CD272 (BTLA), CD276 (B7-H3), CD279 (PD-1), CD314 (NKG2D), CD319 (SLAMF7), CD335 (NK-p46), CD336 (NK-p44), CD337 (NK-p30), CD352 (SLAMF6), CD353 (SLAMF8), CD355(CRTAM), CD357 (TNFRSF18), inducible T cell costimulators (ICOS), LFA-1 (CD11a / CD18), NKG2C, DAP-10, ICAM-1, NKp80 (KLRF1), IL-2Rβ, IL-2Rγ, IL-7Rα, LFA-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1 / CBP, PD-1, CD83 ligand, Fcγ receptor, MHC class 1 molecules, MHC class 2 molecules, TNF receptor protein, immunoglobulin protein, cell mediator receptor, integrin, activated NK cell receptor, Toll ligand receptor, and fragments or combinations thereof.

[0127] The terms "reduction" and "reduction" are used interchangeably and both indicate any change less than the original. "Reduction" and "reduction" are relative terms, requiring a comparison before and after measurement. The terms "reduction" and "reduction" include complete depletion.

[0128] As used herein, the terms "treatment" for an individual and "treatment system" refer to any type of intervention or process performed on an individual, or the administration of an active agent to that individual to reverse, alleviate, improve, suppress, slow, or prevent the onset, progression, development, severity, or recurrence of disease-related symptoms, complications, or signs or biochemical indicators. In one specific embodiment, "treatment" includes partial remission, as defined in the context of a given treatment regimen. In another specific embodiment, "treatment" includes complete remission, as defined in the context of a given treatment regimen.

[0129] As used herein, the term "percentage identity" refers to the percentage of identical residues between amino acids or nucleotides in molecules being compared. In these calculations, gaps in the alignment (if any) must be handled using specific mathematical models or computer programs (i.e., "algorithms"). Methods that can be used to calculate the identity of aligned nucleic acids or peptides include those described in the following literature: [Computational Molecular Biology], (Lesk, ed.), (1988) New York: Oxford University Press; [Biocomputing Informatics and Genome Projects], (Smith, ed.), 1993, New York: Academic Press; [Computer Analysis of Sequence Data, Part I], (Griffin and Griffin, eds.), 1994, New Jersey: Humana Press; von Heinje, (1987) [Sequence Analysis in Molecular Biology], New York: Academic Press; [Sequence Analysis Primer], (Gribskov and Devereux, eds.), 1991, New York: M. Stockton Press; and Carillo et al., (1988) J. Applied Math.48: 1073.

[0130] When calculating the percentage of identity, the compared sequences are typically aligned in a way that maximizes the match between them. Computer programs that can be used to determine the percentage of identity include, for example, MOE (Chemical Computing Group) or DNASTAR (University of Wisconsin, Madison, WI). The GAP algorithm can be used to align two polypeptides or polynucleotides whose percentage of identity is to be determined. Align sequences to ensure optimal matching of individual amino acids or nucleotides (by means of the "match span" determined by the algorithm). A gap-opening penalty (calculated as 3 × mean diagonal, where the "mean diagonal" is the average of the diagonals of the comparison matrix used; "diagonal" is the score or number assigned to each perfect amino acid match by a specific comparison matrix) and a gap-extending penalty (typically 1 / 10 of the gap-opening penalty), along with comparison matrices (such as PAM 250 or BLOSUM 62), are used with the algorithm. In some specific embodiments, the algorithm also uses a standard comparison matrix (see, for example, Dayhoff et al., (1978)). [Atlas of Protein Sequence and Structure] 5: 345-352, used for PAM 250 comparison matrix; Henikoff et al., (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919, used for BLOSUM 62 comparison matrix).

[0131] Some alignment schemes used to align two amino acid sequences can result in a short matching region between the two sequences, and this small aligned region can have very high sequence identity, even if there is no obvious relationship between the two full-length sequences. Therefore, if needed, the selected alignment method (e.g., the GAP program) can be adjusted so that the alignment can span at least 50 consecutive amino acids of the target peptide.

[0132] The various forms disclosed herein are described in more detail in the following sections. II. Antigen-binding molecules and their encoded polynucleotides

[0133] This disclosure targets antigen-binding molecules (including antibodies) that specifically bind to CD70, molecules containing these sequences (CARs and TCRs described herein), and cells presenting such molecules, and / or antigen-binding molecules that cross-compete with one or more of the antigen-binding molecules described herein (i.e., one or more described in Figures 7A to 7H and / or disclosed in the appended sequence listings). Polynucleotides encoding antigen-binding molecules are also provided, forming the configuration of this disclosure. Antigen-binding molecules can form antigen-binding components of one or more of the CARs and TCRs disclosed herein.

[0134] The antibody or antigen-binding molecule encoded by this disclosure may be single-chain or double-chain. In some embodiments, the antibody or antigen-binding molecule is single-chain. In some embodiments, the antigen-binding molecule is selected from the group consisting of: scFv, Fab, Fab', Fv, F(ab')2, dAb, and any combination thereof. In some embodiments, the antibody or antigen-binding molecule comprises scFv. In some embodiments, the antigen-binding molecule comprises a single chain, wherein the heavy chain variable region and the light chain variable region are linked by a linker to form scFv (e.g., the antigen-binding molecule disclosed herein). In some embodiments, VH is located at the N-terminus of the linker and VL is located at the C-terminus of the linker. In other embodiments, VL is located at the N-terminus of the linker and VH is located at the C-terminus of the linker. In some specific embodiments, the linker comprises at least about 5, at least about 8, at least about 10, at least about 13, at least about 15, at least about 18, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, or at least about 100 amino acids. In some specific embodiments, the linker comprises between about 8 amino acids and about 18 amino acids (e.g., 10 amino acids). Examples of suitable linkers for binding VH to VL to form scFv (e.g., the antigen-binding molecule disclosed herein) include (Gly-Gly-Gly-Gly-Ser) 3 (SEQ ID NO: 80) and GSTSGSGGPGSGEGSTKG (SEQ ID NO: 81).

[0135] In some specific embodiments, the antigen-binding molecule disclosed herein specifically binds to CD70, molecules containing this sequence, and cells presenting this molecule. In some specific embodiments, the antigen-binding molecule disclosed herein specifically binds to CD70 with a KD of less than 1×10⁻⁶ M, less than 1×10⁻⁷ M, less than 1×10⁻⁸ M, or less than 1×10⁻⁹ M. In some specific embodiments, the antigen-binding molecule specifically binds to CD70 with a KD of less than 1×10⁻⁷ M. In another specific embodiment, the antigen-binding molecule specifically binds to SEQ ID NO:1, molecules containing this sequence, and cells presenting this molecule with a KD of less than 1×10⁻⁸ M. In some specific embodiments, the antigen-binding molecules are expressed in quantities of approximately 1×10⁻⁷ M, approximately 2×10⁻⁷ M, approximately 3×10⁻⁷ M, approximately 4×10⁻⁷ M, approximately 5×10⁻⁷ M, approximately 6×10⁻⁷ M, approximately 7×10⁻⁷ M, approximately 8×10⁻⁷ M, approximately 9×10⁻⁷ M, approximately 1×10⁻⁸ M, approximately 2×10⁻⁸ M, approximately 3×10⁻⁸ M, approximately 4×10⁻⁸ M, approximately 5×10⁻⁸ M, approximately 6×10⁻⁸ M, approximately 7×10⁻⁸ M, approximately 8×10⁻⁷ M, approximately 1×10⁻⁸ M, approximately 2×10⁻⁹ M, approximately 3×10⁻⁹ M, approximately 4×10⁻⁹ M, approximately 5×10⁻⁹ M, approximately 6×10⁻⁹ M, approximately 7×10⁻⁹ M, approximately 8×10⁻⁹ M, approximately 1 ... KD combined with CD70 at -9M, approximately 9×10⁻⁹M, approximately 1×10⁻¹⁰M, or approximately 5×10⁻¹⁰M. KD can be calculated using the standard methods described herein.

[0136] In specific embodiments, the antigen-binding molecules disclosed herein are antibodies or scFvs formed therefrom, identified herein as strain 8G1, strain 1C8, or 6E9. Each of the disclosed antigen-binding molecules comprises one of the following heavy and light chain amino acids, encoding, variable, and CDR sequences as provided and labeled:

[0137] In one specific embodiment, the antigen-binding molecule disclosed herein is an antibody and its antigen-binding fragment (e.g., scFv). In another specific embodiment, the antibody and antigen-binding molecule disclosed herein comprise at least one CDR listed in FIG6. In yet another embodiment, this disclosure provides a fusion tumor capable of generating the antibody and antigen-binding molecule disclosed herein, as described herein and known in the art, and a method for generating antibodies from the fusion tumor. []

[0138] The antigen-binding molecules disclosed herein can also be humanized monoclonal antibodies, from which scFv can be generated, which can then form components of the CAR or TCR provided herein. In one specific embodiment, the humanized monoclonal antibody comprises a variable domain (or all or part of its antigen-binding site) of a mouse or rabbit antibody and a constant domain derived from a human antibody. Alternatively, the humanized antibody fragment may comprise an antigen-binding site of a mouse or rabbit monoclonal antibody and a variable domain fragment derived from a human antibody (lacking the antigen-binding site). Procedures for generating engineered monoclonal antibodies include those described in the following literature: Riechmann et al., (1988) Nature 332: 323, Liu et al., (1987) Proc. Nat. Acad. Sci. USA 84: 3439, Larrick et al., (1989) Bio / Technology 7: 934, and Winter et al., (1993) TIPS 14: 139. In one specific embodiment, the chimeric antibody is an antibody transplanted with a CDR. The techniques used for humanized antibodies are discussed in, for example, the following literature: U.S. Patent Nos. 5,869,619; 5,225,539; 5,821,337; 5,859,205; 6,881,557; Padlan et al., (1995) FASEB J.9: 133-39; Tamura et al., (2000) J. Immunol. 164: 1432-41; Zhang et al., (2005) Mol. Immunol.42(12): 1445-1451; Hwang et al., Methods. (2005) 36(1): 35-42; Dall'Acqua et al., (2005) Methods36(1): 43-60; and Clark, (2000) Immunology Today21(8): 397-402. []

[0139] The antigen-binding molecule disclosed herein can also be a full-length human monoclonal antibody, in which scFv can be generated, which can then form the CAR or TCR components provided herein. The full-length human monoclonal antibody can be generated by any amount of technology familiar to those skilled in the art to which this invention pertains. Such methods include, but are not limited to: transfection of human peripheral blood cells (e.g., containing B lymphocytes) with Ebola virus (EBV), in vitro immunization of human B cells, fusion of spleen cells from immunized transgenic mice (carrying the inserted human immunoglobulin gene), isolation from a human immunoglobulin V region phage collection library, or other procedures as known in the art and based on the procedures disclosed herein. []

[0140] Procedures for generating human monoclonal antibodies in non-human animals have been developed. For example, mice in which one or more endogenous immunoglobulin genes have been deactivated by various methods have been prepared. Human immunoglobulin genes have been introduced into mice to replace the deactivated mouse genes. In this technique, elements of human heavy and light chain loci are introduced into mouse strains derived from embryonic stem cell lines containing endogenous heavy and light chain loci that have been targeted and disrupted (see also Bruggemann et al., (1997) Curr. Opin. Biotechnol. 8: 455-58). []

[0141] Examples of techniques for producing and using transgenic animals to generate human or partially human antibodies are described in the following documents: U.S. Patent Nos. 5,814,318, 5,569,825, and 5,545,806; Davis et al. [Antibody Engineering: Methods and Protoc]ols, (Lo, ed) Humana Press, NJ, 191-200 (2003); Kellermann et al., (2002) Curr Opin Biotechnol.13: 593-97; Russel et al., (2000) Infect Immun.68: 1820-26; Gallo et al., (2000) Eur J. Immun.30: 534-40; Davis et al., (1999) Cancer Metastasis Rev. 18: 421-25; Green, (1999) J Immunol Methods231:11-23; Jakobovits, (1998) Advanced Drug Delivery Reviews31: 33-42; Green et al., (1998) J Exp Med. 188: 483-95; Jakobovits, (1998) Exp. Opin. Invest. Drugs. 7:607-14; Tsuda et al., (1997) Genomics, 42: 413-21; Mendez et al., (1997) Nat. Genet. 15: 146-56; Jakobovits, (1994) Curr Biol. 4: 761-63; Arbones et al., (1994) Immunity 1: 247-60; Green et al., (1994) Nat. Genet. 7: 13-21; Jakobovits et al., (1993) Nature 362: 255-58; Jakobovits et al., (1993) Proc Natl Acad Sci USA 90: 2551-55; Chen et al., (1993) Intl Immunol 5: 647-656; Choi et al., (1993) Nature Genetics 4: 117-23; Fishwild et al., (1996) Nature Biotechnology 14: 845-51; Lonberg et al., (1994) Nature 368: 856-59; Lonberg, (1994) [Handbook of Experimental Pharmacology] 113: 49-101; Neuberger, (1996) Nature Biotech 14: 826; Taylor et al., (1992) Nucleic Acids Research 20: 6287-95; Taylor et al., (1994) Intl Immunol 6: 579-91; Tomizuka et al., (1997) Nature Genetics 16: 133-43; Tomizuka et al., (2000) Proc Nat Acad Sci USA 97: 722-27; Tuaillon et al., (1993) Proc Nat Acad Sci USA 90: 3720-24; Tuaillon et al., (1994) J Immunol 152: 2912-20; Lonberg et al., (1994) Nature 368: 856; Taylor et al., (1994) Intl Immunol 6: 579; U.S. Patent No. 5,877,397; Bruggemann et al., (1997) Curr. Opin. Biotechnol. 8: 455-58; Jakobovits et al., (1995) Ann. NY Acad. Sci. 764: 525-35. []

[0142] Another method for obtaining the antigen-binding molecules disclosed herein is the use of a phage display library, which has been well-established for this purpose. See, for example, Winter et al., (1994) Ann. Rev. Immunol. 12: 433-55; Burton et al., (1994) Adv. Immunol 57: 191-280. Human or mouse immunoglobulin variable region gene combination libraries can be created in phage vectors, which can be screened to select Ig fragments (Fab, Fv, sFv, or multimers thereof) that bind to scFv FMC63, molecules containing this sequence, and cells presenting such molecules. See, for example, U.S. Patent Application No. 5,223,409; Huse et al., (1989) Science 246: 1275-81; Sastry et al., (1989) Proc. Natl. Acad. Sci. USA 86: 5728-32; Alting-Mees et al., (1990) Strategies in Molecular Biology 3: 1-9; Kang et al., (1991) Proc. Natl. Acad. Sci. USA 88: 4363-66; Hoogenboom et al., (1992) J. Mol. Biol. 227: 381-388; Schlebusch et al., (1997) Hybridoma 16: 47-52 and the references cited therein. For example, a library containing multiple polynucleotide sequences encoding Ig variable region fragments can be inserted into the genome of filamentous phages (such as M13 or λ phages (λImmunoZap™(H) and λImmunoZap™(L) vectors (Stratagene, La Jolla, Calif) or variants thereof can also be used in this method), alongside sequences encoding phage coat proteins.

[0143] In short, mRNA is isolated from B cell populations and used to create aggregate libraries of heavy and light chain immunoglobulin cDNA expression in λImmunoZap™ (H) and λImmunoZap™ (L) vectors. These vectors can be individually screened or co-expressed to form Fab fragments or antibodies. Positive plaques can then be converted into non-lytic plasmids to allow for high expression of monoclonal antibody fragments from E. coli. []

[0144] In one specific embodiment, nucleotide primers are used to amplify the variable regions of genes expressing monoclonal antibodies of interest in fusion tumors. These primers can be synthesized by those skilled in the art to which this invention pertains, or can be purchased from commercial sources that also sell primers for mouse and human variable regions (including, in particular, primers for the VH, VL, CH, and CL regions). These primers can be used to amplify heavy or light chain variable regions, which can then be inserted into vectors. These vectors can then be introduced into E. coli, yeast, or mammalian-based systems for expression. Using these methods, large quantities of single-chain proteins containing fusions of the VH and VL domains can be produced. []

[0145] Once the cells producing antigen-binding molecules described herein are obtained using any of the aforementioned immunochemical and other techniques, the genes for specific antibodies can be selected from their DNA or mRNA by isolation and amplification according to the standard procedures described herein. The resulting antibodies can be sequenced and identified with CDRs, and the DNA encoding the CDR can be used to generate other antibodies according to this disclosure. []

[0146] Those skilled in the art will understand that some proteins (such as antibodies) can undergo various post-translational modifications. The type and extent of these modifications typically depend on the host cell line and culture conditions used to express the protein. Such modifications may include glycosylation, methionine oxidation, diketopigmentation, and other similar modifications. Variants formed, aspartic acid isomerized, and aspartic acid deacetylated. Common modifications include the loss of carboxyl-terminal basic residues (such as lysine or arginine) due to the action of carboxypeptidase (as described in, for example, Harris, (1995) J Chromatog 705: 129-34). []

[0147] Another method for generating mouse monoclonal antibodies involves injecting fusion tumor cells into the peritoneal cavity of syngeneic mice (e.g., pretreated mice sensitized with pristane) to promote the formation of ascites containing the monoclonal antibody. Monoclonal antibodies can be isolated and purified using various well-established techniques. These isolation techniques include affinity chromatography, size exclusion chromatography, and ion exchange chromatography using protein A agarose gel (see, for example, Baines and Thorpe, (1992) in...). [Methods in Molecular Biology], 10: 79-104 (The Humana Press). Monoclonal antibodies can be purified by affinity chromatography using appropriate ligands selected based on the specific properties of the antibody (e.g., heavy or light chain isotype, binding specificity, etc.). Examples of suitable ligands immobilized on solid supports include protein A, protein G, antibodies against constant regions (light or heavy chains), and antibodies against individual genotypes.

