Chimeric antigen-receptor targeting CD70

Abstract

Chimeric antigens receptors (CARs) that specifically bind to CD70 are provided.SOLUTION: Provided is a CD70 specific CAR comprising an extracellular ligand-binding domain, a first trans-membrane domain, and an intracellular signaling domain, wherein said extracellular domain contains a single chain Fv fragment (scFv) that binds to the extracellular domain of CD70, and wherein said intracellular signaling domain contains a 4 - 1BB signaling domain. CD70 specific CAR comprising: SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 775,246, filed December 4, 2018, U.S. Provisional Patent Application No. 62 / 641,869, filed March 12, 2018, U.S. Provisional Patent Application No. 62 / 641,873, filed March 12, 2018, U.S. Provisional Patent Application No. 62 / 625,009, filed February 1, 2018, and U.S. Provisional Patent Application No. 62 / 625,019, filed February 1, 2018, each of which is incorporated herein by reference in its entirety. Reference to sequence listing

[0002] This application has been filed electronically via EFS-Web and contains a Sequence Listing that has been submitted electronically in .txt format. The .txt file contains a Sequence Listing entitled "ALGN_014_03WO_SeqList_ST25.txt," created on January 24, 2019, having a size of approximately 725 kilobytes. The Sequence Listing contained in this .txt file is a part of the present specification and is incorporated herein by reference in its entirety.

[0003] The present disclosure relates to chimeric antigen receptors (CARs). CARs can redirect the specificity and reactivity of immune cells to selected targets utilizing the properties of their ligand-binding domains. In particular, the present disclosure relates to CARs that specifically bind to Cluster of Differentiation 70 (CD70-specific CARs). The present disclosure further relates to polynucleotides encoding CD70-specific CARs and isolated cells expressing CD70-specific CARs on their surface. The present disclosure further relates to methods for engineering immune cells that express CD70-specific CARs on their surface. The present disclosure is particularly useful for treating cancers such as lymphoma, leukemia, glioma, or renal cell carcinoma (RCC). The present disclosure further relates to immune cells comprising a CD70-specific CAR (CD70-specific CAR-T cells), compositions comprising CD70-specific CAR-T cells, and methods of using CD70-specific CAR-T cells to treat conditions associated with malignant cells expressing CD70 (e.g., cancer). [Background technology]

[0004] Adoptive transfer of immune cells genetically engineered to recognize malignant tumor-associated antigens has shown promise as a new approach to cancer treatment (see, e.g., Brenner et al., Current Opinion in Immunology, 22(2):251-257 (2010); Rosenberg et al., Nature Reviews Cancer, 8(4):299-308 (2008)). T cells can be engineered to express chimeric antigen receptors (CARs), which are fusion proteins composed of an antigen-recognition moiety and a T-cell activation domain (see, e.g., Eshhar et al., Proc. Natl. Acad. Sci. USA, 90(2):720-724 (1993), and Sadelain et al., Curr. Opin. Immunol, 21(2):215-223 (2009)).

[0005] Cluster of Differentiation 70 (CD70, CD27LG, or TNFSF7) is a member of the tumor necrosis factor (TNF) superfamily and a ligand for the TNF superfamily receptor CD27. The transient interaction between CD27 and CD70 results in T cell costimulation, which is complementary to that mediated by CD28. CD70 is expressed in hematological cancers, such as non-Hodgkin's lymphoma and Hodgkin's disease, and solid tumors, such as glioblastoma and renal cell carcinoma, and its expression on ccRCC is nearly uniform (see, e.g., Grewal I., et al., Expert Opinion on Therapeutic Targets, 12(3):341-351 (2008)). Adoptive transfer of T cells genetically engineered to recognize malignant tumor-associated antigens has shown promise as a novel approach to cancer treatment (see, e.g., Brenner et al., Current Opinion in Immunology, 22(2):251-257 (2010); Rosenberg et al., Nature Reviews Cancer, 8(4):299-308 (2008)). T cells can be engineered to express chimeric antigen receptors (CARs), fusion proteins composed of an antigen recognition moiety and a T cell activation domain (see, e.g., Eshhar et al., Proc. Natl. Acad. Sci. USA, 90(2):720-724 (1993), and Sadelain et al., Current Opinion in Immunology, 21(2):215-223 (2009)). CD70 expression on normal tissues is restricted to activated T cells, B cells, NK cells, and dendritic cells. However, CD70 expression on activated T cells may raise concerns about the manufacturing of CAR T cells, as it may lead to target-induced T cell differentiation, depletion, and fratricide during the manufacturing process.

[0006] Renal cell carcinoma (RCC) originates in the renal cortex and accounts for approximately 90% of kidney cancers. Histologically, RCC can be classified into several subtypes. Clear cell renal cell carcinoma (ccRCC) is the most common and causes the most deaths. Over 320,000 cases of RCC are reported worldwide each year, resulting in approximately 140,000 deaths. The incidence of RCC has steadily increased over the past decade and now accounts for 2–3% of all adult malignancies. Patients with early-stage, localized tumors can elect to undergo surgical resection. However, metastases can occur if localized disease disseminates hematogenously early. Sites of early metastasis include the lungs, lymph nodes, liver, bone, and brain, and, less commonly, the adrenal gland and contralateral kidney. Patients with advanced disease have a high mortality rate, with a median 5-year survival rate of 53% for stage III disease and only 8% for metastatic disease. Current first-line treatment options for advanced disease include small-molecule tyrosine kinase inhibitors (TKIs) that target the vascular endothelial growth factor (VEGF) receptor, such as sunitinib and pazopanib, monoclonal antibodies targeting VEGF, such as bevacizumab, temsirolimus, an inhibitor of mammalian target of rapamycin (mTOR), and high-dose interleukin-2 (IL-2). While these VEGF-targeting therapies have improved overall survival, long-term drug resistance has led to disease recurrence, and there remains an unmet need for treatments for advanced disease (see, e.g., Zarrabi, K. et al., Journal of Hematology and Oncology, 10:38 (2017)).

[0007] Therefore, there is a need for alternative treatments for cancer, particularly malignant tumors containing abnormal expression of CD70. New immunotherapies, such as CAR T therapy, have the potential to significantly improve the outcomes of patients with cancers that express CD70, such as mRCC. Therefore, the treatment of cancer (e.g., mRCC) using CD70-specific CARs and CD70-specific CAR-T cells would be promising therapeutic agents. Provided herein are methods and compositions that address this need. Summary of the Invention

[0008] Provided herein are chimeric antigen receptors (CARs) that bind to CD70, as well as methods for producing and using the receptors. Also provided herein are immune cells, such as T cells, that contain CD70 CARs. Some CD70-specific CARs have been shown to be effective in activating T cells upon contact with CD70 when expressed in T cells. Advantageously, the CD70-specific CARs provided herein bind to human CD70. Even more advantageously, the CD70-specific CAR-T cells provided herein exhibit cytotoxic activity upon contact with CD70-expressing cells. Also provided herein are antibodies that bind to CD70, as well as methods for producing and using the antibodies. The CD70-specific antibodies provided herein bind to human CD70.

[0009] In one embodiment, the present disclosure provides a Cluster of Differentiation 70 (CD70)-specific chimeric antigen receptor (CAR) comprising an extracellular ligand-binding domain, a first transmembrane domain, and an intracellular signaling domain, wherein the extracellular domain contains a single chain Fv fragment (scFv) that binds to the extracellular domain of CD70.

[0010] In some embodiments, the disclosure provides a CD70-specific CAR, wherein the extracellular domain of the CAR provided herein comprises a sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, or 381. and a light chain variable (VL) region comprising three CDRs from the VL region set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, or 380. In some embodiments, the VH region comprises a sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, or 381, or a variant thereof with one or more conservative amino acid substitutions at a residue not within a CDR; and / or the VL region comprises the amino acid sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378 or 380, or a variant thereof with one or more amino acid substitutions at amino acids not within the CDRs.

[0011] In some embodiments, the VH region contains a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 49, 50 or 51, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 52 or 53, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 54, and the VL region contains a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 193, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 194, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 195.

[0012] In some embodiments, the VH region contains the amino acid sequence set forth in SEQ ID NO:2 and the VL region contains the amino acid sequence set forth in SEQ ID NO:1.

[0013] In some embodiments, the VH region contains a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 55, 56 or 57, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 58 or 59, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 60, and the VL region contains a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 196, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 197, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 198.

[0014] In some embodiments, the VH region contains the amino acid sequence set forth in SEQ ID NO:4, and the VL region contains the amino acid sequence set forth in SEQ ID NO:3.

[0015] In some embodiments, the VH region contains a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 61, 62 or 63, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 64 or 65, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 66, and the VL region contains a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 199, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 200, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 201.

[0016] In some embodiments, the VH region contains the amino acid sequence set forth in SEQ ID NO:6 and the VL region contains the amino acid sequence set forth in SEQ ID NO:5.

[0017] In some embodiments, the VH region contains a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 67, 68 or 69, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 70 or 71, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 72, and the VL region contains a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 202, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 203, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 204.

[0018] In some embodiments, the VH region contains the amino acid sequence set forth in SEQ ID NO:8 and the VL region contains the amino acid sequence set forth in SEQ ID NO:7.

[0019] In some embodiments, the VH region contains a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 73, 74 or 75, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 76 or 77, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 78, and the VL region contains a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 205, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 206, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 207.

[0020] In some embodiments, the VH region contains the amino acid sequence set forth in SEQ ID NO:10 and the VL region contains the amino acid sequence set forth in SEQ ID NO:9.

[0021] In some embodiments, the VH region contains a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 79, 80 or 81, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 82 or 83, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 84, and the VL region contains a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 208, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 209, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 210.

[0022] In some embodiments, the VH region contains the amino acid sequence set forth in SEQ ID NO:12 and the VL region contains the amino acid sequence set forth in SEQ ID NO:11.

[0023] In some embodiments, the VH region contains a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 85, 86 or 87, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 88 or 89, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 90, and the VL region contains a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 211, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 212, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 213. In some embodiments, the VH region contains the amino acid sequence set forth in SEQ ID NO:14, and the VL region contains the amino acid sequence set forth in SEQ ID NO:13.

[0024] In some embodiments, the VH region contains a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 91, 92 or 93, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 94 or 95, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 96, and the VL region contains a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 214, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 215, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 216.

[0025] In some embodiments, the VH region contains the amino acid sequence set forth in SEQ ID NO:16 and the VL region contains the amino acid sequence set forth in SEQ ID NO:15.

[0026] In some embodiments, the VH region contains a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 97, 98 or 99, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 100 or 101, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 102, and the VL region contains a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 217, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 218, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 219.

[0027] In some embodiments, the VH region contains the amino acid sequence set forth in SEQ ID NO:18 and the VL region contains the amino acid sequence set forth in SEQ ID NO:17.

[0028] In some embodiments, the VH region contains a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 103, 104 or 105, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 106 or 107, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 108, and the VL region contains a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 220, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 221, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 222.

[0029] In some embodiments, the VH region contains the amino acid sequence set forth in SEQ ID NO:20, and the VL region contains the amino acid sequence set forth in SEQ ID NO:19.

[0030] In some embodiments, the VH region contains a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 109, 110 or 111, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 112 or 113, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 114, and the VL region contains a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 223, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 224, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 225.

[0031] In some embodiments, the VH region contains the amino acid sequence set forth in SEQ ID NO:22 and the VL region contains the amino acid sequence set forth in SEQ ID NO:21.

[0032] In some embodiments, the VH region contains a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 115, 116 or 117, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 118 or 119, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 120, and the VL region contains a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 226, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 227, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 228.

[0033] In some embodiments, the VH region contains the amino acid sequence set forth in SEQ ID NO:24, and the VL region contains the amino acid sequence set forth in SEQ ID NO:23.

[0034] In some embodiments, the VH region contains a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 121, 122 or 123, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 124 or 125, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 126, and the VL region contains a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 229, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 230, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 231.

[0035] In some embodiments, the VH region contains the amino acid sequence set forth in SEQ ID NO:26, and the VL region contains the amino acid sequence set forth in SEQ ID NO:25.

[0036] In some embodiments, the VH region contains a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 127, 128 or 129, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 130 or 131, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 132, and the VL region contains a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 232, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 233, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 234.

[0037] In some embodiments, the VH region contains the amino acid sequence set forth in SEQ ID NO:28 and the VL region contains the amino acid sequence set forth in SEQ ID NO:27.

[0038] In some embodiments, the VH region contains a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 133, 134 or 135, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 136 or 137, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 138, and the VL region contains a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 235, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 236, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 237.

[0039] In some embodiments, the VH region contains the amino acid sequence set forth in SEQ ID NO:30, and the VL region contains the amino acid sequence set forth in SEQ ID NO:29.

[0040] In some embodiments, the VH region contains a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 139, 140 or 141, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 142 or 143, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 144, and the VL region contains a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 238, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 239, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 240.

[0041] In some embodiments, the VH region contains the amino acid sequence set forth in SEQ ID NO:32, and the VL region contains the amino acid sequence set forth in SEQ ID NO:31.

[0042] In some embodiments, the VH region contains a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 145, 146 or 147, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 148 or 149, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 150, and the VL region contains a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 241, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 242, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 243.

[0043] In some embodiments, the VH region contains the amino acid sequence set forth in SEQ ID NO:34, and the VL region contains the amino acid sequence set forth in SEQ ID NO:33.

[0044] In some embodiments, the VH region contains a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 151, 152 or 153, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 154 or 155, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 156, and the VL region contains a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 244, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 245, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 246.

[0045] In some embodiments, the VH region contains the amino acid sequence set forth in SEQ ID NO:36, and the VL region contains the amino acid sequence set forth in SEQ ID NO:35.

[0046] In some embodiments, the VH region contains a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 157, 158 or 159, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 160 or 161, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 162, and the VL region contains a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 247, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 248, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 249.

[0047] In some embodiments, the VH region contains the amino acid sequence set forth in SEQ ID NO:38, and the VL region contains the amino acid sequence set forth in SEQ ID NO:37.

[0048] In some embodiments, the VH region contains a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 163, 164 or 165, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 166 or 167, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 168, and the VL region contains a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 250, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 251, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 252.

[0049] In some embodiments, the VH region contains the amino acid sequence set forth in SEQ ID NO:40, and the VL region contains the amino acid sequence set forth in SEQ ID NO:39.

[0050] In some embodiments, the VH region contains a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 169, 170 or 171, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 172 or 173, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 174, and the VL region contains a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 253, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 254, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 255.

[0051] In some embodiments, the VH region contains the amino acid sequence set forth in SEQ ID NO:42 and the VL region contains the amino acid sequence set forth in SEQ ID NO:41.

[0052] In some embodiments, the VH region contains a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 175, 176 or 177, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 178 or 179, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 180, and the VL region contains a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 256, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 257, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 258.

[0053] In some embodiments, the VH region contains the amino acid sequence set forth in SEQ ID NO:44, and the VL region contains the amino acid sequence set forth in SEQ ID NO:43.

[0054] In some embodiments, the VH region contains a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 181, 182 or 183, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 184 or 185, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 186, and the VL region contains a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 259, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 260, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 261.

[0055] In some embodiments, the VH region contains the amino acid sequence set forth in SEQ ID NO:46, and the VL region contains the amino acid sequence set forth in SEQ ID NO:45.

[0056] In some embodiments, the VH region contains a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 187, 188 or 189, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 190 or 191, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 192, and the VL region contains a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 262, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 263, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 264.

[0057] In some embodiments, the VH region contains the amino acid sequence set forth in SEQ ID NO:48, and the VL region contains the amino acid sequence set forth in SEQ ID NO:47.

[0058] In some embodiments, each CDR is defined according to the Kabat definition, the Chothia definition, a combination of the Kabat and Chothia definitions, the AbM definition, or the contact definition of a CDR.

[0059] In some embodiments, each CDR is defined according to the Kabat definition, the Chothia definition, the extended definition, a combination of the Kabat and Chothia definitions, the AbM definition, the CDR contact definition, and / or the CDR conformational definition.

[0060] In some embodiments, the intracellular signaling domain contains a CD3ζ signaling domain. In some embodiments, the intracellular signaling domain contains a 4-1BB domain. In some embodiments, the CAR further contains a second intracellular signaling domain. In some embodiments, the second intracellular signaling domain contains a 4-1BB domain. In some embodiments, the CAR contains a first CD3ζ intracellular signaling domain and a second 4-1BB intracellular signaling domain.

[0061] In some embodiments, the intracellular signaling domain contains a CD3ζ signaling domain. In some embodiments, the intracellular signaling domain contains a 4-1BB domain. In some embodiments, the CAR further contains two intracellular signaling domains. In some embodiments, the CAR further contains three, four, five, or six intracellular signaling domains. In some embodiments, the CAR contains a first intracellular signaling domain and a second intracellular signaling domain, wherein the second intracellular signaling domain contains a 4-1BB domain. In some embodiments, the CAR contains a CD3ζ intracellular signaling domain and a 4-1BB intracellular signaling domain.

[0062] In some embodiments, the CAR may contain a stalk domain between the extracellular ligand-binding domain and the first transmembrane domain. In some embodiments, the stalk domain is selected from the group consisting of a human CD8α hinge, an IgG1 hinge, and an FcγRIIIα hinge. In some embodiments, the stalk domain is a human CD8α hinge, a human IgG1 hinge, or a human FcγRIIIα hinge.

[0063] In some embodiments, the CAR may contain an epitope of CD20. In some embodiments, the epitope of CD20 contains the amino acid sequence set forth in SEQ ID NO: 293 or SEQ ID NO: 294 or SEQ ID NO: 609.

[0064] In some embodiments, a CAR may contain the amino acid sequence set forth in SEQ ID NOs: 311-334 listed in Table 5. In some embodiments, a CAR may contain the amino acid sequence set forth in SEQ ID NO: 319 or SEQ ID NO: 327.

[0065] In some embodiments, the first transmembrane domain comprises the transmembrane domain of the CD8 α chain.

[0066] In some embodiments, the CAR may contain another extracellular ligand-binding domain that is not specific for CD70.

[0067] In some embodiments, the extracellular ligand-binding domain, the first transmembrane domain, and the intracellular signaling domain are on a single polypeptide.

[0068] In some embodiments, a CAR may contain a second transmembrane domain, wherein the first transmembrane domain and the extracellular ligand-binding domain are on a first polypeptide, the second transmembrane domain and the intracellular signaling domain are on a second polypeptide, and the first transmembrane domain contains a transmembrane domain derived from the α chain of a high affinity IgE receptor (FcεRI), and the second transmembrane domain contains a transmembrane domain derived from the γ or β chain of FcεRI.

[0069] In some embodiments, the CAR may contain a third polypeptide containing a third transmembrane domain fused to an intracellular signaling domain derived from a costimulatory molecule, wherein the third transmembrane domain contains a transmembrane domain derived from the gamma or beta chain of FcεRI.

[0070] In another aspect, the present disclosure provides an isolated polynucleotide containing a nucleic acid sequence encoding a CD70-specific CAR described herein.

[0071] In another aspect, the present disclosure provides an expression vector containing a polynucleotide encoding a CD70-specific CAR described herein.

[0072] In another aspect, the present disclosure provides engineered immune cells that express the CD70-specific CAR described herein on their cell surface membrane. In some embodiments, the engineered immune cells may contain another CAR that is not specific for CD70. In some embodiments, the engineered immune cells may contain a polynucleotide encoding a suicide polypeptide. In some embodiments, the suicide polypeptide is RQR8.

[0073] In some embodiments, the engineered immunity is derived from inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes, or helper T lymphocytes.

[0074] In some embodiments, the engineered immune cells may have one or more endogenous genes disrupted, where the endogenous genes encode TCRα, TCRβ, CD52, glucocorticoid receptor (GR), deoxycytidine kinase (dCK), CD70, or immune checkpoint proteins such as programmed death-1 (PD-1).

[0075] In some embodiments, the engineered immune cells are obtained from a healthy donor, hi some embodiments, the engineered immune cells are obtained from a patient.

[0076] In another aspect, the present disclosure provides an engineered immune cell that expresses the CD70-specific CAR described herein on its cell surface membrane for use as a pharmaceutical. In some embodiments, the pharmaceutical is a pharmaceutical for use in cancer treatment. In some embodiments, the pharmaceutical is a pharmaceutical for treating renal cell carcinoma, glioblastoma, glioma such as low-grade glioma, non-Hodgkin's lymphoma (NHL), Hodgkin's disease (HD), Waldenstrom's hypergammaglobulinemia, acute myeloid leukemia, multiple myeloma, diffuse large cell lymphoma, follicular lymphoma, or non-small cell lung cancer.

[0077] In another aspect, the disclosure provides a method of engineering an immune cell, the method comprising providing an immune cell and expressing on the cell surface at least one CD70-specific CAR described herein. In some embodiments, the method comprises providing an immune cell, introducing into the cell at least one polynucleotide encoding the CD70-specific CAR, and expressing the polynucleotide in the cell.

[0078] In some embodiments, the method includes providing immune cells, introducing into the cells at least one polynucleotide encoding the CD70-specific CAR, and introducing at least one other CAR that is not specific for CD70.

[0079] In another aspect, the present disclosure provides a method of treating a subject suffering from a condition associated with malignant cells, the method comprising providing immune cells that express a CD70-specific CAR described herein on their surface, and administering the immune cells to the patient.

[0080] In another aspect, the present disclosure provides pharmaceutical compositions containing the engineered immune cells described herein.

[0081] In another aspect, the disclosure provides methods of treating a condition associated with malignant cells that express CD70 in a subject, the method comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising the engineered immune cells described herein. In some embodiments, the condition is cancer. In some embodiments, the cancer is renal cell carcinoma, glioblastoma, glioma, e.g., low-grade glioma, non-Hodgkin's lymphoma (NHL), Hodgkin's disease (HD), Waldenstrom's hypergammaglobulinemia, acute myeloid leukemia, multiple myeloma, diffuse large cell lymphoma, follicular lymphoma, or non-small cell lung cancer.

[0082] In another aspect, the present disclosure provides a method of inhibiting tumor growth or progression in a subject having malignant cells that express CD70, the method comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising the engineered immune cells described herein.

[0083] In another aspect, the present disclosure provides a method of inhibiting metastasis of malignant cells that express CD70 in a subject, the method comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising the engineered immune cells described herein.

[0084] In another aspect, the present disclosure provides a method of inducing tumor regression in a subject having malignant cells that express CD70, the method comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising the engineered immune cells described herein.

[0085] In some embodiments, any of the above methods further comprise administering one or more additional therapies, such as, for example, a monoclonal antibody and / or a chemotherapeutic agent. In some embodiments, the monoclonal antibody may be an antibody that binds to a checkpoint inhibitor, such as, for example, an anti-PD-1 antibody or an anti-PD-L1 antibody. In some embodiments, any of the above methods further comprise administering a receptor tyrosine kinase inhibitor, such as, for example, sunitinib or axitinib.

[0086] In some embodiments, the present disclosure provides a CD70-specific CAR containing an extracellular ligand-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular domain contains a single chain Fv fragment (scFv) that binds to the extracellular domain of CD70 having a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 18. and the VL region contains an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 17, or the VH region contains an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 34, and the VL region contains an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 33.

[0087] In some embodiments, the extracellular domain contains an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 319. In some embodiments, the extracellular domain contains an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 327.

[0088] In some embodiments, the disclosure provides a polynucleotide encoding a CD70-specific CAR, wherein the polynucleotide contains a nucleic acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 297 and at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 298, or contains a nucleic acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 307 and at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 308.

[0089] In some embodiments, the present disclosure provides a CAR containing an antigen-binding molecule that specifically binds to CD70, wherein the antigen-binding molecule is selected from the group consisting of SEQ ID NOs: 49-51, 55-57, 61-63, 67-69, 73-75, 79-81, 85-87, 91-93, 97-99, 103-105, 109-111, 115-117, 121-123, 127-129, 133-135, 139-141, 145-147, 151-153, 157-159, 163-165, 169-171, 175-177, 181-183, 187-189, 190-191, 192-193, 193-194, 194-195, 195-196, 196-197, 197-198, 198-200, 199-200, 201-202, 202-203, 203-204, 204-205, 205-206, 206-207, 207-208, 208-209, 210-211, 212-213, 214-215, 216-217, 218-219, 219-220, 221-222, 222-223, 223-224, 224-225, 225-226, 226-227, 227-22 and variable heavy chain CDR1 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 82-384, 388-390, 394-396, 400-402, 406-408, 412-414, 418-420, 424-426, 430-432, 436-438, 442-444, 448-450, 454-456, 460-462, 466-468, 472-474, 478-480, 484-486, 490-492, 496-498, 502-504, and 508-510. 8, 89, 94, 95, 100, 101, 106, 107, 112, 113, 118, 119, 124, 125, 130, 131, 136, 137, 142, 143, 148, 149, 154, 155, 160, 161, 166, 167, 172, 173, 178, 179, 18 4, 185, 190, 191, 385, 386, 391, 392, 397, 398, 403, 404, 409, 410, 415, 416, 421, 422, 427, 428, 433, 434, 439, 440, 445, 446, 451, 452, 457, 458, 463, 464 , 469, 470, 475, 476, 481, 482, 487, 488, 493, 494, 499, 500, 505, 506, 511, and 512; variable heavy chain CDR2 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 387, 393, 399, 405, 411, 417, 423, 429, 435, 441, 447, 453, 459, 465, 471;477, 483, 489, 495, 501, 507, and 513, and a variable heavy chain CDR3 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 193, 196, 199, 202, 205, 208, 211, 214, 217, 220, 223, 226, 229, 232, 235, 238, 241, 244, 247, 250, 253, 256, 259, 262, 514, 517, 520, 523, 526, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 32, 535, 538, 541, 544, 547, 550, 553, 556, 559, 562, 565, 568, 571, 574, and 577; variable light chain CDR1 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 194, 197, 200, 203, 206, 209, 212, 215, 218, 221, 224, 227, 230, 233, 236, 239, 242, 245, 248, 251, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 31 and a variable light chain CDR2 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 225, 228. , 231, 234, 237, 240, 243, 246, 249, 252, 255, 258, 261, 264, 516, 519, 522, 525, 528, 531, 534, 537, 540, 543, 546, 549, 552, 555, 558, 561, 564, 567, 570, 573, 576, and 579.

[0090] In some embodiments, the antigen-binding molecules are selected from the group consisting of SEQ ID NOs: 49-51, 52-53, 54; 55-57, 58-59, 60; 61-63, 64-65, 66; 67-69, 70-71, 72; 73-75, 76-77, 78; 79-81, 82-83, 84; 85-87, 88-89, 90; 91-93, 94-95, 96; 97-99, 100-101, 102; 103-105, 106-107, 108; 109-111, 112-113, 114; 115-117, 118-119, 120; 121-123, 124-125, 126; 127-128, 129-200, 2010; 129, 130-131, 132; 133-135, 136-137, 138; 139-141, 142-143, 144; 145-147, 148-149, 150; 151-153, 154-155, 156; 157-159, 160-161, 162; 163-165, 166-167, 168; 169-171, 172-173, 174; 175-177, 178-179, 180; 181-183, 184-185, 186; 187-189, 190-191, 192; 382-384, 385-386, 387; 388-390, 391-3 92, 393; 394-396, 397-398, 399; 400-402, 403-404, 405; 406-408, 409-410, 411; 412-414, 415-416, 417; 418-420, 421-422, 423; 424-426, 427-428, 4 29;430-432, 433-434, 435;436-438, 439-440, 441;442-444, 445-446, 447;448-450, 451-452, 453;454-456, 457-458, 459;460-462, 463-464, 465;46 a variable heavy chain domain comprising the amino acid sequences of CDRH1, CDRH2, and CDRH3 selected from one of SEQ ID NOs: 193, 194, 195; 196, 197, 198, respectively;199, 200, 201;202, 203, 204;205, 206, 207;208, 209, 210;211, 212, 213;214, 215, 216;217, 218, 219;220, 221, 222;223, 224, 225;226, 227, 228;229, 230, 231;232, 233, 234;235 , 236, 237;238, 239, 240;241, 242, 243;244, 245, 246;247, 248, 249;250, 251, 252;253, 254, 255;256, 257, 258;259, 260, 261;262, 263, 264;514, 515, 516;517, 518, 519;520, 52 1, 522;523, 524, 525;526, 527, 528;529, 530, 531;532, 533, 534;535, 536, 537;538, 539, 540;541, 542, 543;544, 545, 546;547, 548, 549;550, 551, 552;553, 554, 555;556, 557, 558 and a variable light chain domain containing CDRL1, CDRL2, and CDRL3 amino acid sequences selected from one of: 58; 559, 560, 561; 562, 563, 564; 565, 566, 567; 568, 569, 570; 571, 572, 573; 574, 575, 576; and 577, 578, 579.

[0091] In some embodiments, the antigen-binding molecules are selected from the group consisting of SEQ ID NOs: 49-51, 52-53, 54, 193, 194, 195; 55-57, 58-59, 60, 196, 197, 198; 61-63, 64-65, 66, 199, 200, 201; 67-69, 70-71, 72, 202, 203, 204; 73-75, 76-77, 78, 205, 206, 207; 79-81, 82-83, 84, 208, 209, 210; 85-87, 88-89, 90, 211, 212, 213; 91-93, 94-95, 96, 214, 215, 216; and 97, ~99, 100~101, 102, 217, 218, 219;103~105, 106~107, 108, 220, 221, 222;109~111, 112~113, 114, 223, 224, 225;115~117, 118~119, 120, 226, 227, 228;121~123, 124~125, 126, 229, 230, 231;127~129, 130~131, 132, 232, 233, 234;133~135, 136~137, 138, 235, 236, 237;139~141, 142~143, 144, 238, 239, 240; 145-147, 148-149, 150, 241, 242, 243; 151-153, 154-155, 156, 244, 245, 246; 157-159, 160-161, 162, 247, 248, 249; 163-165, 166-167, 168, 250, 251, 252; 169-171, 172-173, 174, 253, 254, 255; 175-177, 178-179, 180, 256, 257, 258; 181-183, 184-185, 186, 259, 260, 261; 187-189, 190- 191, 192, 262, 263, 264; 382-384, 385-386, 387, 514, 515, 516; 388-390, 391-392, 393, 517, 518, 519; 394-396, 397-398, 399, 520, 521, 522; 400-402, 403-404, 405, 523, 524, 525; 406-408, 409-410, 411, 526, 527, 528; 412-414, 415-416, 417, 529, 530, 531; 418-420, 421-422, 423, 532, 533, 534;424-426, 427-428, 429, 535, 536, 537; 430-432, 433-434, 435, 538, 539, 540; 436-438, 439-440, 441, 541, 542, 543; 442-444, 445-446, 447, 544, 545, 546; 448-450, 451-452, 453, 547, 548, 549; 454-456, 457-458, 459, 550, 551, 552; 460-462, 463-464, 465, 553, 554, 555; 466-468, 469-470, 471, 556, 557, 558; 472-474, 475-476, 4 and 508-510, 511-512, 513, 577, 578, 579, and 579.

[0092] In some embodiments, the antigen binding molecule comprises a variable light chain domain comprising a CDRH1, a CDRH2, and a CDRH3 amino acid sequence selected from one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, and 380, respectively. and a variable heavy chain domain containing the amino acid sequences of CDRL1, CDRL2 and CDRH3 selected from one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, and 381, respectively.

[0093] In some embodiments, the antigen-binding molecules are selected from the group consisting of SEQ ID NOs: 1 and 2; 3 and 4; 5 and 6; 7 and 8; 9 and 10; 11 and 12; 13 and 14; 15 and 16; 17 and 18; 19 and 20; 21 and 22; 23 and 24; 25 and 26; 27 and 28; 29 and 30; 31 and 32; 33 and 34; 35 and 36; 37 and 38; 39 and 40; 41 and 42; 43 and 44; 45 and 46; 47 and 48; 338 and 339; 340 and 341; 342 and 343, respectively. 344 and 345; 346 and 347; 348 and 349; 350 and 351; 352 and 353; 354 ​​and 355; 356 and 357; 358 and 359; 360 and 361; 362 and 363; 364 and 365; 366 and 367; 368 and 369; 370 and 371; 372 and 373; 374 and 375; 376 and 377; 378 and 379; and 380 and 381.

[0094] In another aspect, the present disclosure provides antibodies that specifically bind to Cluster of Differentiation 70 (CD70).

[0095] In some embodiments, the antibody comprises a VH region set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 339, 341, 343, 345, 347, 349, 351, 353, 355, 662, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, or 381. and / or the VL region set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 338, 340, 342, 344, 346, 348, 350, 352, 354, 661, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378 or 380.

