Chimeric antigen receptors and binding agents targeting Claudin 18.2 and uses thereof

Abstract

Provided herein are Claudin 18.2 binding agents and chimeric antigen receptors (CARs) that comprise Claudin 18.2 binding molecules that specifically bind to Claudin 18.2, and the immune cells, such as CAR-T cells, that comprise these Claudin 18.2 specific CARs.Also provided are the methods for producing and using Claudin 18.2 specific CARs and Claudin 18.2 binding agents, and the immune cells that comprise Claudin 18.2 specific CARs.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of priority to U.S. Provisional Patent Application No. a3 / 428,308, filed November 28, 2022, and U.S. Provisional Patent Application No. 63 / 590,719, filed October 16, 2023, the contents of both applications being incorporated herein by reference in their entireties.

[0002] Sequence Listing Reference This application contains a Sequence Listing that has been submitted electronically in XML file format, which is incorporated herein by reference in its entirety. The XML copy, created on November 9, 2023, is named AT-057-03WO_SL.xml and is 236,349 bytes in size.

[0003] The present disclosure relates to Claudin 18.2-binding agents and chimeric antigen receptors (CARs), including antigen-binding molecules that bind to Claudin 18.2, polynucleotides encoding same, and methods of using same to treat cancer in patients. [Background technology]

[0004] There is a need to develop more targeted and potent therapies for proliferative disorders in general, and for gastric, gastroesophageal junction (GEJ), and pancreatic cancer in particular.

[0005] Adoptive transfer of immune cells genetically modified to recognize malignant tumor-associated antigens has shown promise as a new approach to cancer treatment (see, for example, Brenner et al., Current Opinion in Immunology, 22(2):251-257(2010); Rosenberg et al., Nature Reviews Cancer, 8(4):299-308(2008)). Immune cells can be genetically modified to express chimeric antigen receptors (CARs), fusion proteins consisting of the Claudin 18.2 antigen recognition portion and a T cell activation domain (see, for example, 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)). CAR-containing immune cells, e.g., CAR-T cells (CAR-T), are engineered to have antigen specificity while retaining or enhancing their ability to recognize and kill target cells.

[0006] Claudin 18.2, a splice variant of Claudin 18, is a tight junction molecule involved in regulating epithelial cell permeability, barrier function, and polarity. Claudin 18.2 expression is strictly limited to the tight junctions of gastric mucosal cells, thereby making it inaccessible to targeted therapeutic agents. In addition to limited normal tissue expression, Claudin 18.2 is highly expressed in different types of primary and metastatic cancers, including gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, and ovarian cancer (Sahin et al., Clinical Cancer Research 14.23 (2008): 7624-7634). Malignant transformation in cancer cells leads to exposure of the Claudin 18.2 epitope, making it an ideal target for targeted therapy. There is a need for the treatment of cancers, particularly malignant tumors, with Claudin 18.2 expression, such as gastric cancer, gastroesophageal junction (GEJ) cancer, and pancreatic cancer. Provided herein are methods and compositions that address this need. Summary of the Invention

[0007] Provided herein are chimeric antigen receptors (CARs) comprising Claudin 18.2 antigen binding domains that specifically bind to Claudin 18.2, and immune cells, such as CAR-T cells, comprising these Claudin 18.2-specific CARs. Also provided are methods for producing and using these Claudin 18.2-specific CARs and immune cells comprising these Claudin 18.2-specific CARs. The CAR T cells targeting Claudin 18.2 described herein exhibit good transduction efficiency, in vitro phenotype, and potent anti-tumor activity in vitro and in vivo. Also provided herein are anti-Claudin 18.2 binding agents, such as antibodies that bind to Claudin 18.2, and methods for producing and using them. The anti-Claudin 18.2 binding agents provided herein bind to human Claudin 18.2.

[0008] In one aspect, the present disclosure provides a chimeric antigen receptor ("CAR") comprising an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises a Claudin 18.2 antigen-binding domain (e.g., scFv) that specifically binds to Claudin 18.2, and the antigen-binding domain comprises: (a) a variable heavy chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3, 16-18, 31-33, 46-48, 61-63, 76-78, 89-91, 102-104, 115, 116, and 117; (b) a variable heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-5, 19-20, 34-35, 49-50, 64-65, 79-80, 92-93, 105-106, 118, and 119; and (c) a variable heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 21, 36, 51, 66, 78, 89-91, 102-104, 115, 116, and 117. (d) a variable heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 22, 37, 52, 67, 82, 95, 108 and 121; (e) a variable light chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 23, 38, 53, 68, 83, 96, 109 and 122; and (f) a variable light chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 24, 39, 54, 69, 84, 97, 110 and 123.