[0148] This disclosure provides antigen-binding molecules (e.g., scFv) that specifically bind to CD70, contain this sequence, and are present on cells. Antigen-binding molecules that cross-compete with the antigen-binding molecules disclosed herein form another morphology of this disclosure. []

[0149] In some specific embodiments, the antigen-binding molecule cross-competes with a reference antibody comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 5, 7, 9, 11, or 13. In some specific embodiments, the antigen-binding molecule cross-competes with a reference antibody comprising VH CDR1, which comprises the amino acid sequences of SEQ ID NO: 62, 65, 68, 71, 73, 75, 77, 78, and 79. []

[0150] In some specific embodiments, the antigen-binding molecule cross-competes with a reference antibody comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 3, 5, 7, 9, 11 or 13, wherein the reference antibody comprises VH CDR2, which comprises the amino acid sequences of SEQ ID NO: 63, 66, 69, 72, 74, 76, 63, 66 and 69. []

[0151] In some specific embodiments, the antigen-binding molecule cross-competes with a reference antibody comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 3, 5, 7, 9, 11 or 13, wherein the reference antibody comprises VH CDR3, which comprises the amino acid sequences of SEQ ID NO: 64, 67 and 70. []

[0152] In some specific embodiments, the antigen-binding molecule cross-competes with a reference antibody comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 3, 5, 7, 9, 11 or 13, wherein the reference antibody comprises VL CDR1, which comprises the amino acid sequences of SEQ ID NO: 53, 56 and 59. []

[0153] In some specific embodiments, the antigen-binding molecule cross-competes with a reference antibody comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 3, 5, 7, 9, 11 or 13, wherein the reference antibody comprises VL CDR2, which comprises the amino acid sequences of SEQ ID NO: 54, 57 and 60. []

[0154] In some specific embodiments, the antigen-binding molecule cross-competes with a reference antibody comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 3, 5, 7, 9, 11 or 13, wherein the reference antibody comprises VL CDR3, which comprises the amino acid sequences of SEQ ID NO: 55, 58 and 61. []

[0155] In some specific embodiments, antibodies or antigen-binding molecules that specifically bind to CD70, molecules containing this sequence, and cells presenting this molecule bind to the same or overlapping antigenic determinants as the reference antibody disclosed herein (e.g., those containing the sequence presented herein, which may include the CDR shown in FIG6).

[0156] In some specific embodiments, the antibody or antigen-binding molecule binds to the same or overlapping antigen determinants as the reference antibody. II.A. Selected strain 8G1 In some specific embodiments, antigen-binding molecules that specifically bind CD70, molecules containing this sequence, and cells presenting this molecule, such as scFv, or antibodies, include VH CDR1, which comprises, is composed of, or is substantially composed of the following amino acid sequence: GFTFSSY (SEQ ID NO: 71).

[0157] In some specific embodiments, antigen-binding molecules that specifically bind CD70, molecules containing this sequence, and cells presenting this molecule, such as scFv, or antibodies, include VH CDR2, which comprises, is composed of, or is substantially composed of the following amino acid sequence: WYDGSN (SEQ ID NO: 72).

[0158] In some specific embodiments, antigen-binding molecules that specifically bind CD70, molecules containing this sequence, and cells presenting this molecule, such as scFv, or antibodies, include VH CDR3, which comprises, is composed of, or is substantially composed of the following amino acid sequence: DLLRGVKGYAMDV (SEQ ID NO: 64).

[0159] In some specific embodiments, antigen-binding molecules that specifically bind CD70, molecules containing this sequence, and cells presenting this molecule, such as scFv or antibodies, contain a heavy chain VH, which includes (a) VH CDR1, which includes, is composed of, or is substantially composed of the following amino acid sequence: GFTFSSY (SEQ ID NO: 71); and / or (b) VH CDR2, which includes, is composed of, or is substantially composed of the following amino acid sequence: WYDGSN (SEQ ID NO: 72); and / or (c) VH CDR3, which includes, is composed of, or is substantially composed of the following amino acid sequence: DLLRGVKGYAMDV (SEQ ID NO: 64).

[0160] In some specific embodiments, antibodies or antigen-binding molecules, such as scFv, that specifically bind to CD70, molecules containing this sequence, and cells presenting this molecule, include VH CDR1, VH CDR2, and VH CDR3, wherein VH CDR1, VH CDR2, and VH CDR3 contain the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 as shown in Figure 6.

[0161] In some specific embodiments, antibodies or antigen-binding molecules, such as scFv, that specifically bind to CD70, molecules containing this sequence, and cells presenting this molecule, include VH CDR1, VH CDR2, and VH CDR3, wherein VH CDR1, VH CDR2, and VH CDR3 contain the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of the antigen-binding molecules shown in Figures 6 and 7A (SEQ ID NO: 71, 72, and 64, respectively).

[0162] In some specific embodiments, antibodies or antigen-binding molecules, such as scFv, that specifically bind to CD70, molecules containing this sequence, and cells presenting this molecule, include a heavy chain variable region sequence containing the amino acid sequence of Figure 7A. In some specific embodiments, the antibody or antigen-binding molecule includes a heavy chain variable region sequence containing SEQ ID NO:3.

[0163] In some specific embodiments, antibodies or antigen-binding molecules, such as scFv, that specifically bind to CD70, molecules containing this sequence, and cells presenting this molecule, include one or more of any of the VH CDRs listed above or described in Figure 6. In some specific embodiments, the antibody or antigen-binding molecule includes the VH frame region (FR) described herein. In particular, the antibody or antigen-binding molecule includes the sequence shown in Figure 7A (e.g., FR one, two, three, or four in a sequence of Figure 7A) or VH FRs that may be derived from the sequence shown in Figure 7A.

[0164] In some specific embodiments, antibodies or antigen-binding molecules, such as scFv, which specifically bind to CD70, molecules containing this sequence, and cells presenting this molecule, and which contain heavy chain sequences as disclosed herein (e.g., in Figure 7A), are used. In one specific embodiment, the antibody or antigen-binding molecule includes a heavy chain variable region containing the following amino acid sequence.

[0165] In various specific embodiments, the heavy chain variable region and the heavy chain variable region sequence of SEQ ID NO:3 have 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0166] In some specific embodiments, antigen-binding molecules that specifically bind CD70, molecules containing this sequence, and cells presenting this molecule, such as scFv, or antibodies, include VL CDR1, which comprises, is composed of, or is substantially composed of the following amino acid sequence: RASQSLRRIYLA (SEQ ID NO: 53).

[0167] In some specific embodiments, antigen-binding molecules that specifically bind CD70, molecules containing this sequence, and cells presenting this molecule, such as scFv, or antibodies, include VL CDR2, which comprises, is composed of, or is substantially composed of the following amino acid sequence: DVFDRAT (SEQ ID NO: 54).

[0168] In some specific embodiments, antigen-binding molecules that specifically bind CD70, molecules containing this sequence, and cells presenting this molecule, such as scFv, or antibodies, include VL CDR3, which comprises, is composed of, or is substantially composed of the following amino acid sequence: QQYSDSPFT (SEQ ID NO: 55).

[0169] In some specific embodiments, antigen-binding molecules that specifically bind CD70, molecules containing this sequence, and cells presenting this molecule, such as scFv, or antibodies, comprise heavy chain VL, which comprises: (a) VL CDR1, which comprises, is composed of, or is substantially composed of the following amino acid sequence: RASQSLRRIYLA (SEQ ID NO: 53); and / or (b) VL CDR2, which comprises, is composed of, or is substantially composed of the following amino acid sequence: DVFDRAT (SEQ ID NO: 54); and / or (c) VL CDR3, which comprises, is composed of, or is substantially composed of the following amino acid sequence: QQYSDSPFT (SEQ ID NO: 55).

[0170] In some specific embodiments, antibodies or antigen-binding molecules, such as scFv, specifically bind to CD70, molecules containing this sequence, and cells presenting this molecule. The antibody or antigen-binding molecule includes VL CDR1, VL CDR2, and VL CDR3, wherein VL CDR1, VL CDR2, and VL CDR3 contain the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 as shown in Figure 6.

[0171] In some specific embodiments, antibodies or antigen-binding molecules, such as scFv, that specifically bind to CD70, molecules containing this sequence, and cells presenting this molecule, include a light chain variable region sequence containing the amino acid sequence of Figure 7B. In some specific embodiments, the antibody or antigen-binding molecule includes a light chain variable region sequence containing SEQ ID NO: 5.

[0172] In some specific embodiments, antibodies or antigen-binding molecules, such as scFv, specifically bind to CD70, molecules containing this sequence, and cells presenting this molecule, wherein the antibody or antigen-binding molecule contains the VL frame region (FR) as described herein. In certain specific embodiments, the antibody or antigen-binding molecule contains the sequence shown in Figure 7B (e.g., FR one, two, three, or four in a sequence of Figure 7B) or an amino acid sequence that may be derived from the sequence shown in Figure 7B.

[0173] In some specific embodiments, antibodies or antigen-binding molecules, such as scFv, that specifically bind to CD70, molecules containing this sequence, and cells presenting this molecule, contain the light chain sequence disclosed herein (e.g., in Figure 7B). In one specific embodiment, the antibody or antigen-binding molecule contains a light chain variable region containing the following amino acid sequence.

[0174] In various specific embodiments, the light chain variable region and the light chain variable region sequence of SEQ ID NO:4 have 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0175] In some specific embodiments, antibodies or antigen-binding molecules, such as scFv, that specifically bind to CD70, molecules containing this sequence, and cells presenting this molecule, contain one, two, and / or three VH CDR sequences disclosed herein. In some specific embodiments, the antibody or antigen-binding molecule contains VH CDR1, VH CDR2, and VH CDR3, each having an amino acid sequence of one of the VH CDR1, VH CDR2, and VH CDR3 disclosed herein. In some specific embodiments, the antibody or antigen-binding molecule contains one, two, and / or three VL CDR sequences disclosed herein. In some specific embodiments, the antibody or antigen-binding molecule contains VL CDR1, VL CDR2, and VL CDR3, each having an amino acid sequence of one of the VL CDR1, VL CDR2, and VL CDR3 disclosed herein.

[0176] In one specific embodiment, an antibody or antigen-binding molecule, such as scFv, that specifically binds to CD70, a molecule containing this sequence, and a cell presenting this molecule comprises: (a) a VH CDR1 region containing the amino acid sequence of SEQ ID NO: 71; (b) a VH CDR2 region containing the amino acid sequence of SEQ ID NO: 72; (c) a VH CDR3 region containing the amino acid sequence of SEQ ID NO: 64; (d) a VL CDR1 region containing the amino acid sequence of SEQ ID NO: 53; (e) a VL CDR2 region containing the amino acid sequence of SEQ ID NO: 54; and (f) a VL CDR3 region containing the amino acid sequence of SEQ ID NO: 55.

[0177] In one specific embodiment, an antibody or antigen-binding molecule, such as scFv, that specifically binds to CD70, a molecule containing this sequence, and a cell presenting this molecule, comprises: (a) a VH CDR1 region; (b) a VH CDR2 region; (c) a VH CDR3 region; (d) a VL CDR1 region, comprising; (e) a VL CDR2 region; and (f) a VL CDR3 region, wherein the VH and VL CDRs are as shown in Figures 7A and 7B, and Figure 6, respectively.

[0178] In some specific embodiments, antibodies or antigen-binding molecules that specifically bind to CD70, molecules containing this sequence, and cells presenting this molecule, such as scFv, contain the heavy chain variable region sequence disclosed herein (e.g., in Figure 7A) and the light chain variable region sequence disclosed herein (e.g., in Figure 7B).

[0179] In one specific embodiment, an antibody or antigen-binding molecule, such as scFv, comprises: (a) a heavy chain variable region containing the amino acid sequence of SEQ ID NO:3; and (b) a light chain variable region containing the amino acid sequence of SEQ ID NO:5. The nucleotide sequences encoding the heavy chain variable region and the light chain variable region are provided herein and are shown in Figures 7A and 7B, respectively.

[0180] In some specific embodiments, antibodies or antigen-binding molecules that specifically bind to CD70, molecules containing this sequence, and cells presenting this molecule, such as scFv, contain the heavy chain sequence disclosed herein (e.g., in Figure 7A) and the light chain sequence disclosed herein (e.g., in Figure 7B).

[0181] In one specific embodiment, the antibody or antigen-binding molecule, such as scFv, comprises: (a) containing The variable region of the light chain of the amino acid sequence.

[0182] In one specific embodiment, the antibody or antigen-binding molecule comprises: (a) a heavy chain comprising an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:3; and (b) a light chain comprising an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:5. II.B. Selected strain 1C8 In some specific embodiments, antigen-binding molecules that specifically bind to CD70, molecules containing this sequence, and cells presenting this molecule, such as scFv, or antibodies, include VH CDR1, which comprises, is composed of, or is substantially composed of the following amino acid sequence: GDSIISGGY (SEQ ID NO:73).

[0183] In some specific embodiments, antigen-binding molecules that specifically bind to CD70, molecules containing this sequence, and cells presenting this molecule, such as scFv, or antibodies, include VH CDR2, which comprises, is composed of, or is substantially composed of the following amino acid sequence: FYSGS (SEQ ID NO: 74).

[0184] In some specific embodiments, antigen-binding molecules that specifically bind to CD70, molecules containing this sequence, and cells presenting this molecule, such as scFv, or antibodies, include VH CDR3, which comprises, is composed of, or is substantially composed of the following amino acid sequence: SGYSYALFDH (SEQ ID NO: 67).

[0185] In some specific embodiments, antigen-binding molecules that specifically bind CD70, molecules containing this sequence, and cells presenting this molecule, such as scFv, or antibodies, comprise a heavy chain VH, comprising (a) VH CDR1, which comprises, is composed of, or is substantially composed of the following amino acid sequence: GDSIISGGY (SEQ ID NO: 73); and / or (b) VH CDR2, which comprises, is composed of, or is substantially composed of the following amino acid sequence: FYSGS (SEQ ID NO: 74); and / or (c) VH CDR3, which comprises, is composed of, or is substantially composed of the following amino acid sequence: SGYSYALFDH (SEQ ID NO: 67).

[0186] In some specific embodiments, antibodies or antigen-binding molecules, such as scFv, that specifically bind to CD70, molecules containing this sequence, and cells presenting this molecule, include VH CDR1, VH CDR2, and VH CDR3, wherein VH CDR1, VH CDR2, and VH CDR3 contain the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 as shown in Figures 6 and 7C.

[0187] In some specific embodiments, antibodies or antigen-binding molecules, such as scFv, that specifically bind to CD70, molecules containing this sequence, and cells presenting this molecule, include VH CDR1, VH CDR2, and VH CDR3, wherein VH CDR1, VH CDR2, and VH CDR3 contain the amino acid sequences (SEQ ID NO: 73, 74, and 67, respectively) of the antigen-binding molecule shown in Figure 7C.

[0188] In some specific embodiments, antibodies or antigen-binding molecules, such as scFv, that specifically bind to CD70, molecules containing this sequence, and cells presenting this molecule, include a heavy chain variable region sequence containing the amino acid sequence of Figure 7C. In some specific embodiments, the antibody or antigen-binding molecule includes a heavy chain variable region sequence containing SEQ ID NO: 7.

[0189] In some specific embodiments, antibodies or antigen-binding molecules that specifically bind to CD70, molecules containing this sequence, and cells presenting this molecule, such as scFv, comprise one or more of the VH CDRs listed above or described in Figure 6. In some specific embodiments, the antibody or antigen-binding molecule comprises the VH frame region (FR) described herein. In particular, the antibody or antigen-binding molecule comprises the sequence shown in Figure 7C (e.g., FR one, two, three, or four in a sequence of Figure 7C) or VH FRs that may be derived from the sequence shown in Figure 7C.

[0190] In some specific embodiments, antibodies or antigen-binding molecules, such as scFv, that specifically bind to CD70, molecules containing this sequence, and cells presenting this molecule, contain the heavy chain sequence disclosed herein (e.g., in Figure 7C). In one specific embodiment, the antibody or antigen-binding molecule contains a heavy chain variable region containing the following amino acid sequence.

[0191] In various specific embodiments, the heavy chain variable region and the heavy chain variable region sequence of SEQ ID NO:7C have 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0192] In some specific embodiments, antigen-binding molecules that specifically bind CD70, molecules containing this sequence, and cells presenting this molecule, such as scFv, or antibodies, include VL CDR1, which comprises, is composed of, or is substantially composed of the following amino acid sequence: RASQFIGRYFN (SEQ ID NO: 56).

[0193] In some specific embodiments, antigen-binding molecules that specifically bind CD70, molecules containing this sequence, and cells presenting this molecule, such as scFv, or antibodies, include VL CDR2, which comprises, is composed of, or is substantially composed of the following amino acid sequence: AESLQS (SEQ ID NO: 57).

[0194] In some specific embodiments, antigen-binding molecules that specifically bind CD70, molecules containing this sequence, and cells presenting this molecule, such as scFv, or antibodies, include VL CDR3, which comprises, is composed of, or is substantially composed of the following amino acid sequence: QQSYSTPFT (SEQ ID NO: 58).