[0096] In some embodiments, the antibody comprises (i) a sequence identical to that of SEQ ID NO: 49, 50, 51, 55, 56, 57, 61, 62, 63, 67, 68, 69, 73, 74, 75, 79, 80, 81, 85, 86, 87, 91, 92, 93, 97, 98, 99, 103, 104, 105, 109, 110, 111, 115, 116, 117, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 1 22, 123, 127, 128, 129, 133, 134, 135, 139, 140, 141, 145, 146, 147, 151, 152, 153, 157, 158, 159, 163, 164, 165, 169, 170, 171, 175, 176, 177, 181, 182, 183, 187, 188, 189, 382, 383, 384, 388, 389, 390, 394, 395, 396, 400, 401, 402, 406, 407, 408, 412, 413, 414, 418, 419, 420, 424, 425, 426, 430, 431, 432, 663, 664, 665, 436, 437, 438, 442, 443, 444, 448 , 449, 450, 454, 455, 456, 460, 461, 462, 466, 467, 468, 472, 473, 474, 478, 479, 480, 484, 485, 486, 490, 491, 492, 496, 497, 498, 502, 503, 504, 508, 509, or 510. CDR1; (ii) SEQ ID NOs: 52, 53, 58, 59, 64, 65, 70, 71, 76, 77, 82, 83, 88, 89, 94, 95, 100, 101, 106, 107, 112, 113, 118, 119, 124, 125, 130, 131, 136, 137, 142, 143, 148, 149, 154, 155, 160, 161, 166, 167, 172, 173, 178, 179, 184, 185, 190, 191, 385, a VH CDR2 comprising the sequence set forth in 386, 391, 392, 397, 398, 403, 404, 409, 410, 415, 416, 421, 422, 427, 428, 433, 434, 666, 667, 439, 440, 445, 446, 451, 452, 457, 458, 463, 464, 469, 470, 475, 476, 481, 482, 487, 488, 493, 494, 499, 500, 505, 506, 511, or 512;and iii) a VH comprising the sequence set forth in SEQ ID NO: 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 387, 393, 399, 405, 411, 417, 423, 429, 435, 668, 441, 447, 453, 459, 465, 471, 477, 483, 489, 495, 501, 507, or 513. and / or (i) a VL comprising the sequence set forth in SEQ ID NO: 193, 196, 199, 202, 205, 208, 211, 214, 217, 220, 223, 226, 229, 232, 235, 238, 241, 244, 247, 250, 253, 256, 259, 262, 514, 517, 520, 523, 526, 529, 532, 535, 538, 669, 541, 544, 547, 550, 553, 556, 559, 562, 565, 568, 571, 574, or 577. (ii) a VL comprising the sequence set forth in SEQ ID NO: 194, 197, 200, 203, 206, 209, 212, 215, 218, 221, 224, 227, 230, 233, 236, 239, 242, 245, 248, 251, 254, 257, 260, 263, 515, 518, 521, 524, 527, 530, 533, 536, 539, 670, 542, 545, 548, 551, 554, 557, 560, 563, 566, 569, 572, 575, or 578 and (iii) a VL CDR3 comprising the sequence set forth in SEQ ID NO: 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 225, 228, 231, 234, 237, 240, 243, 246, 249, 252, 255, 258, 261, 264, 516, 519, 522, 525, 528, 531, 534, 537, 540, 671, 543, 546, 549, 552, 555, 558, 561, 564, 567, 570, 573, 576, or 579;

[0097] In some embodiments, the antibody comprises a VH CDR1, a VH CDR2, and a VH CDR3 of the VH sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 339, 341, 343, 345, 347, 349, 351, 353, 355, 662, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, or 381. and / or a VL region comprising VL CDR1, VL CDR2, and VL CDR3 of the VL sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 338, 340, 342, 344, 346, 348, 350, 352, 354, 661, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, or 380.

[0098] In further aspects, the disclosure provides nucleic acids, vectors, host cells, pharmaceutical compositions, methods of making, and methods of treating conditions using the disclosed antibodies. [Brief explanation of the drawings]

[0099] [Figure 1] Figure 1 shows tumor volumes in mice treated with various doses of 4F11 CAR T cells in a subcutaneous xenograft model.

[0100] [Figure 2] Figure 2 is a plot showing the body weight of mice treated with various doses of 4F11 CAR T cells in a subcutaneous xenograft model.

[0101] [Figure 3] Figure 3 shows tumor volume in mice treated with 4F11 and P08F08 CAR T cells in a subcutaneous xenograft model with or without CD70 KO.

[0102] [Figure 4] Figure 4 shows the body weight of mice treated with 4F11 and P08F08 CAR T cells in a subcutaneous xenograft model with or without CD70 KO.

[0103] [Figure 5A] Figures 5A-5C are a series of charts showing tumor fluctuation (fluctuation) values ​​for mice treated with control cells, mice treated with cells expressing CAR 4F11 with or without CD70 KO and with or without TCRa KO, and mice treated with cells expressing CAR P08F08 with or without CD70 KO and with or without TCRa KO, from three donors in the ACHN lung metastasis model. [Figure 5B] Same as above. [Figure 5C] Same as above.

[0104] Figure 5A shows results obtained with control cells, cells expressing the 4F11 CAR with KO of CD70, TCRa, or both CD70 and TCRa, and cells expressing the P08F08 CAR with TCR KO. Cells were obtained from donor D419.

[0105] Figure 5B shows results obtained with control cells, cells expressing the 4F11 CAR with or without CD70 KO, and cells expressing the P08F08 CAR. Cells were obtained from donor D710.

[0106] Figure 5C shows results obtained with control cells, cells expressing the 4F11 CAR with or without CD70 KO, and cells expressing the P08F08 CAR. Cells were obtained from donor D503.

[0107] [Figure 6]Figures 6A-6F show five exemplary, non-limiting CAR designs. In each of Figures 6A-6F, the approximate distance between the antigen-binding fragment of the CD70-specific domain and the cell membrane is indicated by a double arrow, and the number of amino acid residues (aa) between the scFv and the transmembrane domain is labeled.

[0108] Figure 6A shows a second-generation CAR design that includes an extracellular domain containing an scFv specific for CD70, a hinge, a transmembrane domain, a first intracellular domain (4-1BB domain), and a second intracellular domain (CD3ζ signaling domain).

[0109] Figure 6B shows the SR2 CAR format, in which a suicide switch is generated by inserting two RTX epitopes (i.e., CD20 epitopes) between the hinge and the scFv.

[0110] Figure 6C shows the RSRQR CAR format, in which a third RTX epitope and a CD34 epitope are added.

[0111] Figure 6D shows the RSR format, in which two RTX epitopes flank the scFv.

[0112] Figure 6E shows a modified version of the RSR CAR format in which the hinge domain is shortened (termed RSR-short).

[0113] Figure 6F shows the R2S CAR format, in which the two RTX epitopes of the R2 CAR format are moved to the N-terminus of the scFv.

[0114] [Figure 7] Figure 7 shows the viability of target cells or non-transduced (NTD) control cells after exposure to the four formats of CD70-specific CAR.

[0115] [Figure 8] Figures 8A-8D are a series of figures showing cell killing of 786-0 cells, ACHN cells, or REH cells using CD70-specific CAR T cells, in which the extracellular domain of the CAR contains the scFv shown in the legend to Figure 8D.

[0116] Figure 8A shows cell killing of 786-0 cells, in which the extracellular domain of the CAR contains the scFv shown in the legend to Figure 8D.

[0117] Figure 8B shows cell killing of ACHN cells, in this case the extracellular domain of the CAR contains the scFv shown in the legend to Figure 8D.

[0118] Figure 8C shows cell killing of REH cells, in which the extracellular domain of the CAR contains the scFv shown in the legend to Figure 8D.

[0119] [Figure 9-1] Figures 9A-9D are a series of figures showing the serial killing of 786-0 cells, ACHN cells, or REH cells using CD70-specific CAR T cells, in which the extracellular domain of the CAR contains the scFv shown in the legend to Figure 9D. [Figure 9-2] Same as above.

[0120] Figure 9A shows the efficacy of a CD70-specific CAR upon repeated exposure to luciferase-labeled 786-O target cells (CAR T cells were transferred to 96-well plates containing fresh targets every 2-3 days). The E:T ratio was 3:1. The CAR was expressed in cells from donor D503.

[0121] Figure 9B shows the efficacy of CD70-specific CAR upon repeated exposure to luciferase-labeled ACHN target cells (CAR T cells were transferred to 96-well plates containing fresh targets every 2-3 days). The E:T ratio was 10:1. The CAR was expressed in cells from donor D503.

[0122] Figure 9C shows the efficacy of CD70-specific CAR upon repeated exposure to luciferase-labeled REH target cells (2x10 at the indicated time points). 6 cells were added). The E:T ratio was 1:5. CAR was expressed in cells from donor D503.

[0123] [Figure 10-1] Figure 10 is a series of diagrams showing the efficacy of CD70-specific CARs in either R2S, SR2, or RSRQR formats upon repeated exposure to luciferase-labeled REH target cells (2x10 cells added at the indicated time points). The E:T ratio was 1:5. The CAR was expressed in cells from donor D772. [Figure 10-2] Same as above. [Figure 10-3] Same as above. [Figure 10-4] Same as above.

[0124] [Figure 11] FIG. 11 shows tumor variation values ​​for mice treated with control cells or cells expressing various CAR scFvs in an ACHN lung metastasis model.

[0125] [Figure 12A] 12A-12C are a series of graphs showing quantification of CD70 expression in terms of CD70 antibody binding capacity (ABC) on various tested cell lines, or cell lines and RCC patient-derived cells. Data from RCC patient-derived cells are also shown. [Figure 12B] Same as above. [Figure 12C]Same as above.

[0126] FIG. 12A shows quantification of CD70 expression in relation to CD70 antibody binding capacity (ABC) on various tested cell lines.

[0127] FIG. 12B shows quantification of CD70 expression in relation to CD70 antibody binding capacity (ABC) on cells derived from RCC patients.

[0128] FIG. 12C shows data from cells derived from an RCC patient.

[0129] [Figure 13-1] 13A-13C are a series of figures showing killing of target cells derived from RCC patient WD-59279, a patient-derived cell line, or ACHN, with antibody binding capacity indicated in each panel. [Figure 13-2] Same as above.

[0130] Figures 13A-B show the killing and antibody binding capacity of target cells from RCC patients.

[0131] FIG. 13C shows the killing and antibody binding capacity of target ACHN cells.

[0132] [Figure 14A] Figures 14A-14B are a series of bar graphs showing quantification of CD70 receptor numbers and heme tumor cell killing for additional cell lines expressing CD70 at various levels and by 4F11 CAR in QR3 format at 1:1 E:T. [Figure 14B] Same as above.

[0133] Figure 14A is a bar graph showing quantification of CD70 receptor numbers of 4F11 CAR in QR3 format at 1:1 E:T on additional cell lines expressing CD70 at various levels.

[0134] Figure 14B is a bar graph showing hematologic tumor cell killing by 4F11 CAR in QR3 format at 1:1 E:T on additional cell lines expressing various levels of CD70.

[0135] [Figure 15A] Figures 15A-15B are a series of graphs showing tumor volume and body weight of mice treated with 4F11 and P08F08 CAR T at a dose of 10x10 6 cells or 5x10 6 cells in a subcutaneous xenograft model. [Figure 15B] Same as above.

[0136] Figure 15A shows the effect of 10x10 6 cells or 5 x 10 6 Figure 1 shows tumor volumes in mice treated with 4F11 and P08F08 CAR T at doses of cells.

[0137] Figure 15B shows the effect of 10x10 6 cells or 5 x 10 6 Figure 1 shows the body weight of mice treated with 4F11 and P08F08 CAR T at a dose of cells. DETAILED DESCRIPTION OF THE INVENTION

[0138] The disclosure herein provides chimeric antigen receptors (CARs) that specifically bind to CD70 (e.g., human CD70), and immune cells (e.g., CAR-T cells) containing the CARs. The disclosure further provides polynucleotides encoding these CARs, compositions containing these CAR-T cells, and methods of making and using these CARs and CAR-T cells. The disclosure also provides methods of treating a condition associated with malignant CD70 expression in a subject, such as cancer.

[0139] general technology The compositions and methods of the present disclosure employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. These techniques include Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JECellis, ed., 1998) Academic Press; Animal Cell Culture(RIFreshney, ed., 1987);Introduction to Cell and Tissue Culture(JPMather and PERoberts, 1998)Plenum Press;Cell and Tissue Culture:Laboratory Procedures(A.Doyle, JBGriffiths, and DGNewell, eds., 1993~1998)J.Wiley and Sons;Methods in Enzymology(Academic Press, Inc.); Handbook of Experimental Immunology (DMWeir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JMMiller and MPCalos, eds., 1987); Current Protocols in Molecular Biology (FMAusubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (JEColigan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty, ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995).

[0140] definition As used herein, the term "extracellular ligand-binding domain" refers to an oligopeptide or polypeptide capable of binding to a ligand. In some exemplary embodiments, the domain will have the ability to interact with a cell surface molecule. For example, the extracellular ligand-binding domain may be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state.

[0141] The terms "stalk domain" or "hinge domain" are used interchangeably herein and refer to any oligopeptide or polypeptide that functions to link the transmembrane domain and the extracellular ligand-binding domain in a CAR. In particular, the stalk domain is used to provide greater flexibility and accessibility to the extracellular ligand-binding domain.

[0142] The term "intracellular signaling domain" refers to the portion of a protein that transmits effector signaling functional signals and instructs the cell to carry out specialized functions.

[0143] As used herein, "costimulatory molecule" refers to a cognate binding partner on an immune cell, such as a T cell, that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the cell, such as, but not limited to, proliferation. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA molecules, and Toll ligand receptors. Examples of costimulatory molecules include CD27, CD28, CD8, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83.

[0144] A "costimulatory ligand" refers to a molecule on an antigen-presenting cell that specifically binds to a cognate costimulatory signal molecule on an immune cell, e.g., a T cell, and generates signals mediated by T cell responses, including but not limited to proliferation, activation, and differentiation, in addition to the primary signal generated by binding of a peptide-loaded MHC molecule to the TCR / CD3 complex. Examples of costimulatory ligands include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BB, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin β receptor, 3 / TR6, ILT3, ILT4, agonists or antibodies that bind to Toll ligand receptors, and ligands that specifically bind to B7-H3. Examples of costimulatory ligands include, but are not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated receptor (IL-1), and IL-2. Also encompassed are antibodies that specifically bind to costimulatory molecules present on T cells, such as ligands that specifically bind to antigen-1 (LFA-1), CD2, CD7, LTGHT, NKG2C, B7-H3, and CD83.

[0145] An "antibody" is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, or polypeptide, located in the variable region of the immunoglobulin molecule through at least one antigen recognition site. As used herein, the term encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), single-chain (scFv) antibodies, and domain antibodies (including, e.g., shark and camelid antibodies), as well as fusion proteins comprising antibodies, and any other modified structure of an immunoglobulin molecule comprising an antigen recognition site. Antibodies include antibodies of any class, such as IgG, IgA, IgE, IgD, or IgM (or subclasses thereof), and antibodies need not be of any particular class. Depending on the antibody amino acid sequence of the constant region of its heavy chain, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins. IgA, IgD, IgE, IgG, and IgM, and some of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of the various classes of immunoglobulins are known.

[0146] As used herein, the term "antigen-binding fragment" or "antigen-binding portion" of an antibody refers to one or more fragments of an intact antibody that retain the ability to specifically bind to a given antigen (e.g., CD70). The antigen-binding function of an antibody can be performed by fragments of an intact antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include Fab, Fab', F(ab')2, an Fd fragment consisting of the VH and CH1 domains, an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, a single-domain antibody (dAb) fragment (Ward et al., Nature 341:544-546, 1989), and isolated complementarity-determining regions (CDRs).

[0147] An antibody, antigen-binding fragment, antibody conjugate, or polypeptide that "preferentially binds" or "specifically binds" (used interchangeably herein) to a target (e.g., a CD70 protein) is a term well understood in the art, and methods for determining such specific or preferential binding are also known in the art. A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or associates with a particular cell or substance more frequently, earlier, for a longer duration, and / or with a higher affinity than with another cell or substance. An antibody "specifically binds" or "preferentially binds" to a target if it binds with higher affinity, avidity, earlier, and / or for a longer duration than it binds to other substances. For example, an antibody that specifically or preferentially binds to a CD70 epitope is an antibody that binds to that epitope with higher affinity, avidity, earlier, and / or for a longer duration than it binds to other CD70 epitopes or non-CD70 epitopes. It will be understood by reading this definition that, for example, an antibody (or moiety or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. Thus, "specific binding" or "preferential binding" does not necessarily require (but can include) exclusive binding. Generally, but not necessarily, reference to binding refers to preferential binding.

[0148] The "variable region" of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. As is known in the art, the variable regions of the heavy and light chains each consist of four framework regions (FRs) connected by three complementarity-determining regions (CDRs), also known as hypervariable regions. The CDRs of each chain are held in close proximity by the FRs and, together with the CDRs of the other chain, contribute to the formation of the antigen-binding site of the antibody. There are at least two techniques for determining CDRs: (1) methods based on cross-species sequence diversity (i.e., Kabat et al., Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda MD); and (2) methods based on crystal studies of antigen-antibody complexes (Al-lazikani et al., 1997, J. Molec. Biol. 273:927-948). As used herein, CDRs may refer to CDRs defined by either method or by a combination of both methods.

[0149] The "CDRs" of a variable domain are amino acid residues in the variable region identified according to the Kabat, Chothia, or both Kabat and Chothia definitions, the AbM, contact, and / or conformational definitions, or any method of CDR determination known in the art. Antibody CDRs may also be defined as hypervariable regions as originally defined by Kabat et al. See, e.g., Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington DC. The locations of CDRs may also be defined as structural loop structures as originally reported by Chothia et al. See, e.g., Chothia et al., Nature 342:877-883, 1989. Other methods of identifying CDRs include the "AbM definition." This method, which lies between the Kabat and Chothia methods, is derived using Oxford Molecular's AbM antibody modeling software (now known as Accelrys®). Alternatively, a "contact definition" of CDRs based on observed antigen contacts is also included, as described in MacCallum et al., J. Mol. Biol., 262:732-745, 1996. Another method, referred to herein as a "conformational definition" of CDRs, can define CDR positions as residues that contribute enthalpic- ically to antigen binding. See, e.g., Makabe et al., Journal of Biological Chemistry, 283:1156-1166, 2008. Still other CDR boundary definitions may not strictly follow one of the above methods, but overlap with at least a portion of the Kabat CDRs. Nevertheless, certain residues or groups of residues, or even entire CDRs, may be too short or too long in light of predictions or experimental results that suggest they do not significantly affect antigen binding. As used herein, CDRs may refer to CDRs defined by any method known in the art, including a combination of methods.The methods used herein may utilize CDRs defined according to any of these methods. For any given embodiment containing multiple CDRs, the CDRs may be defined according to any of the Kabat, Chothia, extended, AbM, contact, and / or conformation definitions.

[0150] As used herein, a "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies. That is, the individual antibodies comprising the population are identical except for possible minor naturally occurring mutations. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as obtained from a substantially homogeneous population of antibodies, but is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present disclosure may be made by the hybridoma method first described by Kohler and Milstein, Nature 256:495, 1975, or may be made by recombinant DNA methods, e.g., as described in U.S. Pat. No. 4,816,567. The monoclonal antibodies may be isolated from phage libraries generated using the techniques described in McCafferty et al., Nature 348:552-554, 1990, for example.

[0151] As used herein, "humanized" antibodies refer to forms of non-human (e.g., murine) antibodies that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. In some exemplary embodiments, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues of the receptor's complementarity-determining regions (CDRs) are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some instances, residues of the Fv framework regions (FRs) of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are present neither in the recipient antibody nor in the imported CDR or framework sequences, but are included to further improve or optimize antibody performance. Generally, a humanized antibody contains substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin, and all or substantially all of the FRs are those of a human immunoglobulin consensus sequence. Furthermore, humanized antibodies optimally contain at least a portion of an immunoglobulin constant region or domain (Fc) of a human immunoglobulin, typically from a human immunoglobulin. An exemplary embodiment is an antibody with an altered Fc region as described in WO 99 / 58572. Other forms of humanized antibodies have one or more CDRs (CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, or CDR H3) that are altered relative to the original antibody, also referred to as one or more CDRs "derived from" one or more CDRs of the original antibody.

[0152] As used herein, "human antibody" refers to an antibody having an amino acid sequence corresponding to that of an antibody produced by a human, and / or an antibody produced using any of the human antibodies known to those skilled in the art or techniques for producing human antibodies disclosed herein. This definition of a human antibody includes antibodies comprising at least one human heavy chain polypeptide or at least one human light chain polypeptide. One such example is an antibody comprising a mouse light chain and a human heavy chain polypeptide. Human antibodies can be produced using a variety of techniques known in the art. In some embodiments, human antibodies are selected from phage libraries that express human antibodies (Vaughan et al., Nature Biotechnology, 14:309-314, 1996; Sheets et al., Proc. Natl. Acad. Sci. (USA) 95:6157-6162, 1998; Hoogenboom and Winter, J. Mol. Biol., 227:381, 1991; Marks et al., J. Mol. Biol., 222:581, 1991). Human antibodies can also be produced by immunization of animals into which human immunoglobulin loci have been transgenically introduced in place of endogenous loci. For example, human antibodies can be produced by immunization of mice in which the endogenous immunoglobulin genes have been partially or completely disrupted or inactivated. This method is described in U.S. Patent Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016. Alternatively, human antibodies may be prepared by immortalizing human B lymphocytes that produce antibodies directed against a target antigen (such B lymphocytes may be obtained from an individual, or may be obtained from single-cell cloning of cDNA, or may be immunized in vitro).See, e.g., Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77, 1985; Boerner et al., J. Immunol., 147(1):86-95, 1991; and U.S. Patent No. 5,750,373.

[0153] The term "chimeric antibody" is intended to refer to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a murine antibody and the constant region sequences are derived from a human antibody.

[0154] The terms "polypeptide," "oligopeptide," "peptide," and "protein" are used interchangeably herein to refer to amino acid chains of any length. In some embodiments, they refer to relatively short amino acid chains (e.g., 10-100 amino acids). The chains may be linear or branched, may include modified amino acids, and / or may be interrupted by non-amino acids. The terms further encompass naturally modified amino acid chains, or amino acids modified by, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as, for example, conjugation with a labeling component. The definition further includes, for example, polypeptides containing one or more amino acid analogs (including, for example, unnatural amino acids), as well as other modifications known in the art. It is understood that polypeptides can occur as single chains or associated chains.

[0155] A "monovalent antibody" contains one antigen-binding site per molecule (e.g., IgG or Fab). In some cases, a monovalent antibody can have multiple antigen-binding sites, although the binding sites are from different antigens.

[0156] A "bivalent antibody" contains two antigen-binding sites per molecule (e.g., IgG). In some cases, the two binding sites have the same antigen specificity. However, a bivalent antibody may also be bispecific.

[0157] The antibodies of the present disclosure may be made using techniques known in the art, such as recombinant techniques, phage display techniques, synthetic techniques, or a combination of such techniques, or other techniques readily known in the art (see, e.g., Jayasena, SD, Clin. Chem., 45:1628-50, 1999 and Fellouse, FA, et al, J. MoI. Biol., 373(4):924-40, 2007).

[0158] As known in the art, the terms "polynucleotide" and "nucleic acid," used interchangeably herein, refer to a chain of nucleotides of any length, including DNA and RNA. The nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a chain by DNA or RNA polymerase. A polynucleotide may also contain modified nucleotides, such as methylated nucleotides and their analogs. If present, modifications to the nucleotide structure are made before or after assembly of the chain. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after multimerization, for example, by conjugation with a label component. Other types of modifications include, for example, substitution of one or more "caps" of natural nucleotides with analogs; internucleotide modifications such as those using uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.); those containing pendant moieties such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.); those using intercalating agents (e.g., acridine, psoralens, etc.); those containing chelators (e.g., metals, radioactive metals, boron, metal oxides, etc.); those containing alkylating agents; those using modified linkages (e.g., alpha-anomeric nucleic acids, etc.); and unmodified forms of polynucleotides. Additionally, any of the hydroxyl groups normally present on the sugar may be substituted, for example, with phosphonate groups, phosphate groups, etc., or may be protected with standard protecting groups or activated to provide for additional linkage to additional nucleotides or attached to a solid support. The 5' and 3' terminal OH may be phosphorylated or substituted with amines or organic capping group moieties of 1 to 20 carbon atoms. Other hydroxyls may be derivatized to standard protecting groups.Polynucleotides may further contain analogs of ribose or deoxyribose sugars commonly known in the art, including, for example, 2'-O-methyl, 2'-O-allyl, 2'-fluoro-, or 2'-azido-ribose, carbocyclic sugar analogs, alpha- or beta-anomeric sugars, epimeric sugars such as arabinose, xylose, or lyxose, pyranose sugars, furanose sugars, sedoheptulose, acyclic analogs, and abasic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages may be replaced by alternative linking groups. Such alternative linking groups include, but are not limited to, embodiments in which the phosphate is replaced by P(O)S (thioate), P(S)S (dithioate), (O)NR2 (amidate), P(O)R, P(O)OR', CO, or CH2 (formacetal), where each R or R' is independently H, or substituted or unsubstituted alkyl (1-20 C), optionally containing an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl. Not all linkages in a polynucleotide need be identical. The foregoing description applies to all polynucleotides referred to herein, including RNA and DNA.

[0159] As known in the art, a "constant region" of an antibody refers to the constant region of the antibody light chain or the constant region of the antibody heavy chain, either alone or in combination.

[0160] As used herein, "substantially pure" refers to a material that is at least 50% pure (i.e., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0161] A "host cell" includes an individual cell or cell culture that may be or has been a recipient for a vector for incorporation of a polynucleotide insert. A host cell includes the progeny of a single host cell, which may not be completely identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells transfected in vivo with a polynucleotide of this disclosure.

[0162] As used herein, "immune cell" refers to a cell of hematopoietic origin that is functionally involved in the initiation and / or execution of the innate and / or adaptive immune response.

[0163] As known in the art, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain. The "Fc region" may be a native-sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the Fc region of a human IgG heavy chain is usually defined to stretch from the amino acid residue at position Cys226, or from the amino acid residue at position Pro230, to the carboxyl-terminus thereof. The numbering of residues in the Fc region is that of the EU index as in Kabat. Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991. The Fc region of an immunoglobulin generally contains two constant domains, CH2 and CH3.

[0164] As used in the art, "Fc receptor" and "FcR" describe a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is a native-sequence human FcR. In further embodiments, the FcR is an FcR (gamma receptor) that binds IgG antibodies, including receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA (activating receptors) and FcγRIIB (inhibitory receptors), which have similar amino acid sequences but differ primarily in their cytoplasmic domains. FcRs are reviewed in Ravetch and Kinet, Ann. Rev. Immunol., 9:457-92, 1991; Capel et al., Immunomethods, 4:25-34, 1994; and de Haas et al., J. Lab. Clin. Med., 126:330-41, 1995. The term "FcR" further includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol., 117:587, 1976; and Kim et al., J. Immunol., 24:249, 1994).

[0165] As used herein with respect to antibodies, the term "compete" means that a first antibody, or antigen-binding fragment (or portion) thereof, binds to an epitope in a manner sufficiently similar to the binding of a second antibody or antigen-binding portion thereof, such that the result of binding of the first antibody to its cognate epitope is detectably reduced in the presence of the second antibody, compared to binding of the first antibody in the absence of the second antibody. Binding of the second antibody to its epitope may, but need not, also be detectably reduced in the presence of the first antibody. That is, the first antibody may inhibit binding of the second antibody to its epitope, but this is not accompanied by inhibition of the first antibody to its respective epitope by the second antibody. However, if each antibody detectably inhibits binding of the other antibody to its cognate epitope or ligand to the same, stronger, or weaker extent, the antibodies are said to "cross-compete" with each other for binding of their respective epitopes. Both competing and cross-competing antibodies are encompassed by the present disclosure. Regardless of the mechanism by which such competition or cross-competition occurs (i.e., steric hindrance, conformational change, or binding to a common epitope or portion thereof), one of skill in the art will recognize, based on the teachings provided herein, that such competing and / or cross-competing antibodies are encompassed and may be useful in the methods disclosed herein.

[0166] As used herein, "autologous" means that the cells, cell line, or cell population used to treat a patient originate from that patient.

[0167] As used herein, "allogeneic" means that the cells or cells used to treat a patient are derived from a donor, rather than originating from the patient.

[0168] As used herein, "treatment" refers to an approach for obtaining beneficial or desired clinical results. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, one or more of the following: reduction in proliferation (or destruction) of tumor or cancer cells, inhibition of metastasis of tumor cells, reduction or decrease in size of a tumor expressing CD70, such as, for example, renal cell carcinoma (RCC), lymphoma, leukemia, or glioma, remission of a CD70-associated disease (e.g., cancer), reduction in symptoms resulting from a CD70-associated disease (e.g., cancer), improvement in the quality of life of a person suffering from a CD70-associated disease (e.g., cancer), reduction in the dosage of other drugs required to treat a CD70-associated disease (e.g., cancer), delay in progression of a CD70-associated disease (e.g., cancer), cure of a CD70-associated disease (e.g., cancer), and / or prolongation of survival of a patient with a CD70-associated disease (e.g., cancer).

[0169] "Ameliorating" refers to a reduction or improvement in one or more symptoms compared to when the CD70-specific CAR or CD70-specific CAR-T cells are not administered. "Ameliorating" also includes a shortening or reduction in the duration of the symptoms.

[0170] As used herein, an "effective dosage" or "effective amount" of a drug, compound, or pharmaceutical composition is an amount sufficient to effect any one or more beneficial or desired results. For prophylactic use, beneficial or desired results include eliminating or reducing the risk of disease, reducing the severity of disease, or delaying the onset of disease, including the biochemical, histological, and / or behavioral manifestations of the disease, its complications, and intermediate pathological phenotypes manifested during the development of the disease. For therapeutic use, beneficial or desired results include clinical results such as reducing the incidence or amelioration of one or more symptoms of various CD70-related diseases or conditions (e.g., multiple myeloma) in a patient, reducing the dosage of other drugs required to treat the disease, enhancing the effectiveness of another drug, and / or delaying the progression of a CD70-related disease. An effective dosage can be administered in one or more administrations. For purposes of this disclosure, an effective dose of a drug, compound, or pharmaceutical composition is an amount sufficient to achieve prophylactic or therapeutic treatment, directly or indirectly. As understood in a clinical context, an effective dosage of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an "effective dosage" may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desired result is likely or is achieved.

[0171] An "individual," "patient," or "subject" is a mammal, and in some embodiments, a human. Mammals include, but are not limited to, humans, monkeys, pigs and other livestock, sport animals, pets, primates, horses, dogs, cats, and rodents, including mice, rats, and guinea pigs. A subject is a mammal, and the terms are used interchangeably herein. In some embodiments, a subject is a human. In some embodiments, a subject is a non-human primate. In some embodiments, a subject is a human or a monkey, such as a cynomolgus monkey.

[0172] As used herein, "vector" refers to a construct capable of delivering one or more genes or sequences of interest and, in some embodiments, expressing one or more genes or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells such as producer cells.

[0173] As used herein, "expression control sequence" means a nucleic acid sequence that directs transcription of a nucleic acid. An expression control sequence can be a promoter, such as a constitutive or inducible promoter, or an enhancer. The expression control sequence is operably linked to the nucleic acid sequence to be transcribed.

[0174] As used herein, the term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" refers to any material that, when combined with an active ingredient, retains biological activity and is non-reactive with the subject's immune system. Examples include, but are not limited to, standard pharmaceutical carriers such as phosphate-buffered saline solution, water, emulsions such as oil / water emulsions, and various types of wetting agents. An exemplary diluent for aerosol or parenteral administration is phosphate-buffered saline (PBS) or normal saline (0.9%). Compositions containing such carriers are formulated by known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and Practice of Pharmacy, 21st Ed., Mack Publishing, 2005).

[0175] As used herein, "kon " refers to the rate constant for association of an antibody or scFv or CAR with an antigen.

[0176] As used herein, "k off " refers to the rate constant for dissociation of an antibody or scFv or CAR from the antibody / antigen complex.

[0177] As used herein, "K D " refers to the equilibrium dissociation constant of an antibody-antigen or scFv-antigen or CAR-antigen interaction.

[0178] References herein to "about" a value or parameter include (and describe) embodiments that inherently direct that value or parameter. For example, a description of "about X" includes a description of "X." Numeric ranges are inclusive of the numbers defining the range.