[0009] In some embodiments of the CARs disclosed herein, the CAR comprises an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises a Claudin 18.2 domain that specifically binds to Claudin 18.2. 18.2 antigen-binding domain, wherein the antigen-binding domain comprises: (a) a variable heavy chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3, 16-18, 31-33, 46-48, 61-63, 76-78, 89-91, 102-104, 115, 116, and 117; (b) a variable heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-5, 19-20, 34-35, 49-50, 64-65, 79-80, 92-93, 105-106, 118, and 119; and (c) a variable heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 21, 36, 51, 66, 81, 94, 107, and 120.

[0010] In some embodiments of the CAR disclosed herein, the CAR comprises an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises a Claudin 18.2 antigen-binding domain that specifically binds to Claudin 18.2, and the antigen-binding domain comprises: (a) a variable light chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 22, 37, 52, 67, 82, 95, 108, and 121; (b) a variable light chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 23, 38, 53, 68, 83, 96, 109, and 122; and (c) a variable light chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 24, 39, 54, 69, 84, 97, 110, and 123.

[0011] In some embodiments of the CAR disclosed herein, the CAR comprises an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises a Claudin 18.2 antigen-binding domain that specifically binds to Claudin 18.2, and the antigen-binding domain comprises at least one of: (a) a variable heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 25, 40, 55, 70, 85, 98, 111, and 124; and (b) a variable light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 26, 41, 56, 71, 86, 99, 112, and 125, wherein the variable heavy chain and the variable light chain are linked by at least one linker.

[0012] In some embodiments of the CAR disclosed herein, the CAR comprises an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises a Claudin 18.2 antigen-binding domain that specifically binds to Claudin 18.2, and the antigen-binding domain comprises (a) a variable heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 25, 40, 55, 70, 85, 98, 111, and 124, and (b) a variable light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 26, 41, 56, 71, 86, 99, 112, and 125, wherein the variable heavy chain and the variable light chain are linked by at least one linker.

[0013] In some embodiments of the CARs disclosed herein, the VH region comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, 2, or 3, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 4 or 5, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6, and the VL region comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9.

[0014] In some embodiments of the CARs disclosed herein, the VH region comprises the amino acid sequence set forth in SEQ ID NO: 10, and the VL region comprises the amino acid sequence set forth in SEQ ID NO: 11.

[0015] In some embodiments of the CARs disclosed herein, the VH region comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 16, 17, or 18, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 19 or 20, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 21, and the VL region comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 22, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 23, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 24.

[0016] In some embodiments of the CARs disclosed herein, the VH region comprises the amino acid sequence set forth in SEQ ID NO:25, and the VL region comprises the amino acid sequence set forth in SEQ ID NO:26.

[0017] In some embodiments of the CARs disclosed herein, the VH region comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 31, 32, or 33, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34 or 35, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 36, and the VL region comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 37, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 38, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 39.

[0018] In some embodiments of the CARs disclosed herein, the VH region comprises the amino acid sequence set forth in SEQ ID NO:40, and the VL region comprises the amino acid sequence set forth in SEQ ID NO:41.

[0019] In some embodiments of the CARs disclosed herein, the VH region comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 46, 47, or 48, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 49 or 50, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 51, and the VL region comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 52, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 53, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 54.

[0020] In some embodiments of the CARs disclosed herein, the VH region comprises the amino acid sequence set forth in SEQ ID NO:55, and the VL region comprises the amino acid sequence set forth in SEQ ID NO:56.

[0021] In some embodiments of the CARs disclosed herein, the VH region comprises 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 comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 67, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 68, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 69.

[0022] In some embodiments of the CARs disclosed herein, the VH region comprises the amino acid sequence set forth in SEQ ID NO:70, and the VL region comprises the amino acid sequence set forth in SEQ ID NO:71.