[0195] In some specific embodiments, antigen-binding molecules that specifically bind CD70, molecules containing this sequence, and cells presenting this molecule, such as scFv or antibodies, comprise heavy chain VL, which comprises: (a) VL CDR1, which comprises, is composed of, or is substantially composed of the following amino acid sequence: RASQFIGRYFN (SEQ ID NO: 56); and / or (b) VL CDR2, which comprises, is composed of, or is substantially composed of the following amino acid sequence: AESLQS (SEQ ID NO: 57); and / or (c) VL CDR3, which comprises, is composed of, or is substantially composed of the following amino acid sequence: QQSYSTPFT (SEQ ID NO: 58).

[0196] In some specific embodiments, antibodies or antigen-binding molecules, such as scFv, specifically bind to CD70, molecules containing this sequence, and cells presenting this molecule. The antibody or antigen-binding molecule includes VL CDR1, VL CDR2, and VL CDR3, wherein VL CDR1, VL CDR2, and VL CDR3 contain the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 as shown in Figures 6 and 7D.

[0197] In some specific embodiments, antibodies or antigen-binding molecules, such as scFv, that specifically bind to CD70, molecules containing this sequence, and cells presenting this molecule, include a light chain variable region sequence containing the amino acid sequence of Figure 7D. In some specific embodiments, the antibody or antigen-binding molecule includes a light chain variable region sequence containing SEQ ID NO: 9.

[0198] In some specific embodiments, antibodies or antigen-binding molecules, such as scFv, specifically bind to CD70, molecules containing this sequence, and cells presenting this molecule, wherein the antibody or antigen-binding molecule includes the VL frame region (FR) as described herein. In certain specific embodiments, the antibody or antigen-binding molecule includes the sequence shown in Figure 7D (e.g., FR one, two, three, or four in a sequence of Figure 7D) or an amino acid sequence that may be derived from the sequence shown in Figure 7D.

[0199] In some specific embodiments, antibodies or antigen-binding molecules, such as scFv, that specifically bind to CD70, molecules containing this sequence, and cells presenting this molecule, contain the light chain sequence disclosed herein (e.g., in Figure 7D). In one specific embodiment, the antibody or antigen-binding molecule contains a light chain variable region comprising the following amino acid sequence.

[0200] In various specific embodiments, the light chain variable region is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the light chain variable region sequence of SEQ ID NO: 9.

[0201] In some specific embodiments, antibodies or antigen-binding molecules, such as scFv, that specifically bind to CD70, molecules containing this sequence, and cells presenting this molecule, contain one, two, and / or three VH CDR sequences disclosed herein. In some specific embodiments, the antibody or antigen-binding molecule contains VH CDR1, VH CDR2, and VH CDR3, each having an amino acid sequence of one of the VH CDR1, VH CDR2, and VH CDR3 disclosed herein. In some specific embodiments, the antibody or antigen-binding molecule contains one, two, and / or three VL CDR sequences disclosed herein. In some specific embodiments, the antibody or antigen-binding molecule contains VL CDR1, VL CDR2, and VL CDR3, each having an amino acid sequence of one of the VL CDR1, VL CDR2, and VL CDR3 disclosed herein.

[0202] In one specific embodiment, an antibody or antigen-binding molecule, such as scFv, that specifically binds to CD70, a molecule containing this sequence, and a cell presenting this molecule comprises: (a) a VH CDR1 region containing the amino acid sequence of SEQ ID NO: 73; (b) a VH CDR2 region containing the amino acid sequence of SEQ ID NO: 74; (c) a VH CDR3 region containing the amino acid sequence of SEQ ID NO: 67; (d) a VL CDR1 region containing the amino acid sequence of SEQ ID NO: 56; (e) a VL CDR2 region containing the amino acid sequence of SEQ ID NO: 57; and (f) a VL CDR3 region containing the amino acid sequence of SEQ ID NO: 58.

[0203] In one specific embodiment, an antibody or antigen-binding molecule, such as scFv, that specifically binds to CD70, a molecule containing this sequence, and a cell presenting this molecule, comprises: (a) a VH CDR1 region; (b) a VH CDR2 region; (c) a VH CDR3 region; (d) a VL CDR1 region, comprising; (e) a VL CDR2 region; and (f) a VL CDR3 region, wherein the VH and VL CDRs are shown in Figures 7C and 7D, and in Figure 6, respectively.

[0204] In some specific embodiments, antibodies or antigen-binding molecules, such as scFv, that specifically bind to CD70, molecules containing this sequence, and cells presenting this molecule, include the heavy chain variable region sequence disclosed herein (e.g., in Figure 7C) and the light chain variable region sequence disclosed herein (e.g., in Figure 7D).

[0205] In one specific embodiment, an antibody or antigen-binding molecule, such as scFv, comprises: (a) a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 7; and (b) a light chain variable region containing the amino acid sequence of SEQ ID NO: 9. The nucleotide sequences encoding the heavy chain variable region and the light chain variable region are provided herein and are shown in Figures 7C and 7D, respectively.

[0206] In some specific embodiments, antibodies or antigen-binding molecules, such as scFv, that specifically bind to CD70, molecules containing this sequence, and cells presenting this molecule, contain the heavy chain sequence disclosed herein (e.g., in Figure 7C) and the light chain sequence disclosed herein (e.g., in Figure 7D).

[0207] In one specific embodiment, an antibody or antigen-binding molecule, such as scFv, comprises: (a) a heavy chain variable region containing the following amino acid sequence.

[0208] In one specific embodiment, the antibody or antigen-binding molecule comprises: (a) a heavy chain comprising an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 7; and (b) a light chain comprising an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 9. II.C. Selected strain 6E9

[0209] In some specific embodiments, antigen-binding molecules that specifically bind to CD70, molecules containing this sequence, and cells presenting this molecule, such as scFv, or antibodies, include VH CDR1, which comprises, is composed of, or is substantially composed of the following amino acid sequence: GYTFTSY (SEQ ID NO: 75).

[0210] In some specific embodiments, antigen-binding molecules that specifically bind CD70, molecules containing this sequence, and cells presenting this molecule, such as scFv, or antibodies, include VH CDR2, which comprises, is composed of, or is substantially composed of the following amino acid sequence: DPSGGS (SEQ ID NO: 76).

[0211] In some specific embodiments, antigen-binding molecules that specifically bind to CD70, molecules containing this sequence, and cells presenting this molecule, such as scFv, or antibodies, include VH CDR3, which comprises, is composed of, or is substantially composed of the following amino acid sequence: DYGDYVFDY (SEQ ID NO: 70).

[0212] In some specific embodiments, antigen-binding molecules that specifically bind CD70, molecules containing this sequence, and cells presenting this molecule, such as scFv or antibodies, contain a heavy chain VH, which includes (a) VH CDR1, which includes, is composed of, or is substantially composed of the following amino acid sequence: GYTFTSY (SEQ ID NO: 75); and / or (b) VH CDR2, which includes, is composed of, or is substantially composed of the following amino acid sequence: DPSGGS (SEQ ID NO: 76); and / or (c) VH CDR3, which includes, is composed of, or is substantially composed of the following amino acid sequence: DYGDYVFDY (SEQ ID NO: 70).

[0213] In some specific embodiments, antibodies or antigen-binding molecules, such as scFv, that specifically bind to CD70, molecules containing this sequence, and cells presenting this molecule, include VH CDR1, VH CDR2, and VH CDR3, wherein VH CDR1, VH CDR2, and VH CDR3 contain the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 as shown in Figures 6 and 7E.

[0214] In some specific embodiments, antibodies or antigen-binding molecules, such as scFv, that specifically bind to CD70, molecules containing this sequence, and cells presenting this molecule, include VH CDR1, VH CDR2, and VH CDR3, wherein VH CDR1, VH CDR2, and VH CDR3 contain the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of the antigen-binding molecules shown in Figure 7E (SEQ ID NO: 75, 76, and 70, respectively).

[0215] In some specific embodiments, antibodies or antigen-binding molecules, such as scFv, that specifically bind to CD70, molecules containing this sequence, and cells presenting this molecule, include a heavy chain variable region sequence containing the amino acid sequence of Figure 7E. In some specific embodiments, the antibody or antigen-binding molecule includes a heavy chain variable region sequence containing SEQ ID NO: 11.

[0216] In some specific embodiments, antibodies or antigen-binding molecules, such as scFv, that specifically bind to CD70, molecules containing this sequence, and cells presenting this molecule, include one or more of any of the VH CDRs listed above or described in Figure 6. In some specific embodiments, the antibody or antigen-binding molecule includes the VH frame region (FR) described herein. In particular, the antibody or antigen-binding molecule includes the sequence shown in Figure 7E (e.g., one, two, three, or four FRs in a sequence of Figure 7E) or VH FRs that may be derived from the sequence shown in Figure 7E.

[0217] In some specific embodiments, antibodies or antigen-binding molecules, such as scFv, that specifically bind to CD70, molecules containing this sequence, and cells presenting this molecule, contain the heavy chain sequence disclosed herein (e.g., in Figure 7E). In one specific embodiment, the antibody or antigen-binding molecule contains a heavy chain variable region containing the following amino acid sequence.

[0218] In various specific embodiments, the heavy chain variable region is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the heavy chain variable region sequence of SEQ ID NO: 7E.

[0219] In some specific embodiments, antigen-binding molecules that specifically bind CD70, molecules containing this sequence, and cells presenting this molecule, such as scFv, or antibodies, include VL CDR1, which comprises, is composed of, or is substantially composed of the following amino acid sequence: SGSSSNIGTNTVN (SEQ ID NO: 59).

[0220] In some specific embodiments, antigen-binding molecules that specifically bind CD70, molecules containing this sequence, and cells presenting this molecule, such as scFv, or antibodies, include VL CDR2, which comprises, is composed of, or is substantially composed of the following amino acid sequence: INNQRPS (SEQ ID NO: 60).

[0221] In some specific embodiments, antigen-binding molecules that specifically bind CD70, molecules containing this sequence, and cells presenting this molecule, such as scFv, or antibodies, include VL CDR3, which comprises, is composed of, or is substantially composed of the following amino acid sequence: ATWDDSLNGPVV (SEQ ID NO: 61).

[0222] In some specific embodiments, antigen-binding molecules that specifically bind CD70, molecules containing this sequence, and cells presenting this molecule, such as scFv or antibodies, comprise heavy chain VL, which comprises: (a) VL CDR1, which comprises, is composed of, or is substantially composed of the following amino acid sequence: SGSSSNIGTNTVN (SEQ ID NO: 59); and / or (b) VL CDR2, which comprises, is composed of, or is substantially composed of the following amino acid sequence: INNQRPS (SEQ ID NO: 60); and / or (c) VL CDR3, which comprises, is composed of, or is substantially composed of the following amino acid sequence: ATWDDSLNGPVV (SEQ ID NO: 61).

[0223] In some specific embodiments, antibodies or antigen-binding molecules, such as scFv, specifically bind to CD70, molecules containing this sequence, and cells presenting this molecule. The antibody or antigen-binding molecule includes VL CDR1, VL CDR2, and VL CDR3, wherein VL CDR1, VL CDR2, and VL CDR3 contain the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 as shown in Figures 6 and 7F.

[0224] In some specific embodiments, antibodies or antigen-binding molecules, such as scFv, that specifically bind to CD70, molecules containing this sequence, and cells presenting this molecule, include a light chain variable region sequence containing the amino acid sequence of Figure 7F. In some specific embodiments, the antibody or antigen-binding molecule includes a light chain variable region sequence containing SEQ ID NO: 13.

[0225] In some specific embodiments, antibodies or antigen-binding molecules, such as scFv, specifically bind to CD70, molecules containing this sequence, and cells presenting this molecule, wherein the antibody or antigen-binding molecule includes the VL frame region (FR) as described herein. In particular, the antibody or antigen-binding molecule includes the sequence shown in Figure 7F (e.g., FR one, two, three, or four in a sequence of Figure 7F) or an amino acid sequence that may be derived from the sequence shown in Figure 7F.

[0226] In some specific embodiments, antibodies or antigen-binding molecules, such as scFv, that specifically bind to CD70, molecules containing this sequence, and cells presenting this molecule, contain the light chain sequence disclosed herein (e.g., in Figure 7F). In one specific embodiment, the antibody or antigen-binding molecule contains a light chain variable region containing the following amino acid sequence.

[0227] In various specific embodiments, the light chain variable region is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the light chain variable region sequence of SEQ ID NO: 13.

[0228] In some specific embodiments, antibodies or antigen-binding molecules, such as scFv, that specifically bind to CD70, molecules containing this sequence, and cells presenting this molecule, contain one, two, and / or three VH CDR sequences disclosed herein. In some specific embodiments, the antibody or antigen-binding molecule contains VH CDR1, VH CDR2, and VH CDR3, each having an amino acid sequence of one of the VH CDR1, VH CDR2, and VH CDR3 disclosed herein. In some specific embodiments, the antibody or antigen-binding molecule contains one, two, and / or three VL CDR sequences disclosed herein. In some specific embodiments, the antibody or antigen-binding molecule contains VL CDR1, VL CDR2, and VL CDR3, each having an amino acid sequence of one of the VL CDR1, VL CDR2, and VL CDR3 disclosed herein.

[0229] In one specific embodiment, an antibody or antigen-binding molecule, such as scFv, that specifically binds to CD70, a molecule containing this sequence, and a cell presenting this molecule comprises: (a) a VH CDR1 region containing the amino acid sequence of SEQ ID NO: 75; (b) a VH CDR2 region containing the amino acid sequence of SEQ ID NO: 76; (c) a VH CDR3 region containing the amino acid sequence of SEQ ID NO: 70; (d) a VL CDR1 region containing the amino acid sequence of SEQ ID NO: 59; (e) a VL CDR2 region containing the amino acid sequence of SEQ ID NO: 60; and (f) a VL CDR3 region containing the amino acid sequence of SEQ ID NO: 61.

[0230] In one specific embodiment, an antibody or antigen-binding molecule, such as scFv, that specifically binds to CD70, a molecule containing this sequence, and a cell presenting this molecule, includes: (a) a VH CDR1 region; (b) a VH CDR2 region; (c) a VH CDR3 region; (d) a VL CDR1 region, including; (e) a VL CDR2 region; and (f) a VL CDR3 region, wherein the VH and VL CDRs are shown in Figures 7E and 7F, respectively, and in Figure 6.

[0231] In some specific embodiments, antibodies or antigen-binding molecules that specifically bind to CD70, molecules containing this sequence, and cells presenting this molecule, such as scFv, contain the heavy chain variable region sequence disclosed herein (e.g., in Figure 7E) and the light chain variable region sequence disclosed herein (e.g., in Figure 7F).

[0232] In one specific embodiment, the antibody or antigen-binding molecule, such as scFv, comprises: (a) a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 11; and (b) a light chain variable region containing the amino acid sequence of SEQ ID NO: 13. The nucleotide sequences encoding the heavy chain variable region and the light chain variable region are provided herein and are shown in Figures 7E and 7F, respectively.

[0233] In some specific embodiments, antibodies or antigen-binding molecules that specifically bind to CD70, molecules containing this sequence, and cells presenting this molecule, such as scFv, contain the heavy chain sequence disclosed herein (e.g., in Figure 7E) and the light chain sequence disclosed herein (e.g., in Figure 7F).

[0234] In one specific embodiment, an antibody or antigen-binding molecule, such as scFv, comprises: (a) a heavy chain variable region containing the following amino acid sequence.

[0235] In one specific embodiment, the antibody or antigen-binding molecule, such as scFv, comprises: (a) a heavy chain containing an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 11; and (b) a light chain containing an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 13. III. Polynucleotides encoding antigen-binding molecules, chimeric antigen receptors, and T-cell receptors containing such antigen-binding molecules.

[0236] This disclosure also relates to polynucleotides encoding antibodies and antigen-binding molecules, such as scFv, that specifically bind to CD70, molecules containing this sequence, and cells that present this molecule.

[0237] In some specific embodiments, the polynucleotides disclosed herein encode antigen-binding molecules (such as scFv), wherein the antigen-binding molecules comprise heavy chain variable region amino acid sequences that are at least about 75%, at least about 85%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the heavy chain variable region amino acid sequences selected from the group consisting of SEQ ID NO: 3, 7, and 11.

[0238] In some specific embodiments, the polynucleotides disclosed herein encode antigen-binding molecules (such as scFv), wherein the antigen-binding molecules comprise a light chain variable region amino acid sequence that is at least about 75%, at least about 85%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the light chain variable region amino acid sequences selected from the group consisting of SEQ ID NO: 5, 9, and 13.

[0239] In some specific embodiments, the polynucleotide comprises a heavy chain coding sequence selected from the following group: SEQ ID NO: 2, 6 and 10.

[0240] In another specific embodiment, the polynucleotide comprises a light chain coding sequence selected from the group consisting of: SEQ ID NO: 4, 8 and 12.

[0241] In another specific embodiment, the polynucleotide comprises a heavy chain coding sequence containing SEQ ID NO:2 and a light chain coding sequence containing SEQ ID NO:4.

[0242] In another specific embodiment, the polynucleotide comprises the heavy chain coding sequence of SEQ ID NO:6 and the light chain coding sequence of SEQ ID NO:8.

[0243] In another specific embodiment, the polynucleotide comprises the heavy chain coding sequence of SEQ ID NO:10 and the light chain coding sequence of SEQ ID NO:12.