[0179] It is understood that for any embodiment described herein using the term "comprising," other similar embodiments described with the terms "consisting of" and / or "consisting essentially of" are also provided.

[0180] When aspects or embodiments of the invention are described in terms of a Markush group or other alternative group, the invention generally encompasses not only the entire group recited, but also each member of the individual group and each member of all possible subgroups of the main group, and the main group lacking one or more of the group members. The invention further contemplates the explicit exclusion of any one or more of the claimed group members of the invention.

[0181] All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs, unless otherwise defined. In case of conflict, the present specification, including definitions, controls. Throughout this specification and claims, variations such as "comprise" or "comprises" are understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group. Unless otherwise required by context, singular terms shall include plural terms, and plural terms shall include the singular.

[0182] Although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, exemplary methods and materials are described herein. The materials, methods, and examples are illustrative only and not intended to be limiting.

[0183] CD70-specific CARs and methods for producing same The present disclosure provides a CAR that binds to CD70 (e.g., human CD70 (e.g., SEQ ID NO: 335)), such as one deposited under the provisions of the Budapest Treaty and assigned accession number P32970-1. CD70-specific CARs provided herein include single-chain CARs and multi-chain CARs. In some embodiments, the CAR has the ability to redirect T cell specificity and reactivity to CD70 in a non-MHC-restricted manner by utilizing the antigen-binding capacity of monoclonal antibodies. Non-MHC-restricted antigen recognition confers on CAR-expressing T cells the ability to recognize antigens independent of antigen processing, thereby bypassing a major mechanism of tumor evasion.

[0184] In some embodiments, the CARs provided herein contain an extracellular ligand-binding domain (e.g., a single-chain variable fragment (scFv)), a transmembrane domain, and an intracellular signaling domain. In some embodiments, the CARs provided herein further contain a "hinge" or "stalk" domain, which may be located between the extracellular ligand-binding domain and the transmembrane domain. In some embodiments, the extracellular ligand-binding domain, the transmembrane domain, and the intracellular signaling domain are in one polypeptide, i.e., in a single chain. Multi-chain CARs and polypeptides are also provided herein. In some embodiments, the multi-chain CAR contains a first polypeptide comprising a transmembrane domain and at least one extracellular ligand-binding domain, and a second polypeptide comprising a transmembrane domain and at least one intracellular signaling domain, where the polypeptides are assembled together to form the multi-chain CAR. In some embodiments, the CAR is induced, for example, by a small molecule (e.g., AP1903) or a protein (e.g., Epo, Tpo, or PD-1). In some embodiments, the CD70-specific multi-chain CAR is based on the high-affinity receptor for IgE (FcεRI). FcεRI, expressed on mast cells and basophils, induces allergic responses. FcεRI is a tetrameric complex composed of one α subunit, one β subunit, and two disulfide-linked γ subunits. The α subunit contains the IgE-binding domain. The β and γ subunits contain ITAMs that mediate signal transduction. In some embodiments, the extracellular domain of the FcR α chain is deleted and replaced with a CD70-specific extracellular ligand-binding domain. In some embodiments, the multi-chain CD70-specific CAR contains an scFv that specifically binds to CD70, a CD8 α hinge, and an ITAM of the FcR β chain. In some embodiments, the CAR may or may not include an FcR γ chain.

[0185] In some embodiments, the extracellular ligand-binding domain comprises an scFv containing the light chain variable (VL) and heavy chain variable (VH) regions of a target antigen (i.e., CD70)-specific monoclonal antibody, connected by a flexible linker. Single-chain variable region fragments are generated by linking the light and / or heavy chain variable regions using a short linking peptide (Bird et al., Science 242:423-426, 1988). An example of a linking peptide is the GS linker having the amino acid sequence (GGGGS)3 (SEQ ID NO: 296), which spans approximately 3.5 nm between the carboxy terminus of one variable region and the amino terminus of the other variable region. Linkers of other sequences can also be designed and used (Bird et al., 1988, supra). Other exemplary linkers generally include other GS linkers generally include (GGGGS)x, where x is 1, 2, 3, 4, 5 (SEQ ID NO: 613). In some embodiments, x is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or any integer less than about 20. In some embodiments, the linker is (GGGGS) 4((SEQ ID NO: 602). In some embodiments, the linker is GSTSGSGKPGSGEGSTKG (SEQ ID NO: 612) and is described in Whitlow et al., Protein Eng. (1993) 6(8):989-895. Generally, linkers can be short, flexible polypeptides, in some embodiments consisting of about 20 or fewer amino acid residues. Linkers can also be modified for additional functionality, such as the attachment of drugs or attachment to solid supports. Single-chain variants can be produced recombinantly or synthetically. For synthetic production of scFvs, automated synthesizers can be used. For recombinant production of scFvs, an appropriate plasmid containing a polynucleotide encoding the scFv can be introduced into a suitable host cell, e.g., a eukaryotic cell such as a yeast cell, a plant cell, an insect cell, or a mammalian cell, or a prokaryotic cell such as E. coli. Polynucleotides encoding the scFv of interest can be produced by routine manipulations, such as polynucleotide ligation. The resulting scFv can be isolated using standard protein purification techniques known in the art.

[0186] In another aspect, a CAR that specifically binds to CD70 is provided, wherein the CAR contains an extracellular ligand-binding domain containing a VH region comprising the VH CDR1, VH CDR2, and VH CDR3 of the VH sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48, and / or a VL region comprising the VL CDR1, VL CDR2, and VL CDR3 of the VL sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, or 47. In some embodiments, the VH and VL are linked to each other by a flexible linker. In some embodiments, the flexible linker contains the amino acid sequence set forth in SEQ ID NO:296.

[0187] In some embodiments, a CAR of the present disclosure contains an extracellular ligand-binding domain having any one of the partial light chain sequences listed in Table 1 and / or any one of the partial heavy chain sequences listed in Table 1. In Table 1, the underlined sequences are CDR sequences according to Kabat, and the bolded sequences are CDR sequences according to Chothia. [Table 1] TIFF2026000897000003.tif240170 TIFF2026000897000004.tif240170 TIFF2026000897000005.tif240170 TIFF2026000897000006.tif240170 TIFF2026000897000007.tif240170

[0188] Also provided herein are CDR portions of the extracellular ligand-binding domain of a CAR against CD70 (including Chothia and Kabat CDRs and CDR contact regions). Determining the CDR regions is within the skill of the art. It should be understood that in some embodiments, the CDRs may be a combination of Kabat and Chothia CDRs (also referred to as "mixed CDRs" or "extended CDRs"). In some embodiments, the CDRs are Kabat CDRs. In other embodiments, the CDRs are Chothia CDRs. In other words, in embodiments involving multiple CDRs, the CDRs may be either Kabat, Chothia, combined CDRs, or a combination thereof. Tables 2A-2B provide examples of CDR sequences provided herein. [Table 2A] TIFF2026000897000009.tif240170 TIFF2026000897000010.tif240170 TIFF2026000897000011.tif240170 TIFF2026000897000012.tif240170 TIFF2026000897000013.tif240170 TIFF2026000897000014.tif240170 [Table 2B] TIFF2026000897000016.tif240170 TIFF2026000897000017.tif240170

[0189] The present disclosure encompasses modifications to CARs and polypeptides comprising the sequences set forth in Tables 1 or 2A-2B, including functionally equivalent CARs with modifications that do not significantly affect their properties, as well as variants with enhanced or decreased activity and / or affinity. For example, amino acid sequences may be mutated to yield antibodies with desired binding affinity for CD70. Polypeptide modifications are routinely performed in the art and need not be described in detail herein. Examples of modified polypeptides include polypeptides with conservative substitutions of amino acid residues, polypeptides with one or more deletions or additions of amino acids that do not significantly adversely alter functional activity or that mature (enhance) the affinity of the polypeptide for its ligand, or polypeptides in which chemical analogs are used.

[0190] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing hundreds or more residues, as well as intersequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue or antibodies fused to an epitope tag. Other insertional variants of antibody molecules include the fusion of enzymes or polypeptides to the antibody N- or C-terminus, thereby extending the half-life of the antibody in the blood circulation.

[0191] Substitutional variants involve removing at least one amino acid residue in an antibody molecule and inserting a different residue in its place. The sites of greatest interest for substitutional mutagenesis include the hypervariable regions, although FR modifications are also anticipated. Conservative substitutions are shown in Table 3 under the heading "Conservative Substitutions." Larger substitution changes can be introduced and the products screened if they result in altered biological activity, as indicated in Table 3 under "exemplary substitutions," or as detailed below for classes of amino acids. [Table 3]

[0192] In some embodiments, the present disclosure provides a CAR that contains an extracellular ligand-binding domain that binds to CD70 and competes for binding to CD70 with a CAR described herein, including 31H1, 63B2, 40E3, 42C3, 45F11, 64F9, 72C2, 2F10, 4F11, 10H10, 17G6, 65E11, P02B10, P07D03, P08A02, P08E02, P08F08 , P08G02, P12B09, P12F02, P12G07, P13F04, P15D02, P16C05, 10A1, 10E2, 11A1, 11C1, 11D1, 11E1, 12A2, 12C4, 12C5, 12D3, 12D6, 12D7, 12F5, 12H4, 8C8, 8F7, 8F8, 9D8, 9E10, 9E5, 9F4 or 9F8.

[0193] In some embodiments, the present disclosure provides a CAR that specifically binds to CD70, wherein the CAR contains a VH region comprising the sequence set forth in SEQ ID NO:20 and / or a VL region comprising the sequence set forth in SEQ ID NO:19. In some embodiments, the present disclosure provides a CAR that specifically binds to CD70, wherein the CAR contains a VH region comprising the sequence set forth in SEQ ID NO:22 and / or a VL region comprising the sequence set forth in SEQ ID NO:21. In some embodiments, the present disclosure provides a CAR that specifically binds to CD70, wherein the CAR contains a VH region comprising the sequence set forth in SEQ ID NO:28 and / or a VL region comprising the sequence set forth in SEQ ID NO:27. In some embodiments, the present disclosure provides a CAR that specifically binds to CD70, wherein the CAR contains a VH region comprising the sequence set forth in SEQ ID NO:36 and / or a VL region comprising the sequence set forth in SEQ ID NO:35. In some embodiments, the present disclosure provides a CAR that specifically binds to CD70, wherein the CAR contains a VH region comprising the sequence set forth in SEQ ID NO:46 and / or a VL region comprising the sequence set forth in SEQ ID NO:45. In some embodiments, the present disclosure provides a CAR that specifically binds to CD70, the CAR containing a VH region comprising the sequence set forth in SEQ ID NO: 18 and / or a VL region comprising the sequence set forth in SEQ ID NO: 17. In some embodiments, the present disclosure provides a CAR that specifically binds to CD70, the CAR containing a VH region comprising the sequence set forth in SEQ ID NO: 34 and / or a VL region comprising the sequence set forth in SEQ ID NO: 33. In some embodiments, the present disclosure also provides a CAR containing a CDR portion of an antibody against CD70 based on the CDR contact regions. CDR contact regions are regions of an antibody that confer specificity for an antigen to the antibody. Generally, CDR contact regions include residue positions in the CDRs and Vernier zones that are constrained to maintain the proper loop structure of an antibody that binds to a specific antigen. See, e.g., Makabe et al., J. Biol. Chem., 283:1156-1166, 2007. Determining CDR contact regions is within the skill of the art.

[0194] The binding affinity (KD) of the ligand-binding domain of a CD70-specific CAR described herein for CD70 (e.g., human CD70) may be, for example, about 0.1 to about 1000 nM, for example, about 0.5 nM to about 500 nM, or for example, about 1 nM to about 250 nM. In some embodiments, the binding affinity is about any of 1000 nm, 750 nm, 500 nm, 400 nm, 300 nm, 250 nm, 200 nm, 100 nm, 90 nm, 80 nM, 70 nM, 60 nm, 50 nM, 45 nM, 40 nM, 35 nM, 30 nM, 25 nM, 20 nM, 19 nM, 18 nM, 17 nM, 16 nM, 15 nM, 10 nM, 8 nM, 7.5 nM, 7 nM, 6.5 nM, 6 nM, 5.5 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.5 nM, 0.3 nM, or 0.1 nM.

[0195] In some embodiments, the binding affinity (KD) of the scFv of the ligand-binding domain of a CD70-specific CAR described herein for CD70 is about 10 nM to about 100 nM, about 10 nM to about 90 nM, about 10 nM to about 80 nM, about 20 nM to about 70 nM, about 25 nM to about 75 nM, or about 40 nM to about 110 nM. In some embodiments, the binding affinity of the scFv described in this paragraph is the binding affinity for human CD70.

[0196] In some embodiments, the binding affinity is less than about any of 1000 nm, 900 nm, 800 nm, 250 nM, 200 nM, 100 nM, 50 nM, 30 nM, 20 nM, 10 nM, 7.5 nM, 7 nM, 6.5 nM, 6 nM, or 5 nM.

[0197] The intracellular signaling domain of the CAR according to the present disclosure is involved in intracellular signal transduction after the binding of the extracellular ligand-binding domain to the target, resulting in the activation of immune cells and an immune response. The intracellular signaling domain has the ability to activate at least one of the normal effector functions of the immune cells in which the CAR is expressed. For example, the effector function of T cells may be cytolytic activity or helper activity, including cytokine secretion.

[0198] In some embodiments, intracellular signaling domains for use in CARs may be, for example, but not limited to, cytoplasmic sequences of T cell receptors and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivatives or variants of these sequences, and any synthetic sequences with the same functional capabilities. The intracellular signaling domain contains two distinct classes of cytoplasmic signaling sequences: sequences that initiate antigen-dependent primary activation, and sequences that act in an antigen-dependent manner to generate secondary or costimulatory signals. Primary cytoplasmic signaling sequences may contain signaling motifs known as ITAMs, for immunoreceptor tyrosine-based activation motifs. ITAMs are well-defined signaling motifs present in the cytoplasmic tails of various receptors that serve as binding sites for tyrosine kinases of the syk / zap70 class. Examples of ITAMs used in the present disclosure include, by way of non-limiting example, ITAMs derived from TCRζ, FcRγ, FcRβ, FcRε, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. In some embodiments, the intracellular signaling domain of a CAR may contain a CD3ζ signaling domain having an amino acid sequence having at least about 70%, at least 80%, at least 90%, 95%, 97%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 272 or 683. In some embodiments, the intracellular signaling domain of a CAR of the present disclosure contains a domain of a costimulatory molecule.

[0199] In some embodiments, the intracellular signaling domain of a CAR of the present disclosure contains a portion of a costimulatory molecule selected from the group consisting of fragments of 41BB (GenBank: AAA53133) and CD28 (NP_006130.1). In some embodiments, the intracellular signaling domain of a CAR of the present disclosure contains an amino acid sequence that contains at least 70%, at least 80%, at least 90%, 95%, 97%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 271 or 682, and SEQ ID NO: 275. In some embodiments, the intracellular signaling domain of a CAR of the present disclosure contains an amino acid sequence that contains at least 70%, at least 80%, at least 90%, 95%, 97%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 271 or 682, and / or at least 70%, at least 80%, at least 90%, 95%, 97%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 276.

[0200] CARs are expressed on the surface membrane of cells. Thus, CARs may contain a transmembrane domain. Suitable transmembrane domains for the CARs disclosed herein include (a) in some embodiments, for example, but not limited to, T helper (T h ) cells, cytotoxic T (T C ) cells, T regulatory (T reg(b) capable of being expressed on the surface of a cell that is an immune cell, such as a CD8+ cell, or a lymphocyte cell, such as a natural killer (NK) cell, and / or (b) capable of interacting with a ligand-binding domain and an intracellular signaling domain to induce a cellular response of an immune cell against a predetermined target cell. The transmembrane domain may be derived from natural or synthetic sources. The transmembrane domain may be derived from any membrane-bound or transmembrane protein. As non-limiting examples, the transmembrane polypeptide may be a subsequence or subunit of a T cell receptor, such as the α, β, γ, or δ polypeptides that constitute the CD3 complex; the IL-2 receptor p55 (α chain), p75 (β chain); or an Fc receptor, particularly the γ chain, a subunit chain of Fcγ receptor III; or a CD protein. Alternatively, the transmembrane domain may be synthetic and contain primarily hydrophobic residues, such as leucine and valine. In some embodiments, the transmembrane domain is derived from the human CD8 α chain (e.g., NP_001139345.1). The transmembrane domain may further comprise a stalk domain between the extracellular ligand-binding domain and the transmembrane domain. The stalk domain may contain up to 300 amino acids, and in some embodiments, 10-100 amino acids, or in some embodiments, 25-50 amino acids. The stalk region may be derived from all or a portion of a naturally occurring molecule, such as all or a portion of the extracellular region of CD8, CD4, CD28, 4-1BB, or IgG (particularly the hinge region of IgG), or all or a portion of an antibody heavy chain constant region. Alternatively, the stalk domain may be a synthetic sequence corresponding to a naturally occurring stalk sequence, or may be an entirely synthetic stalk sequence. In some embodiments, the stalk domain is a portion of the human CD8 α chain (e.g., NP_001139345.1). In another specific embodiment, the hinge and transmembrane domain comprises a portion of the human CD8 alpha chain, which in some embodiments contains at least 70%, at least 80%, at least 90%, 95%, 97%, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 268 and 270.In some embodiments, the stalk domain of a CAR described herein contains a subsequence of CD8α, IgG1, or FcγRIIIα, particularly the hinge region of either CD8α, IgG1, or FcγRIIIα. In some embodiments, the stalk domain contains a human CD8α hinge, a human IgG1 hinge, or a human FcγRIIIα hinge. In some embodiments, a CAR disclosed herein may contain an extracellular ligand-binding domain that specifically binds to CD70. In some embodiments, a CAR disclosed herein contains an scFv, a human CD8α hinge and transmembrane domain, a CD3ζ signaling domain, and a 4-1BB signaling domain.

[0201] Table 4 provides exemplary sequences of domains that can be used in the CARs disclosed herein. [Table 4] TIFF2026000897000020.tif240170

[0202] Downregulation or mutation of target antigens is commonly observed in cancer cells, resulting in antigen-eliminating escape variants. Therefore, to counteract tumor escape and make immune cells more specific to their targets, CD70-specific CARs can contain one or more additional extracellular ligand-binding domains to simultaneously bind to different elements in the target and increase immune cell activation and function. In some embodiments, the extracellular ligand-binding domains can be arranged in tandem on the same transmembrane polypeptide and, optionally, separated by a linker. In some embodiments, the different extracellular ligand-binding domains can be arranged on different transmembrane polypeptides that make up the CAR. In some embodiments, the present disclosure relates to a group of CARs, each containing a different extracellular ligand-binding domain. In certain embodiments, the present disclosure relates to a method of engineering immune cells, comprising providing an immune cell and expressing a group of CARs on the surface of the cell, each containing a different extracellular ligand-binding domain. In another specific embodiment, the present disclosure relates to a method for engineering immune cells, the method comprising providing immune cells and introducing into the cells a polynucleotide encoding a polypeptide containing a group of CARs, each CAR containing a different extracellular ligand-binding domain. A group of CARs refers to at least two, three, four, five, six, or more CARs, each CAR containing a different extracellular ligand-binding domain. The different extracellular ligand-binding domains of the present disclosure can, in some embodiments, simultaneously bind to different elements in a target, thereby enhancing the activation and function of the immune cells. The present disclosure also relates to isolated immune cells containing a group of CARs, each CAR containing a different extracellular ligand-binding domain.

[0203] In another aspect, the disclosure provides polynucleotides encoding any of the CARs and polypeptides described herein. Polynucleotides can be made and expressed by methods known in the art.

[0204] In another aspect, the present disclosure provides a composition (e.g., a pharmaceutical composition) containing any of the cells of the present disclosure. In some embodiments, the composition comprises a cell comprising a polynucleotide encoding any of the CARs described herein. In yet other embodiments, the composition contains any of the polynucleotides set forth in the following SEQ ID NOs: 297 and 298, 299 and 300, 301 and 302, 303 and 304, 305 and 306, 307 and 308, or 309 and 310: 4F11 heavy chain variable region CAGGTCACCTTGAAGGAGTCTGGTCCTGTGCTGGTGAAACCCACAGAGACCCTCACGCTGACCTGCACCGTCTCTGGGTTCTCACTCAGTAATGCTAGAATGGGTGTGACCTGGATCCGTCAGCCCCCAGGGAAGGCCCTGGAGTGGCTTGCACACATTTTTTCGAATGACGAAAAAATCCTACA GTACATCTCTGAAGAGCAGGCTCACCATCTCCAAGGACACTTCCAAAACCCAGGTGGTCCTTACCATGACCAACATGGACCCTGTGGACACAGCCACATATTACTGTGCACGGATACGAGATTACTATGACATTAGTAGTTATTATGACTACTGGGCCAGGGAACCCTGGTCAGCGTCTCCTCA (Sequence number 297) 4F11 light chain variable region GACATCCAGATGACCCAGTCTCCATCTGCCATGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGACATTAGCAATTATTTAGCCTGGTTTCAGCAGAAACCAGGGAAAGTCCCTAAGCGCCTGATCTATGCTGCATCCAGTT TGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCGGGGACAGAATTCACTCTCACAATCAGCAGCCTGCTGCCTGAAGATTTTGCAACTTATTACTGTCTACAGCTTAATAGTTTCCCGTTCACTTTTGGCGGAGGGACCAAGGTGGAGATCAAC (Sequence number 298)

[0205] In yet other embodiments, the composition contains either or both of the polynucleotides set forth in SEQ ID NO:299 and SEQ ID NO:300: 17G6 heavy chain variable region GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGTAGCCTCTGGATTCACCTTTAGTAGTTATTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAGCATAAAGCAAGATGGAAGTGAGAAATACTATGTGGAC TCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCAGTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGGTGTGTATTACTGTGCGAGAGAAGGAGTCAACTGGGGATGGAGACTCTACTGGCACTTCGATCTCTGGGGCCGTGGAACCCTGGTCACTGTCTCCTCA (Sequence number 299) 17G6 light chain variable region GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATACAGCTACAACAATAAGAACTACGTAGCTTGGTACCAGCAGAAACCAGGACAACCTCCTAACCTACTCATTTTCTGG GCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTACTACTGTCAGCAATATTATAGTACGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA (Sequence number 300).

[0206] In yet other embodiments, the composition contains either or both of the polynucleotides set forth in SEQ ID NO: 301 and SEQ ID NO: 302: 10H10 heavy chain variable region GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGTCTCTGGATTCACCTTCAGTAACCATAACATACACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATTTCATACATTAGTCGAAGTAGTAGTACCATATATT ACGCAGACTCTGTGAAGGGCCGATTCACAATCTCCAGAGACAATGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGACGAAGACACGGCTGTGTATTACTGTGCGAGAGATCACGCTCAGTGGTACGGTATGGACGTTTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA (SEQ ID NO: 301). 10H10 light chain variable region GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCGGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGGTCCTGATCTATGCTGCATCCAGTT TGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTTTCAGTTTCCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAA (SEQ ID NO: 302).

[0207] In yet other embodiments, the composition contains either or both of the polynucleotides set forth in SEQ ID NO: 303 and SEQ ID NO: 304: P07D03 Heavy chain variable region GAAGTGCAGCTTGTCCAGAGCGGAGCCGAAGTGAAGAAGCCTGGCGAGAGCCTGAAGATCAGCTGCAAGGGCTCGGATATCGCTTCACAAGTTACTGGATAGGGTGGGTGCGCCAGATGCCTGGTAAGGGACTGGAATGGATGGGCTCTATATATCCTGATGATTCCGACACACGTTATAGC CCAAGCTTTCAGGGCCAGGTCACAATCAGCGCTGACAAGAGCATCAGCACCGCCTACCTTCAGTGGTCGTCTCTGAAGGCCAGCGACACCGCAATGTACTACTGCGCCTCTAGCACAGTTGACTACCCGGGATACAGTTACTTCGACTACTGGGGCCAAGGTACACTGGTCACCGTCAGCAGC (Sequence number 303) P07D03 Light chain variable region GAGCTCCAGAGCGTGCTGACCCAGCCTCCTAGCGCAAGCGGCACCCCTGGACAGCGTGTGACAATTAGCTGTAGCGGAAGTCGTAGCAATATCGGATCAAACTATGTGTATTGGTATCAGCAATTGCCCGGTACAGCACCCAAATTGCTCATATATAGAAATAATCAG AGACCTAGCGGAGTGCCTGATCGTTTTAGCGGTAGCAAAAGCGGCACCAGCGCATCACTGGCAATTTCAGGCCTGCGTAGCGAAGATGAGGCGGATTATTACTGTGCGAGTTGGGATGGTTCGCTGAGTGCTGTTGTGTTCGGCACCGGTACAAAACTGACCGTTCTG (Sequence number 304)

[0208] In yet other embodiments, the composition contains either or both of the polynucleotides set forth in SEQ ID NO: 305 and SEQ ID NO: 306: P08G02 heavy chain variable region GAAGTGCAGCTTGTCCAGAGCGGAGCCGAAGTGAAGAAGCCTGGCGAGAGCCTGAAGATCAGCTGCAAGGGCTCGGATACACCTTTCCTTCATCATGGATAGGTTGGGTGCGCCAGATGCCTGGTAAGGGACTGGAATGGATGGGCATCATATACCCTGATACTAGCCATACCCGTTACAGCCCAAGC TTTCAGGGCCAGGTCACAATCAGCGCTGACAAGAGCATCAGCACCGCCTACCTTCAGTGGTCGTCTCTGAAGGCCAGCGACACCGCAATGTACTACTGTGCCCGTGCGAGCTATTTCGATCGTGGAACAGGGTATAGTTCTTGGTGGATGGATGTGTGGGGCCAAGGTACACTGGTCACCGTCAGCAGC (Sequence number 305) P08G02 light chain variable region GAGCTCGATATTCAGATGACCCAGAGCCCTAGCAGCCTGAGCGCAAGCGTGGGCGATAGAGTGACCATTACCTGTAGGGCCTCACAATCCATATACGACTATTTGCACTGGTATCAGCAGAAACCCGGGAAAGCACCCAAACTGCTGATTTACGATGCTTCCAAC CTACAGAGTGGCGTTCCTTCACGTTTTAGCGGTAGCGGTTCAGGCACCGATTTCACCCTGACCATTAGCAGCCTTCAGCCGAAGATTTCGCTACGTATTATTGCCAGCAATCATACACCACGCCGTTGTTTACATTCGGCCAGGGTACCAAAGTGGAAATCAAA (Sequence number 306)

[0209] In yet other embodiments, the composition contains either or both of the polynucleotides set forth in SEQ ID NO: 307 and SEQ ID NO: 308: P08F08 Heavy Chain Variable Region GAAGTGCAGCTTGTCCAGAGCGGAGCCGAAGTGAAGAAGCCTGGCGAGAGCCTGAAGATCAGCTGCAAGGGCTCGGATACGGATTCACAAGTTATTGGATAGGTTGGGTGCGCCAGATGCCTGGTAAGGGACTGGAATGGATGGGTATCATTCATCCCGATGATAGCGACACCAAATACAGC CCAAGCTTTCAGGGCCAGGTCACAATCAGCGCTGACAAGAGCATCAGCACCGCCTACCTTCAGTGGTCGTCTCTGAAGGCCAGCGACACCGCAATGTACTACTGTGCCTCTAGCTATTTGCGTGGCTTGTGGGGAGGCTATTTTGACTATTGGGGCCAAGGTACACTGGTCACCGTCAGCAG (Sequence number 307) P08F08 light chain variable region GAGCTCCAGAGCGTGCTGACCCAGCCTCCTAGCGCAAGCGGCACCCCTGGACAGCGTGTGACAATTAGCTGTAGCGGATCAAGCTCAAACATTGGCTCAAATTATGTGAATTGGTATCAGCAATTGCCCGGTACAGCACCCAAACTGCTCATTTATGGAGATTATCAAC GACCTAGCGGAGTGCCTGATCGTTTTAGCGGTAGCAAAAGCGGCACCAGCGCATCACTGGCAATTTCAGGCCTGCGTAGCGAAGATGAGGCGGATTATTACTGTGCTACCCGCGACGATTCGTTATCTGGGTCTGTCGTTTTTGGCACCGGTACAAAACTGACCGTGCTG (Sequence number 308)

[0210] In yet other embodiments, the composition contains either or both of the polynucleotides set forth in SEQ ID NO: 309 and SEQ ID NO: 310: P15D02 heavy chain variable region GAAGTGCAGCTTGTCCAGAGCGGAGCCGAAGTGAAGAAGCCTGGCGAGAGCCTGAAGATCAGCTGCAAGGGCTCCGGATACAGTTTTGCCTCATACTGGATCGGTTGGGTGCGCCAGATGCCTGGTAAGGGACTGGAATGGATGGGCGTAATTTACCCCGGAACTAGCGAGACACGTTACAGC CCAAGCTTTCAGGGCCAGGTCACAATCAGCGCTGACAAGAGCATCAGCACCGCCTACCTTCAGTGGTCGTCTCTGAAGGCCAGCGACACCGCAATGTACTACTGCGCTAAAGGGTTGAGTGCGAGTGCAAGTGGATATTTCTTTCCAATATTGGGGCCAAGGTACACTGGTCACCGTCAGCAGC (Sequence number 309) P15D032 light chain variable region GAGCTCGATATTCAGATGACCCAGAGCCCTAGCAGCCTGAGCGCAAGCGTGGGCGATAGAGTGACCATTACCTGTAGGGCCTCACAAAGCATCGACACATATTTAAACTGGTATCAGCAGAAACCCGGGAAAGCACCCAAACTGCTGATTTATTCAGCTAGTAGC CTACACAGTGGCGTTCCTTCACGTTTTAGCGGTAGCGGTTCAGGCACCGATTTCACCCTGACCATTAGCAGCCTTCAGCCGAAGATTTCGCTACGTATTATTGCCAACAATCATACAGCACAACTGCTTGGACATTCGGCCAGGGTACCAAAGTGGAAATCAAA (Sequence number 310)

[0211] Expression vectors and administration of polynucleotide compositions are further described herein.

[0212] In another aspect, the disclosure provides a method of making any of the polynucleotides described herein.

[0213] Polynucleotides complementary to any of these sequences are also encompassed by the present disclosure. Polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be DNA molecules (genomic, cDNA, or synthetic) or RNA molecules. RNA molecules include HnRNA molecules, which contain introns and correspond in a one-to-one manner to DNA molecules, and mRNA molecules, which do not contain introns. Additional coding or non-coding sequences may, but need not, be present within the polynucleotides of the present disclosure, and polynucleotides may, but need not, be linked to other molecules and / or support materials.

[0214] The polynucleotide may comprise a native sequence (i.e., an endogenous sequence encoding an antibody or portion thereof) or may comprise a variant of such a sequence. Polynucleotide variants contain one or more substitutions, additions, deletions, and / or insertions such that the immunoreactivity of the encoded polypeptide is not diminished compared to the native immunoreactive molecule. The effect on the immunoreactivity of the encoded polypeptide may generally be assessed as described herein. Embodiments of variants exhibit at least about 70% identity, at least about 80% identity, at least about 90% identity, or at least about 95% identity to the polynucleotide sequence encoding a native antibody or portion thereof.

[0215] Two polynucleotide or polypeptide sequences are said to be "identical" if the sequences of nucleotides or amino acids in the two sequences are the same when aligned for maximum correspondence as described below. Comparison between two sequences is typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. As used herein, a "comparison window" refers to a segment of at least about 20 contiguous positions, usually 30 to about 75, or 40 to about 50 contiguous positions, within which two sequences can be optimally aligned and then compared to a reference sequence of the same number of contiguous positions.

[0216] Alignment of optimal sequences for comparison may be performed using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, Wis.) using default parameters. This program embodies several alignment schemes described in the following references: Dayhoff, MO, 1978, A model of evolutionary change in proteins—Matrices for detecting distant relationships. In Dayhoff, MO (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358; Hein J., 1990, Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, CA; Higgins, DGand Sharp, PM,1989,CABIOS 5:151-153;Myers,EWand Muller W.,1988,CABIOS 4:11-17;Robinson,ED,1971,Comb.Theor.11:105;Santou,N.,Nes,M.,1987,Mol.Biol.Evol.4:406-425;Sneath,PHAand Sokal, RR, 1973, Numerical Taxonomy the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, WJand Lipman, DJ, 1983, Proc. Natl. Acad. Sci. USA 80:726-730.

[0217] "Percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window of at least 20 positions, where the portion of the polynucleotide or polypeptide sequence in the comparison window may contain 20% or less, typically 5-15%, or 10-12% additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) due to optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue occurs in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the reference sequence (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity.