[0023] In some embodiments of the CARs disclosed herein, the VH region comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 76, 77, or 78, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 79 or 80, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 81, and the VL region comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 82, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 83, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 84.

[0024] In some embodiments of the CARs disclosed herein, the VH region comprises the amino acid sequence set forth in SEQ ID NO:85, and the VL region comprises the amino acid sequence set forth in SEQ ID NO:86.

[0025] In some embodiments of the CARs disclosed herein, the VH region comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 89, 90, or 91, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 92 or 93, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 94, and the VL region comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 95, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 96, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 97.

[0026] In some embodiments of the CARs disclosed herein, the VH region comprises the amino acid sequence set forth in SEQ ID NO:98, and the VL region comprises the amino acid sequence set forth in SEQ ID NO:99.

[0027] In some embodiments of the CARs disclosed herein, the VH region comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 102, 103, or 104, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 105 or 106, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 107, and the VL region comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 108, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 109, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 110.

[0028] In some embodiments of the CARs disclosed herein, the VH region comprises the amino acid sequence set forth in SEQ ID NO:111, and the VL region comprises the amino acid sequence set forth in SEQ ID NO:112.

[0029] In some embodiments of the CARs disclosed herein, the VH region comprises 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 comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 121, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 122, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 123.

[0030] In some embodiments of the CARs disclosed herein, the VH region comprises the amino acid sequence set forth in SEQ ID NO: 124, and the VL region comprises the amino acid sequence set forth in SEQ ID NO: 125.

[0031] In some embodiments of the CARs disclosed herein, the CAR comprises an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises a Claudin 18.2 antigen-binding domain that specifically binds to Claudin 18.2, and wherein the antigen-binding domain comprises a sequence selected from the group consisting of those scFvs presented in Table 1c. In some embodiments, the extracellular domain of the CAR comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to any one of the amino acid sequences of SEQ ID NOs: 12, 27, 42, 57, 72, 187, 189, 191, and 193.

[0032] In some embodiments, the present disclosure provides a Claudin 18.2-specific CAR comprising an extracellular ligand-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular domain comprises a single chain Fv fragment (scFv) that binds to the extracellular domain of Claudin 18.2, and wherein the scFv comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises an amino acid sequence that shares at least about 94%, 95%, 96%, 97%, 98%, 99%, or up to 100% with SEQ ID NO: 10, and the VL region comprises an amino acid sequence that shares at least about 94%, 95%, 96%, 97%, 98%, 99%, or up to 100% with SEQ ID NO: 11; or the VH region comprises an amino acid sequence that shares at least about 94%, 95%, 96%, 97%, 98%, 99%, or 100% with SEQ ID NO:25, and the VL region comprises an amino acid sequence that shares at least about 94%, 95%, 96%, 97%, 98%, 99%, or 100% with SEQ ID NO:26; or the VH region comprises an amino acid sequence that shares at least about 94%, 95%, 96%, 97%, 98%, 99%, or 100% with SEQ ID NO:40, and the VL region comprises an amino acid sequence that shares at least about 94%, 95%, 96%, 97%, 98%, 99%, or 100% with SEQ ID NO:41; or the VH region comprises an amino acid sequence that shares at least about 94%, 95%, 96%, 97%, 98%, 99%, or 100% with SEQ ID NO: 55, and the VL region comprises an amino acid sequence that shares at least about 94%, 95%, 96%, 97%, 98%, 99%, or 100% with SEQ ID NO: 56; or the VH region comprises an amino acid sequence that shares at least about 94%, 95%, 96%, 97%, 98%, 99%, or 100% with SEQ ID NO: 70, and the VL region comprises an amino acid sequence that shares at least about 94%, 95%, 96%, 97%, 98%, 99%, or 100% with SEQ ID NO: 71; or the VH region comprises an amino acid sequence that shares at least about 94%, 95%, 96%, 97%, 98%, 99%, or up to 100% with SEQ ID NO: 85, and the VL region comprises an amino acid sequence that shares at least about 94%, 95%, 96%, 97%, 98%, 99%, or up to 100% with SEQ ID NO: 86; orthe VH region comprises an amino acid sequence that shares at least about 94%, 95%, 96%, 97%, 98%, 99%, or 100% with SEQ ID NO:98, and the VL region comprises an amino acid sequence that shares at least about 94%, 95%, 96%, 97%, 98%, 99%, or 100% with SEQ ID NO:99; or the VH region comprises an amino acid sequence that shares at least about 94%, 95%, 96%, 97%, 98%, 99%, or 100% with SEQ ID NO:111, and the VL region comprises an amino acid sequence that shares at least about 94%, 95%, 96%, 97%, 98%, 99%, or 100% with SEQ ID NO:112; or The VH region comprises an amino acid sequence that shares at least about 94%, 95%, 96%, 97%, 98%, 99%, or up to 100% with SEQ ID NO: 124, and the VL region comprises an amino acid sequence that shares at least about 94%, 95%, 96%, 97%, 98%, 99%, or up to 100% with SEQ ID NO: 125.