[0244] Those skilled in the art will understand that the variations in the disclosed polynucleotide sequences may be due to the degeneracy of the genetic code. These variants of the disclosed polynucleotide sequences thus constitute a pattern of this disclosure. IV. Chimeric antigen receptors and T-cell receptors

[0245] This disclosure also relates to chimeric antigen receptors (CARs) or T-cell receptors (TCRs) and engineered T cells. Chimeric antigen receptors (CARs) or T-cell receptors (TCRs) include antigen-binding molecules (e.g., scFv) that specifically bind CD70 as described herein, and engineered T cells include antigen-binding molecules that specifically bind CD70 as described herein. In some specific embodiments, the anti-CD70 CAR or TCR of this disclosure includes an antigen-binding molecule that specifically binds CD70. In some specific embodiments, the anti-CD70 CAR or TCR further includes a co-stimulatory domain, and / or an extracellular domain (i.e., a "hinge" or "spacer" region), and / or a transmembrane domain, and / or an intracellular (signal transduction) domain, and / or a CD3ζ activation domain. In some specific embodiments, the anti-CD70 CAR or TCR includes the scFv antigen-binding molecule that specifically binds to CD70 (e.g., hCD70) as disclosed herein, a co-stimulatory domain, an extracellular domain, a transmembrane domain, and a CD3ζ activation domain.

[0246] In some specific embodiments, the TCR disclosed herein includes an antigen-binding molecule that specifically binds to CD70, and wherein the TCR further includes a fourth complementarity-determining region (CDR4). In some specific embodiments, the TCR includes an antigen-binding molecule that specifically binds to CD70 and a constant region. In some specific embodiments, the constant region is selected from the constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, and IgM. CAR and TCR designs are further discussed below.

[0247] It will be further recognized that, when needed, the various domains and regions described herein can be represented on separate chains from the antigen-binding molecule (e.g., scFv) and the activation domain, in a so-called "trans-configuration". Thus, in one specific embodiment, the activation domain may be represented on one chain, while the antigen-binding molecule, and / or extracellular domain, and / or transmembrane domain and / or co-stimulatory domain (depending on the desired CAR or TCR configuration) may be represented on separate chains.

[0248] As will be further appreciated as described more fully herein, the sequence of the CAR components disclosed herein can be varied as desired, from N to C-terminus or from extracellular to intracellular. The antigen-binding molecule (scFv) will be extracellular to associate with the target antigen, and a leader or signal peptide may be included at the N-terminal tail of the scFv (at the most distal end of the cell membrane). A preferred orientation and sequence for the CAR disclosed herein is: leader sequence as needed (e.g., CD8a leader sequence) – anti-CD70 scFv – micro-connector as needed, such as GSG or AAA – hinge – micro-connector as needed, such as GSG or AAA – transmembrane region (e.g., CD8a transmembrane region) – micro-connector as needed, such as GSG or AAA – co-stimulatory region (e.g., CD28T or 4-1BB subsequence) – micro-connector as needed, such as GSG or AAA – activation domain (e.g., CD3ζ domain, such as one of the providers herein).

[0249] Another preferred orientation and sequencing of the CAR disclosed herein includes two co-stimulatory domains and is: a demand-dependent leader sequence (e.g., CD8a leader sequence) -- anti-CD70 scFv -- demand-dependent micro-connector, such as GSG or AAA -- hinge -- demand-dependent micro-connector, such as GSG or AAA -- transmembrane region (e.g., CD8a transmembrane region) -- demand-dependent micro-connector, such as GSG or AAA -- co-stimulatory region (e.g., CD28T or 4-1BB subsequence) -- co-stimulatory region (e.g., CD28T or 4-1BB subsequence) -- demand-dependent micro-connector, such as GSG or AAA -- activation domain (e.g., CD3ζ domain, such as one of the present invention).

[0250] The following provides a more detailed description of the components of CAR. IV.A. Extracellular or "hinge-like" domain

[0251] In one specific embodiment, the CAR or TCR disclosed herein comprises an “extracellular” or “hinge” or “spacer” domain or region, the terms of which are used interchangeably herein. In another specific embodiment, the extracellular domain is derived from (e.g., comprising all or fragments of the following) CD2, CD3δ, CD3ε, CD3γ, CD4, CD7, CD8α, CD8β, CD11a (ITGAL), CD11b (ITGAM), CD11c (ITGAX), CD11d (ITGAD), CD18 (ITGB2), CD19 (B4), CD27 (TNFRSF7), CD28, CD28T, CD29 (ITGB1), CD30 (TNFRSF8), CD40 (TNFRSF5), CD48 (SLAMF2), CD49a (ITGA1), CD49d (ITGA4), CD49f (ITGA6), CD66a (CEACAM1), CD66b (CEACAM8), CD66c (CEACAM6), CD66d (CEACAM3), CD66e (CEACAM5), CD69 (CLEC2), CD79A (β-chain of B-cell antigen receptor complex), CD79B (β-chain of B-cell antigen receptor complex), CD84 (SLAMF5), CD96 (Tactile), CD100 (SEMA4D), CD103 (ITGAE), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD158A (KIR2DL1), CD158B1 (KIR2DL2), CD158B2 (KIR2DL3), CD158C (KIR3DP1), CD158D (KIRDL4), CD158F1 (KIR2DL5A), CD158F2 (KIR2DL5B), CD158K (KIR3DL2), CD160 (BY55), CD162 (SELPLG), CD226 (DNAM1), CD229 (SLAMF3), CD244 (SLAMF4), CD247 (CD3-ζ), CD258 (LIGHT), CD268 (BAFFR), CD270 (TNFSF14), CD272 (BTLA), CD276 (B7-H3), CD279 (PD-1), CD314 (NKG2D), CD319 (SLAMF7), CD335 (NK-p46), CD336 (NK-p44), CD337 (NK-p30), CD352 (SLAMF6), CD353 (SLAMF8), CD355(CRTAM), CD357 (TNFRSF18), inducible T cell costimulators (ICOS), LFA-1 (CD11a / CD18), NKG2C, DAP-10, ICAM-1, NKp80 (KLRF1), IL-2Rβ, IL-2Rγ, IL-7Rα, LFA-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1 / CBP, CD83 ligand, Fcγ receptor, MHC class 1 molecules, MHC class 2 molecules, TNF receptor proteins, immunoglobulin proteins, cell mediator receptors, integrins, activated NK cell receptors, Toll ligand receptors, and fragments or combinations thereof. The extracellular, hinge, or spacer domain or region may be derived from natural or synthetic sources.

[0252] In some embodiments, the "extracellular" or "hinge" or "spacer" domain or region is located between the antigen-binding molecule (e.g., scFv) and the transmembrane domain. In this orientation, the hinge domain provides the distance between the antigen-binding molecule and the cell membrane surface on which the CAR or TCR is expressed. In some embodiments, the "extracellular" or "hinge" or "spacer" domain or region is derived from or derived from immunoglobulins. In some embodiments, the "extracellular" or "hinge" or "spacer" domain or region is selected from the hinge regions of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, and IgM, or fragments thereof. In other embodiments, the "extracellular" or "hinge" or "spacer" domain or region includes, is derived from, or is derived from the hinge region of CD8α. In some specific embodiments, the “extracellular” or “hinge” or “spacer” domain or region comprises, is derived from, or is derived from the hinge region of CD28T. In some specific embodiments, the “extracellular” or “hinge” or “spacer” domain or region comprises a segment of the hinge region of CD8α or a segment of the hinge region of CD28T, wherein the segment is anything smaller than the entire hinge region. In some specific embodiments, the CD8α hinge region segment or the CD28 hinge region segment comprises an amino acid sequence excluding at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 amino acids at the N-terminus or C-terminus of the CD8α hinge region or the CD28T hinge region.

[0253] In some specific embodiments, the "extracellular," "hinge," or "spacer" domain or region contains an amino acid sequence, which is related to the amino acid sequence. The fragment thereof is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical. In some specific embodiments, the “extracellular” or “hinge” or “spacer” domain or region contains the amino acid sequence of SEQ ID NO:15 or a fragment thereof.

[0254] In some specific embodiments, the "extracellular," "hinge," or "spacer" domain or region contains an amino acid sequence, which is related to the amino acid sequence. The fragment thereof is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical. In some specific embodiments, the “extracellular” or “hinge” or “spacer” domain or region contains the amino acid sequence of SEQ ID NO:83, or a fragment thereof.

[0255] In some specific embodiments, the “extracellular” or “hinge” or “spacer” domain or region is encoded by a nucleotide sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the following nucleotide sequences or fragments thereof. In some specific embodiments, the “extracellular” or “hinge” or “spacer” domain or region is encoded by a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO:14 or a fragment thereof.

[0256] In some specific embodiments, the “extracellular” or “hinge” or “spacer” domain or region is encoded by a nucleotide sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the following nucleotide sequences or fragments thereof. In some specific embodiments, the “extracellular” or “hinge” or “spacer” domain or region is encoded by a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO:82 or a fragment thereof.

[0257] In some specific embodiments, the "extracellular," "hinge," or "spacer" domain or region contains an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the following amino acid sequences or fragments thereof. In some specific embodiments, the "extracellular," "hinge," or "spacer" domain or region contains an amino acid sequence of SEQ ID NO:17 or a fragment thereof.

[0258] In some specific embodiments, the “extracellular” or “hinge” or “spacer” domain or region is encoded by a nucleotide sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the following nucleotide sequences or fragments thereof. In some specific embodiments, the “extracellular” or “hinge” or “spacer” domain or region is encoded by a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO:16 or a fragment thereof.

[0259] In some specific embodiments, the "extracellular," "hinge," or "spacer" domain or region contains an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the following amino acid sequences or fragments thereof. In some specific embodiments, the "extracellular" or "hinge" or "spacer" domain or region contains the amino acid sequence of SEQ ID NO:85 or a fragment thereof.

[0260] In some specific embodiments, the "extracellular," "hinge," or "spacer" domain or region contains an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the following amino acid sequences or fragments thereof. In some specific embodiments, the "extracellular" or "hinge" or "spacer" domain or region contains the amino acid sequence of SEQ ID NO:85 or a fragment thereof.

[0261] In some specific embodiments, the “extracellular” or “hinge” or “spacer” domain or region is encoded by a nucleotide sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the following nucleotide sequences or fragments thereof. In some specific embodiments, the “extracellular” or “hinge” or “spacer” domain or region is encoded by a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO:84 or a fragment thereof.

[0262] In some embodiments, the CD28T domain is derived from the human CD28T hinge region and may include SEQ ID NO:15. In other embodiments, the CD28T domain is derived from the rodent, mouse, or primate (e.g., non-human primates) CD28T hinge region. In some embodiments, the CD28T domain is derived from the chimeric CD28T hinge region.

[0263] In some embodiments, the CD28T domain is derived from the human CD28T hinge region and may include SEQ ID NO:83. In other embodiments, the CD28T domain is derived from the rodent, mouse, or primate (e.g., non-human primates) CD28T hinge region. In some embodiments, the CD28T domain is derived from the chimeric CD28T hinge region.

[0264] In some embodiments, the CD8 domain is derived from the human CD8 hinge region and may include SEQ ID NO:17. In other embodiments, the CD8 domain is derived from the rodent, rat, or primate (e.g., non-human primates) CD8 hinge region. In some embodiments, the CD8 domain is derived from the chimeric CD8 hinge region.

[0265] In some embodiments, the CD8 domain is derived from the human CD8 hinge region and may include SEQ ID NO:85. In other embodiments, the CD8 domain is derived from the CD8 hinge region of rodents, mice, or primates (e.g., non-human primates). In some embodiments, the CD8 domain is derived from the chimeric CD8 hinge region.

[0266] In some specific embodiments, the extracellular domain contains some or all of the members of the immunoglobulin family, such as IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, or fragments thereof.

[0267] As needed, short peptide or polypeptide linkers, preferably between 2 and 10 amino acids in length, can form links between the hinge domain and the antigen-binding domain of the CAR, or between the hinge domain and the transmembrane domain. As needed, short oligo- or polypeptide linkers, preferably between 2 and 10 amino acids in length, can form links. Glycine-serine dual (GS), glycine-serine-glycine triple (GSG), or alanine-alanine-alanine triple (AAA) linkers provide particularly suitable options. IV.B. Transmembrane domain

[0268] The CAR or TCR disclosed herein may further include a transmembrane domain. The transmembrane domain may be designed to fuse with a hinge domain. Similarly, it may fuse with an intracellular domain such as a co-stimulatory domain. In one specific embodiment, a transmembrane domain naturally associated with one of the domains in the CAR may be used. For example, the transmembrane domain may include a natural transmembrane region of a co-stimulatory domain (e.g., the TM region of CD28T or 4-1BB, as a co-stimulatory domain) or a natural transmembrane domain of a hinge region (e.g., the TM region of CD8α or CD28T, as a hinge domain).

[0269] In some cases, transmembrane domains can be selectively or modified by amino acid substitution to prevent them from binding to transmembrane domains of the same or different surface membrane proteins, thereby minimizing their interactions with other members of the receptor complex. Transmembrane domains can be derived from natural or synthetic sources. When transmembrane domains are derived from natural sources, they can be derived from any membrane-bound or transmembrane protein. In some specific embodiments, the transmembrane structural domains are derived from: CD2, CD3δ, CD3ε, CD3γ, CD4, CD7, CD8α, CD8β, CD11a (ITGAL), CD11b (ITGAM), CD11c (ITGAX), CD11d (ITGAD), CD18 (ITGB2), CD19 (B4), CD27 (TNFRSF7), CD28, CD28T, CD29 (ITGB1), CD30 (TNFRSF8), CD40 (TNFRSF5), CD48 (SLAMF2), CD49a (ITGA1), CD49d (ITGA4), CD49f (ITGA6), CD66a (CEACAM1), CD66b (CEACAM8), CD66c (CEACAM6), CD66d (CEACAM3), CD66e (CEACAM5), CD69 (CLEC2), CD79A (β-chain of B-cell antigen receptor complex-associated), CD79B (β-chain of B-cell antigen receptor complex-associated), CD84 (SLAMF5), CD96 (Tactile), CD100 (SEMA4D), CD103 (ITGAE), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD158A (KIR2DL1), CD158B1 (KIR2DL2), CD158B2 (KIR2DL3), CD158C (KIR3DP1), CD158D (KIRDL4), CD158F1 (KIR2DL5A), CD158F2 (KIR2DL5B), CD158K (KIR3DL2), CD160 (BY55), CD162 (SELPLG), CD226 (DNAM1), CD229 (SLAMF3), CD244 (SLAMF4), CD247 (CD3-ζ), CD258 (LIGHT), CD268 (BAFFR), CD270 (TNFSF14), CD272 (BTLA), CD276 (B7-H3), CD279 (PD-1), CD314 (NKG2D), CD319 (SLAMF7), CD335(NK-p46), CD336 (NK-p44), CD337 (NK-p30), CD352 (SLAMF6), CD353 (SLAMF8), CD355 (CRTAM), CD357 (TNFRSF18), inducible T cell costimulators (ICOS), LFA-1 (CD11a / CD18), NKG2C, DAP-10, ICAM-1, NKp80 (KLRF1), IL-2Rβ, IL-2Rγ, IL-7Rα, LFA-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1 / CBP, CD83 ligand, Fcγ receptor, MHC class 1 molecules, MHC class 2 molecules, TNF receptor protein, immunoglobulin protein, cell mediator receptor, integrin, activated NK cell receptor, Toll ligand receptor, and combinations thereof.

[0270] In some specific embodiments, a transmembrane domain may include a sequence that spans the cell membrane but extends into the cytoplasm and / or extracellular space. For example, a transmembrane domain may include a membrane-crossing sequence that may further include amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more that extend into the cytoplasm and / or extracellular space. Thus, a transmembrane domain includes a membrane-crossing region and may further include amino acid(groups) extending beyond the inner or outer surface of the membrane itself; this sequence may also be considered a “transmembrane domain”.

[0271] In one specific embodiment, the transmembrane domain of the CAR disclosed herein includes a CD28T transmembrane domain. In another specific embodiment, the CD28T transmembrane domain includes a transmembrane portion of a protein encoded by the following nucleic acid sequence:

[0272] For example, a transmembrane domain can be encoded by a nucleic acid sequence containing the following nucleic acid sequence.

[0273] In another specific embodiment, the CD28T transmembrane domain comprises a transmembrane amino acid sequence containing the following amino acid sequence:

[0274] For example, transmembrane domains may contain amino acid sequences.

[0275] In one specific embodiment, the transmembrane domain of the CAR disclosed herein includes a CD8 transmembrane domain. In one specific embodiment, the CD8 transmembrane domain includes a transmembrane portion of a protein encoded by the following nucleic acid sequence:

[0276] For example, a transmembrane domain can be encoded by a nucleic acid sequence containing the following nucleic acid sequence.

[0277] In another specific embodiment, the CD8 transmembrane domain comprises a transmembrane amino acid sequence containing the following amino acid sequence:

[0278] For example, transmembrane domains may contain amino acid sequences.

[0279] In some specific embodiments, the transmembrane domain is a CD28T transmembrane domain. In some specific embodiments, the transmembrane domain contains amino acid sequences. An amino acid sequence with at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or 100% identity.

[0280] In some specific embodiments, the transmembrane domain contains the amino acid sequence of SEQ ID NO:19.

[0281] In some specific embodiments, the CD28T transmembrane domain is composed of nucleotide sequences. Encoding nucleotide sequences with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity.