[0218] Variants may also, or alternatively, be substantially homologous to a native gene, or a portion thereof, or a complement thereof, and such polynucleotide variants have the ability to hybridize under moderately stringent conditions to a native DNA sequence encoding the native antibody (or a complementary sequence).

[0219] Suitable "moderately stringent conditions" include prewashing in a solution of 5X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing overnight at 50°C to 65°C in 5X SSC; and then washing twice at 65°C for 20 minutes, each with 2X, 0.5X, and 0.2X SSC containing 0.1% SDS.

[0220] As used herein, "highly stringent conditions" or "highly stringent conditions" refers to (1) the use of low ionic strength and high temperature for washing, e.g., 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at 50°C; (2) the use of a denaturing agent such as formamide, e.g., 750 mM sodium chloride, 75 mM sodium citrate, at 42°C during hybridization, with 50% (v / v) formamide containing 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer, pH 6.5; or (3) 50% formamide, 5x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x It employs Denhardt's solution, sonicated salmon sperm DNA (50 μg / ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes in 0.2×SSC (sodium chloride / sodium citrate) at 42° C. and 50% formamide at 55° C., followed by a high stringency wash consisting of 0.1×SSC containing EDTA at 55° C. One of skill in the art will know how to adjust temperature, ionic strength, etc. as needed to accommodate factors such as probe length.

[0221] Those skilled in the art will recognize that, as a result of the degeneracy of the genetic code, numerous nucleotide sequences exist that encode the polypeptides described herein. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any native gene. Nevertheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by this disclosure. Additionally, alleles of genes comprising the polynucleotide sequences provided herein are within the scope of this disclosure. Alleles are endogenous genes that vary as a result of one or more mutations, such as, for example, nucleotide deletions, additions, and / or substitutions. The resulting mRNA and protein may, but need not, be altered in structure or function. Alleles may be identified using standard techniques (e.g., hybridization, amplification, and / or database sequence comparison).

[0222] The polynucleotide of the present disclosure can be obtained by chemical synthesis, recombinant method or PCR.Chemical polynucleotide synthesis method is known in the art and does not need to be described in detail herein.Those skilled in the art can use the sequence provided herein and commercially available DNA synthesizer to generate desired DNA sequence.

[0223] As discussed further herein, to prepare a polynucleotide using recombinant methods, a polynucleotide containing a desired sequence may be inserted into an appropriate vector, which may then be introduced into a suitable host cell and replicated and amplified. Polynucleotides can be inserted into host cells by any means known in the art. Cells are transformed by introducing an exogenous polynucleotide via direct uptake, endocytosis, transfection, F mating, or electroporation. Once introduced, the exogenous polynucleotide may be maintained within the cell as a non-integrated vector (e.g., a plasmid) or integrated into the host cell genome. Such amplified polynucleotide can be isolated from the host cell by methods known in the art. See, e.g., Sambrook et al., 1989.

[0224] Alternatively, DNA sequences can be reproduced by PCR. PCR techniques are known in the art and are described in U.S. Patent Nos. 4,683,195, 4,800,159, 4,754,065 and 4,683,202, and in PCR: The Polymerase Chain Reaction, Mullis et al. eds., Birkauer Press, Boston, 1994.

[0225] RNA can be obtained by using the isolated DNA in a suitable vector and inserting it into a suitable host cell, for example, as described in Sambrook et al., 1989, supra, when the cell replicates and the DNA is transcribed into RNA, which can be isolated using methods known to those skilled in the art.

[0226] Suitable cloning vectors may be constructed according to standard techniques or may be selected from a large number of cloning vectors available in the art. While the cloning vector selected may vary depending on the host cell intended for use, useful cloning vectors generally possess the ability to autonomously replicate, may possess a single target for a particular restriction endonuclease, and / or may carry a marker gene that can be used to select clones containing the vector. Suitable examples include plasmids and bacterial viruses, such as pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, and shuttle vectors such as pSA3 and pAT28. These and many other cloning vectors are available from commercial sources, such as BioRad, Strategene, and Invitrogen.

[0227] An expression vector is generally a replicable polynucleotide construct containing a polynucleotide according to the present disclosure. It is implied that an expression vector must be replicable in a host cell either as an episome or as an integral part of chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, viral vectors including adenoviruses, adeno-associated viruses, and retroviruses, cosmids, and the expression vector disclosed in PCT Publication WO 87 / 04462 or the lentiviral pLVX vector available from Clonetech. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, and appropriate transcription control elements (e.g., promoters, enhancers, and terminators). For expression (i.e., translation), one or more translation control elements are also usually required, such as a ribosome binding site, a translation initiation site, and a stop codon.

[0228] A vector containing a polynucleotide of interest can be introduced into a host cell by any of a number of suitable means, including electroporation, transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances, microprojectile bombardment, lipofection, and infection (where the vector is an infectious entity such as, for example, vaccinia virus). The choice of vector or polynucleotide introduction often depends on characteristics of the host cell.

[0229] A polynucleotide encoding a CD70-specific CAR disclosed herein may be present in an expression cassette or expression vector (e.g., a plasmid for introduction into a bacterial host cell, or a viral vector such as a baculovirus vector for transfection of an insect host cell, or a viral vector such as a plasmid or lentivirus for transfection of a mammalian host cell). In some embodiments, the polynucleotide or vector may contain a nucleic acid sequence encoding a ribosomal skipping sequence, such as, but not limited to, a sequence encoding a 2A peptide. The 2A peptide, identified in the aphthovirus subgroup of picornaviruses, causes the ribosome to "skip" from one codon to the next without forming a peptide bond between the two amino acids encoded by the codon (see (Donnelly and Elliott 2001; Atkins, Wills et al. 2007; Doronina, Wu et al. 2008)). By "codon" is meant three nucleotides in an mRNA (or the sense strand of a DNA molecule) that are translated by the ribosome into a single amino acid residue. Thus, two polypeptides can be synthesized from one continuous open reading frame within an mRNA when the polypeptides are separated in frame by a 2A oligopeptide sequence. Such ribosomal skipping mechanisms are known in the art and are known to be used in several vectors for the expression of several proteins encoded by a single messenger RNA.

[0230] In some embodiments, a secretory signal sequence (also known as a leader sequence, prepro sequence, or pre sequence) is provided in the polynucleotide or vector sequence to direct the transmembrane polypeptide into the secretory pathway of the host cell. The secretory signal sequence is operably linked to the transmembrane nucleic acid sequence; i.e., the two sequences are joined in the correct reading frame and positioned to direct the newly synthesized polypeptide into the secretory pathway of the host cell. Secretory signal sequences are typically positioned 5' to the nucleic acid sequence encoding the polypeptide of interest. However, particular secretory signal sequences may be positioned elsewhere in the nucleic acid sequence of interest (see, e.g., U.S. Pat. No. 5,037,743 to Welch et al. and U.S. Pat. No. 5,143,830 to Holland et al.). In some embodiments, the signal peptide contains the amino acid sequence set forth in SEQ ID NO:266 or SEQ ID NO:277. Those skilled in the art will recognize that considerable sequence variation is possible in these polynucleotide molecules given the degeneracy of the genetic code. In some embodiments, the nucleic acid sequences of the present disclosure are codon-optimized for expression in mammalian cells, or, in some embodiments, for expression in human cells. Codon optimization refers to the replacement of a subject sequence of codons that are generally rare in highly expressed genes of a given species with codons that are generally frequent in highly expressed genes of that species, where the codons encode the same amino acid as the replaced codon.

[0231] CD70-specific antibodies and methods for producing same Provided herein are CD70 antibodies.

[0232] In some embodiments, the CD70 antibodies of the disclosure contain any one of the partial light chain sequences listed in Table 1 and / or any one of the partial heavy chain sequences listed in Table 1. In Table 1, the underlined sequences are CDR sequences according to Kabat, and the bolded sequences are CDR sequences according to Chothia.

[0233] Tables 2A-2B provide examples of CDR sequences for the CD70 antibodies provided herein.

[0234] In some embodiments, the disclosure provides antibodies (including antibody fragments, such as single chain variable fragments (scFv) that specifically bind to Cluster of Differentiation 70 (CD70)), wherein the antibody is selected from the group consisting of: (a)(i) SEQ ID NOs: 49, 50, 51, 55, 56, 57, 61, 62, 63, 67, 68, 69, 73, 74, 75, 79, 80, 81, 85, 86, 87, 91, 92, 93, 97, 98, 99, 103 , 104, 105, 109, 110, 111, 115, 116, 117, 121, 122, 123, 127, 128, 129, 133, 134, 135, 139, 140, 141, 145, 146, 147, 151, 152, 153, 157, 158, 159, 163, 164, 165, 169, 170, 171, 175, 176, 177, 181, 182, 183, 187, 188, 189, 382, ​​383, 384, 388, 389, 390, 394, 395, 396, 400, 401, 402, 406, 407, 408, 412, 413, 414, 418, 419, 420, 424, 425, 426, 430, 431, 432, 663, 664, 665, 436, 437, 438, 442, 443, 444, a VH complementarity determining region 1 (CDR1) comprising the sequence set forth in 448, 449, 450, 454, 455, 456, 460, 461, 462, 466, 467, 468, 472, 473, 474, 478, 479, 480, 484, 485, 486, 490, 491, 492, 496, 497, 498, 502, 503, 504, 508, 509, or 510;(ii) SEQ ID NOs: 52, 53, 58, 59, 64, 65, 70, 71, 76, 77, 82, 83, 88, 89, 94, 95, 100, 101, 106, 107, 112, 113, 118, 119, 124, 125, 130, 131, 136, 137, 142, 143, 148, 149, 154, 155, 160, 161, 166, 167, 172, 173, 178, 179, 184, 185, 190, 191, 385, 386 , 391, 392, 397, 398, 403, 404, 409, 410, 415, 416, 421, 422, 427, 428, 433, 434, 666, 667, 439, 440, 445, 446, 451, 452, 457, 458, 463, 464, 469, 470, 475, 476, 481, 482, 487, 488, 493, 494, 499, 500, 505, 506, 511, or 512. CDR2; and iii) a VH comprising the sequence set forth in SEQ ID NO: 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 387, 393, 399, 405, 411, 417, 423, 429, 435, 668, 441, 447, 453, 459, 465, 471, 477, 483, 489, 495, 501, 507, or 513. and / or (i) a VL CDR1 comprising the sequence set forth in SEQ ID NO: 193, 196, 199, 202, 205, 208, 211, 214, 217, 220, 223, 226, 229, 232, 235, 238, 241, 244, 247, 250, 253, 256, 259, 262, 514, 517, 520, 523, 526, 529, 532, 535, 538, 669, 541, 544, 547, 550, 553, 556, 559, 562, 565, 568, 571, 574, or 577;(ii) a VL comprising the sequence set forth in SEQ ID NO: 194, 197, 200, 203, 206, 209, 212, 215, 218, 221, 224, 227, 230, 233, 236, 239, 242, 245, 248, 251, 254, 257, 260, 263, 515, 518, 521, 524, 527, 530, 533, 536, 539, 670, 542, 545, 548, 551, 554, 557, 560, 563, 566, 569, 572, 575, or 578 and (iii) a VL CDR3 comprising the sequence set forth in SEQ ID NO: 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 225, 228, 231, 234, 237, 240, 243, 246, 249, 252, 255, 258, 261, 264, 516, 519, 522, 525, 528, 531, 534, 537, 540, 671, 543, 546, 549, 552, 555, 558, 561, 564, 567, 570, 573, 576, or 579;

[0235] In some embodiments, the disclosure provides an antibody (e.g., scFv) that specifically binds to Cluster of Differentiation 70 (CD70), wherein the antibody comprises a VH CDR1, a VH CDR2, and a VH CDR3 of the VH sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 339, 341, 343, 345, 347, 349, 351, 353, 355, 662, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, or 381. and / or a light chain variable (VL) region comprising VL CDR1, VL CDR2, and VL CDR3 of the VL sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 338, 340, 342, 344, 346, 348, 350, 352, 354, 661, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, or 380.

[0236] In some embodiments, the present disclosure provides an isolated antibody that specifically binds to CD70 and competes with any of the aforementioned antibodies.

[0237] In some embodiments, the present invention provides antibodies that bind to CD70 and are selected from the group consisting of 31H1, 63B2, 40E3, 42C3, 45F11, 64F9, 72C2, 2F10, 4F11, 10H10, 17G6, 65E11, P02B10, P07D03, P08A02, P08E02, P08F08, P08G02, P12B09, P12F02, P12G07, P Antibodies that compete with the antibodies described herein are provided, including 13F04, P15D02, P16C05, 10A1, 10E2, 11A1, 11C1, 11D1, 11E1, 12A2, 12C4, 12C5, 12D3, 12D6, 12D7, 12F5, 12H4, 8C8, 8F7, 8F8, 9D8, 9E10, 9E5, 9F4 or 9F8.

[0238] In some embodiments, the present invention further provides CDR portions of antibodies against CD70 antibodies based on the CDR contact regions. CDR contact regions are regions of an antibody that confer specificity to the antibody for an antigen. Generally, CDR contact regions include residue positions in the CDRs and Vernier zones that are constrained to maintain the proper loop structure of an antibody binding to a specific antigen. See, for example, Makabe et al., J. Biol. Chem., 283:1156-1166, 2007. Determining CDR contact regions is within the skill of the art.

[0239] The binding affinity (K) of the CD70 antibodies described herein to CD70 (e.g., human CD70 (e.g., SEQ ID NO: 335)) D) may be about 0.001 to about 5000 nM. In some embodiments, the binding affinity is approximately 5000 nM, 4500 nM, 4000 nM, 3500 nM, 3000 nM, 2500 nM, 2000 nM, 1789 nM, 1583 nM, 1540 nM, 1500 nM, 1490 nM, 1064 nM, 1000 nM, 933 nM, 894 nM, 750 nM, 705 nM, 678 nM, 532 nM, 500 nM, 494 nM, 400 nM, 349 nM, 340 nM, 353 nM, 300 nM, 250 nM, 244 nM, 231 nM, 225 nM, 207 nM, 200 nM, 186 nM, 172 nM, 100 nM, 90 nM, 83 nM, 79 nM, 74 nM, 54 nM, 50 nM, 45 nM, 42 nM, 40 nM, 35 nM, 32 nM, 30 nM, 25 nM, 24 nM, 22 nM, 20 nM, 19 nM, 18 nM, 17 nM, 16 nM, 15 nM, 12 nM, 10 nM, 9 nM, 8 nM, 7.5 nM, 7 nM, 6.5 nM, 6 nM, 5.5 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.5 nM, 0.3 nM, 0.1 nM, 0.01 nM, or 0.001 nM. In some embodiments, the binding affinity is less than approximately any of 5000 nM, 4000 nM, 3000 nM, 2000 nM, 1000 nM, 900 nM, 800 nM, 250 nM, 200 nM, 100 nM, 50 nM, 30 nM, 20 nM, 10 nM, 7.5 nM, 7 nM, 6.5 nM, 6 nM, 5 nM, 4.5 nM, 4 nM, 3.5 nM, 3 nM, 2.5 nM, 2 nM, 1.5 nM, 1 nM, or 0.5 nM.

[0240] In some embodiments, the disclosure provides a nucleic acid encoding any of the foregoing isolated antibodies. In some embodiments, the disclosure provides a vector comprising the nucleic acid. In some embodiments, the disclosure provides a host cell comprising the nucleic acid.

[0241] The present disclosure further provides any of the foregoing antibodies for use as a medicament. In some embodiments, the medicament is for use in treating a CD70-associated cancer selected from the group consisting of renal cell carcinoma, glioblastoma, glioma such as low-grade glioma, non-Hodgkin's lymphoma (NHL), Hodgkin's disease (HD), Waldenstrom's hypergammaglobulinemia, acute myeloid leukemia, multiple myeloma, diffuse large cell lymphoma, follicular lymphoma, or non-small cell lung cancer.

[0242] In some embodiments, the present disclosure provides a method of treating a subject in need thereof, the method comprising providing any of the aforementioned antibodies and administering the antibody to the subject.

[0243] In some embodiments, the present disclosure provides a pharmaceutical composition comprising any of the foregoing antibodies.

[0244] In some embodiments, the present disclosure provides a method of treating a condition associated with malignant cells that express CD70 in a subject, the method comprising administering to a subject in need thereof an effective amount of any one of the aforementioned antibodies or an effective amount of a pharmaceutical composition comprising any one of the aforementioned antibodies. In some embodiments, the condition is cancer. In some embodiments, the cancer is a CD70-associated cancer selected from the group consisting of renal cell carcinoma, glioblastoma, glioma, e.g., low-grade glioma, non-Hodgkin's lymphoma (NHL), Hodgkin's disease (HD), Waldenstrom's hypergammaglobulinemia, acute myeloid leukemia, multiple myeloma, diffuse large cell lymphoma, follicular lymphoma, or non-small cell lung cancer.

[0245] In some embodiments, the present disclosure provides a method of inhibiting tumor growth or progression in a subject having malignant cells that express CD70, the method comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition of the present disclosure.

[0246] In some embodiments, the present disclosure provides a method of inhibiting metastasis of malignant cells that express CD70 in a subject, the method comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition of the present disclosure.

[0247] In some embodiments, the present disclosure provides a method of inducing tumor regression in a subject having malignant cells that express CD70, the method comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition of the present disclosure.

[0248] In some embodiments, an antibody, comprising culturing a host cell of the present disclosure under conditions that result in the production of the antibody, and isolating the antibody from the host cell or culture.

[0249] Antibodies useful in the present invention may include monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab', F(ab')2, Fv, Fc, etc.), chimeric antibodies, bispecific antibodies, heteroconjugate antibodies, single-chain (ScFv), variants thereof, fusion proteins containing antibody portions (e.g., domain antibodies), humanized antibodies, and any other modified structure of an immunoglobulin molecule containing an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. Antibodies may be murine, rat, human, or any other origin (including chimeric or humanized antibodies).

[0250] In some embodiments, the CD70 monospecific antibodies described herein are monoclonal antibodies, e.g., they are human monoclonal antibodies.

[0251] The present disclosure further provides the following exemplary embodiments. 1. An isolated antibody that specifically binds to Cluster of Differentiation 70 (CD70), the antibody comprising: (a) (i) SEQ ID NOs: 49, 50, 51, 55, 56, 57, 61, 62, 63, 67, 68, 69, 73, 74, 75, 79, 80, 81, 85, 86, 87, 91, 92, 93, 97, 98, 99, 103, 104, 105, 109, 110, 111, 115, 116, 117, 121, 122, 123, 127, 128, 129, 133, 134, 135, 139, 140, 141, 145, 146, 147, 151, 152, 153, 157, 158, 159, 163, 164, 165, 169, 170, 171, 175, 176, 177, 181, 182, 183, 187, 188, 189, 382, ​​383, 384, 388, 389, 390, 394, 395, 396, 400, 401, 402, 406, 407, 408, 412, 413, 414, 418, 419, 420, 424, 425, 426, 430, 431, 432, 663, 664, 665, 436, 437, 438, 442, 443, 444, 448, 449, 450, 454, 455, 456, 460, 461, 462, 466, 467, 468, 472, 473, 474, 478 , 479, 480, 484, 485, 486, 490, 491, 492, 496, 497, 498, 502, 503, 504, 508, 509, or 510; (ii) a VH complementarity determining region 1 (CDR1) comprising the sequence set forth in SEQ ID NOs: 52, 53, 58, 59, 64, 65, 70, 71, 76, 77, 82, 83, 88, 89, 94, 95, 100, 101, 106, 107, 112, 113, 118, 119, 124, 125, 130, 131, 136, 137, 142, 143, 148, 149, 154, 155, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 205, 206, 207, 208, 209, VH comprising the sequence shown in 1, 166, 167, 172, 173, 178, 179, 184, 185, 190, 191, 385, 386, 391, 392, 397, 398, 403, 404, 409, 410, 415, 416, 421, 422, 427, 428, 433, 434, 666, 667, 439, 440, 445, 446, 451, 452, 457, 458, 463, 464, 469, 470, 475, 476, 481, 482, 487, 488, 493, 494, 499, 500, 505, 506, 511 or 512a heavy chain variable (VH) region comprising a CDR2, and iii) a VH CDR3 comprising the sequence set forth in SEQ ID NO: 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 387, 393, 399, 405, 411, 417, 423, 429, 435, 668, 441, 447, 453, 459, 465, 471, 477, 483, 489, 495, 501, 507 or 513; and / or (b)(i) a VL comprising the sequence set forth in SEQ ID NO: 193, 196, 199, 202, 205, 208, 211, 214, 217, 220, 223, 226, 229, 232, 235, 238, 241, 244, 247, 250, 253, 256, 259, 262, 514, 517, 520, 523, 526, 529, 532, 535, 538, 669, 541, 544, 547, 550, 553, 556, 559, 562, 565, 568, 571, 574, or 577 CDR1, (ii) a VL comprising the sequence set forth in SEQ ID NO: 194, 197, 200, 203, 206, 209, 212, 215, 218, 221, 224, 227, 230, 233, 236, 239, 242, 245, 248, 251, 254, 257, 260, 263, 515, 518, 521, 524, 527, 530, 533, 536, 539, 670, 542, 545, 548, 551, 554, 557, 560, 563, 566, 569, 572, 575, or 578 and (iii) a VL CDR3 comprising the sequence set forth in SEQ ID NO: 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 225, 228, 231, 234, 237, 240, 243, 246, 249, 252, 255, 258, 261, 264, 516, 519, 522, 525, 528, 531, 534, 537, 540, 671, 543, 546, 549, 552, 555, 558, 561, 564, 567, 570, 573, 576, or 579. 2. An isolated antibody that specifically binds to Cluster of Differentiation 70 (CD70), the antibody comprising: a) a VH region comprising the VH CDR1, VH CDR2 and VH CDR3 of the VH sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 339, 341, 343, 345, 347, 349, 351, 353, 355, 662, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379 or 381, and / or (b) a VL region comprising VL CDR1, VL CDR2 and VL CDR3 of the VL sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 338, 340, 342, 344, 346, 348, 350, 352, 354, 661, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378 or 380. 3. An isolated antibody that specifically binds to CD70 and competes with the antibody of embodiment 1. 4. A nucleic acid encoding the antibody according to any one of embodiments 1 to 3. 5. A vector comprising the nucleic acid of embodiment 4. 6. A host cell comprising the nucleic acid of embodiment 4. 7. The antibody according to any one of embodiments 1 to 3 for use as a pharmaceutical. 8. The antibody of embodiment 7, wherein the medicament is for use in the treatment of a CD70-associated cancer selected from the group consisting of renal cell carcinoma, glioblastoma, glioma, such as low-grade glioma, non-Hodgkin's lymphoma (NHL), Hodgkin's disease (HD), Waldenstrom's hypergammaglobulinemia, acute myeloid leukemia, multiple myeloma, diffuse large cell lymphoma, follicular lymphoma, or non-small cell lung cancer. 9. A method of treating a subject in need thereof, comprising: a. providing an antibody according to any one of embodiments 1 to 3; and b. administering said antibody to said subject. 10. A pharmaceutical composition comprising the antibody according to any one of embodiments 1 to 3. 11. A method of treating a condition associated with malignant cells expressing CD70 in a subject, comprising administering to a subject in need thereof an effective amount of an antibody described in any one of embodiments 1 to 3, or an effective amount of the pharmaceutical composition described in embodiment 10. 12. The method of embodiment 11, wherein the condition is cancer. 13. The method of embodiment 12, wherein the cancer is a CD70-associated cancer selected from the group consisting of renal cell carcinoma, glioblastoma, glioma, such as low-grade glioma, non-Hodgkin's lymphoma (NHL), Hodgkin's disease (HD), Waldenstrom's hypergammaglobulinemia, acute myeloid leukemia, multiple myeloma, diffuse large cell lymphoma, follicular lymphoma, or non-small cell lung cancer. 14. A method for inhibiting tumor growth or progression in a subject having malignant cells that express CD70, comprising administering to said subject in need thereof an effective amount of the pharmaceutical composition of embodiment 10. 15. A method for inhibiting metastasis of malignant cells expressing CD70 in a subject, comprising administering to said subject in need thereof an effective amount of the pharmaceutical composition of embodiment 10. 16. A method for inducing tumor regression in a subject having malignant cells that express CD70, comprising administering to said subject in need thereof an effective amount of the pharmaceutical composition of embodiment 10. 17. A method of producing an antibody, comprising culturing a host cell of embodiment 6 under conditions that result in the production of said antibody, and isolating said antibody from said host cell or culture.

[0252] How to manipulate immune cells Provided herein are methods for preparing immune cells for use in immunotherapy. In some embodiments, the methods include introducing a CAR according to the present disclosure into immune cells and expanding the cells. In some embodiments, the present disclosure relates to a method for engineering immune cells, the method including providing cells and expressing at least one of the above-described CARs on the cell surface. Methods for engineering immune cells are described, for example, in PCT Publications WO / 2014 / 039523, WO / 2014 / 184741, WO / 2014 / 191128, WO / 2014 / 184744, and WO / 2014 / 184143, which are incorporated by reference in their entireties. In some embodiments, the method includes transfecting cells with at least one polynucleotide encoding the above-described CAR and expressing the polynucleotide in the cells.

[0253] In some embodiments, the polynucleotide is present in a lentiviral vector for stable expression in cells.

[0254] In some embodiments, the method may further comprise genetically modifying the cells by disrupting or inactivating at least one gene expressing, for example, but not limited to, a TCR component, a target of an immunosuppressant, an HLA gene, CD70, and / or an immune checkpoint protein, such as PDCD1 or CTLA-4. By disrupting or inactivating a gene, it is intended that the gene of interest is not expressed in the form of a functional protein. In some embodiments, the gene that is disrupted or inactivated is selected from the group consisting of, for example, but not limited to, TCRα, TCRβ, CD52, GR, PD-1, CD70, and CTLA-4. In some embodiments, the method comprises disrupting or inactivating one or more genes by introducing into the cells a rare-cutting endonuclease capable of selectively inactivating genes by selective DNA cleavage. In some embodiments, the slow-cutting endonuclease may be, for example, a zinc finger nuclease (ZFN), a megaTAL nuclease, a meganuclease, a transcription activator-like effector nuclease (TALE-nuclease), or a CRISPR-associated endonuclease.

[0255] In some embodiments, an additional catalytic domain is used together with the low-frequency-cutting endonuclease to enhance the ability to inactivate the target gene. For example, the additional catalytic domain may be a DNA end-processing enzyme. Non-limiting examples of DNA end-processing enzymes include 5-3' exonucleases, 3-5' exonucleases, 5-3' alkaline exonucleases, 5' flap endonucleases, helicases, phosphatases, hydrolases, and template-independent DNA polymerases. Non-limiting examples of such catalytic domains include a protein domain selected from the group consisting of hExoI (EXO1_HUMAN), yeast ExoI (EXO1_YEAST), E. coli ExoI, human TREX2, mouse TREX1, human TREX1, bovine TREX1, rat TREX1, TdT (terminal deoxynucleotidyl transferase), human DNA2, and yeast DNA2 (DNA2_YEAST), or a catalytically active derivative of such a protein domain. In some embodiments, the additional catalytic domain may have 3'-5'-exonuclease activity, and in some embodiments, the additional catalytic domain is a TREX, such as the TREX2 catalytic domain (WO2012 / 058458). In some embodiments, the catalytic domain is encoded by a single-chain TREX polypeptide. The additional catalytic domain may be fused to a nuclease fusion protein or a chimeric protein. In some embodiments, the additional catalytic domain is fused, for example, using a peptide linker.

[0256] In some embodiments, the method further includes introducing into the cell an exogenous nucleic acid that has sequence homology to at least a portion of the target nucleic acid sequence, thereby allowing homologous recombination to occur between the target nucleic acid sequence and the exogenous nucleic acid. In some embodiments, the exogenous nucleic acid includes a first portion and a second portion, each homologous to the 5' and 3' regions of the target nucleic acid sequence. The exogenous nucleic acid may also include a third portion located between the first and second portions, which portion does not share homology with the 5' and 3' regions of the target nucleic acid sequence. After cleavage of the target nucleic acid sequence, a homologous recombination event is stimulated between the target nucleic acid sequence and the exogenous nucleic acid. In some embodiments, a homologous sequence of at least about 50 bp, a homologous sequence greater than about 100 bp, or a homologous sequence greater than about 200 bp may be used in the donor matrix. The exogenous nucleic acid may be, for example, but not limited to, about 200 bp to about 6000 bp, or about 1000 bp to about 2000 bp. The shared nucleic acid homology is located in the regions adjacent to the upstream and downstream of the cleavage site, and the nucleic acid sequence to be introduced is located between the two arms.

[0257] In some embodiments, the nucleic acid sequentially contains: a first region of homology to a sequence upstream of the cleavage site; a sequence that inactivates a target gene selected from the group consisting of TCRα, TCRβ, CD52, CD70, glucocorticoid receptor (GR), deoxycytidine kinase (DCK), and immune checkpoint proteins such as programmed death-1 (PD-1); and a second region of homology to a sequence downstream of the cleavage site. The polynucleotide introduction step may be performed simultaneously with, before, or after the introduction or expression of the rare-cutting endonuclease. Depending on the location of the target nucleic acid sequence where the cleavage event occurs, the exogenous nucleic acid can be used to knock out a gene, for example, when the exogenous nucleic acid is placed within the open reading frame of the gene, or to introduce a new sequence or gene of interest. Sequence insertion using such exogenous nucleic acids can be used to modify or replace a gene (such as, but not limited to, allele swapping), to alter an existing target gene, or to up-regulate or down-regulate the expression of a target gene (such as, but not limited to, promoter swapping), target gene correction, or target gene replacement. In some embodiments, inactivation of a gene selected from the group consisting of TCRα, TCRβ, CD52, CD70, GR, DCK, and immune checkpoint proteins is carried out at a precise genomic location targeted by a specific TALE-nuclease, where the specific TALE-nuclease catalyzes cleavage, and where the exogenous nucleic acid contains, consecutively, at least a homologous region and a sequence that inactivates one target gene selected from the group consisting of TCRα, TCRβ, CD52, CD70, GR, DCK, and immune checkpoint proteins that are integrated by homologous recombination. In some embodiments, several genes may be disrupted or inactivated consecutively or simultaneously using several TALE-nucleases that each specifically target one defined gene, and several specific polynucleotides for specific gene inactivation.

[0258] In some embodiments, the method includes inactivating one or more additional genes selected from the group consisting of TCR alpha, TCR beta, CD52, CD70, GR, DCK, and an immune checkpoint protein. In some embodiments, gene inactivation can be achieved by introducing into the cell at least one rare-cutting endonuclease, whereby the rare-cutting endonuclease specifically catalyzes cleavage at a target sequence in the cellular genome, and optionally introducing into the cell an exogenous nucleic acid containing, consecutively, a first region of homology to a sequence upstream of the cleavage, a sequence to be inserted into the cellular genome, and a second region of homology to a sequence downstream of the cleavage, wherein the introduced exogenous nucleic acid inactivates the gene and integrates at least one exogenous polynucleotide sequence encoding at least one recombinant protein of interest. In some embodiments, the exogenous polynucleotide sequence is integrated into a gene encoding a protein selected from the group consisting of TCR alpha, TCR beta, CD52, CD70, GR, DCK, and an immune checkpoint protein.

[0259] In another embodiment, the step of genetically modifying the cells can include modifying T cells by disrupting or inactivating at least one gene expressing an immunosuppressant target, and optionally expanding the cells in the presence of the immunosuppressant. Immunosuppressants are agents that suppress immune function through one of several mechanisms. Immunosuppressants can reduce the extent of immune responses and / or phagocytosis. Non-limiting examples of immunosuppressants include calcineurin inhibitors, rapamycin targets, interleukin-2α chain blockers, inhibitors of inosine monophosphate dehydrogenase, inhibitors of dihydrofolate reductase, corticosteroids, and immunosuppressive antimetabolites. Some cytotoxic immunosuppressants act by inhibiting DNA synthesis. Others may act through T cell activation or by inhibiting helper cell activation. The method of the present disclosure can render T cells immune-resistant to immunotherapy by disrupting or inactivating the immunosuppressant target in T cells. Non-limiting example targets of immunosuppressants can be receptors for immunosuppressants such as, but not limited to, CD52, glucocorticoid receptor (GR), members of the FKBP family of genes, and members of the cyclophilin family of genes.