[0033] In some embodiments of the CARs disclosed herein, the CAR comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 13, 28, 43, 58, 73, 87, 100, 113, 126, 128, 130, 132, 179-183, 184-186, and 195-198, with or without a signal sequence. In some embodiments, the chimeric antigen receptor comprises the amino acid sequence of any one of SEQ ID NOs: 13, 28, 43, 58, 73, 87, 100, 113, 126, 128, 130, 132, 179-183, 184-186, 195-198, 200-201, and 208-211, with or without a signal sequence. The present disclosure provides the amino acid sequences of the CARs disclosed herein, with and without a signal sequence.

[0034] In some embodiments, the chimeric antigen receptor described herein further comprises a hinge domain. In some embodiments, the hinge domain and transmembrane domain comprise the hinge domain and transmembrane domain of human CD8α. In some embodiments, the hinge domain and transmembrane domain comprise the hinge domain and transmembrane domain of human CD28.

[0035] In some embodiments, the intracellular domain of the chimeric antigen receptor comprises at least one costimulatory domain. In some embodiments, the CARs disclosed herein comprise one costimulatory domain. In some embodiments, the CARs disclosed herein comprise two costimulatory domains.

[0036] In some embodiments, the costimulatory domain of the chimeric antigen receptor is selected from the group consisting of CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1 (CD1 la / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class I molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, signaling lymphocyte activation molecule (SLAM protein), activating NK cell receptor, BTLA, Toll ligand receptor Body, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE, CD103, ITGAL, CD1 1a, LFA-1, ITGAM, CD1 1b, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAMI(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT The signal transduction domains are ligands that specifically bind to AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83, or any combination thereof.

[0037] In some embodiments, the costimulatory domain comprises the signaling region of CD28, or a variant thereof. In some embodiments, the CD28 costimulatory domain comprises the amino acid sequence of SEQ ID NO: 158 or 174.

[0038] In some embodiments, the costimulatory domain comprises the CD28.YMFM intracellular domain (SEQ ID NO: 216). In some embodiments, the CD28.YMFM intracellular domain (SEQ ID NO: 216) comprises the amino acid sequence of SEQ ID NO: 159.

[0039] In some embodiments, the costimulatory domain comprises the signaling region of 4-1BB / CD137. In some embodiments, the 4-1BB / CD137 costimulatory domain comprises SEQ ID NO: 137.

[0040] In some embodiments, the intracellular domain comprises at least one activation domain. In some embodiments, the activation domain comprises CD3. In some embodiments, the activation domain comprises the CD3 activation domain CD3 zeta. In some embodiments, the CD3 zeta comprises the amino acid sequence of SEQ ID NO: 138. In some embodiments, the CD3 zeta comprises the amino acid sequence of SEQ ID NO: 139.

[0041] In some embodiments, the chimeric antigen receptor is encoded by the polynucleotide sequence of any one of SEQ ID NOs: 15, 30, 45, 60, 75, 88, 101, 114, and 127.

[0042] In some embodiments, the disclosure provides a polynucleotide encoding a Claudin 18.2-specific CAR, wherein the polynucleotide comprises a nucleic acid sequence that shares at least 95%, 96%, 97%, 98%, 99%, or 100% with any one of SEQ ID NOs: 14, 15, 29, 30, 44, 45, 59, 60, 74, 75, 88, 101, 114, and 127.