[0282] In some specific embodiments, the transmembrane domain is encoded by a nucleic acid sequence containing the nucleic acid sequence of SEQ ID NO:18.

[0283] In another specific embodiment, the transmembrane domain is a CD8 transmembrane domain. In some specific embodiments, the transmembrane domain comprises an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the following amino acid sequences:

[0284] In some specific embodiments, the transmembrane domain contains the amino acid sequence of SEQ ID NO:21.

[0285] In another specific embodiment, the transmembrane domain is a CD8 transmembrane domain. In some specific embodiments, the transmembrane domain comprises an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the following amino acid sequences:

[0286] In some specific embodiments, the transmembrane domain contains the amino acid sequence of SEQ ID NO:94.

[0287] In some specific embodiments, the CD8 transmembrane domain is encoded by a nucleotide sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the following nucleotide sequences:

[0288] In some specific embodiments, the transmembrane domain is encoded by a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO:20.

[0289] As noted herein, the various domains of the disclosed CAR may, as needed, include amino acids other than those sequences provided herein. For example, the transmembrane domain may include an amino acid that extends into the cytoplasm and / or extracellular space, which may occur spontaneously within the molecule from which the transmembrane domain is derived. Thus, in some specific embodiments, the CD8 transmembrane domain further includes a continuous amino acid sequence. Encoded by the following nucleic acid sequence Furthermore, it extends its transmembrane domain into the cytoplasm or intracellular space.

[0290] As needed, short peptide or polypeptide linkers, particularly those between 2 and 10 amino acids in length, can form links between the transmembrane domains and proximal cytoplasmic signaling domains of the CAR, such as co-stimulatory or activating domains, or to antigen-binding molecules (e.g., anti-CD70 scFv). As needed, short oligo- or polypeptide linkers, particularly those between 2 and 10 amino acids in length, can form links. Glycine-serine dual (GS), glycine-serine-glycine triple (GSG), or alanine-alanine-alanine triple (AAA) linkers provide particularly suitable linkers. IV.C. Co-stimulatory domain

[0291] In some specific embodiments, this disclosure includes a CAR, wherein the CAR comprises an antigen-binding molecule that specifically binds to CD70 (one or more antigen-binding molecules provided herein, as shown in the figures or in the appended sequence listing), and wherein the CAR further comprises a co-stimulatory domain. In some specific embodiments, the co-stimulatory domain is located between the antigen-binding molecule (e.g., scFv) and the activation domain. In some specific embodiments, the co-stimulatory domain may, but does not need to, include an extracellular domain and / or a transmembrane domain in addition to an intracellular signaling domain. In some specific embodiments, the co-stimulatory domain may include a transmembrane domain and an intracellular signaling domain. In some specific embodiments, the co-stimulatory domain may include an extracellular domain and a transmembrane domain. In some specific embodiments, the co-stimulatory domain may include an intracellular signaling domain. The CAR, TCR, or engineered T cell disclosed herein may include one, two, or three co-stimulatory domains, which may be configured in tandem or laterally to one or more other components of the CAR.

[0292] The co-stimulatory domains of CARs, TCRs, and engineered T cells disclosed in this paper can provide signal transduction to an activation domain, which then activates at least one normal effector function of an immune cell. The effector functions of T cells may include, for example, cytolytic activity or helper activity, including the secretion of cellular mediators.

[0293] In some specific embodiments, suitable co-stimulatory domains include (i.e., contain), but are not limited to, CD2, CD3δ, CD3ε, CD3γ, CD4, CD7, CD8α, CD8β, CD11a (ITGAL), CD11b (ITGAM), CD11c (ITGAX), CD11d (ITGAD), CD18 (ITGB2), CD19 (B4), CD27 (TNFRSF7), CD28, CD28T, CD29 (ITGB1), CD30 (TNFRSF8), CD40 (TNFRSF5), CD48 (SLAMF2), CD49a (ITGA1), CD49d (ITGA4), CD49f (ITGA6), CD66a (CEACAM1), CD66b (CEACAM8), CD66c (CEACAM6), CD66d (CEACAM3), CD66e (CEACAM5), CD69 (CLEC2), and CD79A. (β-chain of B-cell antigen receptor complex-associated), CD79B (β-chain of B-cell antigen receptor complex-associated), CD84 (SLAMF5), CD96 (Tactile), CD100 (SEMA4D), CD103 (ITGAE), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD158A (KIR2DL1), CD158B1 (KIR2DL2), CD158B2 (KIR2DL3), CD158C (KIR3DP1), CD158D (KIRDL4), CD158F1 (KIR2DL5A), CD158F2 (KIR2DL5B), CD158K (KIR3DL2), CD160 (BY55), CD162 (SELPLG), CD226 (DNAM1), CD229 (SLAMF3), CD244 (SLAMF4), CD247 (CD3-ζ), CD258 (LIGHT), CD268 (BAFFR), CD270 (TNFSF14), CD272 (BTLA), CD276 (B7-H3), CD279 (PD-1), CD314 (NKG2D), CD319 (SLAMF7), CD335 (NK-p46), CD336 (NK-p44), CD337 (NK-p30), CD352 (SLAMF6), CD353 (SLAMF8), CD355 (CRTAM), CD357 (TNFRSF18), Inducible T cell costimulators (ICOS), LFA-1(CD11a / CD18), NKG2C, DAP-10, ICAM-1, NKp80 (KLRF1), IL-2Rβ, IL-2Rγ, IL-7Rα, LFA-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1 / CBP, CD83 ligand, Fcγ receptor, MHC class 1 molecules, MHC class 2 molecules, TNF receptor protein, immunoglobulin protein, cell mediator receptor, integrin, activated NK cell receptor, Toll ligand receptor, and fragments or combinations thereof.

[0294] Examples of nucleotide sequences encoding co-stimulatory domains are listed in SEQ ID NO. 22:

[0295] In one specific embodiment, the polynucleotide encoding the co-stimulatory signal transduction domain includes and The nucleotide sequence is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical.

[0296] An example of a co-stimulation signal transduction structure domain is

[0297] In some specific embodiments, the intracellular signal transduction domain within the co-stimulatory domain includes... An amino acid sequence with at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 96%, at least 97%, at least approximately 98%, at least approximately 99%, or at least 100% identity.

[0298] Another example of a nucleotide sequence encoding a signal transduction domain within the cell is

[0299] In one specific embodiment, the polynucleotide encoding the co-stimulatory signal transduction domain comprises a nucleotide sequence that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the following nucleotide sequences.

[0300] Another example of an intracellular signal transduction domain is serialized in SEQ ID NO. 25:

[0301] In some specific embodiments, the intracellular signal transduction domain includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the following amino acid sequences.

[0302] The CAR co-stimulatory signaling sequences disclosed herein can be directly linked, randomly or sequentially, to another co-stimulatory domain, to an activation domain, to a transmembrane domain, or to other components of the CAR. Short oligo- or polypeptide linkers, particularly those between 2 and 10 amino acids in length, can form links as needed. Glycine-serine dual (GS), glycine-serine-glycine triple (GSG), or alanine-alanine-alanine triple (AAA) linkers provide particularly suitable linkers.

[0303] It is further noted that multiple co-stimulatory domains can be incorporated into the CAR or TCR disclosed herein. For example, both the CD28T co-stimulatory domain and the 4-1BB co-stimulatory domain can be incorporated into the CAR or TCR disclosed herein, and can still target CD70 and cells expressing CD70 on their surface by means of the antigen-binding component of the CAR or TCR. IV.D Activation Domain

[0304] In some specific embodiments, the intracellular domains of the engineered T cells used in this disclosure include cytoplasmic sequences of the T cell receptor (TCR) and co-receptor that work synergistically to initiate signal transduction after antigen / receptor binding, as well as any derivatives or variants of these sequences and any synthetic sequences having the same functional capabilities. CD3 is an element of the T cell receptor on natural T cells and has been shown to be an important intracellular activating element in CARs. In one specific embodiment, the activation domain is CD3, such as CD3ζ, whose nucleotide sequence is listed in SEQ ID NO:26:

[0305] In some specific embodiments, the polynucleotide encoding the activation domain includes... The nucleotide sequence is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical.

[0306] The corresponding amino acid for intracellular CD3ζ is

[0307] In some specific embodiments, the activation domain includes and The amino acid sequence is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence.

[0308] In some specific embodiments, the activation domain includes and The amino acid sequence is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence. IV.E. precursor peptide

[0309] In some specific embodiments, the polynucleotide of this disclosure encodes a CAR or TCR, wherein the CAR or TCR comprises an antigen-binding molecule that specifically binds to CD70 and wherein the CAR or TCR further comprises a leader peptide (also referred to herein as a "signal peptide" or "signal sequence"). A signal sequence may be included in the CAR or TCR of this disclosure as needed. If a leader sequence is included in the CAR or TCR of this disclosure, it may be represented at the N-terminus of the CAR or TCR. Thus, depending on which domain is located at the N-terminus of the CAR or TCR, the leader sequence may associate with the VH or VL component of the antigen-binding molecule of the CAR or TCR of this disclosure.

[0310] If a leader sequence is to be included, the leader sequence can be synthesized or derived from naturally occurring molecules. For example, the naturally occurring 21-residue leader sequence of CD8 (see, for example, Littman et al., (1985) Cell40:237-46) can be used as a leader sequence in the disclosed CAR and TCR constructs.

[0311] Therefore, in some specific embodiments, the precursor peptide comprises an amino acid sequence. An amino acid sequence with at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or 100% identity.

[0312] In some specific embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO:28. In some specific embodiments, the leader peptide is encoded by a nucleotide sequence that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the following:

[0313] In some specific embodiments, the polynucleotide of this disclosure encodes a CAR, wherein the CAR comprises a leader peptide (P), an antigen-binding molecule (such as scFv linked to human CD70) (B), a hinge domain (H), a transmembrane domain (T), one or more co-stimulatory regions (C), and an activation domain (A), wherein the CAR is configured as follows: PBHTCA. In some specific embodiments, the components of the CAR are linked as needed via linker sequences (such as AAA or GSG). In some specific embodiments, the antigen-binding molecule comprises VH and VL, wherein the CAR is configured as follows: P-VH-VL-HTCA or P-VL-VH-HTCA. In some specific embodiments, VH and VL are linked by a linker (L) (such as SEQ ID NO: 80 or 81), wherein the CAR from the N-terminus to the C-terminus is configured as follows: P-VH-L-VL-HTCA or P-VH-L-VL-HTCA.

[0314] In some specific embodiments, the polynucleotide of this disclosure encodes a CAR, wherein the CAR comprises an amino acid sequence that is at least about 75%, at least about 85%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the amino acid sequences shown in Figures 8A to 8L and Table 2. In some specific embodiments, the polynucleotide of this disclosure encodes a CAR, wherein the CAR comprises the amino acid sequences shown in Figures 8A to 8L and Table 2.

[0315]

[0316] In some specific embodiments, the disclosed polynucleotide encodes a CAR, wherein the CAR comprises an amino acid sequence that is at least about 75%, at least about 85%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO: 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, and 52. In some specific embodiments, the CAR comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, and 52. In one specific embodiment, the CAR comprises the amino acid sequence of SEQ ID NO: 30. In another specific embodiment, the CAR comprises the amino acid sequence of SEQ ID NO: 32. In yet another specific embodiment, the CAR comprises the amino acid sequence of SEQ ID NO: 34. In another embodiment, the CAR comprises the amino acid sequence of SEQ ID NO:36. In another embodiment, the CAR comprises the amino acid sequence of SEQ ID NO:38. In another embodiment, the CAR comprises the amino acid sequence of SEQ ID NO:40. In another embodiment, the CAR comprises the amino acid sequence of SEQ ID NO:42. In another embodiment, the CAR comprises the amino acid sequence of SEQ ID NO:44. In another embodiment, the CAR comprises the amino acid sequence of SEQ ID NO:46. In another embodiment, the CAR comprises the amino acid sequence of SEQ ID NO:48. In another embodiment, the CAR comprises the amino acid sequence of SEQ ID NO:50. In another embodiment, the CAR comprises the amino acid sequence of SEQ ID NO:52.

[0317] In some specific embodiments, the polynucleotide encoding CAR disclosed herein comprises a nucleotide sequence that is at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 85%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO: 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, and 51. In some specific embodiments, the polynucleotide comprises a nucleotide sequence selected from the group consisting of: SEQ ID NO: 29. In one specific embodiment, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 31. In another specific embodiment, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 33. In another specific embodiment, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 35. In another specific embodiment, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 37. In another embodiment, the polynucleotide comprises the nucleotide sequence of SEQ ID NO:39. In another embodiment, the polynucleotide comprises the nucleotide sequence of SEQ ID NO:41. In another embodiment, the polynucleotide comprises the nucleotide sequence of SEQ ID NO:43. In another embodiment, the polynucleotide comprises the nucleotide sequence of SEQ ID NO:45. In another embodiment, the polynucleotide comprises the nucleotide sequence of SEQ ID NO:47. In another embodiment, the polynucleotide comprises the nucleotide sequence of SEQ ID NO:49. In another embodiment, the polynucleotide comprises the nucleotide sequence of SEQ ID NO:51. IV. Carriers, cells, and pharmaceutical ingredients

[0318] In some embodiments, the present invention provides vectors comprising the polynucleotides disclosed herein. In some specific embodiments, the disclosure pertains to vectors or groups of vectors comprising polynucleotides encoding CARs or TCRs as described herein. In other specific embodiments, the disclosure pertains to vectors or groups of vectors comprising polynucleotides encoding antibodies or antigen-binding molecule specifically binding to CD70 as disclosed herein.

[0319] Any vector known in the art may be suitable for use in this disclosure. In some specific embodiments, the vector is a viral vector. In some specific embodiments, the vector is a retroviral vector, a DNA vector, a murine leukemia virus vector, an SFG vector, a plastid, an RNA vector, an adenovirus vector, a baculoviral vector, an Epstein-Barr viral vector, a papovaviral vector, a vaccinia viral vector, a herpes simplex viral vector, an adenovirus-associated vector (AAV), a lentiviral vector, or any combination thereof. In some specific embodiments, one, two, or more vectors disclosed herein may be used. For example, in one specific embodiment, one or more components of a CAR or TCR may be configured in one vector, while one or more different components of a CAR or TCR may be configured in different vectors.

[0320] In other embodiments, the present invention provides cells comprising the polynucleotides or vectors disclosed herein. In some specific embodiments, the disclosure pertains to host cells, such as in vitro cells, comprising polynucleotides encoding CARs or TCRs as described herein. In some specific embodiments, the disclosure pertains to host cells, such as in vitro cells, comprising polynucleotides encoding antibodies or antigen-binding molecule specifically binding to CD70 as disclosed herein.

[0321] In other specific embodiments, this disclosure pertains to in vitro cells comprising polypeptides encoded by polynucleotides encoding CAR or TCR as disclosed herein. In other specific embodiments, this disclosure pertains to cells, or in vitro cells, comprising polypeptides encoded by polynucleotides encoding antigen-binding molecule specifically binding to CD70 as disclosed herein.

[0322] Any cell can serve as the host cell for the polynucleotides, vectors, or polypeptides disclosed herein. In some specific embodiments, the cell may be a prokaryotic cell, fungal cell, yeast cell, or higher eukaryotic cell, such as a mammalian cell. Suitable prokaryotic cells include, but are not limited to, eubacteria, such as Gram-negative or Gram-positive microorganisms, such as Enterobacteriaceae, such as Escherichia, for example, E. coli; Enterobacter; Erwinia; Klebsiella; Proteus; Salmonella, for example, Salmonella typhimurium; Serratia, for example, Serratia marcescans and Shigella; bacilli, such as Bacillus subtilis and Bacillus licheniformis; pseudomonads, such as Pseudomonas aeruginosa; and Streptomyces. In some specific embodiments, the host cell is a human cell. In some specific embodiments, the cell is an immune cell. In some embodiments, the immune cell line is selected from the group consisting of: T cells, B cells, tumor-infiltrating lymphocytes (TILs), TCR-expressing cells, natural killer (NK) cells, dendritic cells, granulocytes, intrinsic lymphoid cells, megakaryocytes, monocytes, macrophages, platelets, thymocytes, and myeloid cells. In one embodiment, the immune cell is a T cell. In another embodiment, the immune cell is an NK cell. In some embodiments, the T cell is a tumor-infiltrating lymphocyte (TIL), autologous T cells, engineered autologous T cells (eACT™), allogeneic T cells, xenogeneic T cells, or any combination thereof.

[0323] The cells disclosed herein can be obtained from any source known in the art. For example, T cells can be differentiated in vitro from hematopoietic stem cell populations, or T cells can be obtained from an individual. T cells can be obtained from, for example, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors. Furthermore, T cells can be derived from one or more T cell lines available in the art. T cells can also be obtained from blood units collected from an individual using any number of techniques known to those skilled in the art, such as FICOLL™ ablation and / or blood component separation. In some specific embodiments, cell lines collected by blood component separation are washed to remove the plasma fraction and placed in appropriate buffers or culture media for further processing. In some specific embodiments, cell lines are washed with PBS. It is understood that the washing step can be performed using, for example, a semi-automated flowthrough centrifuge, such as the Cobe™ 2991 cell processor, Baxter CytoMate™, etc. In some specific embodiments, the washed cell lines are resuspended in one or more biocompatible buffer solutions, or other saline solutions with or without buffer solutions. In some specific embodiments, unwanted components of the blood component separation sample are removed. Additional methods for isolating T cells for T-cell therapy are disclosed in U.S. Patent Publication No. 2013 / 0287748 (the entirety of which is incorporated herein by reference).