[0260] In some embodiments, the genetic modification of the method comprises expressing a rare-cutting endonuclease in the provided engineered cells, such that the rare-cutting endonuclease specifically catalyzes cleavage in a target gene, destroying or inactivating the target gene. In some embodiments, the method of engineering cells comprises at least one of the following steps: providing T cells, e.g., from a cell culture or a blood sample; selecting a gene in the T cells that expresses a target of an immunosuppressant; introducing into the T cells a rare-cutting endonuclease that can selectively inactivate a gene encoding the target of the immunosuppressant by DNA cleavage (in some embodiments, a double-strand break); and optionally expanding the cells in the presence of the immunosuppressant.

[0261] In some embodiments, the method includes providing T cells, e.g., from a cell culture or from a blood sample; selecting a gene in the T cells that expresses a target of an immunosuppressant; transfecting the T cells with a nucleic acid encoding a rare-cutting endonuclease that can selectively inactivate a gene encoding the target of the immunosuppressant by DNA cleavage (in some embodiments, a double-strand break); expressing the rare-cutting endonuclease in the T cells; and optionally expanding the cells in the presence of the immunosuppressant.

[0262] In some embodiments, the rare-cutting endonuclease specifically targets CD52 or GR. In some embodiments, the gene selected for inactivation encodes CD52 and the immunosuppressive therapeutic agent comprises a humanized antibody targeting the CD52 antigen. In some embodiments, the gene selected for inactivation encodes GR and the immunosuppressive therapeutic agent comprises a corticosteroid, such as dexamethasone. In some embodiments, the gene selected for inactivation is a member of the FKBP family of genes or a variant thereof, and the immunosuppressive therapeutic agent comprises FK506, also known as tacrolimus or fujimycin. In some embodiments, the member of the FKBP family of genes is FKBP12 or a variant thereof. In some embodiments, the gene selected for inactivation is a member of the cyclophilin family of genes or a variant thereof, and the immunosuppressive therapeutic agent comprises cyclosporine.

[0263] In some embodiments, the infrequently cutting endonuclease may be, for example, a zinc finger nuclease (ZFN), a megaTAL nuclease, a meganuclease, a transcription activator-like effector nuclease (TALE-nuclease), or a CRISPR-associated endonuclease. In some embodiments, the infrequently cutting endonuclease is a TALE-nuclease. In some embodiments, the infrequently cutting endonuclease is a CRISPR nuclease, and the guide RNA is at least partially complementary or fully complementary to the target site.

[0264] Typically, a CRISPR-associated nuclease is provided with a guide RNA (gRNA) or its functional equivalent. The gRNA consists of two parts: a crispr-RNA (crRNA) specific to the target genomic DNA sequence and a transactivating RNA (tracrRNA) that promotes Cas binding to DNA. In some embodiments, the crRNA and tracrRNA may be present in the same RNA oligonucleotide, referred to as a single guide RNA (sgRNA). In some embodiments, the crRNA and tracrRNA may be present as separate RNA oligonucleotides. As used herein, the term "guide RNA" or "gRNA" refers to the combination of tracrRNA and crRNA present as an sgRNA or as a crRNA:tracrRNA duplex. In some embodiments, the CRISPR-associated nuclease is a Cas9 nuclease. In some embodiments, the Cas9 protein may be derived from Streptococcus pyogenes (SpCas9). In some embodiments, the Cas9 protein may be derived from other bacterial strains, including Staphylococcus aureus (SaCas9). In some embodiments, the Cas endonuclease is selected from the group consisting of SpCas9, SpCas9-HF1, SpCas9-HF2, SpCas9-HF3, SpCas9-HF4, SaCas9, FnCpf, FnCas9, eSpCas9, C2C1, C2C3, Cpf1, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csx12), Cas10, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, Csx10, Csx16, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, or Csf4.

[0265] Studies have shown that the undifferentiated state (i.e., T SCM or T CM Adoptive transfer of T cells derived from a subset of CD70 has been proposed to result in long-term persistence in vivo (see, e.g., Berger, C. et al., The Journal of Clinical Investigation, 118(1):294-305 (2008)). Therefore, genetic knockdown of CD70 in CAR T products is an important consideration for inhibiting T cell differentiation.

[0266] In some embodiments, the genetic modification of the method comprises expressing a rare-cutting endonuclease in the engineered provided cells, such that the rare-cutting endonuclease specifically catalyzes cleavage in the CD70 gene, disrupting or inactivating the CD70 gene. In some embodiments, the method of engineering cells comprises at least one of the following steps: providing T cells, e.g., from a cell culture or from a blood sample, introducing into the T cells a rare-cutting endonuclease that can selectively inactivate the gene encoding CD70 by DNA cleavage (in some embodiments, a double-strand break), and expanding the cells.

[0267] In some embodiments, the method includes providing T cells, e.g., from a cell culture or from a blood sample, transfecting the T cells with a nucleic acid encoding a rare-cutting endonuclease that can selectively inactivate the gene encoding CD70 by DNA cleavage (in some embodiments, a double-strand break), expressing the rare-cutting endonuclease in the T cells, and expanding the cells.

[0268] In some embodiments, the low-frequency cutting endonuclease may be, for example, meganuclease, zinc finger nuclease, or TALE-nuclease (TALEN). In some embodiments, the low-frequency cutting endonuclease is a TALE-nuclease. In some embodiments, the low-frequency cutting endonuclease is a CRISPR-associated nuclease, and the guide RNA is at least partially complementary or fully complementary to the target site.

[0269] Further provided herein are methods of engineering T cells suitable for immunotherapy, wherein the methods comprise genetically modifying T cells by disrupting or inactivating at least one immune checkpoint protein. In some embodiments, the immune checkpoint protein is, for example, PD-1 and / or CTLA-4. In some embodiments, the method of genetically modifying cells comprises modifying T cells by disrupting or inactivating at least one immune checkpoint protein and expanding the cells. Immune checkpoint proteins include, but are not limited to, Programmed Death 1 (PD-1, also known as PDCD1 or CD279, Accession Number: NM - 005018), Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4, also known as CD152, GenBank accession number AF414120.1), LAG3 (also known as CD223, accession number NM - 002286.5), Tim3 (also known as HAVCR2, GenBank accession number: JX049979.1), BTLA (also known as CD272, accession number: NM - 181780.3), BY55 (also known as CD160, GenBank accession number: CR541888.1), TIGIT (also known as VSTM3, accession number: NM -173799), B7H5 (also known as C10orf54, homolog of mouse vista gene, accession number: NM - 022153.1), LAIR1 (also known as CD305, GenBank accession number: CR542051.1), SIGLEC10 (GeneBank accession number: AY358337.1), 2B4 (also known as CD244, accession number: NM - 001166664.1), which directly inhibit immune cells. For example, CTLA-4 is a cell surface protein expressed on certain CD4 T cells and CD8 T cells, which inhibits T cell activation and effector function when it engages with its ligands (B7-1 and B7-2) on antigen-presenting cells.

[0270] In some embodiments, the method of engineering cells comprises at least one of the following steps: providing T cells, e.g., from a cell culture or from a blood sample, transfecting the T cells with a rare-cutting endonuclease that can selectively inactivate a gene encoding an immune checkpoint protein by DNA cleavage (in some embodiments, a double-strand break), and expanding the cells. In some embodiments, the method comprises providing T cells, e.g., from a cell culture or from a blood sample, transfecting the T cells with a nucleic acid encoding a rare-cutting endonuclease that can selectively inactivate a gene encoding an immune checkpoint protein by DNA cleavage (in some embodiments, a double-strand break), expressing the rare-cutting endonuclease in the T cells, and expanding the cells. In some embodiments, the infrequent-cutting endonuclease specifically targets a gene selected from the group consisting of PD-1, CTLA-4, LAG3, Tim3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10, 2B4, TCRα, and TCRβ. In some embodiments, the infrequent-cutting endonuclease may be a meganuclease, a zinc finger nuclease, or a TALE-nuclease. In some embodiments, the infrequent-cutting endonuclease is a TALE-nuclease. In some embodiments, the infrequent-cutting endonuclease is a Cas9 nuclease, and the guide RNA is at least partially complementary or fully complementary to the target site.

[0271] In some embodiments, the present disclosure may be particularly suitable for allogeneic immunotherapy. In such embodiments, cells may be modified by a method comprising: disrupting or inactivating at least one gene encoding a component of a T cell receptor (TCR) in a T cell; and expanding the T cell. In some embodiments, the genetic modification of the method relies on expressing a rare-cutting endonuclease in the engineered donor cell, whereby the rare-cutting endonuclease specifically catalyzes cleavage in a target gene, disrupting or inactivating the target gene. In some embodiments, the method of engineering cells comprises at least one of the following steps: providing T cells, for example, from a cell culture or a blood sample; introducing into the T cell a rare-cutting endonuclease capable of selectively inactivating at least one gene encoding a component of a T cell receptor (TCR) by DNA cleavage (in some embodiments, a double-strand break); and expanding the cell.

[0272] In some embodiments, the method includes providing T cells, e.g., from a cell culture or from a blood sample; transfecting the T cells with a nucleic acid encoding a rare-cutting endonuclease that can selectively inactivate at least one gene encoding a component of a T cell receptor (TCR) by DNA cleavage (in some embodiments, a double-strand break); expressing the rare-cutting endonuclease in the T cells; sorting transformed T cells that do not express a TCR on their cell surface; and expanding the cells.

[0273] In some embodiments, the infrequently cutting endonuclease may be a meganuclease, a zinc finger nuclease, or a TALE-nuclease. In some embodiments, the infrequently cutting endonuclease is a TALE-nuclease. In some embodiments, the TALE-nuclease recognizes and cleaves a sequence encoding TCRα or TCRβ. In some embodiments, the TALE-nuclease contains a polypeptide sequence selected from the amino acid sequences set forth in SEQ ID NOs: 281, 282, 283, 284, 285, 286, 287, 288, 289, or 290. TALE-nuclease polypeptide sequence: Repeat TRAC_T01-L LTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLC QAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLP VLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNGGGKQALETVQ RLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALE (SEQ ID NO: 281). Repeat TRAC_T01-R LTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALE (SEQ ID NO: 282). Repeat TRBC_T01-L LTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNGGGRPALE (SEQ ID NO: 283). Repeat TRBC_T01-R NPQRSTVWYLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQ RLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETV QRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALE (sequence number 284). Repeat TRBC_T02-L LTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLC QAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLP VLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQVVAIASNGGGRPALE (sequence number 285). Repeat TRBC_T02-R LTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLC QAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLP VLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGRPALE (sequence number 286). Repeat CD52_T02-L LTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNGGGRPALE (SEQ ID NO: 287). Repeat CD52_T02-R LTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLC QAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLP VLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPQVVAIASNIGGKQALETVQALLPVLCQAHGLTPPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALE (sequence number 288). Repeat CD70-L LTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLC QAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLP VLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALE (sequence number 289) Repeat CD70-R LTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTQQVVAIASNNGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLC QAHGLTPQQVVAIASNNGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLP VLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNNGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQ RLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALE (Sequence number 290)

[0274] In another aspect, another step of genetically modifying a cell can be a method of expanding TCRα-deficient T cells, the method comprising introducing pTα (also known as preTCRα) or a functional variant thereof into a T cell and, optionally, expanding the cell via stimulation of the CD3 complex. In some embodiments, the method comprises: a) transfecting a cell with a nucleic acid encoding at least a fragment of pTα to support surface expression of CD3, b) expressing the pTα in the cell, and c) optionally expanding the cell via stimulation of the CD3 complex.

[0275] Also provided are methods for preparing T cells for immunotherapy, comprising steps of the method for expanding the T cells. In some embodiments, the pTα polynucleotide sequence can be introduced randomly or by homologous recombination. In some embodiments, the insertion can be associated with inactivation of the TCRα gene.

[0276] Different functional variants of pTα may be used. A "functional variant" of a peptide refers to a molecule substantially similar to either the entire peptide or a fragment thereof. A "fragment" of pTα or a functional variant thereof refers to any molecular subset, i.e., a peptide shorter than full-length pTα. In some embodiments, pTα or a functional variant may be, for example, full-length pTα or a C-terminally truncated pTα. A C-terminally truncated pTα lacks one or more residues at the C-terminus. As a non-limiting example, a C-terminally truncated pTα lacks 18, 48, 62, 78, 92, 110, or 114 residues from the C-terminus of the protein. Amino acid sequence variants of the peptide may be prepared by mutations in the DNA encoding the peptide. Such functional variants include, for example, deletions, insertions, or substitutions of residues within the amino acid sequence. Any combination of deletions, insertions, and substitutions may be made to arrive at a final construct that retains the desired activity, particularly restoration of a functional CD3 complex. In some embodiments, at least one mutation affecting dimerization is introduced into the different pTα forms as described above. As a non-limiting example, the mutated residue may be at least W46R, D22A, K24A, R102A, or R117A at a position aligned using the CLUSTALW method to the human pTα protein or to pTα family or homologous members. In some embodiments, the pTα or variants described above contain the mutated residue W46R, or the mutated residues D22A, K24A, R102A, and R117A. In some embodiments, the pTα or variants are also fused to signaling domains, such as, but not limited to, CD28, OX40, ICOS, CD27, CD137 (4-1BB), and CD8. The extracellular domain of the pTα or variants described above may be fused to a fragment of the TCRα protein, particularly the transmembrane and intracellular domains of TCRα. The pTα variants may also be fused to the intracellular domain of TCRα.

[0277] In some embodiments, the pTα form may be fused to an extracellular ligand-binding domain, hi some embodiments, the pTα or a functional variant thereof is fused to a single-chain antibody fragment (scFv) containing the variable fragments of the light and heavy chains of a target antigen-specific monoclonal antibody joined by a flexible linker.

[0278] The term "TCRα-deficient T cells" refers to isolated T cells that lack expression of a functional TCRα chain. Such cells may be achieved, by way of non-limiting example, by engineering T cells so that they do not express any functional TCRα on their cell surface, by engineering T cells so that they produce little functional TCRα on their cell surface, or by engineering T cells to express a mutant or truncated version of the TCRα chain. TCRα-deficient cells can no longer be expanded through the CD3 complex. Therefore, to overcome this problem and expand TCRα-deficient cells, pTα or a functional variant thereof is introduced into the cells, restoring functional CD3 complexes. In some embodiments, the method comprises introducing into the T cells a rare-cutting endonuclease capable of selectively inactivating a gene encoding a component of the T cell receptor (TCR) by DNA cleavage. In some embodiments, the rare-cutting endonuclease is a TALE-nuclease.

[0279] In another embodiment, engineered T cells obtained by the methods described herein may be contacted with a bispecific antibody. For example, T cells may be contacted with the bispecific antibody ex vivo before administration to a patient, or in vivo after administration to a patient. Bispecific antibodies contain two variable regions with different antigenic specificities, thereby facilitating the proximity of the engineered cells to the target antigen. As a non-limiting example, bispecific antibodies may be directed against a tumor marker and a lymphocyte antigen, such as, but not limited to, CD3, and may have the ability to redirect and activate any circulating T cells to tumors.

[0280] In some embodiments, a polynucleotide encoding a polypeptide according to the present disclosure may be mRNA that is directly introduced into cells, for example, by electroporation. In some embodiments, live cells may be transiently permeabilized using the cytoPulse method to deliver substances into the cells. Parameters may be modified to determine conditions that result in high transfection efficiency with minimal failure rates.

[0281] Also provided herein are methods for transfecting T cells. In some embodiments, the methods include contacting a T cell with RNA and applying to the T cell an agile pulse sequence consisting of: (a) an electric pulse in a voltage range of about 2250-3000 V per centimeter, (b) a pulse width of 0.1 milliseconds, (c) a pulse interval of about 0.2-10 milliseconds between the electric pulses of steps (a) and (b), (d) an electric pulse in a voltage range of about 2250-3000 V, a pulse width of about 100 milliseconds, and a pulse interval of about 100 milliseconds between the electric pulse of step (b) and the first electric pulse of step (c), and (e) four electric pulses of about 0.2 millisecond pulse width and a voltage of about 325 V, with a pulse interval of 2 milliseconds between each of the four electric pulses. In some embodiments, a method of transfecting a T cell comprises contacting the T cell with RNA and subjecting the T cell to an agile pulse sequence consisting of: (a) an electrical pulse of about 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2400, 2450, 2500, 2600, 2700, 2800, 2900, or 3000 V per centimeter; (b) a pulse width of 0.1 milliseconds; and (c) about 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, or a 10-millisecond pulse interval; (d) a voltage range from about 2250 V to 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2400, 2450, 2500, 2600, 2700, 2800, 2900, or 3000 V, with one electric pulse having a 100-millisecond pulse width and a 100-millisecond pulse interval between the electric pulse in step (b) and the first electric pulse in step (c); and (e) four electric pulses having a pulse width of about 0.2 milliseconds and a voltage of about 325 V with a pulse interval of about 2 milliseconds between each of the four electric pulses. All values ​​within the above ranges are disclosed herein. The electroporation medium may be any suitable medium known in the art, such as BTXpress Cytoporation® Media T4 available from BTX Corporation. In some embodiments, the electroporation medium has a conductivity ranging from about 0.01 to about 1.0 millisiemens.

[0282] In some embodiments, by way of non-limiting example, the RNA encodes one monomer of a rare-cutting endonuclease, such as a half-TALE-nuclease, a CAR, at least one component of a multi-chain chimeric antigen receptor, pTα or a functional variant thereof, an exogenous nucleic acid, and / or an additional catalytic domain.

[0283] Engineered immune cells The present disclosure also provides engineered immune cells comprising any of the CAR polynucleotides described herein.In some embodiments, the CAR can be introduced into immune cells as a transgene via a plasmid vector.In some embodiments, the plasmid vector can also contain a selection marker, for example, to identify and / or select cells that have received the vector.

[0284] The CAR polypeptide may be synthesized in situ in a cell after introduction of a polynucleotide encoding the CAR polypeptide into the cell. Alternatively, the CAR polypeptide may be produced outside the cell and then introduced into the cell. Methods for introducing a polynucleotide construct into a cell are known in the art. In some embodiments, a stable transformation method may be used to integrate the polynucleotide construct into the cell genome. In other embodiments, a transient transformation method may be used to express the polynucleotide construct, and the polynucleotide construct is not integrated into the cell genome. In other embodiments, a viral-mediated method may be used. The polynucleotide may be introduced into the cell by any suitable means, such as a recombinant viral vector (e.g., retrovirus, adenovirus), liposome, etc. Transient transformation methods include, but are not limited to, microinjection, electroporation, or particle bombardment. The polynucleotide may be contained in a vector, such as a plasmid vector or a viral vector.

[0285] Also provided herein are isolated cells and cell lines obtained by the cell manipulation methods provided herein. In some embodiments, the isolated cells contain at least one of the above-mentioned CARs. In some embodiments, the isolated cells contain a group of CARs, each CAR containing a different extracellular ligand-binding domain.

[0286] Also provided herein are isolated immune cells obtained according to any one of the above-described methods. Any immune cells capable of expressing heterologous DNA can be used for the purpose of expressing a target CAR. In some embodiments, the immune cells are T cells. In some embodiments, the immune cells can be derived from stem cells, for example, but not limited to, adult stem cells, non-human embryonic stem cells, more specifically non-human stem cells, umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells, or hematopoietic stem cells. A representative human cell is a CD34+ cell. The isolated cells can also be dendritic cells, killer dendritic cells, mast cells, NK cells, B cells, or T cells selected from the group consisting of inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes, or helper T lymphocytes. In some embodiments, the cells can be derived from the group consisting of CD4+ T lymphocytes and CD8+ T lymphocytes.

[0287] In some embodiments, engineered immune cells expressing a CD70-specific CAR of the present disclosure on their cell surface membrane constitute greater than 10%, 20%, 30%, 40%, 50%, or 60% of stem cell memory cells and central memory cells. In some embodiments, engineered immune cells expressing a CD70-specific CAR of the present disclosure on their cell surface membrane constitute about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 15% to about 50%, about 15% to about 40%, about 20% to about 60%, or about 20% to about 70% of stem cell memory cells and central memory cells.

[0288] In some embodiments, the immune cells are inflammatory T lymphocytes that express any one of the CARs described herein. In some embodiments, the immune cells are cytotoxic T lymphocytes that express any one of the CARs described herein. In some embodiments, the immune cells are regulatory T lymphocytes that express any one of the CARs described herein. In some embodiments, the immune cells are helper T lymphocytes that express any one of the CARs described herein.

[0289] Prior to expansion and genetic modification, cell sources can be obtained from subjects through various non-limiting methods. Cells can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, any number of T cell lines are available and known to those skilled in the art and can be used. In some embodiments, cells can be derived from healthy donors, patients diagnosed with cancer, or patients diagnosed with infectious diseases. In some embodiments, cells can be part of a mixed cell population that exhibits different phenotypes.

[0290] Also provided herein are cell lines obtained from T cells transformed according to any one of the above methods. Also provided herein are modified cells that are resistant to immunosuppressive treatment. In some embodiments, the isolated cells according to the present disclosure contain a polynucleotide encoding a CAR.

[0291] The immune cells of the present disclosure can be activated and expanded either before or after genetic modification of the T cells using methods outlined in, for example, but not limited to, U.S. Patent Nos. 6,352,694, 6,534,055, 6,905,680, 6,692,964, 5,858,358, 6,887,466, 6,905,681, 7,144,575, 7,067,318, 7,172,869, 7,232,566, 7,175,843, 5,883,223, 6,905,874, 6,797,514, 6,867,041, and U.S. Patent Application Publication No. 20060121005. T cells can be expanded in vitro or in vivo. Generally, the T cells of the present disclosure can be expanded by contacting them with an agent that stimulates the CD3 TCR complex and costimulatory molecules on the T cell surface, thereby generating an activation signal for the T cell. For example, chemicals such as calcium ionophore A23187, phorbol 12-myristate 13-acetate (PMA), or mitogenic lectins such as phytohemagglutinin (PHA) can be used to generate an activation signal for the T cell.

[0292] In some embodiments, a population of T cells may be stimulated in vitro, for example, by contacting them with an anti-CD3 antibody or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contacting them with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore. For costimulation of an accessory molecule on the surface of T cells, a ligand that binds to the accessory molecule is used. For example, a population of T cells may be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions suitable for stimulating T cell proliferation. The anti-CD3 antibody and the anti-CD28 antibody may be disposed on beads or a plate or other substrate. Suitable conditions for T cell culture include an appropriate medium (e.g., Minimal Essential Media or RPMI Media 1640 or X-vivo 5 (Lonza)) that may contain factors necessary for growth and activity, including serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-2, IL-15, TGFp, and TNF, or any other additives for cell growth known to those of skill in the art. Other additives for cell growth include, but are not limited to, detergents, plasmanate, and reducing agents, such as N-acetyl-cysteine ​​and 2-mercaptoethanol. Culture media include RPMI 1640, A1M-V, DMEM, MEM, α-MEM, F-12, X-Vivo 1, X-Vivo 20, and Optimizer, supplemented with amino acids, sodium pyruvate, and vitamins, and either serum-free or supplemented with an appropriate amount of serum (or plasma) or a combination of hormones and / or cytokines (e.g., IL-7 and / or IL-15) sufficient for T cell proliferation and expansion. Antibiotics, such as penicillin and streptomycin, are included only in experimental cultures and not in cell cultures intended for infusion into subjects. Target cells are maintained under conditions necessary to support proliferation, such as an appropriate temperature (e.g., 37°C) and atmosphere (e.g., air plus 5% CO2). T cells exposed to various stimulation times may exhibit different characteristics.

[0293] In some embodiments, the cells of the present disclosure can be expanded by co-culture with tissue or cells. The cells can also be expanded in vivo, for example, in the blood of a subject after administration of the cells to the subject.

[0294] In some embodiments, the isolated cells according to the present disclosure comprise one disrupted or inactivated gene selected from the group consisting of CD52, CD70, GR, PD-1, CTLA-4, LAG3, Tim3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10, 2B4, HLA, TCRα and TCRβ, and / or express a CAR, multi-chain CAR, and / or pTα transgene. In some embodiments, the isolated cells comprise a polynucleotide encoding a polypeptide comprising a multi-chain CAR. In some embodiments, the isolated cells according to the present disclosure comprise two disrupted or inactivated genes selected from the group consisting of: CD52 and GR, CD52 and TCRα, CDR52 and TCRβ, CD70 and CD52, CD70 and TCRα, CD70 and TCRβ, GR and TCRα, GR and TCRβ, TCRα and TCRβ, PD-1 and TCRα, PD-1 and TCRβ, CTLA-4 and TCRα, CTLA-4 and TCRβ, LAG3 and TCRα, LAG3 and TCRβ, Expressing Tim3 and TCRα, Tim3 and TCRβ, BTLA and TCRα, BTLA and TCRβ, BY55 and TCRα, BY55 and TCRβ, TIGIT and TCRα, TIGIT and TCRβ, B7H5 and TCRα, B7H5 and TCRβ, LAIR1 and TCRα, LAIR1 and TCRβ, SIGLEC10 and TCRα, SIGLEC10 and TCRβ, 2B4 and TCRα, 2B4 and TCRβ, and / or CAR, multi-chain CAR, and pTα transgenes.

[0295] In some embodiments, the isolated cells according to the present disclosure comprise three disrupted or inactivated genes selected from CD52, CD70 and TCRα, or CD52, CD70 and TCRβ, and / or express a CAR, a multi-chain CAR and a pTα transgene.

[0296] In some embodiments, the TCR is not functional in cells according to the present disclosure due to disruption or inactivation of the TCR alpha and / or TCR beta genes. In some embodiments, methods are provided for obtaining modified cells derived from an individual, wherein the cells are capable of proliferation independent of the major histocompatibility complex (MHC) signaling pathway. Modified cells capable of proliferation independent of the MHC signaling pathway are amenable to obtaining by the present methods and are encompassed within the scope of the present disclosure. The modified cells disclosed herein can be used to treat host-versus-graft (HvG) rejection and graft-versus-host disease (GvHD) in a patient in need thereof. Accordingly, within the scope of the present disclosure, a method for treating a patient in need thereof for host-versus-graft (HvG) rejection and graft-versus-host disease (GvHD) comprises administering to the patient an effective amount of modified cells comprising disrupted or inactivated TCR alpha and / or TCR beta genes, thereby treating the patient.

[0297] In some embodiments, immune cells are engineered to be resistant to one or more chemotherapeutic agents. The chemotherapeutic agents may be, for example, purine nucleotide analogs (PNAs), making the immune cells suitable for cancer treatment combining adoptive immunotherapy and chemotherapy. Exemplary PNAs include, for example, clofarabine, fludarabine, cyclophosphamide, and cytarabine, used alone or in combination. PNAs are metabolized by deoxycytidine kinase (dCK) to monophosphate PNA, diphosphate PNA, and triphosphate PNA. These triphosphate forms compete with ATP for DNA synthesis and act as proapoptotic agents. They are potent inhibitors of ribonucleotide reductase (RNR), which is involved in trinucleotide production. Provided herein are CD70-specific CAR-T cells containing a disrupted or inactivated dCK gene. In some embodiments, dCK knockout cells are generated by transfecting T cells with a polynucleotide encoding a specific TAL-nuclease directed against the dCK gene, e.g., by electroporation of mRNA. dCK knockout CD70-specific CAR-T cells can be resistant to PNAs, including, e.g., chlorofarabine and / or fludarabine, while maintaining the cytotoxic activity of T cells against CD70-expressing cells.

[0298] In some embodiments, the isolated cells or cell lines of the present disclosure may contain pTα or a functional variant thereof. In some embodiments, the isolated cells or cell lines may be further genetically modified by disrupting or inactivating the TCRα gene.

[0299] Monoclonal antibody specific epitope In some embodiments, the extracellular domain of any one of the CD70-specific CARs disclosed herein may contain one or more epitopes specific for (i.e., specifically recognized by) a monoclonal antibody. These epitopes are also referred to herein as mAb-specific epitopes. Examples of mAb-specific epitopes are disclosed in International Patent Application Publication WO2016 / 120126, which is incorporated herein in its entirety. In these embodiments, the extracellular domain contains a VH polypeptide and a VL polypeptide that specifically bind to CD70 and one or more epitopes that bind to one or more monoclonal antibodies (mAbs). CARs containing mAb-specific epitopes can be single-chain or multi-chain.

[0300] The inclusion of a monoclonal antibody-specific epitope in the extracellular domain of the CAR described herein allows for the sorting and depletion of engineered immune cells expressing the CAR. In some embodiments, this property also facilitates the recovery of endogenous CD70-expressing cells that are depleted by administration of engineered immune cells expressing the CAR.

[0301] Thus, in some embodiments, the present disclosure relates to methods for sorting and / or depleting engineered immune cells comprising a CAR comprising a mAb-specific epitope, and for promoting the recovery of endogenous CD70-expressing cells, such as, for example, myeloid progenitor cells.

[0302] In particular, some epitope-monoclonal antibody combinations, such as, for example, CD20 epitope / rituximab, which are already approved for medical use, can be used to generate CARs containing monoclonal antibody-specific epitopes.

[0303] The disclosure further encompasses methods for sorting engineered immune cells containing CD70-specific CARs that express mAb-specific epitopes, and therapeutic methods in which the activation of engineered immune cells containing these CARs is modulated by depleting the cells using antibodies that target the external ligand-binding domain of the CAR. [Table A]

[0304] In some embodiments, the CAR-T cells contain a polynucleotide encoding a suicide polypeptide, such as RQR8. See, for example, WO2013153391A, which is incorporated herein by reference in its entirety. In the CAR-T cells containing the polynucleotide, the suicide polypeptide is expressed on the surface of the CAR-T cells. In some embodiments, the suicide polypeptide contains the amino acid sequence set forth in SEQ ID NO:291. CPYSNPSLCSGGGGSELPTQGTFSNVSTNVSPAKPTTTACPYSNPSLCSGGGGSP APRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLS LVITLYCNHRNRRRVCKCPRPVV (SEQ ID NO: 291).

[0305] The suicide polypeptide may further contain a signal peptide at the amino terminus, such as, for example, MGTSLLCWMALCLLGADHADA (SEQ ID NO: 611). In some embodiments, the suicide polypeptide contains the amino acid sequence set forth in SEQ ID NO: 292, including the signal sequence of SEQ ID NO: 611. MGTSLLCWMALCLLGADHADACPYSNPSLCSGGGGSELPTQGTFSNVSTNVSPAKPTTTACPYSNPSLCSGGGGSPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVV (SEQ ID NO: 292).

[0306] In some embodiments, the suicide polypeptide contains the amino acid sequence set forth in SEQ ID NO:611.

[0307] When a suicide polypeptide is expressed on the surface of a CAR-T cell, binding of rituximab to the R epitope of the polypeptide results in cell lysis. Multiple molecules of rituximab may bind to each polypeptide expressed on the cell surface. Each R epitope of the polypeptide may bind to a separate rituximab molecule. For example, administration of rituximab to a patient may result in in vivo depletion of CD70-specific CAR-T cells. The decision to deplete the transferred cells may be made when undesirable effects caused by the transferred cells are detected in the patient, such as unacceptable levels of toxicity.

[0308] In some embodiments, upon administration to a patient, engineered immune cells expressing any one of the CD70-specific CARs described herein on their cell surface can kill or lyse the patient's endogenous CD70-expressing cells. In one embodiment, the rate of depletion or lysis of endogenous CD70-expressing cells or cells of a CD70-expressing cell line by engineered immune cells expressing any one of the CD70-specific CARs described herein is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more. In one embodiment, the rate of depletion or lysis of endogenous CD70-expressing cells or cells of a cell line expressing CD70 by engineered immune cells expressing any one of the CD70-specific CARs described herein is about 5% to about 95%, about 10% to about 95%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 25% to about 75%, or about 25% to about 60%. In one embodiment, the endogenous CD70-expressing cells are endogenous CD70-expressing bone marrow cells.

[0309] In one embodiment, the rate of depletion or lysis of target cells, e.g., cell lines expressing CD70, by engineered immune cells expressing a CD70-specific CAR of the present disclosure on their cell surface membrane can be measured using the assays disclosed herein.

[0310] Methods for sorting CAR-positive immune cells In one embodiment, a method for in vitro sorting of an immune cell population is provided, wherein a subset of the immune cell population contains engineered immune cells that express any one of the CD70-specific CARs that comprise an epitope specific for a monoclonal antibody described herein. The method includes contacting the immune cell population with a monoclonal antibody specific for the epitope, and selecting immune cells that bind to the monoclonal antibody to obtain a cell population enriched for engineered immune cells that express the CD70-specific CAR.

[0311] In some embodiments, the monoclonal antibody specific for the epitope is optionally conjugated to a fluorophore. In this embodiment, selecting cells that bind to the monoclonal antibody can be performed by fluorescence-activated cell sorting (FACS). In some embodiments, the monoclonal antibody specific for the epitope is optionally conjugated to a magnetic particle. In this embodiment, selecting cells that bind to the monoclonal antibody can be performed by magnetic-activated cell sorting (MACS).