[0043] For all polynucleotide sequences disclosed herein, alternative versions of the disclosed sequences may be substituted (in whole or in part) to optimize the sequence according to the codon preferences of the organism in which the sequence is expressed, or according to any other known method of codon optimization, to avoid recombination of polynucleotide sequences encoding similar molecules (e.g., two different versions of the CD3 zeta domain), and / or for any other practical reason. Those skilled in the art will recognize the degeneracy and codon preferences of the genetic code of various organisms, e.g., laboratory model organisms, and cell lines used for small-scale (e.g., laboratory) and commercial-scale production of CARs and antibodies such as those disclosed herein. For example, CHKim et al., Codon optimization for high-level expression of human erythropoietin(EPO) in mammalian cells.Gene.1997 Oct 15;199(1-2):293-301.doi:10.1016 / s0378-1119(97)00384-3.PMID:9358069, S.Guedan et al.,Engineering and Design of Chimeric Antigen Receptors.Mol Ther Methods Clin Dev.2018 Dec 31;12:145-156.doi:10.1016 / j.omtm.2018.12.009.PMID:30666307, PMCID:PMC6330382..

[0044] In some embodiments, the chimeric antigen receptor further comprises a safety switch.

[0045] In some embodiments, the safety switch comprises a CD20 mimotope, or a QBEND-10 epitope.

[0046] In some embodiments, the safety switch comprises one or more CD20 mimotopes or one or more QBEND-10 epitopes, or a combination thereof.

[0047] In some embodiments, the chimeric antigen receptor comprises one or more safety switches in the form of QR3, SR2, RSR, or R2S.

[0048] In some embodiments, the chimeric antigen receptor comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 128, 130, 132, and 184-186.

[0049] In some embodiments, the chimeric antigen receptor comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 128, 130, 132, with or without a signal sequence.

[0050] In some aspects, the present disclosure provides an isolated polynucleotide encoding any one of the chimeric antigen receptors described herein.

[0051] In another aspect, the present disclosure provides a vector comprising a polynucleotide encoding any one of the chimeric antigen receptors described herein.

[0052] In some embodiments, the vector is a retroviral vector, a DNA vector, a plasmid, an RNA vector, an adenoviral vector, an adeno-associated viral vector, a lentiviral vector, or any combination thereof.

[0053] In some embodiments, the extracellular domain of a chimeric antigen receptor described herein further comprises an anti-CD70 scFv that specifically binds to CD70. In some embodiments, the anti-CD70 scFv comprises the amino acid sequence of SEQ ID NO: 204, 205, and / or 206.

[0054] In another aspect, the present disclosure provides engineered immune cells that comprise or express (e.g., express on their cell surface membrane) the Claudin 18.2-specific chimeric antigen receptor described herein. In some embodiments, the engineered immune cells comprise another CAR that is not specific for Claudin 18.2. In some embodiments, the engineered immune cells comprise a polynucleotide encoding a suicide polypeptide. In some embodiments, the suicide polypeptide is RQP8.

[0055] In some aspects, the present disclosure provides engineered immune cells that contain or express a polynucleotide or vector encoding any one of the chimeric antigen receptors described herein.

[0056] In some embodiments, the engineered immune cells further comprise or express a CD70 binding protein. In some embodiments, the CD70 binding protein comprises an anti-CD70 antibody or antigen-binding fragment thereof, a transmembrane domain, and optionally a hinge domain. In some embodiments, the anti-CD70 antibody comprises the amino acid sequence of SEQ ID NO: 204, 205, and / or 206. In some embodiments, the CD70 binding protein further comprises a CD3z signaling domain or a CD3z signaling domain and does not comprise a costimulatory domain. In some embodiments, the CD70 binding protein comprises the amino acid sequence of SEQ ID NO: 207.

[0057] In some embodiments, the engineered immune cells further comprise or express a dominant negative receptor. In some embodiments, the dominant negative receptor can attenuate immune inhibitory signals present in the tumor microenvironment. In some embodiments, the dominant negative receptor is a PD1 or TGFβ receptor (TGFβR) dominant negative receptor. In some embodiments, the dominant negative receptor comprises a PD1 or TGFβR extracellular domain, a transmembrane domain, and does not comprise a functional intracellular signaling domain. In some embodiments, the PD1 or TGFβR extracellular domain comprises an extracellular domain derived from WT PD1 or WT TGFβR, or a variant thereof. In some embodiments, the dominant negative receptor comprises a CD8 or CD28 transmembrane domain, or a transmembrane domain derived from PD1 or TGFβR. In some embodiments, exemplary PD1 and TGFβR dominant negative receptors may comprise the amino acid sequence of SEQ ID NO: 212, 213, or 214.