[0324] In some embodiments, T cells are isolated from PBMCs by lysing red blood cells and depleting monocytes, for example, by centrifugation using a PERCOLL™ gradient. In some embodiments, specific subsets of T cells, such as CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, can be further isolated using positive or negative selection techniques known in the art. For example, enrichment of T cell populations by negative selection can be accomplished using a combination of antibodies targeting surface markers specific to the negatively selected cells. In some embodiments, cell sorting and / or selection via negative magnetic immunoadhesion or flow cytometry (which uses a mixture of monoclonal antibodies targeting cell surface markers present on the negatively selected cells) can be used. For example, to enrich CD4+ cells by negative selection, the monoclonal antibody mixture typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some specific embodiments, flow cytometry and cell sorting are used to isolate the cell populations of interest used in this disclosure. Using these standard techniques, when performing the methods provided herein, the engineered T cells delivered to a patient can contain any desired cell ratio. For example, it can be to provide the patient with only engineered CD8+ cells, only engineered CD4+ cells, or a desired ratio of CD4+ to CD8+ cells, such as equal amounts of CD4+ and CD8+ cells.

[0325] In some specific embodiments, PBMCs can be used directly for genetic modification with immune cells (such as CARs or TCRs) using methods as described herein. In some specific embodiments, after isolating PBMCs, T lymphocytes can be further isolated, and cytotoxic and helper T lymphocytes can be sorted into naive T cells, memory T cells, and effector T cell subsets before or after genetic modification and / or expansion.

[0326] In some embodiments, CD8+ cells are further sorted into initial cells, central memory cells, and effector cells by identifying cell surface antigens associated with each of the initial, central memory, and effector cell types of CD8+ cells. In some embodiments, the phenotypic markers of central memory T cells include CD3, CD28, CD44, CD45RO, CD45RA, and CD127, and are granzyme B negative. In some embodiments, central memory T cells are CD3+, CD28+, CD44 hi, CD45RO hi, CD45RA low, and CD127 hi CD8+ T cells. In some embodiments, effector T cells are CD62L, CCR7, CD28, and CD127 negative, and are granzyme B and perforin positive. In some embodiments, CD4+ T cells are further sorted into subsets. For example, CD4+ T helper cells can be sorted into initial cells, central memory cells, and effector cells by identifying cell populations with cell surface antigens.

[0327] In some specific embodiments, immune cells, such as T cells, are genetically modified using known methods after isolation, or the immune cell line is activated and expanded in vitro (or, in the case of progenitor cells, differentiated) prior to genetic modification. In another specific embodiment, immune cells, such as T cell lines, are genetically modified with chimeric antigen receptors as described herein (e.g., transduced with a viral vector containing one or more nucleotide sequences encoding CARs), and then activated and / or expanded in vitro. Methods for activating and expanding T cells are known in the art and described, for example, in U.S. Patent Nos. 6,905,874; 6,867,041; and 6,797,514; and PCT Publication No. WO 2012 / 079000 (the entire contents of which are incorporated herein by reference). Generally, these methods involve contacting PBMCs or isolated T cells in a culture medium containing appropriate cell mediators (such as IL-2) with stimulants and co-stimulants (such as anti-CD3 and anti-CD28 antibodies, typically adhered to beads or other surfaces). The anti-CD3 and anti-CD28 antibody system adhered to the same beads serves as "alternative antigen-presenting cells" (APCs). One example is the Dynabeads® system, a CD3 / CD28 activator / stimulator system for physiologically activating human T cells. In other specific embodiments, the T cell line uses methods described in, such as U.S. Patent Nos. 6,040,177 and 5,827,642 and PCT Publication No. WO 2012 / 129514 (the entire contents of which are incorporated herein by reference), to feed the cells and activate and stimulate them for proliferation with appropriate antibodies and cell mediators.

[0328] In some embodiments, the T cell line is obtained from a donor individual. In some embodiments, the donor individual is a human patient with cancer or tumor. In other embodiments, the donor individual is a human patient without cancer or tumor. In yet another embodiment, the T cell line is derived from pluripotent stem cells maintained under conditions conducive to stem cell differentiation into T cells.

[0329] Other embodiments disclosed herein relate to compositions comprising the polynucleotides, carriers, peptides, or in vitro cells provided herein. In some embodiments, the compositions comprise pharmaceutically acceptable carriers, diluents, solubilizers, emulsifiers, preservatives, and / or adjuvants. In some embodiments, the compositions comprise excipients. In one embodiment, the composition comprises a polynucleotide encoding a CAR or TCR, wherein the CAR or TCR comprises an antigen-binding molecule that specifically binds to CD70. In another embodiment, the composition comprises a CAR or TCR encoded by a polynucleotide provided herein, wherein the CAR or TCR comprises an antigen-binding molecule that specifically binds to CD70. In another embodiment, the composition comprises T cells containing a polynucleotide encoding a CAR or TCR, wherein the CAR or TCR comprises an antigen-binding molecule that specifically binds to CD70. In yet another embodiment, the composition comprises an antibody or its antigen-binding molecule encoded by a polynucleotide provided herein. In another specific embodiment, the composition comprises in vitro cells containing a polynucleotide encoding an antibody or antigen-binding molecule encoded by a polynucleotide provided herein. In yet another specific embodiment, the composition comprises a CAR or TCR provided herein, wherein the CAR or TCR comprises a component containing an antigen-binding molecule that specifically binds to CD70.

[0330] In some specific embodiments, the composition includes more than one different CAR or TCR containing a component comprising an antigen-binding molecule that specifically binds to CD70. In some specific embodiments, the composition includes more than one CAR or TCR containing a component comprising an antigen-binding molecule that specifically binds to CD70, wherein the antigen-binding molecule to CD70 binds to more than one antigenic determinant site. In some specific embodiments, the CARs or TCRs containing components comprising antigen-binding molecules that specifically bind to CD70 will not compete with each other to bind to CD70. In some specific embodiments, any CAR or TCR comprising components comprising antigen-binding molecules that specifically bind to CD70 provided herein are combined together in a pharmaceutical composition.

[0331] In other embodiments, the composition is selected for parenteral delivery. The preparation of this pharmaceutically acceptable composition is within the capabilities of those skilled in the art to which this invention pertains. In some embodiments, a buffer solution is used to maintain the composition at a physiological pH or slightly lower, typically in the pH range of about 5 to about 8. In some embodiments, when considering parenteral administration, the composition is in the form of a pyrogen-free, parenteral-acceptable aqueous solution containing a desired CAR or TCR in a pharmaceutically acceptable carrier, the desired CAR or TCR comprising a component containing an antigen-binding molecule that specifically binds to CD70, with or without an additional therapeutic agent. In some embodiments, the carrier for parenteral injection is sterile distilled water comprising a CAR or TCR containing a component containing an antigen-binding molecule that specifically binds to CD70, with or without at least one additional therapeutic agent, formulated as a properly preserved sterile isotonic solution. In some specific embodiments, preparation involves formulating a CAR or TCR comprising a component containing an antigen-binding molecule that specifically binds to CD70, and a polymeric compound (such as polylactic acid or polyglycolic acid), beads, or liposomes that provide controlled or sustained release of the product, which is then delivered via depot injection. In some specific embodiments, an implantable drug delivery device is used to introduce the desired molecule. V. Methods using the disclosed antigen-binding molecules, CARs, and TCRs

[0332] Another aspect of this disclosure pertains to a method for manufacturing cells expressing a CAR or TCR, comprising transducing cells with the polynucleotides disclosed herein under suitable conditions. In some embodiments, the method comprises transducing cells with polynucleotides encoding a CAR or TCR, wherein the CAR or TCR comprises an antigen-binding molecule that specifically binds CD70 as disclosed herein. In some embodiments, the method comprises transducing cells with a vector comprising a polynucleotide encoding a CAR or TCR, wherein the CAR or TCR comprises an antigen-binding molecule that specifically binds CD70. In other embodiments, the method comprises transducing cells with polynucleotides encoding a CAR or TCR comprising an antigen-binding molecule that specifically binds CD70 as disclosed herein. In some embodiments, the method comprises transducing cells with a vector containing a polynucleotide encoding a CAR or TCR comprising an antigen-binding molecule that specifically binds CD70 as described herein. In some embodiments, the method further comprises single-cell transduction.

[0333] Another aspect of this disclosure pertains to a method for inducing anti-tumor immunity, comprising administering to an individual an effective amount of cells comprising a polynucleotide as described herein, a vector as described herein, or a CAR or TCR as described herein. In one embodiment, the method comprises administering to an individual cells comprising a polynucleotide encoding a CAR or TCR, wherein the CAR or TCR comprises an antigen-binding molecule that specifically binds to CD70 as disclosed herein. In another embodiment, the method comprises administering to an individual cells comprising a vector containing a polynucleotide encoding a CAR or TCR, wherein the CAR or TCR comprises an antigen-binding molecule that specifically binds to CD70 as disclosed herein. In yet another embodiment, the method comprises administering to an individual cells comprising a CAR or TCR encoded by a polynucleotide disclosed herein, wherein the CAR or TCR comprises an antigen-binding molecule that specifically binds to CD70.

[0334] In other embodiments, the method includes administering an effective amount of cells to an individual, the cells containing a polynucleotide encoding a CAR or TCR comprising an antigen-binding molecule that specifically binds to CD70 as disclosed herein. In another embodiment, the method includes administering an effective amount of cells comprising a vector to an individual, the vector containing a polynucleotide encoding a CAR or TCR comprising an antigen-binding molecule that specifically binds to CD70 as disclosed herein. In yet another embodiment, the method includes administering an effective amount of cells to an individual, the cells containing an antibody or antigen-binding molecule (e.g., CAR or TCR) encoded by a polynucleotide disclosed herein, wherein the antibody or antigen-binding molecule specifically binds to CD70.

[0335] Another aspect of the present invention pertains to a method for inducing an immune response in an individual, comprising administering an effective amount of the engineered immune cells of this application. In some embodiments, the immune response is a T-cell-mediated immune response. In some embodiments, the T-cell-mediated immune response is directed against one or more target cells. In some embodiments, the engineered immune cells comprise CARs or TCRs, such as those provided herein. In some embodiments, the target cells are tumor cells.

[0336] Another aspect of this disclosure relates to a method for treating or preventing malignant tumors, which involves administering to an individual in need an effective amount of at least one isolated antigen-binding molecule as described herein or at least one immune cell, wherein the immune cell comprises at least one CAR, TCR and / or isolated antigen-binding molecule as described herein.

[0337] Another aspect of this disclosure is a method for treating hyperplastic disorders or inflammatory diseases in individuals in need, comprising administering to the individual the polynucleotides, vectors, CARs or TCRs disclosed herein, cells, or components disclosed herein. In some specific embodiments, the inflammatory disease is selected from the group consisting of: rheumatoid arthritis, psoriasis, allergies, asthma, autoimmune diseases (such as Crohn's disease), IBD, fibromyalgia, mast cell disease, celiac disease, and any combination thereof.

[0338] Another aspect of this disclosure pertains to a method for treating cancer in an individual in need, comprising administering to the individual the polynucleotide, vector, CAR or TCR disclosed herein, cell, or composition disclosed herein. In one embodiment, the method comprises administering a polynucleotide encoding a CAR or TCR, wherein the CAR or TCR comprises an antigen-binding molecule that specifically binds to CD70 as disclosed herein. In another embodiment, the method comprises administering to a vector containing a polynucleotide encoding a CAR or TCR, wherein the CAR or TCR comprises an antigen-binding molecule that specifically binds to CD70 as disclosed herein. In another embodiment, the method comprises administering a CAR or TCR encoded by a polynucleotide disclosed herein, wherein the CAR or TCR comprises an antigen-binding molecule that specifically binds to CD70 as disclosed herein. In yet another embodiment, the method comprises administering to a cell comprising a polynucleotide encoding a CAR or TCR or a vector comprising a polynucleotide encoding a CAR or TCR, wherein the CAR or TCR comprises an antigen-binding molecule that specifically binds to CD70 as disclosed herein. In other embodiments, the method includes administering a polynucleotide encoding a CAR or TCR as disclosed herein, wherein the CAR or TCR comprises an antigen-binding molecule that specifically binds to CD70. In another embodiment, the method includes administering a vector containing a polynucleotide encoding a CAR or TCR as disclosed herein, wherein the CAR or TCR comprises an antigen-binding molecule that specifically binds to CD70. In another embodiment, the method includes administering an antibody, CAR, or TCR, wherein the CAR or TCR comprises an antigen-binding molecule that specifically binds to CD70 and is encoded by a polynucleotide disclosed herein. In another embodiment, the method includes administering cells having a polynucleotide encoding an antibody, CAR, or TCR as disclosed herein, or administering a vector containing a polynucleotide encoding an antibody, CAR, or TCR, wherein the CAR or TCR comprises an antigen-binding molecule that specifically binds to CD70.

[0339] In some specific embodiments, antibodies, CARs, or TCRs are administered individually, wherein the CAR or TCR contains an antigen-binding molecule that specifically binds to CD70. In these immune cells, antibodies, CARs, or TCRs may be under the control of the same promoter region or different promoters, wherein the CAR or TCR contains an antigen-binding molecule that specifically binds to CD70. In some specific embodiments, the genes encoding protein agents and / or antibodies, CARs, or TCRs may be in different vectors, wherein the CAR or TCR contains an antigen-binding molecule that specifically binds to CD70.

[0340] In some specific embodiments, methods for treating cancer in individuals with a need include T-cell therapy. In one specific embodiment, the T-cell therapy disclosed herein is an engineered autologous cell therapy (eACT™). According to this specific embodiment, the method may include collecting blood cells from a patient. The isolated blood cells (e.g., T cells) may then be engineered to represent the disclosed anti-CD70 CAR (“anti-CD70 CAR T cells”). In a particular specific embodiment, the anti-CD70 CAR T cell line is administered to the patient. In some specific embodiments, the anti-CD70 CAR T cells treat the patient’s tumor or cancer. In one specific embodiment, the anti-CD70 CAR T cells shrink the size of the tumor or cancer.

[0341] In some embodiments, the donor T cell line for T cell therapy is obtained from the patient (e.g., for autologous T cell therapy). In other embodiments, the donor T cell line for T cell therapy is obtained from an individual who is not the patient (e.g., for allogeneic T cell therapy).

[0342] T cells can be administered in a therapeutically effective dose. For example, a therapeutically effective dose of T cells may be at least about 10⁴ cells, at least about 10⁵ cells, at least about 10⁶ cells, at least about 10⁷ cells, at least about 10⁸ cells, at least about 10⁹ cells, at least about 10¹⁰ cells, or at least about 10¹¹ cells. In another specific embodiment, a therapeutically effective dose of T cells is about 10⁴ cells, about 10⁵ cells, about 10⁶ cells, about 10⁷ cells, or about 10⁸ cells. In some specific embodiments, the therapeutically effective doses of anti-CD70 CAR T cells are approximately 1 x 10⁵ cells / kg, 2 x 10⁵ cells / kg, 3 x 10⁵ cells / kg, 4 x 10⁵ cells / kg, 5 x 10⁵ cells / kg, 1 x 10⁶ cells / kg, 2 x 10⁶ cells / kg, approximately 3 x 10⁶ cells / kg, approximately 4 x 10⁶ cells / kg, approximately 5 x 10⁶ cells / kg, approximately 6 x 10⁶ cells / kg, approximately 7 x 10⁶ cells / kg, approximately 8 x 10⁶ cells / kg, approximately 9 x 10⁶ cells / kg, approximately 1 x 10⁷ cells / kg, approximately 2 x 10⁷ cells / kg, approximately 3 x 10⁷ cells / kg, and approximately 4 x 10⁶ cells / kg. 7 cells / kg, approximately 5 x 10⁷ cells / kg, approximately 6 x 10⁷ cells / kg, approximately 7 x 10⁷ cells / kg, approximately 8 x 10⁷ cells / kg, or approximately 9 x 10⁷ cells / kg.

[0343] Another aspect of this disclosure pertains to methods for diagnosis, detection, or validation. In some embodiments, antigen-binding molecules serve as diagnostic or validation tools. In some embodiments, the antigen-binding molecules disclosed herein are used to analyze the amount of CD70 present in a sample and / or individual. In some embodiments, the diagnostic antigen-binding molecule is not neutralized. In some embodiments, the antigen-binding molecules disclosed herein are used or provided in analytical kits and / or methods for detecting CD70 in mammalian tissues or cells to screen / diagnose diseases or disorders related to changes in CD70 levels. In some embodiments, the kit includes an antigen-binding molecule that binds to CD70, along with means for indicating the binding of the antigen-binding molecule to CD70 (if present), and, if necessary, the amount of CD70 protein. Various means for indicating the presence of antigen-binding molecules can be used. For example, fluorophores, other molecular probes, or enzymes can be attached to the antigen-binding molecule and its presence can be observed in various ways. Examples of fluorophores include luciferin, rose red, tetramethylrose red, eosin, red luciferin, coumarin, methyl-coumarin, pyrene, malachite green, violet, Lucifer yellow, Cascade blue, Texas red, IAEDANS, EDANS, BODIPY FL, LC red 640, Cy 5, Cy 5.5, LC red 705, Oregon green, Alexa-Fluor dyes (Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680), Cascade blue, Cascade yellow, and R-phycoerythrin (PE) (molecular red). Probes), FITC, rose red, and Texas red (Pierce), Cy5, Cy5.5, Cy7 (Amersham Life Science). Those skilled in the art will understand that the degree of binding of antigen-binding molecules can be used to determine the amount of CD70 in a sample. VA Cancer Treatment []

[0344] The methods disclosed herein can be used to treat cancer in an individual, reduce tumor size, kill tumor cells, prevent tumor cell proliferation, prevent tumor growth, remove tumors from a patient, prevent tumor recurrence, prevent tumor metastasis, induce patient remission, or any combination thereof. In some embodiments, the methods induce a complete response. In other embodiments, the methods elicit a partial response.