[0312] In some embodiments, the extracellular binding domain of the CAR contains one or more mAb-specific epitopes of SEQ ID NO: 294 or 601-610. In some embodiments, the extracellular binding domain of the CAR contains the mAb-specific epitope of SEQ ID NO: 609. In some embodiments, the extracellular binding domain of the CAR contains the mAb-specific epitope of SEQ ID NO: 295. In some embodiments, the extracellular binding domain of the CAR contains the mAb-specific epitope of SEQ ID NO: 609 and the antibody used to contact the immune cell population is QBEND-10. In some embodiments, the extracellular binding domain of the CAR contains the mAb-specific epitope of SEQ ID NO: 295 and the antibody used to contact the immune cell population is QBEND-10.

[0313] In some embodiments, the extracellular binding domain of the CAR contains the mAb-specific epitope of SEQ ID NO: 294. In some embodiments, the extracellular binding domain of the CAR contains the mAb-specific epitope of SEQ ID NO: 294, and the antibody used to contact the immune cell population is rituximab.

[0314] In some embodiments, the population of CAR-expressing immune cells obtained when using the above-described in vitro sorting methods for immune cells contains at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% CAR-expressing immune cells. In some embodiments, the population of CD70 CAR-expressing immune cells obtained when using the above-described in vitro sorting methods for CAR-expressing immune cells contains at least 85% CAR-expressing immune cells.

[0315] According to the present disclosure, cells administered to a recipient may be enriched in vitro from a source population. Methods for expanding a source population are known in the art and may include selecting cells expressing an antigen, such as the CD34 antigen, using a combination of density centrifugation, immuno-magnetic bead purification, affinity chromatography, and fluorescence-activated cell sorting known to those skilled in the art.

[0316] Flow cytometry is widely used in the field and is known to those skilled in the art, and is used to sort and quantify specific cell types in a cell population.Generally, flow cytometry is a method that mainly uses optical means to quantify the components or structural characteristics of cells.By quantifying structural characteristics, different cell types can be distinguished, so that flow cytometry and cell sorting methods can be used to count and sort cells with different phenotypes in a mixture.

[0317] Flow cytometry analysis involves two basic steps: 1) labeling selected cell types with one or more labeling markers, and 2) determining the number of labeled cells relative to the total number of cells in the population.

[0318] The primary method for labeling cell types is to bind labeled antibodies to markers expressed by specific cell types. The antibodies are either directly labeled with fluorescent compounds or indirectly labeled, for example, using a fluorescently labeled secondary antibody that recognizes the primary antibody.

[0319] In some embodiments, the method used to sort CAR-expressing immune cells is magnetic-activated cell sorting (MACS). Magnetic-activated cell sorting (MACS) uses superparamagnetic nanoparticles and columns to separate various cell populations according to their surface antigens (CD molecules). A few simple steps result in a pure cell population. Cells in a single-cell suspension are magnetically labeled with microbeads. The sample is passed through a column composed of ferromagnetic spheres. The column is coated with a cytophilic coating, allowing for rapid and gentle cell separation. Unlabeled cells pass through, while magnetically labeled cells remain within the column. The effluent can be collected as the unlabeled cell fraction. After a short wash step, the column is removed from the separator, and the magnetically labeled cells are eluted.

[0320] In some embodiments, the mAb used in the method of sorting immune cells expressing a CAR is selected from the group consisting of alemtuzumab, ibritumomab tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, bevacizumab, certolizumab pegol, and ribosomal. pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab, palivizumab, ranibizumab, tocilizumab, trastuzumab, vedolizumab The mAb is selected from vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab, ofatumumab, panitumumab, QBEND-10, and / or ustekinumab. In some embodiments, the mAb is rituximab. In other embodiments, the mAb is QBEND-10.

[0321] In some embodiments, the CAR-T cells contain a selected epitope within the scFv, providing specificity for recognition by a particular antibody. See, e.g., WO2016 / 120216, which is incorporated herein by reference in its entirety. Such an epitope facilitates sorting of CAR-T cells and / or facilitates depletion of CAR-T cells. The epitope can be selected from any number of epitopes known in the art. In some embodiments, the epitope may be the target of a monoclonal antibody approved for medical use, such as, but not limited to, the CD20 epitope recognized by rituximab. In some embodiments, the epitope contains the amino acid sequence of CPYSNPSLC (SEQ ID NO: 293).

[0322] In some embodiments, the epitope is placed within the CAR. For example, but not limited to, the epitope can be placed between the scFv and the hinge of the CAR. In some embodiments, two identical epitopes separated by a linker can be used in the CAR. For example, a polypeptide containing the amino acid sequence set forth in SEQ ID NO: 294, SEQ ID NO: 609, or SEQ ID NO: 295 can be used in the CAR and placed between the light chain variable region and the hinge. GSGGGGSCPYSNPSLCSGGGGSCPYSNPSLCSGGGGS (SEQ ID NO: 294) ELPTQGTFSNVSTNVS (SEQ ID NO: 609) ELPTQGTFSNVSTNVSPAKPTTTA (SEQ ID NO: 295)

[0323] In some embodiments, the extracellular binding domain of the CAR contains the following sequence: V1-L1-V2-(L) x -Epitope 1-(L) x -; V1-L1-V2-(L) x -Epitope 1-(L) x -Epitope 2-(L) x -; V1-L1-V2-(L)x -Epitope 1-(L) x -Epitope 2-(L) x -Epitope 3-(L) x -; (L) x -Epitope 1-(L) x -V1-L1-V2; (L) x -Epitope 1-(L) x -Epitope 2-(L) x -V1-L1-V2; Epitope 1-(L) x -Epitope 2-(L) x -Epitope 3-(L) x -V1-L1-V2; (L) x -Epitope 1-(L) x -V1-L1-V2-(L) x -Epitope 2-(L) x ; (L) x -Epitope 1-(L) x -V1-L1-V2-(L) x -Epitope 2-(L) x -Epitope 3-(L) x -; (L) x -Epitope 1-(L) x -V1-L1-V2-(L) x -Epitope 2-(L) x -Epitope 3-(L) x -Epitope 4-(L) x -; (L) x -Epitope 1-(L) x -Epitope 2-(L) x -V1-L1-V2-(L) x -Epitope 3-(L) x -; (L) x -Epitope 1-(L) x -Epitope 2-(L) x -V1-L1-V2-(L) x -Epitope 3-(L) x -Epitope 4-(L) x -; V1-(L) x -Epitope 1-(L) x -V2; V1-(L) x -Epitope 1-(L) x -V2-(L) x -Epitope 2-(L) x ; V1-(L) x -Epitope 1-(L) x -V2-(L) x -Epitope 2-(L) x -Epitope 3-(L) x ; V1-(L) x -Epitope 1-(L) x -V2-(L) x -Epitope 2-(L) x -Epitope 3-(L) x -Epitope 4-(L) x ; (L) x -Epitope 1-(L) x -V1-(L) x -Epitope 2-(L) x -V2; or (L) x -Epitope 1-(L) x -V1-(L) x -Epitope 2-(L) x -V2-(L) x -Epitope 3-(L) x ; During the ceremony, V1 is V L and V2 is V H or V1 is V H and V2 is V L is; L1 is V H Chain V L a linker suitable for attachment to a chain; L is a linker comprising glycine and serine residues, each occurrence of L in the extracellular binding domain may be the same or different from other occurrences of L in the same extracellular binding domain, and in embodiments contains or is SGGGG (SEQ ID NO: 614), GGGGS (SEQ ID NO: 615), or SGGGGS (SEQ ID NO: 616); and x is 0 or 1 or 2, each occurrence of x being selected independently of the others; and Epitope 1, epitope 2, epitope 3 and epitope 4 are mAb-specific epitopes, which may be the same or different, and V H is the heavy chain variable fragment, V L is a light chain variable fragment. In some embodiments, epitope 1, epitope 2, and epitope 4 are mAb-specific epitopes having the amino acid sequence of SEQ ID NO: 293, and epitope 3 is a mAb-specific epitope having the amino acid sequence of SEQ ID NO: 295.

[0324] In some embodiments, the extracellular binding domain of the CAR contains the following sequence: V1-L1-V2-(L) x -Epitope 1-(L) x -; V1-L1-V2-(L) x -Epitope 1-(L) x -Epitope 2-(L) x -; V1-L1-V2-(L) x -Epitope 1-(L) x -Epitope 2-(L) x -Epitope 3-(L) x -; (L) x -Epitope 1-(L) x -V1-L1-V2; (L) x -Epitope 1-(L) x -Epitope 2-(L) x -V1-L1-V2; Epitope 1-(L) x -Epitope 2-(L) x-Epitope 3-(L) x -V1-L1-V2; (L) x -Epitope 1-(L) x -V1-L1-V2-(L) x -Epitope 2-(L) x ; (L) x -Epitope 1-(L) x -V1-L1-V2-(L) x -Epitope 2-(L) x -Epitope 3-(L) x -; (L) x -Epitope 1-(L) x -V1-L1-V2-(L) x -Epitope 2-(L) x -Epitope 3-(L) x -Epitope 4-(L) x -; (L) x -Epitope 1-(L) x -Epitope 2-(L) x -V1-L1-V2-(L) x -Epitope 3-(L) x -; (L) x -Epitope 1-(L) x -Epitope 2-(L) x -V1-L1-V2-(L) x -Epitope 3-(L) x -Epitope 4-(L) x -; V1-(L) x -Epitope 1-(L) x -V2; V1-(L) x -Epitope 1-(L) x -V2-(L) x -Epitope 2-(L) x ; V1-(L) x -Epitope 1-(L) x -V2-(L) x -Epitope 2-(L) x -Epitope 3-(L) x ; V1-(L) x -Epitope 1-(L) x -V2-(L) x -Epitope 2-(L) x -Epitope 3-(L) x -Epitope 4-(L) x ; (L) x -Epitope 1-(L) x -V1-(L) x -Epitope 2-(L) x -V2; or (L) x -Epitope 1-(L) x -V1-(L) x -Epitope 2-(L) x -V2-(L) x -Epitope 3-(L) x ; During the ceremony, V1 is V L and V2 is V H or V1 is V H and V2 is V L is; L1 is V H Chain V L a linker suitable for attachment to a chain; L is a linker comprising glycine and serine residues, each occurrence of L in the extracellular binding domain may be the same or different from other occurrences of L in the same extracellular binding domain, and in embodiments contains or is SGGGG (SEQ ID NO: 614), GGGGS (SEQ ID NO: 615), or SGGGGS (SEQ ID NO: 616); and x is 0 or 1 or 2, each occurrence of x being selected independently of the others; and Epitope 1, epitope 2, epitope 3 and epitope 4 are mAb-specific epitopes, which may be the same or different, and V H is the heavy chain variable fragment, V Lis a light chain variable fragment. In some embodiments, epitope 1, epitope 2, and epitope 4 are mAb-specific epitopes having the amino acid sequence of SEQ ID NO: 293, and epitope 3 is a mAb-specific epitope having the amino acid sequence of SEQ ID NO: 609.

[0325] In some embodiments, the extracellular binding domain of the CAR contains the following sequence: V1-L1-V2-(L) x -Epitope 1-(L) x -Epitope 2-(L) x -;or (L) x -Epitope 1-(L) x -V1-L1-V2-(L) x -Epitope 2-(L) x -Epitope 3-(L) x -Epitope 4-(L) x wherein V1, V2, L1, L, x and epitope 1, epitope 2, epitope 3 and epitope 4 are as defined above.

[0326] In some embodiments, the extracellular binding domain of the CAR contains the following sequence: (L) x -Epitope 1-(L) x -V1-L1-V2-(L) x -Epitope 2-(L) x -Epitope 3-(L) x -Epitope 4-(L) x wherein V1, V2, L1, L, x are as defined above, and wherein (L) x -Epitope 1-(L) x is GGGGSCPYSNPSLCSGGGGSGGGGS (SEQ ID NO: 617), (L) x -Epitope 2-(L) x -Epitope 3-(L) x- Epitope 4 is GSGGGGSCPYSNPSLCSGGGGSELPTQGTFSNVSTNVSPAKPTTTACPYSNPSLC (SEQ ID NO: 618), and V1, V2, L1, L, x are as defined above.

[0327] In some embodiments, epitope-specific antibodies may be conjugated to cytotoxic agents. Furthermore, engineered antibodies grafted with components of the complement system may be used to promote CDC cytotoxicity. In some embodiments, CAR-T cell activation can be regulated by depleting the cells using antibodies that recognize the epitope.

[0328] Therapeutic applications The isolated cells obtained by the above-mentioned method or cell lines derived from such isolated cells can be used as pharmaceuticals. In some embodiments, such pharmaceuticals can be used to treat cancer. In some embodiments, the cancer is renal cell carcinoma, glioblastoma, glioma such as low-grade glioma, non-Hodgkin's lymphoma (NHL), Hodgkin's disease (HD), Waldenstrom's hypergammaglobulinemia, acute myeloid leukemia, multiple myeloma, diffuse large cell lymphoma, follicular lymphoma, or non-small cell lung cancer.

[0329] In some embodiments, the cancer is a cancer of hematopoietic origin, such as, for example, lymphoma or leukemia. In some embodiments, the cancer is multiple myeloma, malignant plasma cell neoplasm, Hodgkin's lymphoma, nodular lymphocyte-predominant Hodgkin's lymphoma, Kahler's disease and myelomatosis, plasma cell leukemia, plasmacytoma, B-cell prolymphocytic leukemia, hairy cell leukemia, B-cell non-Hodgkin's lymphoma (NHL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), chronic myeloid leukemia (CML), follicular lymphoma, Burkina Fasciitis, leukemia ... B-cell lymphoma, marginal zone lymphoma, mantle cell lymphoma, large cell lymphoma, precursor B-lymphoblastic lymphoma, myeloid leukemia, Waldenstrom's hypergammaglobulinemia, diffuse large B-cell lymphoma, follicular lymphoma, marginal zone lymphoma, mucosa-associated lymphoid tissue lymphoma, small cell lymphocytic lymphoma, mantle cell lymphoma, Burkitt's lymphoma, primary mediastinal (thymic) large B-cell lymphoma, lymphoplasmacytic lymphoma, Waldenstrom's Trehm's hypergammaglobulinemia, nodal marginal zone B-cell lymphoma, splenic marginal zone lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, T-cell / histiocytocyte-rich large B-cell lymphoma, primary central nervous system lymphoma, primary cutaneous diffuse large B-cell lymphoma (lower limb type), EBV-positive diffuse large B-cell lymphoma of the elderly, inflammation-associated diffuse large B-cell lymphoma, intravascular large B-cell lymphoma, The cancer is selected from the group consisting of ALK-positive large B-cell lymphoma, plasmablastic lymphoma, large B-cell lymphoma occurring in HHV8-associated multicentric Castleman disease, unclassified B-cell lymphoma with properties intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma, unclassified B-cell lymphoma with properties intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma, and other hematopoietic cell-associated cancers, such as ALL or AML.

[0330] In some embodiments, the isolated cells according to the present disclosure, or cell lines derived from the isolated cells, can be used in the manufacture of a medicament for the treatment of cancer in a patient in need thereof.

[0331] Also provided herein are methods of treating a patient. In some embodiments, the methods comprise providing immune cells of the present disclosure to a patient in need thereof. In some embodiments, the methods comprise administering transformed immune cells of the present disclosure to a patient in need thereof.

[0332] In some embodiments, the T cells of the present disclosure are capable of stable in vivo T cell expansion and can persist for long periods of time.

[0333] The disclosed therapeutic methods can ameliorate, cure, or prevent disease. The disclosed methods can be part of either an autoimmunotherapy treatment or an alloimmunotherapy treatment. The disclosure is particularly suited to alloimmunotherapy. Donor T cells can be transformed into non-alloreactive cells using standard protocols and, if necessary, regenerated to produce CAR-T cells that can be administered to one or more patients. The CAR-T cell therapy will be available as an "off-the-shelf" therapeutic product.

[0334] Cells that can be used with the disclosed methods are described in the sections above. The treatment can be used, for example, to treat patients diagnosed with cancer. Cancers that can be treated include, but are not limited to, cancers involving B lymphocytes, including any of the cancers listed above. Cancer types that can be treated with the CARs and CAR-T cells of the present disclosure include, but are not limited to, certain leukemias or lymphoid malignancies. Adult tumors / cancers and pediatric tumors / cancers are also included. In some embodiments, the treatment can be combined with one or more treatments for cancer selected from the group consisting of antibody therapy, chemotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy, laser phototherapy, and radiation therapy.

[0335] In some embodiments, the treatment may be administered to a patient undergoing immunosuppressive therapy. Indeed, in some embodiments, the disclosed methods rely on a cell or population of cells rendered resistant to at least one immunosuppressant drug by inactivation of a gene encoding a receptor for that immunosuppressant drug. In this embodiment, the immunosuppressive therapy assists in the selection and expansion of T cells in the patient according to the present disclosure. Administration of the cells or population of cells according to the present disclosure may be carried out by any convenient method, including aerosol inhalation, injection, ingestion, infusion, transfusion, or implantation or transplantation. The compositions described herein may be administered to a patient by subcutaneous, intradermal, intratumoral, intranodal, intramedullary, intramuscular, intravenous, or intralymphatic injection or infusion, or intraperitoneal. In some embodiments, the cell compositions of the present disclosure are administered by intravenous injection or infusion.

[0336] In some embodiments, the administration of the cells or cells is at a dose of, for example, about 10 per kg of body weight. 4 ~about 10 9 In some embodiments, the administration of a cell or cells may be at least about 10 cells per kg of body weight, including all integer values ​​within the range. 5 ~about 10 6The range may include administration of individual cells, including all integer values ​​within the range of cell numbers. The cells or cells can be administered in one or more administrations. In some embodiments, an effective amount of the cells can be administered as a single administration. In some embodiments, an effective amount of the cells can be administered as multiple administrations over a period of time. The timing of administration is within the discretion of the attending physician and depends on the clinical condition of the patient. The cells or cells may be obtained from any source, such as the patient, a blood bank, or a donor. While individual needs vary, determining the optimal range of effective amounts of a given cell type for a particular disease or condition is within the skill of one of ordinary skill in the art. An effective amount refers to an amount that provides a therapeutic or prophylactic benefit. The dose administered depends on the age, health, and weight of the recipient, the type of concomitant treatment, if any, the frequency of treatment, and the nature of the desired effect. In some embodiments, an effective amount of cells or an effective amount of a composition comprising the cells is administered parenterally. In some embodiments, administration may be intravenous. In some embodiments, administration may be directly via intratumoral injection.

[0337] In some embodiments, the method includes (a) administering to a subject having a disease a first dose of allogeneic CAR-T cells. In some embodiments, the first dose is about 1 x 10 4 cells, approximately 5 x 10 4 cells, approximately 1 x 10 5 cells, approximately 5 x 10 5 cells, approximately 1 x 10 6 cells, approximately 5 x 10 6 cells, approximately 6 x 10 6 cells, approximately 1 x 10 7 cells, approximately 6 x 10 7 cells, approximately 1 x 10 8 , about 1.8×10 8 cells, or approximately 4.8 x 10 8 In some embodiments, the method further comprises (b) administering to the subject a subsequent dose of CAR-T cells at least about 5 weeks, or more than about 5 weeks, after initiation of administration in (a) and less than about 24 weeks after initiation of administration in (a).

[0338] In some embodiments, a subject with relapsed / refractory disease (e.g., relapsed / refractory RCC) is administered a first dose and subsequent doses of allogeneic CAR-T cells, each dose containing 6×10 6 cells, and the subsequent dose of CAR-T cells in (b) is administered about 99 days after the start of administration of (a).

[0339] In some embodiments, the method further includes administering additional subsequent or subsequent doses, such that the first and multiple subsequent doses are administered, e.g., according to a specified dosing and timing schedule for the first and subsequent doses. In some embodiments, the first of the one or more subsequent doses is administered at least 5 weeks or more than 5 weeks after initiation of administration of the subsequent doses. In some embodiments, administration of the first, subsequent, and further subsequent doses includes administering at least three of the doses within 5 weeks or about 5 weeks. In some embodiments, the first dose is administered about 16 weeks after initiation of administration, and the additional subsequent or subsequent dose is administered 17 weeks after initiation of administration of the first dose. In some embodiments, the additional subsequent dose is administered 17 and / or 34 weeks after initiation of administration of the first dose.

[0340] In some embodiments, the subsequent dose is administered after the subject has not mounted an immune response, e.g., has not mounted a specific, detectable adaptive host immune response to the first (or previous) administration of CAR-T.

[0341] In some embodiments, the time between administration of the first dose (initial dose), e.g., start of administration of the first or previous dose, and start of administration of the subsequent dose (e.g., start of administration of the subsequent dose) is more than about 4 weeks, e.g., more than about 5, 6, 7, 8, or 9 days, e.g., more than about 20 weeks, e.g., between about 9 weeks and about 35 weeks, between about 14 weeks and about 28 weeks, between 15 weeks and 27 weeks, or between 16 weeks and about 18 weeks, and / or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 weeks. In some embodiments, the administration of a subsequent dose (e.g., its initiation) is more than about 5 weeks and less than about 24 weeks after the administration of the first or previous dose (e.g., its initiation). In some embodiments, the administration of a subsequent dose begins 17 weeks after the initiation of the first dose. In some embodiments, the interval between the administration of a first dose and a subsequent dose (e.g., its initiation), or between the administration of a previous dose and the next subsequent dose, is more than about 5 weeks and less than about 24 weeks, e.g., between 10 and 24 weeks, e.g., about 17 weeks. In some embodiments, the time between the administration of the first dose and the administration of a subsequent dose (e.g., its initiation) is about 17 weeks.

[0342] In some embodiments of the present disclosure, the cells are administered to the patient in combination with (e.g., before, simultaneously with, or after) any number of relevant treatment modalities, including, but not limited to, monoclonal antibody therapy, CCR2 antagonists (e.g., INC-8761), antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or nataliziimab treatment for MS patients, or efaliztimab treatment for psoriasis patients, or other treatments for PML patients. In some embodiments, the CD70-specific CAR-T cells are administered to a patient in combination with one or more of the following: an anti-PD-1 antibody (e.g., nivolumab, pembrolizumab, or PF-06801591), an anti-PD-L1 antibody (e.g., avelumab, atezolizumab, or durvalumab), an anti-OX40 antibody (e.g., PF-04518600), an anti-4-1BB antibody (e.g., PF-05082566), an anti-MCSF antibody (e.g., PD-0360324), an anti-GITR antibody, and / or an anti-TIGIT antibody. In some embodiments, a CD70-specific CAR comprising the amino acid sequence set forth in SEQ ID NO: 319 or 327 is administered to a patient in combination with the anti-PD-L1 antibody avelumab. In further embodiments, the T cells of the present disclosure may be used in combination with chemotherapy, radiation therapy, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, i.e., other immunoablative agents such as CAMPATH, anti-CD3 antibodies or other antibody therapies, cytoxin, fludaribine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and / or radiation therapy.These drugs inhibit the calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or the p70S6 kinase (rapamycin), which is important for growth factor-induced signal transduction (Henderson, Naya et al. 1991; Liu, Albers et al. 1992; Bierer, Hollander et al. 1993). In further embodiments, the T cells of the present disclosure may be used in combination with receptor tyrosine kinase inhibitors, such as, for example, Midostaurin, Sunitinib, and Axitanib; mTOR inhibitors, such as, for example, Rapamacyn and Everolimus; epigenetic modulators, such as, for example, Vormostat; proteasome inhibitors, such as, for example, Bortezomib; immunomodulators, such as, for example, Lenalidomide; Hedgehog inhibitors, such as, for example, Erismodegib and PF-04449913; or isocitrate dehydrogenase (IDH) inhibitors, such as, for example, AG-120 and AG-221. In further embodiments, the cell compositions of the present disclosure are administered to patients in combination with (e.g., before, simultaneously with, or after) bone marrow transplantation, e.g., T cell ablative therapy using fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In some embodiments, the cell compositions of the present disclosure are administered after B cell depletion therapy, e.g., agents that react with CD20, such as Rituxan. For example, in some embodiments, the subject may undergo standard treatment with high-dose chemotherapy followed by a peripheral blood stem cell transplant. In certain embodiments, after transplantation, the subject receives an infusion of expanded immune cells of the present disclosure. In some embodiments, the expanded cells are administered before or after surgery.

[0343] In some embodiments, methods are provided for depleting engineered immune cells expressing a CD70-specific CAR from a subject to which the cells have been administered. Depletion can be by inhibition or removal.

[0344] In one embodiment, a method of depleting engineered immune cells expressing a CD70-specific CAR that comprises an epitope specific for a monoclonal antibody comprises contacting the engineered immune cells with a monoclonal antibody specific for the epitope.

[0345] In some embodiments, a method for depleting engineered immune cells expressing a CD70-specific CAR comprising an epitope specific for a monoclonal antibody from a recipient subject comprises administering to the subject a monoclonal antibody specific for the epitope. In these embodiments, administering to the subject a monoclonal antibody specific for an epitope present in the extracellular domain of the CAR removes or inhibits the activity of the engineered CAR-expressing immune cells from the subject. In one aspect, depletion of the engineered CAR-expressing immune cells allows for the restoration of an endogenous population of CD70-expressing cells.

[0346] In some aspects, the present disclosure relates to a method of promoting recovery of endogenous CD70-expressing cells in a subject administered engineered immune cells expressing a CD70-specific CAR on their surface, the CAR comprising an epitope specific for a monoclonal antibody, the method comprising administering the monoclonal antibody specific for the epitope to the subject. In some embodiments, the endogenous CD70-expressing cells are endogenous CD70-expressing bone marrow cells. In one aspect, the term "recovery" refers to an increase in the number of endogenous CD70-expressing cells. The number of endogenous CD70-expressing cells may be increased by an increase in proliferation of endogenous CD70-expressing cells and / or by a decrease in elimination of endogenous CD70-expressing cells by the engineered immune cells expressing the CAR. In some embodiments, administering the monoclonal antibody to the subject depletes the engineered immune cells expressing the CAR and increases the number of endogenous CD70-expressing cells, e.g., endogenous CD70-expressing bone marrow progenitor cells, in the subject. In one embodiment, administration of the monoclonal antibody to a subject increases the number of endogenous CD70-expressing cells by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% compared to the number of endogenous CD70-expressing cells prior to administration of the monoclonal antibody.

[0347] In one embodiment, a method of treating a CD70-mediated condition in a subject is provided, the method comprising: (a) administering to the subject engineered immune cells that express a CD70-specific CAR on their cell surface, the CAR comprising one or more epitopes specific for one or more monoclonal antibodies; and (b) subsequently depleting the engineered immune cells from the subject by administering to the subject one or more monoclonal antibodies specific for the epitopes.

[0348] In some embodiments, the mAb used in the method of depleting engineered immune cells expressing a CAR is selected from the group consisting of alemtuzumab, ibritumomab tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, bevacizumab, certolizumab pegol, and ribosomal isoforms. pegol), daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab, palivizumab, ranibizumab, tocilizumab, trastuzumab, vedolizumab vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab, ofatumumab, panitumumab, QBEND-10, ustekinumab, and combinations thereof.

[0349] In some embodiments, the epitope specific for the monoclonal antibody (mAb-specific epitope) is an epitope or mimotope of CD20, such as SEQ ID NO: 609, SEQ ID NO: 294 or SEQ ID NO: 295, and the mAb specific for the epitope is rituximab.

[0350] In some embodiments, administering the monoclonal antibody to the subject comprises infusing the monoclonal antibody into the subject. In some embodiments, the amount of epitope-specific mAb administered to the subject is sufficient to eliminate at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of CAR-expressing immune cells in the subject.

[0351] In some embodiments, administering the monoclonal antibody to the subject comprises administering 375 mg / m2 of rituximab intravenously to the subject once or several times per week.

[0352] In some embodiments, immune cells expressing a CAR comprising a mAb-specific epitope (CAR-expressing immune cells) are depleted in a CDC assay using an epitope-specific mAb, and the amount of active CAR-expressing immune cells is reduced, for example, by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

[0353] In some embodiments, a cytotoxic agent is conjugated to an epitope-specific mAb and used to deplete CAR-expressing immune cells. By combining the targeting capabilities of a monoclonal antibody with the cell-killing capabilities of a cytotoxic agent, antibody-drug conjugates (ADCs) can discriminate between healthy and diseased tissue with greater sensitivity than the agent alone. Several ADCs have received marketing approval; methods for their preparation, particularly with regard to linkers, are abundant in the prior art (Payne, G. (2003) Cancer Cell 3:207-212; Trail et al (2003) Cancer Immunol. Immunother. 52:328-337; Syrigos and Epenetos (1999) Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drug Del. Rev. 26:151-172; US Pat. No. 4,975,278).

[0354] In some embodiments, the epitope-specific mAb to be infused is pre-bound to a molecule capable of promoting complement-dependent cytotoxicity (CDC). Thus, the complement system assists or complements the ability of antibodies to eliminate pathogens from the organism. When stimulated by one of several factors, an activation cascade is triggered, resulting in a significant amplification of the cell-killing membrane attack complex response and activation. Different molecules, such as glycans, can be used to conjugate the mAb (Courtois, A., Gac-Breton, S., Berthou, C., Guezennec, J., Bordron, A., and Boisset, C. (2012). Complement-dependent cytotoxicity activity of therapeutic antibody fragments is acquired by immunogenic glycan coupling, Electronic Journal of Biotechnology ISSN: 0717-3458; http: / / www.ejbiotechnology.info DOI: 10.2225 / voll5-issue5).

[0355] kit The present disclosure further provides kits for use in the present methods. The kits of the present disclosure contain one or more containers containing a polynucleotide encoding a CD70-specific CAR described herein, or engineered immune cells comprising a polynucleotide encoding a CD70-specific CAR, and instructions for use according to any of the disclosed methods described herein. Typically, these instructions include instructions for administering the engineered immune cells for the above-mentioned therapeutic treatment. The kit may also include one or more agents for lymphodepletion (e.g., alemtuzumab, cytoxan, fludarabine, cyclophosphamide, or temozolomide).

[0356] Instructions for use of the engineered immune cells described herein generally include information regarding the dose, dosing schedule, and route of administration for the intended treatment. Containers may be unit doses, bulk packages (e.g., multi-dose packages), or sub-unit doses. Instructions provided in kits of the present disclosure are often written instructions on a label or package insert (e.g., a paper sheet included in the kit), although machine-readable instructions (e.g., instructions embodied on a magnetic or optical storage disk) are also acceptable. In some embodiments, the containers are identifiable (e.g., by a label, by a bar code, or by radio-frequency identification (RFID)), traceable, or printed with a machine-readable container identifier.

[0357] The kits of the present disclosure are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar bags or plastic bags), and the like. In some embodiments, the container (e.g., plastic bag) is suitable for intravenous infusion. Packaging for use in combination with specific devices, such as inhalers, nasal administration devices (e.g., atomizers), or infusion devices, such as minipumps, is also contemplated. The kit may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle). The container may also have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is a CD70-specific CAR. The container may further contain a second pharmaceutically active agent.

[0358] The kit may optionally provide additional components, such as buffers and instructional information. Typically, the kit comprises a container and a label or package insert on or associated with the container.

[0359] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present disclosure in any way. Indeed, various modifications of the disclosure in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

[0360] The deposit was made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and its Regulations (Budapest Treaty), which ensures the maintenance of viable cultures of the deposit for 30 years from the date of deposit. The deposit will be made available by ATCC under the terms of the Budapest Treaty, and an agreement will be entered into between Pfizer Inc. and ATCC, which will ensure perpetual, unrestricted public availability of the progeny of the cultures of the deposit upon the issuance of an appropriate U.S. patent or the public disclosure of any U.S. patent application or foreign patent application, whichever occurs first, and availability of the progeny to which rights are granted as determined by the Director of Patents and Trademarks in accordance with 35 U.S.C. § 122 and corresponding Director regulations (including, in particular, 37 CFR 1.14 regarding 886 OG 638).