[0058] In some embodiments, the engineered immune cells are or are derived from T cells, tumor-infiltrating lymphocytes (TILs), NK cells, TCR-expressing cells, dendritic cells, or NK-T cells. In some embodiments, the engineered immune cells are or are derived from inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes, or helper T lymphocytes.

[0059] In some embodiments, the engineered immune cells are autologous T cells. In some embodiments, the engineered immune cells are allogeneic T cells. In some embodiments, the engineered immune cells are obtained from a healthy donor. In some embodiments, the engineered immune cells are obtained from a patient.

[0060] In some embodiments, the engineered immune cells can comprise a disruption (e.g., knockout) of one or more endogenous genes encoding TCRα, TCRβ, CD52, glucocorticoid receptor (GR), deoxycytidine kinase (dCK), CD70, or immune checkpoint proteins such as, for example, programmed death-1 (PD-1).

[0061] In another aspect, the disclosure relates to a method of engineering an immune cell, the method comprising providing an immune cell and expressing at least one Claudin 18.2-specific CAR on the surface of the cell as described herein. In some embodiments, the method comprises providing an immune cell, introducing into the cell at least one polynucleotide encoding a Claudin 18.2-specific CAR as described herein, and expressing the polynucleotide in the cell, or expressing the polynucleotide in the cell, e.g., by providing in the cell appropriate elements (e.g., one or more transcription promoters and / or enhancers) that direct expression of the polynucleotide encoding the CAR.

[0062] In some embodiments, the method includes providing an immune cell, introducing into the cell at least one polynucleotide encoding a Claudin 18.2-specific CAR as described herein, introducing at least one other polynucleotide encoding a second polypeptide, e.g., a CAR that is not specific for Claudin 18.2, and expressing the polynucleotide in the cell, or expressing the polynucleotide in the cell, e.g., by providing the cell with appropriate elements that direct expression of the polynucleotide (e.g., one or more transcription promoters and / or enhancers).

[0063] In one aspect, the present disclosure provides pharmaceutical compositions. In some embodiments, the pharmaceutical compositions comprise engineered immune cells disclosed herein and at least one pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical compositions comprise an anti-Claudin 18.2 binding agent disclosed herein and at least one pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical compositions comprise engineered immune cells expressing an anti-Claudin 18.2 chimeric antigen receptor disclosed herein and at least one pharmaceutically acceptable excipient.

[0064] In some aspects, the present disclosure provides methods of treating a disease or disorder in a subject in need thereof, comprising administering to the subject an engineered immune cell disclosed herein or a pharmaceutical composition disclosed herein, e.g., an engineered immune cell expressing an anti-Claudin 18.2 chimeric antigen receptor disclosed herein, and at least one pharmaceutically acceptable excipient. In some embodiments, the engineered immune cell expresses an anti-Claudin 18.2 chimeric antigen receptor disclosed herein and expresses a second polypeptide, e.g., a second CAR.

[0065] In some embodiments, the disease or disorder is cancer.

[0066] In some embodiments, the disease or disorder is gastric cancer, gastroesophageal junction (GEJ) cancer, or pancreatic cancer.

[0067] In some embodiments, the disease or disorder is an autoimmune disease.

[0068] In some aspects, the present disclosure provides a method for treating a disease or disorder in a subject in need thereof, comprising administering to the subject an anti-Claudin 18.2 binding agent or a pharmaceutical composition comprising an anti-Claudin 18.2 binding agent as disclosed herein. In some embodiments, the disease or disorder is cancer. In some embodiments, the disease or disorder is gastric cancer, gastroesophageal junction (GEJ) cancer, or pancreatic cancer.

[0069] In another aspect, the present disclosure provides a method of inhibiting tumor growth or progression in a subject having malignant cells that express Claudin 18.2, the method comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition disclosed herein, e.g., a pharmaceutical composition comprising an engineered immune cell described herein and at least one pharmaceutically acceptable excipient, or a pharmaceutical composition comprising an anti-Claudin 18.2 binding agent described herein and at least one pharmaceutically acceptable excipient.