[0345] Cancers that can be treated with the CAR, YCR, and antibody methods described herein include unvascularized, substantially non-vascularized, or vascularized tumors. Cancers may also include solid or non-solid tumors. In some embodiments, the cancer is a blood cancer. In some embodiments, the cancer is a cancer of white blood cells. In other embodiments, the cancer is a cancer of plasma cells. In some embodiments, the cancer is leukemia, lymphoma, or myeloma. In some specific embodiments, the cancer is multiple myeloma, Hodgkin's disease, non-Hodgkin's lymphoma (NHL), primary mediastinal large B-cell lymphoma (PMBC), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), transformed follicular lymphoma, splenic marginal zone lymphoma (SMZL), chronic or acute leukemia, myeloid disorders, including, but not limited to, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL) (including non-T-cell ALL), chronic lymphocytic leukemia (CLL), T-cell lymphoma, one or more B-cell acute lymphoblastic leukemias (“BALL”), T-cell acute lymphoblastic leukemia. TALL, Acute Lymphoblastic Leukemia (ALL), Chronic Myeloid Leukemia (CML), B-cell Lymphoblastic Leukemia, Plasmacytoid Dendritic Cell Tumor, Burkitt's Lymphoma, Germ Cell Leukemia, Small Cell or Large Cell Follicular Lymphoma, Malignant Lymphoproliferative Conditions, MALT Lymphoma, Mantle Cell Lymphoma, Marginal Zone Lymphoma, Myelodysplastic Dysplasia and Myelodysplastic Syndrome (MDS), Hemophagocytic Syndrome (Macrophage Activation Syndrome (MAS) and Hemophagocytic Lymphohistioglobulin Hyperplasia (HLH)), Chronic or Acute Granulomatosis, Large Cell Granuloma, Leukocyte Adhesion Defect, Plasmoblastic Lymphoma, Plasmacytoid Dendritic Cell Tumor, Waldestron Macroglobulinemia Macroglobulinemia, plasma cell proliferation disorders (e.g., asymptomatic myeloma (accumulated multiple myeloma or painless myeloma)), monoclonal gammopathy of undetermined significance (MGUS), plasmacytomas (e.g., plasma cell cachexia, solitary myeloma, solitary plasmacytoma, extramedullary plasmacytoma, and multiple plasmacytoma), systemic amyloid light chain amyloidosis, POEMS syndrome (Crow-Fukase syndrome, Takatsuki disease, PEP syndrome), or combinations thereof. In one specific embodiment, the cancer is myeloma. In some specific embodiments, the cancer is multiple myeloma. In another specific embodiment, the cancer is T- or B-cell lymphoma. []

[0346] In some specific embodiments, the method further includes administration of a chemotherapeutic agent. In some specific embodiments, the selected chemotherapeutic agent is a lymphocyte-clearing (pre-modulation) chemotherapeutic agent. Advantageous pre-modulation treatment regimens, together with relevant advantageous biomarkers, are described in U.S. Patent Application Nos. 15 / 167,977 and 15 / 295,931 and published U.S. Patent Application No. PCT / US2016 / 034885 (the entirety of which is incorporated herein by reference). These documents describe, for example, methods for regulating patients requiring T-cell therapy, including administering specific beneficial doses of cyclophosphamide alone (between approximately 100 mg / m² / day and approximately 2000 mg / m² / day; for example, approximately 100 mg / m² / day, approximately 200 mg / m² / day, approximately 300 mg / m² / day, approximately 400 mg / m² / day, approximately 500 mg / m² / day, approximately 600 mg / m² / day, approximately 700 mg / m² / day, approximately 800 mg / m² / day, approximately 900 mg / m² / day, approximately 1000 mg / m² / day, approximately 1500 mg / m² / day, or approximately 2000 mg / m² / day) or in combination with specific doses of fludarabine (between approximately 10 mg / m² / day and approximately 900 mg / m² / day). Between 2 mg / m² / day; for example, combinations of approximately 10 mg / m² / day, approximately 20 mg / m² / day, approximately 30 mg / m² / day, approximately 40 mg / m² / day, approximately 50 mg / m² / day, approximately 60 mg / m² / day, approximately 70 mg / m² / day, approximately 80 mg / m² / day, approximately 90 mg / m² / day, approximately 100 mg / m² / day, approximately 500 mg / m² / day, or approximately 900 mg / m² / day.

[0347] In some specific embodiments, the dosage regimen involves treating the patient by administering approximately 500 mg / m² / day of cyclophosphamide and approximately 60 mg / m² / day of fludarabine for 3 days prior to administering a therapeutically effective amount of engineered T cells to the patient.

[0348] In some specific embodiments, the dosage regimen involves treating the patient with approximately 600 mg / m² / day of cyclophosphamide and approximately 30 mg / m² / day of fludarabine for 3 days prior to administering a therapeutically effective amount of engineered T cells to the patient.

[0349] In some specific embodiments, the dosage regimen involves treating the patient with approximately 500 mg / m² / day of cyclophosphamide for 2 days and approximately 30 mg / m² / day of fludarabine for 4 days before administering a therapeutically effective amount of engineered T cells to the patient.

[0350] In some specific embodiments, the dosage regimen involves treating the patient with approximately 600 mg / m² / day of cyclophosphamide for 3 days and approximately 30 mg / m² / day of fludarabine for 3 days prior to administering a therapeutically effective amount of engineered T cells to the patient.

[0351] In some specific embodiments, the dosage regimen involves treating the patient with approximately 550 mg / m² / day of cyclophosphamide for 3 days and approximately 30 mg / m² / day of fludarabine for 3 days before administering a therapeutically effective amount of engineered T cells to the patient.

[0352] In some specific embodiments, the dosage regimen involves treating the patient with approximately 500 mg / m² / day of cyclophosphamide for 3 days and approximately 30 mg / m² / day of fludarabine for 3 days prior to administering a therapeutically effective amount of engineered T cells to the patient.

[0353] In some specific embodiments, the dosage regimen involves treating the patient with approximately 450 mg / m² / day of cyclophosphamide for 3 days and approximately 30 mg / m² / day of fludarabine for 3 days before administering a therapeutically effective amount of engineered T cells to the patient.

[0354] In some specific embodiments, the dosage regimen involves treating the patient with approximately 400 mg / m² / day of cyclophosphamide for 3 days and approximately 30 mg / m² / day of fludarabine for 3 days prior to administering a therapeutically effective amount of engineered T cells to the patient.

[0355] In some specific embodiments, the dosage regimen involves treating the patient with approximately 440 mg / m² / day of cyclophosphamide for 2 days and approximately 100 mg / m² / day of etoposide for 2 days before administering a therapeutically effective amount of engineered T cells to the patient.

[0356] In some specific embodiments, the dosage regimen involves treating the patient with approximately 2-4 g / m² / day of cyclophosphamide for 3 days and approximately 200 mg / m² / day of etoposide for 3 days prior to administering a therapeutically effective amount of engineered T cells to the patient.

[0357] In some specific embodiments, the dosage regimen involves treating the patient with approximately 300 mg / m² / day of cyclophosphamide for 3 days and approximately 30 mg / m² / day of fludarabine for 3 days before administering a therapeutically effective amount of engineered T cells to the patient.

[0358] In some specific embodiments, the dosage regimen involves treating the patient with approximately 30-60 mg / kg (approximately 1100 mg / m²-2200 mg / m²) of cyclophosphamide for 3 to 5 days and approximately 25 mg / m² of fludarabine for 3 to 5 days before administering a therapeutically effective amount of engineered T cells to the patient.

[0359] In some specific embodiments, the dosing regimen involves treating the patient with cyclophosphamide (approximately 1 g / m²) daily before administering a therapeutically effective amount of engineered T cells to the patient.

[0360] In some specific embodiments, the dosage regimen involves treating the patient with cyclophosphamide (approximately 1.2 g / m²) daily for up to 4 days prior to administering a therapeutically effective amount of engineered T cells to the patient.

[0361] In some specific embodiments, the protocol involves treating a patient by administering cyclophosphamide (approximately 2 g / m²) daily before administering a therapeutically effective amount of engineered T cells to the patient.

[0362] In some specific embodiments, the dosage regimen involves treating the patient with approximately 25 mg / m² of fludarabine daily for up to 3 days prior to administering a therapeutically effective amount of engineered T cells to the patient.

[0363] In some specific embodiments, the dosage regimen involves treating the patient by administering approximately 90 mg / m² of bendamustine daily for up to 2 days prior to administering a therapeutically effective amount of engineered T cells to the patient.

[0364] In some specific embodiments, the dosage regimen involves treating the patient with approximately 500 mg / m² / day of cyclophosphamide for 2 days and approximately 30 mg / m² / day of fludarabine for 4 days before administering a therapeutically effective amount of engineered T cells to the patient.

[0365] In some specific embodiments, the dosing regimen involves treating the patient with methotrexate on day 1, and with approximately 1000 mg / m² every 12 hours on days 2 and 3, prior to administering a therapeutically effective amount of engineered T cells to the patient.

[0366] Another preferred dosing regimen involves treating the patient with approximately 300 mg / m² of cyclophosphamide every 12 hours on days 1, 2, and 3, 2 mg of vincristine on day 3, and 50 mg / m² of adenomycin on day 3, prior to administering a therapeutically effective dose of engineered T cells.

[0367] Another preferred dosing regimen involves treating the patient with approximately 200 mg / m² / day of cyclophosphamide for 3 days and approximately 20 mg / m² / day of fludarabine for 3 days before administering a therapeutically effective dose of engineered T cells.

[0368] In some specific embodiments, antigen-binding molecules, transduced (or otherwise engineered) cells (such as CARs or TCRs), and chemotherapeutic agents are each administered in amounts that are effective in treating the individual’s disease or condition.

[0369] In some specific embodiments, a composition comprising immune effector cells exhibiting CAR and / or TCR expression as disclosed herein may be administered in combination with any number of chemotherapeutic agents. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN™); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedepa, and uredopa; ethyleneimines and methylmelamines, including hexamethylmelamine, triethylenemelamine, triethylenephosphonamine, triethylenethiophosphonamine, and trimethylolmelamine; and nitrogen mustards such as chlorambucil, naphthiamethoxam, cyclophosphamide, estradiol, ifosfamide, mechlorethamine, and mechlorethamine oxide. Hydrochloride, melphalan, novelbichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;Antibiotics, such as aclacinomycin, actinomycin, autramycin, azoserine, bleomycin, cactinomycin, calicheamicin, carminomycin, carzinophilin, chromomycin, daunorubicin, detorubicin, 6-diaza-5-sideoxy-L-leucine, doxorubicin, epirubicin, etc. Sorboxine, Idarubicin, Marcellomycin, Mitomycin, Mycophenolic acid, Nogalamycin, Olivomycin, Peplomycin, Potfiromycin, Purinemycin, Quelamycin, Rodorubicin, Streptonigrin, Streptozocin, Tuberculin Bercidin, ubenimex, zinostatin, zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidines Analogs, such as ancitabine, azacitidine, azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, fluxuridine, and 5-FU; androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone.Anti-adrenergic drugs, such as aminoglutethimide, mitotane, and trilostane; folic acid supplements, such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defosfamine; demecolcine; diaziquone; elformithine; elliptinium acetate); etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; sizofiran; spirogermanium; tenuazonic acid acid); triaziquone; 2,2',2ˮ-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactalol; piperobroman; gacytosine; arabinoside (Ara-C); cyclophosphamide; thiotepa; paclitaxel-like substances, such as paclitaxel (TAXOL™, Bristol-Myers Squibb) and docetaxel (TAXOTERE) ® (Rorner-Planck); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vincristine; platinum; etoposide (VP-16);Ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunorubicin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DFMO); retinoic acid derivatives, such as TARGRETIN™ (bexarotene), PANRETIN™ (alvitamin A acid); ONTAK™ (denileukin diftitox); esperamicin; capecitabine; and any pharmaceutically acceptable salts, acids, or derivatives thereof. In some specific embodiments, the components of immune effector cells containing CAR and / or TCR expression disclosed herein may be administered in combination with anti-hormonal agents used to regulate or inhibit the effects of hormones on tumors, such as anti-estrogens, including, for example, tamoxifen, raloxifene, aromatase inhibitors of 4(5)-imidazole, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (FARESTON™); and anti-androgens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids, or derivatives of any of the above. When appropriate, combinations of chemotherapy agents may also be administered, including, but not limited to, CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone); EPOCH (etoposide, prednisone, vincristine, and doxorubicin hydrochloride); and carboplatin and gemcitabine.

[0370] In some embodiments, the chemotherapeutic agent is administered at the same time as or within one week of the administration of the engineered cells or nucleic acid. In other embodiments, the chemotherapeutic agent is administered 1 to 4 weeks, 1 week to 1 month, 1 week to 2 months, 1 week to 3 months, 1 week to 6 months, 1 week to 9 months, or 1 week to 12 months after the administration of the engineered cells or nucleic acid. In some embodiments, the chemotherapeutic agent is administered at least one month before the administration of the cells or nucleic acid. In some embodiments, the method further includes administering two or more chemotherapeutic agents.

[0371] A variety of additional therapeutic agents can be used in conjunction with the components described herein. For example, potentially useful additional therapeutic agents include PD-1 inhibitors such as nivolumab (OPDIVO®), pembrolizumab (KEYTRUDA®), pidilizumab (CureTech), and atezolizumab (TECENTRIQ®).

[0372] Additional therapeutic agents suitable for use in combination with this disclosure include, but are not limited to, ibrutinib (IMBRUVICA®), ofatumumab (ARZERRA®), rituximab (RITUXAN®), bevacizumab (AVASTIN®), trastuzumab (HERCEPTIN®), trastuzumab emtansine (KADCYLA®), imatinib (GLEEVEC®), cetuximab (ERBITUX®), panitumumab (VECTIBIX®), catumaxomab (REMOVAB®), and ibritumomab (ZEVALIN®). (®), tositumomab, lentuximab (ADCETRIS®), alemtuzumab (LEMTRADA®), gemtuzumab, erlotinib (TARCEVA®), gefitinib (IRESSA®), vandetanib (CAPRELSA®), afatinib (GIOTRIF®), lapatinib (TYKERB®), neratinib, axitinib (INLYTA®), masitinib (MASIVET®), pazopanib (VOTRIENT®), sunitinib (SUTENT®), sorafenib (NEXAVAR®), lestaurtinib, cediranib, lenvatinib (LENVIMA®), nintedanib (OFEV®), regorafenib (STIVARGA®), semaxanib, tivozanib, entrectinib, cabozantinib (CABOMETYX®), dasatinib (SPRYCEL®), nilotinib(TASIGNA®), Ponatinib (ICLUSIG®), Radotinib (SUPECT®), Bosutinib (BOSULIF®), Ruxolitinib (JAKAVI®), Papritinib, Cobimetinib (COTELLIC®), Selumetinib, Trametinib (MEKINIST®), Binimetinib, Alectinib (ALECENSA®), Ceritinib (ZYKADIA®), Crizotinib (XALKORI®), Aflibercept (EYELEA®), Adipotide, Denileukin Diftitox (ONTAK®), mTOR inhibitors, such as everolimus (AFINITOR®) and sirolimus (TORISEL®), hedgehog inhibitors, such as sonidegib (ODOZMO®) and vismodegib (ERIVEDGE®), and CDK inhibitors, such as CDK inhibitors (palbociclib; IBRANCE®).

[0373] In some specific embodiments, the immune system comprising CAR and / or TCR is administered together with an anti-inflammatory agent. The anti-inflammatory agent or drug may include, but is not limited to: steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, methylprednisolone, prednisolone, prednisone, and triamcinolone), nonsteroidal anti-inflammatory drugs (NSAIDs) including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF drugs, cyclophosphamide, and mycophenolate mofetil. Exemplary NSAIDs include ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors, and sialic acid salts. Exemplary analgesics include acetaminophen, oxycodone, tramadol propoxyphenol hydrochloride, and others. Exemplary glucocorticoids include cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Exemplary bioresponse modifiers include molecules targeting cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors such as TNF antagonists (e.g., etanercept (ENBREL®), adalimumab (HUMIRA®), and infliximab (REMICADE®), chemokine inhibitors, and adhesion molecule inhibitors. These bioresponse modifiers include monoclonal antibodies and molecules in recombinant forms. Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, Gold (orally (auranofin) and intramuscularly), and minocycline.