[0361] The assignee of this application will, upon notice, promptly replace any culture of the deposited material with another identical one if the culture dies, is lost, or is destroyed when cultured under appropriate conditions. Use of the deposited material shall not be construed as a license under any government authority to practice the present disclosure in contravention of the rights granted pursuant to its patent laws. [Example]

[0362] Example 1. Generation of CD70-specific CAR-T cells The following codon-optimized CD70 CAR sequences listed in Table 5 below were synthesized and subcloned into the following lentiviral vectors pLVX-EF1a-TurboGFP-P2A-CD70 CAR (Clontech) or pCLS-EF1a-BFP-P2A-CD70 CAR (Cellectis) using the XmaI (5') and MluI (3') restriction enzyme sites (i.e., the CAR was cloned after the P2A site). [Table 5] TIFF2026000897000023.tif240170 TIFF2026000897000024.tif240170 TIFF2026000897000025.tif240170 TIFF2026000897000026.tif240170 TIFF2026000897000027.tif240170

[0363] CARs containing ScFvs based on the 10A1, 10E2, 11A1, 11C1, 11D1, 11E1, 12A2, 12C4, 12C5, 12D3, 12D6, 12D7, 12F5, 12H4, 8C8, 8F7, 8F8, 9D8, 9E10, 9E5, 9F4 or 9F8 sequences have also been prepared, which CARs include the sequences set forth in SEQ ID NOs: 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600 and 601.

[0364] Example 2: Jurkat screening for in vitro characterization of anti-CD70 CAR Jurkat cells are an immortalized human T cell line that closely resemble primary T cells and express CD70 upon activation or transduction. These cells were then selected for transduction with a CD70 CAR, and their activation profile was examined. Jurkat cells engineered using CRISPR / Cas9 to knock out CD70 (KO Jurkat) or parental Jurkat (WT Jurkat) were transduced with a CD70 CAR. The autoactivation profile was determined by comparing the ratio of CD69 expression on WT Jurkat (WT Jurkat expresses the target, i.e., CD70, and thus autoactivates and activates in a target-dependent manner upon transduction) with the ratio of CD69 expression on KO Jurkat (only autoactivation in the absence of the target, i.e., CD70). Clones that showed target-specific activation and minimal autoactivation were then selected based on the "activation ratio" (see below for a definition of this term). Autoactivation is a term used to describe target-dependent clustering and activation of the CAR. CAR autoactivation can range from minimal / none to high and is thought to be an inherent property or tendency of scFvs to aggregate and cluster. Autoactivation can result in chronic signaling, leading to T cell differentiation, exhaustion, and reduced cytolytic activity. Screening and elimination of highly autoactivating CARs is an important step in identifying optimal CARs. An ideal CAR is one with minimal autoactivation.

[0365] Binding of CARs to recombinant antigens is one way to characterize and classify CARs into unique groups. Strong binding may indicate that the CAR is highly expressed on the cell surface and has moderate to high affinity for its target antigen. Low or minimal binding may indicate low expression and / or weak affinity. Because optimal affinity and cell surface expression are unknown, CARs with high and low binding will be further characterized. material and method CRISPR / Cas9 knockout of CD70 in Jurkat cells

[0366] Jurkat cells were transfected with a CD70-targeting guide RNA vector containing Cas9 and GFP obtained from DNA2.0 using Lipofectamine 3000 (Invitrogen). Forty-eight hours after transfection, fluorescence-activated single-cell sorting was performed to select GFP+CD70- cells. Individual clones were then expanded, and genomic DNA was obtained for PCR using crude cell lysates. PCR products were sequenced to identify clones with indels or frameshifts, indicative of CD70 knockout.

[0367] Transduction of Jurkat cells with CD70 CAR On day 0, 0.5 million HEK293T cells were seeded per well of a 6-well plate in 2 mL of DMEM (Gibco) supplemented with 10% FBS (Hyclone or JR Scientific). On day 1, lentivirus was prepared (DNA mix) by mixing the lentiviral packaging vectors 1.5 μg psPAX2, 0.5 μg pMD2G, and 2 μg of the appropriate transfer CAR vector containing a GFP tag in 250 μL of Opti-MEM (Gibco) per well of a 6-well plate. 10 μL of Lipofectamine 2000 (Invitrogen) was incubated in 250 μL of Opti-MEM for 5 minutes at room temperature and then added to the DNA mix. The virus was incubated for 20 minutes at room temperature, and a total volume of 500 μL was slowly added to the side of the well containing the HEK293T cells. On day 2, the medium from each well of the 6-well plate was replaced with 2 mL of Jurkat cell medium, i.e., RPMI (Gibco) supplemented with 10% FBS, per well. On day 3, Jurkat cells (KO or WT) were resuspended at 0.5 million cells per mL in 2 mL of RPMI supplemented with 10% FBS per well of the 6-well plate. Lentiviral supernatant from HEK293T cells was collected and filtered through a 0.45 micron filter (EMD Millipore) to remove cellular debris before being added to the Jurkat cells. On day 6, transduction efficiency was determined by detecting GFP signal via flow cytometry. Cells were further expanded in larger flasks as needed using RPMI supplemented with 10% FBS.

[0368] Activation profile and ability to bind to CD70 protein On day 5, transduced cells were stained with PE-Cy7-conjugated human anti-CD69 antibody and acquired on a flow cytometer to obtain the percentage of the CD69+ population for each CAR. Cells were further incubated with recombinant biotinylated human CD70 protein, stained with PE-conjugated streptavidin, and acquired on a flow cytometer to determine protein binding. Activation ratios (WT activation / KO activation) and protein binding were used to rank the CARs. [Table 6]

[0369] The activation ratio and protein binding of hybridoma CARs were determined in Jurkat screens and used for in vitro characterization of the CARs. The activation ratio was determined by calculating the ratio of CD69 expression on WT Jurkat cells transduced with CD70 CAR (WT) to that on CD70 knockout Jurkat cells transduced with CD70 CAR (KO). A high activation ratio indicates target-dependent activation. Protein binding was determined by binding to recombinant biotinylated hCD70 protein and detected using streptavidin conjugated to phycoerythrin (PE) dye via flow cytometry. Protein binding values ​​represent the percentage of CD3+ CAR T cells that bind human CD70 protein. [Table 7]

[0370] Table 7: Activation ratios and protein binding of phage CARs were determined in Jurkat screening. Activation ratios were used for in vitro characterization of CARs. Activation ratios were determined by calculating the ratio of CD69 expression on WT Jurkat cells transduced with CD70 CAR (WT) to that on CD70 knockout Jurkat cells transduced with CD70 CAR (KO). A high activation ratio suggests target-dependent activation. Protein binding was determined by binding to recombinant biotinylated hCD70 protein and detected using streptavidin conjugated to phycoerythrin (PE) dye via flow cytometry. Protein binding values ​​indicate the percentage of CD3+ CAR T cells that bind human CD70 protein.

[0371] CARs that showed minimal autoactivation, i.e., minimal CD69 expression when transduced in CD70 KO Jurkat, were considered more desirable compared to CARs that were highly activated even in the absence of target, potentially resulting in a highly exhaustible CAR phenotype. Similarly, CARs with higher human CD70 protein binding were considered more desirable, as this suggests proper expression and folding of the CAR on the surface.

[0372] Example 3: Screening of primary T cells for in vitro characterization of phage and hybridoma CARs Primary human T cells were transduced with a CD70 CAR to determine transduction efficiency, CD70 expression on T cells in culture, and T cell subsets. Transduced CAR T cells were then frozen and used for functional assays.

[0373] The transduction efficiency of CAR constructs can vary greatly between different clones.High transduction efficiency leads to a larger number of CAR T cells and a more efficient manufacturing process.On the other hand, CARs with low transduction efficiency may not be suitable for large-scale manufacturing.High transduction efficiency is beneficial in some embodiments.

[0374] T cells have a wide range of phenotypes or subsets that are indicative of differentiation state and antigen exposure. Cell surface markers help distinguish T cell subsets, and the CD62L marker is generally present on naive stem cell memory T cells and central memory T cells. These cells are less differentiated than other subsets, such as effector memory T cells and effector T cells, and therefore, a CAR T cell population with a high percentage of CD62L-positive cells is beneficial in some embodiments.

[0375] Surface expression of CD70 also varies among different transduced CARs, with some CAR T cells expressing CD70 (20–40%) upon T cell activation and transduction, whereas other T cells do not.

[0376] material and method Isolation of primary T cells T cells were purified from buffy coat samples obtained from Stanford University using Ficoll gradient density medium (Ficoll Paque PLUS / GE Healthcare Life Sciences). The PBMC layer was collected and T cells were purified using a commercially available T cell isolation kit (Miltenyi Biotec).

[0377] Transduction of T cells with CD70 CAR On day 0, 0.5 million HEK293T cells were seeded per well of a 6-well plate in 2 mL of DMEM (Gibco) supplemented with 10% FBS (Hyclone or JR Scientific). On day 1, lentivirus was prepared (DNA mix) by mixing the lentiviral packaging vectors 1.5 μg psPAX2, 0.5 μg pMD2G, and 2 μg of the appropriate transfer CAR vector containing a GFP tag in 250 μL of Opti-MEM (Gibco) per well of a 6-well plate. 10 μL of Lipofectamine 2000 (Invitrogen) was incubated in 250 μL of Opti-MEM for 5 minutes at room temperature and then added to the DNA mix. The virus was incubated for 20 minutes at room temperature, and a total volume of 500 μL was slowly added to the side of the well containing the HEK293T cells. Purified T cells were activated in X-Vivo-15 medium (Lonza) supplemented with 100 IU / mL human IL-2 (Miltenyi Biotec), 10% FBS (Hyclone or JR Scientific), and human T-activating CD2 / CD3 / CD28 beads (Miltenyi Biotec) at a bead-to-cell ratio of 1:2. On day 2, the medium from each well of a 6-well plate was replaced with 2 mL per well of T cell transduction medium, i.e., X-Vivo-15 supplemented with 10% FBS. On day 3, T cells were resuspended at 0.5 million cells per mL in 2 mL of T cell transduction medium per well of a 6-well plate. Lentiviral supernatant from HEK293T cells was collected and passed through a 0.45 micron filter (EMD Millipore) to remove cellular debris, then added to T cells along with 100 IU / mL human IL-2. On day 6, transduction efficiency was determined by detecting GFP signal via flow cytometry. Cells were expanded in larger flasks as needed or in G-Rex vessels (Wilson Wolf) using T cell expansion medium, i.e., X-Vivo-15 supplemented with 5% human AB serum (Gemini Bio).

[0378] CD70 expression on CAR T cells and T cell subsets On day 13 after activation, transduced CAR T cells were stained with PE-conjugated anti-human CD70 and BV605-conjugated anti-human CD62L antibodies and acquired on a flow cytometer to obtain the percentage of CD70+ and CD62L+ populations for each CAR. Expanded CAR T cells were then frozen in FBS containing 10% DMSO (Sigma Aldrich) and subsequently used in functional assays. [Table 8]

[0379] Table 8: CAR T cell phenotypes were used for in vitro characterization of phage and hybridoma CARs at day 13 post-activation. CAR+ populations were determined by gating on CD3+ cells. CD70-expressing populations were determined by gating on live CD3+ cells. CD62L-expressing populations were determined by gating on live CD3+ CAR+ CD8+ cells.

[0380] The validated CAR constructs showed varying levels of transduction efficiency. Clones with lower transduction efficiency were considered less promising because they were less suitable for large-scale manufacturing. CAR T cell products also displayed different phenotypes (measured by CD62L expression) and showed varying levels of CD70 expression on their surface. In general, CAR T cells that expressed higher levels of CD62L were considered more promising, as they may be less differentiated.

[0381] Example 4: Stress test with phage CAR CAR T cells were generated from 12 different scFvs from the phage library as described in the previous examples and frozen.These CAR T cells were then lysed and mixed with Raji target cells known to express CD70 in RPMI supplemented with 10% FBS at an effector:target (E:T) ratio of 1:1.Raji cells were then added to the CAR T cells every two days, and the E:T ratio was then maintained at 1:1.The lysis rate of target cells and the expansion fold of effector CAR T cells were determined at each time point.

[0382] Stress testing is a screening assay that involves repeated exposure of CAR T cells to targets, which allows the CAR to expand and, in certain cases, differentiate and deplete. Stress testing was used to select optimal clones with high target cell lysis and expansion capacity after several rounds of exposure to target cells.

[0383] material and method On day 0, CAR T cells derived from 12 different scFvs from the phage library were lysed and mixed with Raji cells at a 1:1 E:T ratio. On day 2, 200 μL of cells from the assay were mixed with 50 μL of cell counting beads (CountBright, Invitrogen) and acquired on a flow cytometer. Using the counting beads, the total number of GFP+ CAR T cells (gated on CD3+) and Raji cells (gated on CD3-) for each CAR treatment was determined. Based on the total CAR T cell count, the number of Raji cells required to maintain a 1:1 E:T ratio was calculated and added to each well of the assay. This process was repeated on days 5 and 7. The percentage of live Raji cells for each CAR treatment was calculated and normalized to the untransduced control to calculate the percentage of target cell lysis at each time point. The fold expansion of CAR T cells relative to the number of CAR T cells seeded on day 0 was calculated at each time point.

[0384] The optimal clone was the one with the highest target cell lysis and the greatest fold expansion at the end of the assay on day 7, such as P08F08. [Table 9]

[0385] Table 9: Target cell lysis and CAR T cell expansion folds in stress tests were used for in vitro characterization of phage CARs. CAR T cell phenotypes were determined on day 13 after activation, followed by lysis on day 14. Stress tests were performed using lysed CAR T cells and target Raji cells at a 1:1 E:T ratio. Target cell lysis was determined via flow cytometry by gating on CD3- target cells after 2, 5, and 7 days of coculture with CAR T cells. CAR T cell expansion folds were determined by using cell counting beads and calculating the cell number on days 2, 5, and 7 compared to the number of cells added at the beginning of the assay. CARs highlighted in bold are CARs that exhibit high target cell lysis and expansion folds.

[0386] Example 5: Repeat stress test with optimal CAR generated from a second donor CAR T cells were generated and frozen from four scFvs (P07D03, P08F08, P08G02, P15D02) from the phage library and two scFvs (4F11, 17G6) from the hybridoma library, as described in Example 3. These CAR T cells were then lysed and mixed with Raji target cells known to express CD70 in a 1:1 effector:target (E:T) ratio in RPMI supplemented with 10% FBS. Raji cells were then added to the CAR T cells every two days, and the 1:1 E:T ratio was maintained thereafter. The percentage of target cell lysis and the fold expansion of effector CAR T cells were determined at each time point.

[0387] All clones performed well in terms of target cell lysis. The best clones based on fold expansion were P08F08 and 4F11. [Table 10]

[0388] Table 10: Target cell lysis and fold expansion of CAR T cells in stress tests were used for in vitro characterization of optimal CARs. CAR T cell phenotypes were determined 14 days after activation, and then cells were frozen the same day. Stress tests were performed using lysed CAR T cells and target Raji cells at a 1:1 E:T ratio. Target cell lysis was determined via flow cytometry by gating on CD3- target cells after 2, 5, and 7 days of co-culture with CAR T cells. CAR T cell expansion was determined by using cell counting beads and calculating the cell number on days 2, 5, and 7 compared to the number of cells added at the beginning of the assay. CARs highlighted in bold are CARs that exhibit high target cell lysis and expansion.

[0389] Example 6: Dose-dependent in vivo efficacy of CAR T in an RCC SC tumor model CAR T cells were generated from 4F11 scFv as described in Example 3. NOD scid gamma (NSG) mice were implanted subcutaneously with 786-O tumors until the tumor volume reached 200 mm 3 Upon reaching 4x10 serotonin B (Sb), mice were treated with various intravenous doses of 4F11 CAR T cells via tail vein injection to determine the optimal CAR T dose. 786-O cells are available from ATCC® as CRL-1932™. 4F11 CAR T cells were highly effective in vivo, reaching 5x10 serotonin B (Sb). 6 One dose of CAR T resulted in complete tumor regression.

[0390] material and method Fifty NOD scid gamma (NSG) mice were shaved and prepped for tumor implantation subcutaneously on the right flank. 786-O tumor cells, known to express CD70, were expanded in RPMI supplemented with 10% FBS. On day 0, 786-O cells were resuspended in serum-free RPMI at the required concentration and injected at 5 million cells per animal. Tumor cells were injected subcutaneously in 100 μL of serum-free RPMI mixed with 100 μL of Matrigel (Corning) per animal. Immediately after tumor implantation, baseline body weights on day 0 were recorded for all animals. Tumors were measured twice weekly starting on day 9 using Digimatic Calipers (Mitutoyo), and body weights were recorded. By day 14, tumors had grown to 200 mm 3 When the tumor-bearing mice reached a mass index (MSI) of 8.39 (standard error), 40 tumor-bearing mice were randomized into four groups of 10 mice per group. 4F11 CAR T cells were lysed in RPMI supplemented with 10% FBS and resuspended in serum-free RPMI at the required concentration, and 1, 3, or 5 million CAR+ T cells were injected per animal (calculated based on the 57.2% transduction efficiency of 4F11). The number of non-transduced T cells (NTD) required to maintain an equal number of total T cells in each group was calculated and added to each sample. CAR T cells or non-transduced T cell controls were injected intravenously via the tail vein in 200uL of serum-free RPMI per animal. The NTD group reached the study endpoint (1500mm 3 Tumors were measured and body weights recorded twice weekly until day 43, when tumor volume reached 100 μg / kg.

[0391] Tumor volumes (mean and SEM) were plotted on GraphPad Prism, and statistics were calculated using one-way ANOVA with repeated measures (see Figures 1 and 2). A dose of 5 million CAR+ cells completely eliminated tumors. Meanwhile, a dose of 1 million CAR+ cells showed no efficacy compared with the NTD group. Therefore, a dose of 3 million CAR+ cells was selected as the optimal dose for future in vivo studies.

[0392] Example 7: In vivo comparison of CD70 CAR with or without CD70 TALEN knockout in 786-O cells 4F11 CAT T cells and P08F08 CAT T cells were generated with or without CD70 TALEN DNA electroporation on day 6 post-activation as described in Example 1. NSG mice were implanted subcutaneously with 786-O tumors, and tumor volumes reached 200 mm 3 When the mice reached 100% CAR T cells per 1000 mg / kg, they were treated with CAR T cells via tail vein injection to determine the CAR and conditions with optimal efficacy. P08F08 CAR T cells were highly effective in vivo, achieving a CAR count of 3x10 despite CD70 knockout (KO). 6 A single CAR T dose resulted in complete tumor regression. 4F11 CAR T cells showed good efficacy when CD70 was knocked out, but simply controlled tumor growth in the absence of CD70 knockout. These results suggest that CD70 knockout can improve the activity of CD70 CARs.

[0393] material and method Seventy-five NSG mice were shaved and prepped for tumor implantation subcutaneously on the right flank. 786-O tumor cells, known to express CD70, were expanded in RPMI supplemented with 10% FBS. On day 0, 786-O cells were resuspended in serum-free RPMI at the required concentration and injected at 5 million cells per animal. Tumor cells were injected subcutaneously in 100 μL of serum-free RPMI mixed with 100 μL of Matrigel (Corning) per animal. Immediately after tumor implantation, baseline body weights on day 0 were recorded for all animals. Tumors were measured twice weekly starting on day 7 using Digimatic Calipers (Mitutoyo), and body weights were recorded. By day 20, tumors had grown to 200 mm 3When the tumor-bearing mice reached the target mass index (MSI) (standard error 9.69), 60 tumor-bearing mice were randomized into 6 groups of 10 mice per group. On day 21, CAR T cells were lysed in RPMI supplemented with 10% FBS and resuspended in serum-free RPMI at 3 million CAR+ T cells per animal (calculated based on individual transduction efficiency). The number of NTD cells required to maintain an equal proportion of CAR+ T cells and an equal number of total T cells in each group was calculated and added to each sample. CAR T cells or NTD control were injected intravenously via the tail vein in 200uL of serum-free RPMI per animal. The NTD group was administered at the study endpoint (1500mm 3 Tumors were measured and body weights recorded twice weekly until day 56, when tumors reached a tumor volume of 1000 μg / kg (see Figures 3 and 4).

[0394] Tumor volumes (mean and SEM) were plotted on GraphPad Prism, and statistics were calculated using one-way ANOVA with repeated measures. Both the P08F08 CAR T groups with and without CD70 knockout resulted in complete tumor regression at a CAR+ dose of 3 million cells. The 4F11 CAR T group with CD70 knockout also resulted in complete tumor regression at a CAR+ dose of 3 million cells. However, the 4F11 CAR T group without CD70 knockout did not result in complete regression.

[0395] In Figures 3 and 4, statistical significance relative to the corresponding NTD control is indicated to the right of the legend (e.g., P08F08 with CD70 KO versus NTD with CD70 KO). Statistical significance of each CAR group with CD70 KO relative to the corresponding CAR group without CD70 KO is indicated to the left of the legend. Statistics represent RM one-way ANOVA with Dunnett's post-hoc test (ns p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0396] Example 8: In vivo comparison of CD70 CAR with or without CD70 TALEN knockout in an ACHN metastasis model The 4F11 and P08F08 CAR T cells of Example 7 were further validated in ACHN cells, a cell line derived from renal cell carcinoma (RCC). ACHN cells are described in Simmons et al. Animal Models of Bone Metastasis. Veterinary Pathology 52:827-841, 834 (2015). NSG mice were intravenously implanted with 1 million ACHN tumor cells, and 15 days after tumor cell injection, the mice were treated with CAR T cells via tail vein injection to determine the CAR and condition with optimal efficacy. 4F11 CAR T cells administered at 3 million CAR+ cells per animal were effective across all three donors tested, regardless of CD70 knockout (KO). P08F08 CAR T cells showed little or no efficacy and were only validated in the absence of CD70 knockout.

[0397] material and method Forty-five NSG mice were prepared for intravenous tumor injection via the tail vein. ACHN tumor cells, known to express CD70, were expanded in MEM supplemented with 10% FBS. On day 0, ACHN tumor cells were resuspended in serum-free MEM at the required concentration and 1 million cells were injected per animal. ACHN tumor cells were injected intravenously in 200 μL of serum-free MEM. Baseline body weights were recorded for all animals on day 4. Tumor growth was measured twice weekly starting on day 7 using bioluminescence (IVIS Spectrum Imager™, PerkinElmer™, autoexposure with a maximum exposure time of 120 seconds), and body weights were recorded. By day 20, tumors had grown to 200 mm 3When tumor size reached 9.69 (standard error 9.69), 20 tumor-bearing mice were randomized into four groups of five mice per group. On day 15, CAR T cells were lysed in MEM supplemented with 10% FBS and resuspended in serum-free MEM at 3 million CAR T cells per animal (calculated based on individual transduction efficiencies). The number of NTD cells required to maintain equal proportions of CAR T cells and equal numbers of total T cells in each group was calculated and added to each sample. CAR T cells or NTD control were injected intravenously via the tail vein in 200 μL of serum-free MEM per animal. Tumor size was measured and body weight was recorded twice weekly until days 35–49, when the NTD group reached the study endpoint (greater than 20% weight loss) (see Figures 5a, 5b, and 5c).

[0398] Bioluminescence (mean and error SEM) was plotted on GraphPad Prism, and statistics were calculated using one-way ANOVA with repeated measures. Both 4F11 CAR T groups with and without CD70 knockout demonstrated antitumor efficacy at the 3 million CAR+ dose. The P08F08 CAR T group without CD70 knockout showed little to no efficacy at the 3 million CAR+ dose.

[0399] Example 9: Activity of CD70-specific CAR T cells expressing CD20 epitopes Part A: In vitro activity CD70-specific CAR T cells expressing the CD20 epitope are effective against 786-0 target cells in cell killing assays.

[0400] Six CD70-specific CAR formats were designed (Figures 6A-6F). The sequences of the constructed CARs are shown in Tables 11A-11F. [Table 11A] TIFF2026000897000034.tif240170 TIFF2026000897000035.tif240170 [Table 11B] TIFF2026000897000037.tif240170 TIFF2026000897000038.tif240170 [Table 11C] TIFF2026000897000040.tif240170 TIFF2026000897000041.tif240170 [Table 11D] TIFF2026000897000043.tif240170 TIFF2026000897000044.tif240170 [Table 11E] TIFF2026000897000046.tif240170 TIFF2026000897000047.tif240170

[0401] Tests were performed on the following: NTD control, the RSRQR format of the 4F11 CAR (described above and shown schematically in Figure 6C), the SR2 format of the P08F08 CAR (described above and shown schematically in Figure 6B), the R2S format of the P08F08 CAR (described above and shown schematically in Figure 6F), and the RSR-short format of the P08F08 CAR (described above and shown schematically in Figure 6E).

[0402] CAR T cells expressing each CAR format were generated. These CAR T cells were then lysed and mixed with 786-0 target cells, known to express CD70, at effector:target (E:T) ratios of 3:1, 1:1, 1:3, or 1:9 (or 0 control) in RPMI supplemented with 10% FBS. The percentage of target cells lysed and the fold expansion of effector CAR T cells were determined (Figure 7).

[0403] Percent target cell lysis was calculated at each time point by calculating the percentage of live 786-O cells for each CAR treatment and then normalizing to the untreated control. Fold expansion of CAR T cells relative to the number of CAR T cells seeded on day 0 was calculated at each time point.

[0404] These results indicate that CD70-specific CAR-T cells expressing the CD20 epitope can efficiently kill target cells at E:T ratios of 3:1, 1:1, 1:3, and 1:9, as well as ratios in between, and potentially ratios outside the 3:1 to 9:1 range that were not tested in this experiment.

[0405] Part B: Sensitivity to rituximab in vivo Depletion of CD70-specific CAR T cells after rituximab administration allows recovery of CD70-expressing lymphocytes in NSG mice.

[0406] The ability of the anti-CD20 antibody rituximab to mediate depletion of CD70-specific CAR T cells expressing the CD20 epitope and promote lymphocyte recovery was tested in mice. In this experiment, mice were treated with T cells expressing either a CD70-specific CAR (α-murine CD70 CAR Ts) containing the CD20 epitope recognized by rituximab, which can bind to the murine CD70 protein on the lymphocyte surface, or a control CAR that weakly binds to the murine FLT3 protein. Flow cytometry analysis of lymphocytes was used to demonstrate the cytotoxic activity of CAR T cells against CD70-expressing lymphocytes. This activity was observed as a reduction in lymphocytes compared to mice administered control CAR T cells or untreated mice. After confirmation of CAR T cell killing activity, mice were given rituximab for four consecutive days, and residual circulating CAR T cells were enumerated by flow cytometry on day 13. CD70-specific CAR T cells in the blood of these mice are depleted compared to controls that did not receive rituximab.Finally, flow cytometry analysis of lymphocytes shows that only mice that received rituximab (day 20) showed lymphocyte recovery.

[0407] This experiment demonstrates that rituximab-dependent depletion of CD70-specific CAR T cells expressing the CD20 epitope reduces damage to CD70-expressing tissues and allows rapid lymphocyte recovery.

[0408] Example 10: Activity of CD70-specific CAR T cells Target cell killing was evaluated using the same assay and similar experimental parameters as described in Part A of Example 9. The cell lines tested were 786-0, ACHN, and REH (human acute lymphocytic leukemia cell lines). The REH cell line exhibits the best CD70-specific CAR T cell differentiation. Therefore, this line will be used to rank scFvs in all future experiments. Figures 8A-8D show cell killing of 786-0 cells (Figure 8A), ACHN cells (Figure 8B), or REH cells (Figure 8C) using CD70-specific CAR T cells. In this case, the extracellular domain of the CAR contains the scFv indicated in the legend (Figure 8D). For all experiments, the naked CAR format was used.

[0409] Table 12 provides the data underlying Figures 8A-8D, and additionally provides the measured percentage of cells positive for fluorescently labeled anti-CD70 antibody, CD25, and 4-1BB (activation markers); the percentage of stem memory T cells (TSCM) before cytotoxicity assays, and the percentage of BFP (CAR+). [Table 12]

[0410] Figure 9A shows the efficacy of a CD70-specific CAR upon repeated exposure to luciferase-labeled 786-O target cells (CAR T cells were transferred to 96-well plates containing fresh targets every 2-3 days). The E:T ratio was 3:1. The CAR was expressed in cells from donor D503. Similar to the results described in Example 4, target cell lysis in a stress test was used for in vivo characterization of the CAR scFv.

[0411] Figure 9B shows the efficacy of the CD70-specific CAR upon repeated exposure to luciferase-labeled ACHN target cells (CAR T cells were transferred to 96-well plates containing fresh targets every 2-3 days). The E:T ratio was 10:1. The CAR was expressed in cells from donor D503. Similar to the results described in Example 4, target cell lysis in a stress test was used for in vivo characterization of the CAR scFv.

[0412] Figure 9C shows the efficacy of the CD70-specific CAR upon repeated exposure to luciferase-labeled REH target cells (2x10 at the indicated time points). 6 (cells were added). The E:T ratio was 1:5. CAR was expressed in cells from donor D503. Similar to the results described in Example 4, lysis of target cells in a stress test was used for in vivo characterization of the CAR scFv.

[0413] Figure 10 shows the efficacy of CD70-specific CARs in various formats (referenced in Example 9, part A) upon repeated exposure to luciferase-labeled REH target cells (2x10 at the indicated time points). 6 cells were added). The E:T ratio was 1:5. Similar to the results described in Example 4, lysis of target cells in a stress test was used for in vivo characterization of CAR scFv.

[0414] Example 11: In vivo comparison of CD70 CAR in an ACHN metastasis model CAR T cells containing different CD70 scFvs were generated and tested in ACHN cells, a renal cell carcinoma (RCC)-derived cell line. ACHN cells are described in Simmons et al., Animal Models of Bone Metastasis. Veterinary Pathology 52:827-841, 834 (2015). NSG mice were intravenously implanted with 1 million ACHN tumor cells. 15 days after tumor cell injection, the mice were treated with CAR T cells via tail vein injection to determine the CAR and conditions with optimal efficacy. 12C5 CAR T cells administered at 3 million CAR+ cells per mouse demonstrated the best efficacy.

[0415] material and method Forty-five NSG mice were prepared for intravenous tumor injection via the tail vein. ACHN tumor cells, known to express CD70, were expanded in MEM supplemented with 10% FBS. On day 0, ACHN tumor cells were resuspended in serum-free MEM at the required concentration and 1 million cells were injected per animal. ACHN tumor cells were injected intravenously in 200 μL of serum-free MEM. Baseline body weights were recorded for all animals on day 4. Tumor growth was measured twice weekly starting on day 7 using bioluminescence (IVIS Spectrum Imager™, PerkinElmer™, autoexposure with a maximum exposure time of 120 seconds), and body weights were recorded. By day 20, tumors had grown to 200 mm 3When the tumor-bearing mice reached a mass index (SEM) of 9.69 (standard error 9.69), 35 tumor-bearing mice were randomized into 7 groups, with 5 mice per group. On day 15, CAR T cells were lysed in MEM supplemented with 10% FBS and resuspended in serum-free MEM at 3 million CAR+ T cells per animal (calculated based on individual transduction efficiency). The number of NTD cells required to maintain an equal proportion of CAR+ T cells and an equal number of total T cells in each group was calculated and added to each sample. CAR T cells or NTD control were intravenously injected via the tail vein in 200 μL of serum-free MEM per animal. Tumor size was measured and body weight was recorded twice weekly until day 32, when the NTD group reached the study endpoint (greater than 20% weight loss) (see Figure 11).

[0416] Bioluminescence (mean and error SEM) was plotted on GraphPad Prism and statistics were calculated using one-way ANOVA with repeated measures. At the 3 million CAR+ dose, the 12C5 CAR T group demonstrated antitumor efficacy.

[0417] Example 12: Expression levels of CD70 on patient-derived RCC samples and lysis of patient-derived RCC samples by CD70-specific CAR T cells Primary RCC patient samples were obtained from Conversant Bio (frozen dissociated tumor cells) or CHTN Western / NDRI (fresh tumor fragments subsequently dissociated by the inventors using the Miltenyi MACS human tumor dissociation kit and GentleMACS). Primary tumor cells were maintained in RPMI supplemented with 20% FBS. To determine the cell surface expression of CD70 protein in these cells along with related RCC cell lines, we performed flow cytometry using an anti-CD70 antibody conjugated to phycoerythrin at a 1:1 ratio to quantify the receptor. Cell surface receptors were measured using BD Biosciences Quantibrite beads, and antibody binding capacity (ABC) was calculated according to the manufacturer's recommendations. CD70-specific CAR T cells were generated as described above, and their cytotoxicity against primary RCC cells and ACHN RCC cell lines was assessed using the same assay and similar experimental parameters as those described in Example 9, Part A.

[0418] result It was confirmed that primary RCC cells express CD70 with ABC values ​​ranging from approximately 2,000 CD70 receptors per cell (receptors / cell) to approximately 25,000 receptors / cell (Figure 12B), and expression on RCC cell lines ranged from approximately 25,000 receptors / cell to approximately 400,000 receptors / cell (Figure 12A). Furthermore, the inventors confirmed that cells expressing 7,000 CD70 receptors per cell (receptors / cell) (Figure 13B), 24,000 receptors / cell (Figure 13A), or 40,000 receptors / cell (Figure 13C) were efficiently killed by CD70-specific CAR T cells, where the CAR was generated from either 4F11 or P08F08 scFv.