[0374] In some specific embodiments, the constituent systems described herein are administered together with cellular mediators. As used herein, “cellular mediator” refers to a protein released from one cell population that acts as an intercellular mediator on another cell. Examples of cellular mediators include lymphotropic mediators, mononuclear factors, and conventional polypeptide hormones. Cellular mediators include growth hormones (such as human growth hormone), N-methionine human growth hormone, and bovine growth hormone; parathyroid hormones; thyroxine; insulin; proinsulin; relaxin; pro-relaxin; glycoprotein hormones such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH); hepatocyte growth factor (HGF); fibroblast growth factor (FGF); prolactin; placental lactogen. lactogen; Müllerian duct inhibitory substance; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factor (NGF), such as NGF-β; platelet growth factor; transforming growth factor (TGF), such as TGF-α and TGF-β; insulin-like growth factor-I and insulin-like growth factor-II; erythropoietin (EPO); bone-inducing factors; interferons, bone-inducing factors such as interferon-α, β and γ; colony-stimulating factor (CSF), such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs), such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, and IL-12; IL-15; tumor necrosis factor, such as TNF-α or TNF-β; and other polypeptide factors, including LIF and kit ligand (KL). As used herein, the term cell mediator includes proteins of natural origin or from recombinant cell cultures, as well as bioactive equivalents of naturally occurring cell mediators.

[0375] All publications, patents, and patent applications mentioned in this patent specification are incorporated herein by reference to the extent that each individual publication, patent, or patent application is specifically and individually indicated to be incorporated herein by reference. However, references herein should not be construed as an admission that such references are prior art to this disclosure. Where any definitions or terms provided in any references incorporated herein differ from those provided and discussed herein, the terms and definitions of this invention shall prevail.

[0376] This disclosure is further illustrated by the following examples, which should not be construed as further limitation. All references cited throughout this application are expressly incorporated herein by reference. Example Example 1

[0377] CD70 expression was measured in various cell lines. CD70 expression (fragment / kilobase-length exon / million-corresponding sequencing reads (FPKM) greater than 35) was found in 99% of the multiple myeloma cell lines tested.

[0378] To further characterize CD70 expression, CEM (ATCC), EoL-1 (Sigma), HL-60 (ATCC), KG-1a (ATCC), MV4;11 (ATCC), NAMALWA (ATCC), Raji (ATCC), Toledo (ATCC), and U-937 (ATCC) cells were stained for 30 minutes at 4°C with an anti-CD70 antibody conjugated with PE (BD Pharmingen) in staining buffer. Then, prior to data acquisition, the cells were washed and resuspended in staining buffer containing propidium iodide (BD Pharmingen). Samples were then obtained by flow cytometry and data were analyzed (Figure 2). CD70 expression was observed in cell lines EoL-1, MV4;11, NAMALWA, Raji, Toledo, and U-937 (Figure 2), but not in cell lines CEM, HL-60, and KG-1a (Figure 2). Example 2

[0379] A third-generation lentiviral transfer vector containing the CD70 CAR construct (shown in Figures 1A to 1H, with sequences shown in Figures 8A to 8L, and in the appended sequence listing (SEQ ID NO: 29 to 52)) was used together with ViraPower lentiviral packaging mixture (Life Technologies) to generate the lentiviral supernatant. Briefly, a transfection mixture was generated by mixing 15 μg of DNA and 22.5 μl of polyethileneimine (Polysciences, 1 mg / ml) in 600 μl of OptiMEM medium. The transfection mixture was incubated at room temperature for 5 minutes. Simultaneously, 293T cells (ATCC) were treated with trypsin and counted. Then, a total of 10 × 10⁶ 293T cells were placed in a T75 flask with the transfection mixture. After three days of incubation, the supernatant was collected, filtered through a 0.45 μm filter, and stored at -80°C.

[0380] Peripheral blood mononuclear cells (PBMCs) were isolated from leukocyte concentrates (hemacare) from two different healthy donors using Ficoll®-Paque density centrifugation. PBMCs were stimulated with OKT3 (Muromonab-CD3, 50 ng / ml, Miltenyi Biotec) in R10 medium supplemented with IL-2 (300 IU / ml, Proleukin®, Prometheus® Therapeutics and Diagnostics). Forty-eight hours post-stimulation, cells were transduced with lentiviruses containing different CD70 CAR constructs at a multiplicity of infection (MOI) of 10. Cells were maintained at concentrations ranging from 0.5 × 10⁶ to 2.0 × 10⁶ cells / ml prior to viability analysis.

[0381] On day 14 post-stimulation, transduced T cells were stained for 30 minutes at 4°C with recombinant CD70-Fc (Sino Biological) in staining buffer (BD Pharmingen). Cells were then washed and stained for 30 minutes at 4°C with goat anti-human IgG Fc PE (Jackson ImmunoResearch) in staining buffer. Cells were then washed again and resuspended in staining buffer containing propidium iodide (BD Pharmingen) before data acquisition. All experiments were performed using two different donors. CD70 CAR expression of each construct was observed in transduced cells from both donor 1 (Fig. 3A) and donor 2 (Fig. 3B).

[0382] Fourteen days after T cell stimulation, effector cells (e.g., anti-CD70 CAR T cells) and target cells were cultured in R10 medium at a 1:1 effector cell to target cell (E:T) ratio. Cell lines tested included KG-1a, NAMALWA, and Raji. Sixteen hours after co-culture, the supernatant was analyzed using a Luminex (EMD Millipore) analyzer to detect the production of cell mediators IFNγ (Fig. 4A-4B), TNFα (Fig. 4C-4D), and IL-2 (Fig. 4E-4F). For each anti-CD70 CAR T cell tested in two donors (Fig. 4A-4F), IFNγ (Fig. 4A-4B), TNFα (Fig. 4C-4D), and IL-2 (Fig. 4E-4F) were observed in the supernatant of the NAMALWA and Raji target cell co-cultures. However, lower levels of IFNγ (Fig. 4A-4B), TNFα (Fig. 4C-4D), and IL-2 (Fig. 4E-4F) were observed in the supernatant of KG-1a target cells co-cultured with various CD70 CAR T cells (Fig. 4A-4F).

[0383] Flow cytometry analysis of propidium iodide (PI) uptake by CD3-negative cells was used to assess target cell viability. Anti-CD70 CAR T cells were co-cultured with KG-1a (Fig. 5A-5B), Raji (Fig. 5C), or NAMALWA (Fig. 5D) target cells for 16, 40, 64, 88, or 112 hours. At any time point, limited cell lysis was observed in the KG-1a co-culture with anti-CD70 CAR T cells (Fig. 5A-5B). Example 3

[0384] Donor CD4+ and CD8+ T cells were thawed and washed twice with OpTmizer medium (Life Technologies) (containing 1X penicillin-streptomycin-L-glutamic acid (PSQ) (Gibco)). Cells were counted using a Vi-Cell cell analyzer, and the cell density was adjusted to 1×10⁶ cells / mL in OpTmizer medium (containing 1X PSQ and 300 IU / mL IL-2) (Proleukin®, Prometheus® Therapeutics and Diagnostics). After counting and resuspending the T cells in the medium, soluble anti-CD28 antibody was added to a final concentration of 1 μg / mL. The cells were then transferred to T25 flasks coated with anti-CD3 antibody (Muromonab-CD3, 50 ng / mL, Miltenyi Biotec) (diluted to a final concentration of 1.23 μg / mL with HBSS).

[0385] 24 hours after stimulation (day 1), CD70 lentiviral vectors containing CAR constructs (e.g., SEQ ID NO: 29 and 30) were added to each T cell culture to an MOI of 10. For non-transduced T cells, cells were stimulated and expanded using the same procedure in the presence of 300 IU / mL IL-2, but no viral vector was added to the cells on day 1. 24 hours after transduction, T cells were washed with culture medium (containing pre-specified components), counted, and reseeded at 0.5 × 10⁶ cells / mL.

[0386] On day 14 post-stimulation, transduced T cells were stained for 30 minutes at 4°C with recombinant CD70-Fc (Sino Biological) (in staining buffer (BD Pharmingen)). Cells were then washed and stained for 30 minutes at 4°C with goat anti-human IgG Fc PE (Jackson ImmunoResearch) (in staining buffer). Prior to data acquisition, cells were washed and resuspended in staining buffer containing propidium iodide (BD Pharmingen). All experiments were performed using two different donors. CD70 CAR expression was observed in cells transduced from both donor 3 and donor 4 (Figure 9).

[0387] Following T cell stimulation, effector cells, such as anti-CD70 CAR T cells, were cultured with target cells in R10 medium at a 1:1 or 4:1 effector cell to target cell (E:T) ratio for 14 days. Cell lines tested included KG-1a and Raji. Sixteen hours after co-culture, the supernatant was analyzed using Luminex (EMD Millipore) according to the manufacturer's instructions regarding the production of cell mediators IFNγ (Fig. 11A-11D), TNFα (Fig. 13A-13D), and IL-2 (Fig. 12A-12D). IFNγ (Fig. 11A-11D), TNFα (Fig. 13A-13D), and IL-2 (Fig. 12A-12D) were observed in the supernatant of the Raji target cell co-culture for each anti-CD70 CAR T cell tested in both donors (Fig. 11A-12D, 12A-12D, and 13A-13D). However, in the supernatant of KG-1a target cells co-cultured with various CD70 CAR T cells (Figs. 11A-12D, 12A-12D, and 13A-13D), IFNγ (Fig. 11A-11D), TNFα (Fig. 13A-13D), and IL-2 (Fig. 12A-12D) were observed in lower amounts.

[0388] Flow cytometry analysis of propidium iodide (PI) uptake by CD3-negative cells was used to assess target cell viability. Anti-CD70 CAR T cells were co-cultured with KG-1a or Raji target cells. A small amount of cell lysis was observed in the KG-1a co-culture of anti-CD70 CAR T cells (Figs. 10A-10D).

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Claims

1. A monoisocyanate polynucleotide encoding a chimeric antigen receptor (CAR) or T-cell receptor (TCR) comprising an antigen-binding molecule that specifically binds to CD70 and a CD8α transmembrane domain, wherein, The antigen-binding molecule includes the amino acid sequence of SEQ ID NO:

34.

2. The polynucleotide as described in claim 1, wherein, The antigen-binding molecule is a single-chain.

3. The polynucleotide as described in claim 1, wherein, The antigen-binding molecule contains scFv.

4. The polynucleotide as described in claim 1, wherein, The antigen-binding molecule comprises a variable heavy chain (VH) and a variable light chain (VL), and the VH and VL are linked by a linker.

5. The polynucleotide as described in claim 4, wherein, The VH system is located at the N end of the connector and the VL system is located at the C end of the connector.

6. The polynucleotide as described in claim 4, wherein, The VL system is located at the N end of the connector, and the VH system is located at the N end of the connector.

7. The polynucleotide as described in claim 4, wherein, The linker contains 5 to 100 amino acids.

8. The polynucleotide as described in claim 4, wherein, The linker contains an amino acid sequence that is at least 75%, at least 85%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to one of the amino acid sequences in SEQ ID NO: 80 and 81.

9. The polynucleotide as requested in claim 1, wherein, The antigen-binding molecule binds CD70 with a KD of less than about 1×10⁻⁶ M, less than about 1×10⁻⁷ M, less than about 1×10⁻⁸ M, or less than about 1×10⁻⁹ M.

10. The polynucleotide as requested in claim 1, wherein, The CAR or TCR further includes a constant region.

11. The polynucleotide as claimed in claim 1, wherein, The CD8 α transmembrane domain contains an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 17, 21, or 94.

12. The polynucleotide as described in claim 1, wherein, The CD8 α transmembrane domain is encoded by a nucleotide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 16 or 20.

13. The polynucleotide as requested in claim 1, wherein, The CAR or TCR further includes a hinge region between the transmembrane domain and the antigen-binding molecule.

14. The polynucleotide as described in claim 13, wherein, The hinge region contains all or a fragment of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, CD28, or CD8α.

15. The polynucleotide as requested in claim 14, wherein, The hinge area is CD28T.

16. The polynucleotide as requested in claim 15, wherein, The hinge region contains an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence selected from the group consisting of SEQ ID NO: 15 and 83.

17. The polynucleotide as requested in claim 15, wherein, The hinge region is encoded by a nucleotide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence selected from the group consisting of SEQ ID NO: 14 and 82.

18. The polynucleotide as requested in claim 14, wherein, The hinge region is CD8 α.

19. The polynucleotide as requested in claim 18, wherein, The hinge region contains an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence selected from the group consisting of SEQ ID NO: 17 and 85.

20. The polynucleotides as requested in claim 18, wherein, The hinge region is encoded by a nucleotide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence selected from the group consisting of SEQ ID NO: 16 and 84.

21. The polynucleotide as described in claim 1, wherein, The CAR or TCR further includes a co-stimulation region.

22. The polynucleotide as requested in claim 21, wherein, The co-stimulatory regions are CD2, CD3δ, CD3ε, CD3γ, CD4, CD7, CD8a, CD86, CD11a (ITGAL), CD11b (ITGAM), CD11c (ITGAX), CD11d (ITGAD), CD18 (ITGB2), CD19 (B4), CD27 (TNFRSF7), CD28, CD28T, CD29 (ITGB1), CD30 (TNFRSF8), CD40 (TNFRSF5), CD48 (SLAMF2), CD49a (ITGA1), CD49d (ITGA4), CD49f (ITGA6), CD66a (CEACAM1), CD66b (CEACAM8), CD66c (CEACAM6), CD66d (CEACAM3), CD66e (CEACAM5), CD69 (CLEC2), CD79A (B-cell antigen receptor complex-related α chain), and CD79B. (B-cell antigen receptor complex-associated β chain), CD84 (SLAMF5), CD96 (Tactile), CD100 (SEMA4D), CD103 (ITGAE), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD158A (KIR2DL1), CD158B1 (KIR2DL2), CD158B2 (KIR2DL3), CD158C (KIR3DP1), CD158D (KIRDL4), CD158F1 (KIR2DL5A), CD158F2 (KIR2DL5B), CD158K (KIR3DL2), CD160 (BY55), CD162 (SELPLG), CD226 (DNAM1), CD229 (SLAMF3), CD244 (SLAMF4), CD247 (CD3-ζ), CD258 (LIGHT), CD268 (BAFFR), CD270 (TNFSF14), CD272 (BTLA), CD276 (B7-H3), CD279 (PD-1), CD314 (NKG2D), CD319 (SLAMF7), CD335 (NK-p46), CD336 (NK-p44), CD337 (NK-p30), CD352 (SLAMF6), CD353 (SLAMF8), CD355 (CRTAM), CD357 (TNFRSF18), Inducible T cell costimulators (ICOS), LFA-1 (CD11a / CD18), NKG2C, DAP-10, ICAM-1, NKp80(KLRF1), IL-2Rβ, IL-2Rγ, IL-7Rα, LFA-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1 / CBP, CD83 ligand, Fcγ receptor, MHC class 1 molecules, MHC class 2 molecules, TNF receptor protein, immunoglobulin protein, cell mediator receptor, integrin, activated NK cell receptor, Toll ligand receptor, or signal transduction regions of fragments or combinations thereof.

23. The polynucleotides as described in claim 22, wherein, This co-stimulatory region is the CD28 co-stimulatory region.

24. The polynucleotides as requested in claim 23, wherein, The co-stimulatory region contains an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO:

23.

25. The polynucleotides as requested in claim 23, wherein, The co-stimulatory region is encoded by a nucleotide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO:

22.

26. The polynucleotides as described in claim 22, wherein, The co-stimulatory region is the CD137 (4-1BB) co-stimulatory region.

27. The polynucleotides as described in claim 26, wherein, The co-stimulatory region contains an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO:

25.

28. The polynucleotides as requested in claim 26, wherein, The co-stimulatory region is encoded by a nucleotide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO:

24.

29. The polynucleotide of claim 1, wherein, The CAR or TCR further includes an activation domain.

30. The polynucleotides as described in claim 29, wherein, The activation domain is the CD3ζ domain.

31. The polynucleotides as requested in claim 30, wherein, The activated domain contains an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 27 or 92.

32. The polynucleotides as requested in claim 30, wherein, The activation domain is encoded by a nucleotide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO:

26.

33. The polynucleotide as described in claim 1, wherein, The CAR or TCR further contains a lead peptide.

34. The polynucleotides as described in claim 33, wherein, The precursor peptide contains an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO:

28.

35. The polynucleotides as claimed in claim 33, wherein, The leader peptide is encoded by a nucleotide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO:

95.

36. A polynucleotide encoding a CAR or TCR, wherein, The polynucleotide contains the nucleotide sequence of SEQ ID NO:

33.

37. A vector comprising the polynucleotide as claimed in claim 1.

38. The vector as claimed in claim 37 is a DNA vector, an RNA vector, or any combination thereof.

39. A CAR encoded by a polynucleotide as claimed in claim 1.

40. A TCR encoded by a polynucleotide as claimed in claim 1.

41. A cell comprising the polynucleotide as claimed in claim 1.

42. The cell as requested in claim 41, wherein, This cell contains immune cells.

43. The cell as requested in claim 42, wherein, This cell is a T cell.

44. As in request item 43, of which, The T cell can be an autologous T cell, an allogeneic T cell, or any combination thereof.

45. The cell as requested in claim 41, wherein, This cell is an in vitro cell.

46. ​​A composition comprising a polynucleotide as claimed in claim 1.

47. Components of claim 46, which are dispensed for delivery to individuals.

48. A method for producing cells that express CAR or TCR, comprising transducing cells with a polynucleotide as claimed in claim 1 under appropriate conditions.

49. The method of claim 48 further includes isolating the cell.

50. A CAR, wherein, The CAR contains SEQ ID NO: 34, where the leading sequence of SEQ ID NO: 34 is absent.

51. A TCR, wherein, The TCR contains SEQ ID NO: 34, where the leading sequence of SEQ ID NO: 34 is absent.

52. A TCR, wherein, The TCR contains the amino acid sequence of SEQ ID NO: 34.

Images