[0419] Example 13: Expression levels of CD70 on various hematological tumor cell lines and lysis of these cells by CD70-specific CAR T cells The feasibility of targeting CD70 in various hematologic malignancies, including lymphoma, leukemia, and myeloma, was analyzed. Characterization included analysis of both CD70 RNA and cell surface protein expression in multiple malignancies, followed by efficacy of CAR T cells against cell lines.

[0420] result RNA expression analysis of acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma (NHL), and multiple myeloma (NM cell) lines using The Cancer Genome Atlas (TCGA) showed that CD70 expression could be observed in all four cancer types. This suggests the potential utility of targeting these cancers with CD70 CAR T cells. To determine the cell surface expression of CD70 protein in these cancers, we performed flow cytometry and receptor quantification on a panel of cell lines originating from selected tumor types. The cell surface protein expression pattern was similar to that of the RNA analysis, confirming that CD70 expression was widely observed in cell lines from all tumor types (Figure 14A). Next, we generated CD70-specific CAR T cells and tested their efficacy against the same cell lines in an in vitro cytotoxicity assay. CD70 CAR T cells exhibited stable specific activity against target cells expressing the CD70 antigen (Figure 14B). The ability of CD70-specific CARs to kill a variety of cell lines (Figure 14B) suggests that they can kill even cells expressing low levels of CD70. This indicates that the activity of CD70-specific CARs is not limited to a specific cell type. Finally, we demonstrated that these CARs were effective against the MM1S cell line in in vivo assays performed as in Example 7 (Figures 15A and 15B). MM1S is a multiple myeloma cell line that expresses a moderate number of CD70 receptors per cell (Figure 14A). In conclusion, we observed that CD70 has a broad expression profile across various hematological malignancies. The use of CD70 CAR T cells, alone or in combination with other hematological targets, offers the opportunity to target or inhibit tumor antigen escape in various hematological malignancies. Example 14: Determination of the kinetics and affinity of human CD70 / CD70 antibody interactions at 37°C

[0421] This example determines the binding kinetics and / or affinity of various anti-CD70 antibodies to human CD70. ScFvs were generated by cloning the variable regions of anti-CD70 antibodies and flanking them with a (GGGGS)4 linker (SEQ ID NO: 602) followed by the hinge and Fc portions of modified human IgG2 sequences to generate scFv-Fc fusions. These were expressed using Expi293 and purified by Protein A affinity chromatography. Recombinant human CD70 was generated by fusing a polyhistidine tag, AviTag™, and the trimerization domain of chicken tenascin to the N-terminus of the human CD70 extracellular domain (ECD). These were expressed using Expi293 and purified by immobilized metal affinity chromatography (IMAC), followed by size exclusion chromatography (SEC) if necessary.

[0422] Antibody binding kinetics were determined by surface plasmon resonance (Biacore 8K, GE Healthcare Bio-Sciences, Pittsburgh, PA) at 37°C in HBS-T+ (0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.05% v / v Tween 20, 1 mg / mL BSA). Recombinant human CD70 diluted in HBS-T+ was captured on a C1 chip with immobilized anti-AviTag™ antibodies. Purified anti-CD70 scFv-Fc fusions were serially diluted in HBS-T+ and injected at 30 μL / min for 2–4 min. Dissociation was monitored for 10 min, and the surface was regenerated with 75 mM phosphoric acid between injections. Buffer cycles were collected for each anti-CD70 scFv-Fc fusion for double-referencing purposes (double-referencing as described in Myszka, DGI Improving biosensor analysis. J. Mol. Recognit. 12, 279-284 (1999)). The kinetic association rate (K on ) and dissociation rate (k off) were simultaneously obtained by fitting the double-referenced sensorgrams to a 1:1 Langmuir with a mass transport model using 8K Evaluation Software (GE Healthcare Bio-Sciences, Pittsburgh, PA), and these were then used to calculate the kinetic rate constants (K D =k off / k on ) to the equilibrium dissociation constant (K D ) was calculated. Data were fitted to a 1:1 steady-state affinity model using Biacore 8K Evaluation Software, and the steady-state equilibrium dissociation constant (SS K) was calculated as needed. D ) was decided.

[0423] The binding kinetics and affinity parameters of the tested anti-CD70 antibodies are shown in Table 13. The antibodies shown in Table 13 share the same scFv sequences as the CARs with the same names shown in Tables 5 and 11. [Table 13]

[0424] While the disclosed teachings have been described with reference to various applications, methods, kits, and compositions, it will be understood that various changes and modifications can be made without departing from the teachings herein and the invention as claimed below. The foregoing examples are provided to better illustrate the disclosed teachings and are not intended to limit the scope of the teachings presented herein. While the present teachings have been described in terms of these exemplary embodiments, those skilled in the art will readily appreciate that numerous variations and modifications of these exemplary embodiments are possible without undue experimentation. All such variations and modifications are within the scope of the present teachings.

[0425] All references cited herein, including patents, patent applications, articles, textbooks, etc., and the references cited therein, to the extent they have not already been referenced, are incorporated herein by reference in their entirety. In the event that one or more of the incorporated literature and similar materials differs from or contradicts this application, this application controls, including, but not limited to, defined terms, term usage, described techniques, or the like.

[0426] The foregoing description and examples detail certain embodiments of the present disclosure and describe the best mode contemplated by the inventors. However, no matter how detailed the foregoing appears in text, it will be understood that the invention may be practiced in many ways and that the invention should be construed in accordance with the appended claims and any equivalents thereof.

Claims

1. A Cluster of Differentiation 70 (CD70)-specific chimeric antigen receptor (CAR) comprising an extracellular ligand-binding domain, a first transmembrane domain, and an intracellular signaling domain, wherein the extracellular domain contains a single-chain Fv fragment (scFv) that binds to the extracellular domain of CD70, and the intracellular signaling domain contains a 4-1BB signaling domain.

2. The extracellular domain comprises a heavy chain variable (VH) region comprising three CDRs from a VH region comprising the sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, or 381; 3. The CD70-specific CAR of claim 1, comprising a single-chain Fv fragment (scFv) containing a light chain variable (VL) region comprising three CDRs derived from the VL region set forth in

3. The VH region contains the sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, or 381, and the light chain variable (VL) region contains the sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378 or 380, or a variant thereof with one or more amino acid substitutions in amino acids not within the CDRs.

4. The CD70-specific CAR of claim 1, wherein the VH region contains a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 97, 98 or 99, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 100 or 101, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 102, and the light chain variable region (VL) contains the following CDRs: a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 217, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 218, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

219.

5. The CD70-specific CAR of claim 1, wherein the VH region contains the amino acid sequence shown in SEQ ID NO: 18, and the VL region contains the amino acid sequence shown in SEQ ID NO:

17.

6. The CD70-specific CAR of claim 1, wherein the VH region contains a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 145, 146 or 147, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 148 or 149, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 150, and the light chain variable region (VL) contains the following CDRs: a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 241, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 242, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

243.

7. The CD70-specific CAR of claim 1, wherein the VH region contains the amino acid sequence shown in SEQ ID NO: 34, and the VL region contains the amino acid sequence shown in SEQ ID NO:

33.

8. The CD70-specific CAR of claim 2 or 3, wherein each CDR is defined according to the Kabat definition, the Chothia definition, a combination of the Kabat definition and the Chothia definition, the AbM definition, or the contact definition of a CDR.

9. The CD70-specific CAR according to any one of claims 1 to 8, wherein the intracellular signaling domain comprises a CD3ζ signaling domain.

10. The CD70-specific CAR according to any one of claims 1 to 9, wherein the intracellular signaling domain comprises a 4-1BB domain.

11. The CD70-specific CAR according to any one of claims 1 to 10, further comprising a second intracellular signaling domain.

12. The CD70-specific CAR according to claim 11, wherein the second intracellular signaling domain contains a 4-1BB domain.

13. The CD70-specific CAR of any one of claims 1 to 12, further comprising a stalk domain between the extracellular ligand-binding domain and the first transmembrane domain.

14. 14. The CD70-specific CAR of claim 13, wherein the stalk domain is selected from the group consisting of a human CD8α hinge, an IgG1 hinge, and an FcγRIIIα hinge.

15. The CD70-specific CAR according to any one of claims 1 to 14, further comprising a CD20 epitope.

16. The CD70-specific CAR of claim 15, wherein the CD20 epitope comprises the amino acid sequence set forth in SEQ ID NO: 293 or SEQ ID NO: 294 or SEQ ID NO:

609.

17. The CD70-specific CAR according to claim 1, wherein the CD70-specific CAR comprises an amino acid sequence shown in any of SEQ ID NOs: 311 to 334.

18. The CD70-specific CAR of claim 1, wherein the CD70-specific CAR comprises the amino acid sequence set forth in SEQ ID NO: 319 or 327.

19. The CD70-specific CAR according to any one of claims 1 to 18, wherein the first transmembrane domain contains a CD8 α chain transmembrane domain.

20. The CD70-specific CAR according to any one of claims 1 to 19, further comprising another extracellular ligand-binding domain that is not specific for CD70.

21. The CD70-specific CAR according to any one of claims 1 to 19, wherein the extracellular ligand-binding domain, the first transmembrane domain, and the intracellular signaling domain are located on a single polypeptide.

22. The CD70-specific CAR according to any one of claims 1 to 19, further comprising a second transmembrane domain, wherein the first transmembrane domain and the extracellular ligand-binding domain are on a first polypeptide, and the second transmembrane domain and the intracellular signaling domain are on a second polypeptide, wherein the first transmembrane domain comprises a transmembrane domain derived from the α chain of a high-affinity IgE receptor (FcεRI), and the second transmembrane domain comprises a transmembrane domain derived from the γ chain or the β chain of FcεRI.

23. The CD70-specific CAR of claim 22, further comprising a third polypeptide comprising a third transmembrane domain fused to an intracellular signaling domain derived from a costimulatory molecule, wherein the third transmembrane domain comprises a transmembrane domain derived from the gamma or beta chain of FcεRI.

24. A polynucleotide comprising a nucleic acid sequence encoding the CD70-specific CAR according to any one of claims 1 to 23.

25. 25. The polynucleotide of claim 24, wherein the polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO: 336 or 337.

26. 26. An expression vector comprising the polynucleotide of claim 24 or 25.

27. An engineered immune cell that expresses the CD70-specific CAR according to any one of claims 1 to 23 on its cell surface membrane.

28. 28. The engineered immune cell of claim 27, further comprising another CAR that is not specific for CD70.

29. 29. The engineered immune cell of claim 27 or 28, further comprising a polynucleotide encoding a suicide polypeptide.

30. 30. The engineered immune cell of claim 29, wherein the suicide polypeptide is RQR8.

31. 31. The engineered immune cell of any one of claims 27 to 30, wherein the immune cell is derived from an inflammatory T lymphocyte, a cytotoxic T lymphocyte, a regulatory T lymphocyte, or a helper T lymphocyte.

32. 32. The engineered immune cell of any one of claims 27-31, further comprising disruption of one or more endogenous genes, wherein the endogenous genes encode TCR alpha, TCR beta, CD52, glucocorticoid receptor (GR), deoxycytidine kinase (dCK), CD70, or an immune checkpoint protein, such as programmed death-1 (PD-1).

33. 32. The engineered immune cell of any one of claims 27 to 31, further comprising disruption of TCRα and CD52, or TCRα, CD52 and CD70.

34. 34. The engineered immune cell of any one of claims 27 to 33, wherein the immune cell is obtained from a healthy donor.

35. The engineered immune cell of any one of claims 27 to 33, wherein the immune cell is obtained from a patient.

36. The engineered immune cell of any one of claims 27 to 35 for use as a medicament.

37. 37. The engineered immune cell of claim 36, wherein the medicament is for use in the treatment of cancer.

38. 38. The engineered immune cell of claim 37, wherein the cancer is selected from the group consisting of renal cell carcinoma, glioblastoma, glioma such as low-grade glioma, non-Hodgkin's lymphoma (NHL), Hodgkin's disease (HD), Waldenstrom's hypergammaglobulinemia, acute myeloid leukemia, multiple myeloma, diffuse large cell lymphoma, follicular lymphoma, and non-small cell lung cancer.

39. A population of cells according to any one of claims 27 to 38, a) the cell population comprises greater than 20%, 30% or 40% of stem cell memory cells and central memory cells; and / or b) said cell population achieves a lysis rate of CD70-expressing cells relative to recurrent CD70-expressing cells of greater than 10%, 20%, 30% or 40% at day 6 as measured using the assay disclosed in Example 4.

40. 40. The cell population of claim 39, wherein the cell population achieves a lysis rate of CD70-expressing cells relative to recurrent CD70-expressing cells of greater than 20% at day 6 as measured using the assay disclosed in Example 4.

41. 1. A method for manipulating immune cells, comprising: a) providing immune cells; and b) expressing at least one CD70-specific CAR according to any one of claims 1 to 23 on the surface of the cell.

42. a) providing immune cells; b) introducing into said cell at least one polynucleotide encoding said CD70-specific CAR; and 42. The method of claim 41, comprising: c) expressing said polynucleotide in said cell.

43. a) providing immune cells; b) introducing into said cell at least one polynucleotide encoding said CD70-specific CAR; and c) introducing at least one other CAR that is not specific for CD70.

44. 1. A method of treating a subject in need thereof, comprising: a) providing an immune cell expressing the CD70-specific CAR according to any one of claims 1 to 23 on its surface; and b) administering said immune cells to said patient.

45. A pharmaceutical composition comprising the engineered immune cell of any one of claims 27 to 39.

46. A method for treating a condition associated with malignant cells expressing CD70 in a subject, comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition of claim 45.

47. 47. The method of claim 46, wherein the condition is cancer.

48. 48. The method of claim 47, wherein the cancer is selected from the group consisting of renal cell carcinoma, glioblastoma, glioma such as low-grade glioma, non-Hodgkin's lymphoma (NHL), Hodgkin's disease (HD), Waldenstrom's hypergammaglobulinemia, acute myeloid leukemia, multiple myeloma, diffuse large cell lymphoma, follicular lymphoma, and non-small cell lung cancer.

49. A method for inhibiting tumor growth or progression in a subject having malignant cells that express CD70, comprising administering to the subject in need thereof an effective amount of the pharmaceutical composition of claim 45.

50. A method for inhibiting metastasis of malignant cells expressing CD70 in a subject, comprising administering to the subject in need thereof an effective amount of the pharmaceutical composition described in claim 45.

51. A method for inducing tumor regression in a subject having malignant cells that express CD70, comprising administering to the subject in need thereof an effective amount of the pharmaceutical composition of claim 45.

52. A Cluster of Differentiation 70-specific chimeric antigen receptor (CD70-specific CAR) comprising an extracellular ligand-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular domain contains a single-chain Fv fragment (scFv) that binds to an extracellular domain of CD70 having a heavy chain variable (VH) region and a light chain variable (VL) region; a) the VH region contains an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 18, and the VL region contains an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 17; or b) A CD70-specific CAR, wherein the VH region contains an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 34, and the VL region contains an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:

33.

53. 53. The CD70-specific CAR of claim 52, wherein the extracellular domain contains an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:

319.

54. 53. The CD70-specific CAR of claim 52, wherein the extracellular domain contains an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:

327.

55. A polynucleotide encoding a CD70-specific CAR, the polynucleotide comprising: a) contains a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 297 and at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 298; or b) A polynucleotide containing a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 307 and at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:

308.

56. A chimeric antigen receptor (CAR) comprising an antigen-binding molecule that specifically binds to CD70, wherein the antigen-binding molecule comprises: a) SEQ ID NOs: 49-51, 55-57, 61-63, 67-69, 73-75, 79-81, 85-87, 91-93, 97-99, 103-105, 109-111, 115-117, 121-123, 127-129, 133-135, 139-141, 145-147, 151-153, 157-159, 163-165, 169-171, 175-177, 181-183, 187-189, 382-384, 388-39 a variable heavy chain CDR1 comprising an amino acid sequence selected from the group consisting of: 0, 394-396, 400-402, 406-408, 412-414, 418-420, 424-426, 430-432, 436-438, 442-444, 448-450, 454-456, 460-462, 466-468, 472-474, 478-480, 484-486, 490-492, 496-498, 502-504, and 508-510; b) SEQ ID NOs: 52, 53, 58, 59, 64, 65, 70, 71, 76, 77, 82, 83, 88, 89, 94, 95, 100, 101, 106, 107, 112, 113, 118, 119, 124, 125, 130, 131, 136, 137, 142, 143, 148, 149, 154, 155, 160, 161, 166, 167, 172, 173, 178, 179, 184, 185, 190, 191, 385, 386, 391, 3 a variable heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of: 92, 397, 398, 403, 404, 409, 410, 415, 416, 421, 422, 427, 428, 433, 434, 439, 440, 445, 446, 451, 452, 457, 458, 463, 464, 469, 470, 475, 476, 481, 482, 487, 488, 493, 494, 499, 500, 505, 506, 511, and 512; c) a variable heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 387, 393, 399, 405, 411, 417, 423, 429, 435, 441, 447, 453, 459, 465, 471, 477, 483, 489, 495, 501, 507, and 513; d) a variable light chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 193, 196, 199, 202, 205, 208, 211, 214, 217, 220, 223, 226, 229, 232, 235, 238, 241, 244, 247, 250, 253, 256, 259, 262, 514, 517, 520, 523, 526, 529, 532, 535, 538, 541, 544, 547, 550, 553, 556, 559, 562, 565, 568, 571, 574, and 577; e) a variable light chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 194, 197, 200, 203, 206, 209, 212, 215, 218, 221, 224, 227, 230, 233, 236, 239, 242, 245, 248, 251, 254, 257, 260, 263, 515, 518, 521, 524, 527, 530, 533, 536, 539, 542, 545, 548, 551, 554, 557, 560, 563, 566, 569, 572, 575, and 578; and f) a variable light chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 225, 228, 231, 234, 237, 240, 243, 246, 249, 252, 255, 258, 261, 264, 516, 519, 522, 525, 528, 531, 534, 537, 540, 543, 546, 549, 552, 555, 558, 561, 564, 567, 570, 573, 576, and 579.

57. The antigen-binding molecule a) respectively, SEQ ID NO: i) 49-51, 52-53, 54; ii) 55-57, 58-59, 60; iii) 61-63, 64-65, 66; iv) 67-69, 70-71, 72; v) 73-75, 76-77, 78; vi) 79-81, 82-83, 84; vii) 85-87, 88-89, 90; viii) 91-93, 94-95, 96; ix) 97-99, 100-101, 102; x) 103-105, 106-107, 108; xi) 109-111, 112-113, 114; xii) 115-117, 118-119, 120; xiii) 121-123, 124-125, 126; xiv) 127-129, 130-131, 132; xv) 133-135, 136-137, 138; xvi) 139-141, 142-143, 144; xvii) 145-147, 148-149, 150; xviii) 151-153, 154-155, 156; xix) 157-159, 160-161, 162; xx) 163-165, 166-167, 168; xxi) 169-171, 172-173, 174; xxii) 175-177, 178-179, 180; xxiii) 181-183, 184-185, 186; xxiv) 187-189, 190-191, 192; xxv) 382-384, 385-386, 387; xxvi) 388-390, 391-392, 393; xxvii) 394-396, 397-398, 399; xxviii) 400-402, 403-404, 405; xxix) 406-408, 409-410, 411; xxx) 412-414, 415-416, 417; xxxi) 418-420, 421-422, 423; xxxii) 424-426, 427-428, 429; xxxiii) 430-432, 433-434, 435; xxxiv) 436-438, 439-440, 441; xxxv) 442-444, 445-446, 447; xxxvi) 448-450, 451-452, 453; xxxvii) 454-456, 457-458, 459; xxxviii) 460-462, 463-464, 465; xxxix) 466-468, 469-470, 471; xl) 472-474, 475-476, 477; xli) 478-480, 481-482, 483; xlii) 484-486, 487-488, 489; xliii) 490-492, 493-494, 495; xliv) 496-498, 499-500, 501; xlv) 502-504, 505-506, 507; or xlvi) a variable heavy chain domain comprising a CDRH1, CDRH2, and CDRH3 amino acid sequence selected from one of: 508-510, 511-512, 513; and b) respectively, the sequence number i) 193, 194, 195; ii) 196, 197, 198; iii) 199, 200, 201; iv) 202, 203, 204; v) 205, 206, 207; vi) 208, 209, 210; vii) 211, 212, 213; viii) 214, 215, 216; ix) 217, 218, 219; x) 220, 221, 222; xi) 223, 224, 225; xii) 226, 227, 228; xiii) 229, 230, 231; xiv) 232, 233, 234; xv) 235, 236, 237; xvi) 238, 239, 240; xvii) 241, 242, 243; xviii) 244, 245, 246; xix) 247, 248, 249; xx) 250, 251, 252; xxi) 253, 254, 255; xxii) 256, 257, 258; xxiii) 259, 260, 261; xxiv) 262, 263, 264; xxv) 514, 515, 516; xxvi) 517, 518, 519; xxvii) 520, 521, 522; xxviii) 523, 524, 525; xxix) 526, 527, 528; xxx) 529, 530, 531; xxxi) 532, 533, 534; xxxii) 535, 536, 537; xxxiii) 538, 539, 540; xxxiv) 541, 542, 543; xxxv) 544, 545, 546; xxxvi) 547, 548, 549; xxxvii) 550, 551, 552; xxxviii) 553, 554, 555; xxxix) 556, 557, 558; xl) 559, 560, 561; xli) 562, 563, 564; xlii) 565, 566, 567; xliii) 568, 569, 570; xliv) 571, 572, 573; xlv) 574, 575, 576; and xlvi) a variable light chain domain comprising the amino acid sequences of CDRL1, CDRL2, and CDRL3 selected from one of 577, 578, and 579.

58. The antigen-binding molecules each have the sequence represented by SEQ ID NO: a) 49-51, 52-53, 54, 193, 194, 195; b) 55-57, 58-59, 60, 196, 197, 198; c) 61-63, 64-65, 66, 199, 200, 201; d) 67-69, 70-71, 72, 202, 203, 204; e) 73-75, 76-77, 78, 205, 206, 207; f) 79-81, 82-83, 84, 208, 209, 210; g) 85-87, 88-89, 90, 211, 212, 213; h) 91-93, 94-95, 96, 214, 215, 216; i) 97-99, 100-101, 102, 217, 218, 219; j) 103-105, 106-107, 108, 220, 221, 222; k) 109-111, 112-113, 114, 223, 224, 225; l) 115-117, 118-119, 120, 226, 227, 228; m) 121-123, 124-125, 126, 229, 230, 231; n) 127-129, 130-131, 132, 232, 233, 234; o)133~135、136~137、138、235、236、237; p)139~141、142~143、144、238、239、240; q)145~147、148~149、150、241、242、243; r)151~153、154~155、156、244、245、246; s)157~159、160~161、162、247、248、249; t)163~165、166~167、168、250、251、252; u)169~171、172~173、174、253、254、255; v)175~177、178~179、180、256、257、258; w)181~183、184~185、186、259、260、261; x)187~189、190~191、192、262、263、264; y)382~384、385~386、387、514、515、516; z)388~390、391~392、393、517、518、519; aa)394~396、397~398、399、520、521、522; bb)400~402、403~404、405、523、524、525; cc)406~408、409~410、411、526、527、528; dd)412~414、415~416、417、529、530、531; ee)418~420、421~422、423、532、533、534; ff)424~426、427~428、429、535、536、537; gg)430~432、433~434、435、538、539、540; hh)436~438、439~440、441、541、542、543; ii)442~444、445~446、447、544、545、546; jj)448~450、451~452、453、547、548、549; kk)454~456、457~458、459、550、551、552; ll)460~462、463~464、465、553、554、555; mm)466~468、469~470、471、556、557、558; nn)472~474、475~476、477、559、560、561; oo)478~480、481~482、483、562、563、564; pp)484~486、487~488、489、565、566、567; qq) 490-492, 493-494, 495, 568, 569, 570; rr) 496-498, 499-500, 501, 571, 572, 573; ss) 502-504, 505-506, 507, 574, 575, 576; and tt) 508-510, 511-512, 513, 577, 578, 579. The CD70-specific CAR of claim 57, comprising the amino acid sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 selected from one of:

59. The antigen-binding molecule a) a variable light chain domain comprising the amino acid sequences of CDRH1, CDRH2, and CDRH3, respectively, selected from one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, and 380; and and b) a variable heavy chain domain comprising the amino acid sequences of CDRL1, CDRL2, and CDRH3 selected from one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, and 381, respectively.

60. The antigen-binding molecules each have the sequence represented by SEQ ID NO: a) 1 and 2; b) 3 and 4; c) 5 and 6; d) 7 and 8; e) 9 and 10; f) 11 and 12; g) 13 and 14; h) 15 and 16; i) 17 and 18; j) 19 and 20; k) 21 and 22; l) 23 and 24; m) 25 and 26; n) 27 and 28; o) 29 and 30; p) 31 and 32; q) 33 and 34; r) 35 and 36; s) 37 and 38; t) 39 and 40; u) 41 and 42; v) 43 and 44; w) 45 and 46; x) 47 and 48; y) 338 and 339; z) 340 and 341; aa) 342 and 343; bb) 344 and 345; cc) 346 and 347; dd) 348 and 349; ee) 350 and 351; ff) 352 and 353; gg) 354 and 355; hh) 356 and 357; ii) 358 and 359; jj) 360 and 361; kk) 362 and 363; ll) 364 and 365; mm) 366 and 367; nn) 368 and 369; oo) 370 and 371; pp) 372 and 373; qq) 374 and 375; rr) 376 and 377; ss) 378 and 379; and 60. The CD70-specific CAR of claim 59, comprising an amino acid sequence of a light chain variable domain and a heavy chain variable domain selected from one of:

61. The CD70-specific CAR according to any one of claims 56 to 60, wherein the intracellular signaling domain comprises a CD3ζ signaling domain.

62. The CD70-specific CAR according to any one of claims 56 to 61, wherein the intracellular signaling domain contains a 4-1BB domain.

63. The CD70-specific CAR according to any one of claims 56 to 62, further comprising a second intracellular signaling domain.

64. The CD70-specific CAR of claim 63, wherein the second intracellular signaling domain contains a 4-1BB domain.

65. The CD70-specific CAR of any one of claims 56 to 64, further comprising a stalk domain between the extracellular ligand-binding domain and the first transmembrane domain.

66. 66. The CD70-specific CAR of claim 65, wherein the stalk domain is selected from the group consisting of a human CD8α hinge, an IgG1 hinge, and an FcγRIIIα hinge.

67. The CD70-specific CAR according to any one of claims 56 to 66, further comprising a CD20 epitope.

68. The CD70-specific CAR of claim 67, wherein the CD20 epitope comprises the amino acid sequence set forth in SEQ ID NO: 293 or SEQ ID NO: 294 or SEQ ID NO:

609.

69. The CD70-specific CAR according to any one of claims 56 to 68, wherein the first transmembrane domain contains a CD8 α chain transmembrane domain.

70. The CD70-specific CAR according to any one of claims 56 to 69, further comprising another extracellular ligand-binding domain that is not specific for CD70.

71. The CD70-specific CAR according to any one of claims 56 to 70, wherein the extracellular ligand-binding domain, the first transmembrane domain, and the intracellular signaling domain are on a single polypeptide.

72. The CD70-specific CAR according to any one of claims 56 to 70, further comprising a second transmembrane domain, wherein the first transmembrane domain and the extracellular ligand-binding domain are on a first polypeptide, and the second transmembrane domain and the intracellular signaling domain are on a second polypeptide, wherein the first transmembrane domain comprises a transmembrane domain derived from the α chain of a high-affinity IgE receptor (FcεRI), and the second transmembrane domain comprises a transmembrane domain derived from the γ chain or the β chain of FcεRI.

73. The CD70-specific CAR of claim 72, further comprising a third polypeptide comprising a third transmembrane domain fused to an intracellular signaling domain derived from a costimulatory molecule, wherein the third transmembrane domain comprises a transmembrane domain derived from the gamma or beta chain of FcεRI.

74. A polynucleotide comprising a nucleic acid sequence encoding the CD70-specific CAR according to any one of claims 56 to 73.

75. 75. An expression vector comprising the polynucleotide of claim 74.

76. An engineered immune cell that expresses the CD70-specific CAR of any one of claims 56 to 73 on its cell surface membrane.

77. 77. The engineered immune cell of claim 76, further comprising another CAR that is not specific for CD70.

78. 78. The engineered immune cell of claim 76 or 77, further comprising a polynucleotide encoding a suicide polypeptide.

79. 79. The engineered immune cell of claim 78, wherein the suicide polypeptide is RQR8.

80. 80. The engineered immune cell of any one of claims 76-79, wherein the immune cell is derived from an inflammatory T lymphocyte, a cytotoxic T lymphocyte, a regulatory T lymphocyte, or a helper T lymphocyte.

81. 81. The engineered immune cell of any one of claims 76-80, further comprising disruption of one or more endogenous genes, wherein the endogenous genes encode TCR alpha, TCR beta, CD52, glucocorticoid receptor (GR), deoxycytidine kinase (dCK), CD70, or an immune checkpoint protein such as, for example, programmed death-1 (PD-1).

82. 82. The engineered immune cell of any one of claims 76-81, further comprising disruption of TCRα and CD52, or TCRα, CD52 and CD70.

83. 83. The engineered immune cell of any one of claims 76 to 82, wherein the immune cell is obtained from a healthy donor.

84. 83. The engineered immune cell of any one of claims 76 to 82, wherein the immune cell is obtained from a patient.

85. 85. The engineered immune cell of any one of claims 76 to 84 for use as a medicament.

86. 86. The engineered immune cell of claim 85, wherein the medicament is for use in the treatment of cancer.

87. 87. The engineered immune cell of claim 86, wherein the cancer is selected from the group consisting of renal cell carcinoma, glioblastoma, glioma such as low-grade glioma, non-Hodgkin's lymphoma (NHL), Hodgkin's disease (HD), Waldenstrom's hypergammaglobulinemia, acute myeloid leukemia, multiple myeloma, diffuse large cell lymphoma, follicular lymphoma, and non-small cell lung cancer.

88. A population of cells according to any one of claims 76 to 87, a) the cell population comprises greater than 20%, 30% or 40% of stem cell memory cells and central memory cells; and / or b) said cell population achieves a lysis rate of CD70-expressing cells relative to recurrent CD70-expressing cells of greater than 10%, 20%, 30% or 40% at day 6 as measured using the assay disclosed in Example 4.

89. 89. The cell population of claim 88, wherein the cell population achieves a lysis rate of CD70-expressing cells relative to recurrent CD70-expressing cells of greater than 20% at day 6 as measured using the assay disclosed in Example 4.

90. 1. A method for manipulating immune cells, comprising: a) providing immune cells; and b) introducing into the cell at least one polynucleotide encoding the CD70-specific CAR of any one of claims 56 to 73.

91. 91. The method of manipulating immune cells of claim 90, further comprising introducing at least one other CAR that is not specific for CD70.

92. 1. A method of treating a subject in need thereof, comprising: a) providing an immune cell expressing the CD70-specific CAR according to any one of claims 56 to 73 on its surface; and b) administering said immune cells to said patient.

93. A pharmaceutical composition comprising the engineered immune cell of any one of claims 76 to 87.

94. A method for treating a condition associated with malignant cells expressing CD70 in a subject, comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition of claim 93.

95. 95. The method of claim 94, wherein the condition is cancer.

96. 96. The method of claim 95, wherein the cancer is selected from the group consisting of renal cell carcinoma, glioblastoma, glioma such as low-grade glioma, non-Hodgkin's lymphoma (NHL), Hodgkin's disease (HD), Waldenstrom's hypergammaglobulinemia, acute myeloid leukemia, multiple myeloma, diffuse large cell lymphoma, follicular lymphoma, and non-small cell lung cancer.

97. A method for inhibiting tumor growth or progression in a subject having malignant cells that express CD70, comprising administering to the subject in need thereof an effective amount of the pharmaceutical composition of claim 93.

98. A method for inhibiting metastasis of malignant cells expressing CD70 in a subject, comprising administering to the subject in need thereof an effective amount of the pharmaceutical composition of claim 93.

99. A method for inducing tumor regression in a subject having malignant cells that express CD70, comprising administering to said subject in need thereof an effective amount of the pharmaceutical composition of claim 93.

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