[0070] In another aspect, the present disclosure provides a method for inhibiting metastasis of malignant cells expressing Claudin 18.2 in a subject, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition disclosed herein, e.g., a pharmaceutical composition comprising an engineered immune cell described herein and at least one pharmaceutically acceptable excipient, or a pharmaceutical composition comprising an anti-Claudin 18.2 binding agent described herein and at least one pharmaceutically acceptable excipient.

[0071] In another aspect, the present disclosure provides a method of inducing tumor regression in a subject having malignant cells that express Claudin 18.2, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition disclosed herein, e.g., a pharmaceutical composition comprising an engineered immune cell described herein and at least one pharmaceutically acceptable excipient, or a pharmaceutical composition comprising an anti-Claudin 18.2 binding agent described herein and at least one pharmaceutically acceptable excipient.

[0072] In some embodiments, the engineered immune cells or pharmaceutical compositions are administered to the subject intravenously, subcutaneously, or intraperitoneally, or by intravenous injection, subcutaneous injection of intraperitoneal injection.

[0073] 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 chemotherapy. In some embodiments, the monoclonal antibody can 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 sunitinib or axitinib.

[0074] In another aspect, the present disclosure provides engineered immune cells that express the Claudin 18.2-specific CAR described herein on its cell surface membrane for use as a medicament. In some embodiments, the medicament is for use in treating cancer. In some embodiments, the medicament is for use in treating gastric cancer, gastroesophageal junction (GEJ) cancer, and pancreatic cancer. In some embodiments, the medicament is for use in treating autoimmune diseases.

[0075] In another aspect, the present disclosure provides an anti-Claudin 18.2 binding agent described herein for use as a medicament. In some embodiments, the medicament is for use in the treatment of cancer. In some embodiments, the medicament is for the treatment of gastric cancer, gastroesophageal junction (GEJ) cancer, and pancreatic cancer. In some embodiments, the anti-Claudin 18.2 binding agent is an antibody, antibody conjugate, or antigen-binding fragment thereof, optionally an F(ab')2 fragment, an Fab' fragment, an Fab fragment, an Fv fragment, an scFv fragment, a dsFv fragment, or a domain antibody (dAb) fragment, or a monoclonal antibody comprising an IgG constant region.

[0076] In some aspects, the present disclosure provides an article of manufacture comprising an engineered immune cell disclosed herein or a pharmaceutical composition disclosed herein, e.g., an engineered immune cell expressing a chimeric antigen receptor described herein or a pharmaceutical composition comprising an engineered immune cell.

[0077] In some embodiments, the present disclosure provides anti-Claudin 18.2 binding agents. In some embodiments, the anti-Claudin 18.2 binding agents comprise: (a) a variable heavy chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3, 16-18, 31-33, 46-48, 61-63, 76-78, 89-91, 102-104, and 115-117; (b) a variable heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-5, 19-20, 34-35, 49-50, 64-65, 79-80, 92-93, 105-106, and 118-119; and (c) a variable heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 21, 36, 51, 66, 81, 94, 107, 118-119. 20; (d) a variable heavy chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 22, 37, 52, 67, 82, 95, 108, 121; (e) a variable light chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 23, 38, 53, 68, 83, 96, 109, 122; and (f) a variable light chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 24, 39, 54, 69, 84, 97, 110, 123.

[0078] In some embodiments, the anti-Claudin 18.2 binding agent is an antibody, antibody conjugate, or antigen-binding fragment thereof, optionally an F(ab')2 fragment, a Fab' fragment, a Fab fragment, an Fv fragment, an scFv fragment, a dsFv fragment, or a domain antibody (dAb) fragment.

[0079] In some embodiments, the anti-Claudin 18.2 binding agent is a monoclonal antibody comprising an IgG constant region.

[0080] In some embodiments, the anti-Claudin 18.2 binding agent comprises a variable heavy (VH) chain sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 25, 40, 55, 70, 85, 98, 111, and 124.

[0081] In some embodiments, the anti-Claudin 18.2 binding agent comprises a variable light (VL) chain sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 26, 41, 56, 71, 86, 99, 112, and 125.

[0082] In some embodiments, the anti-Claudin 18.2 binding agent comprises a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 27, 42, 57, 72, 187, 189, 191, and 193.

[0083] In some embodiments, the anti-Claudin 18.2 binding agent is a fusion protein comprising an scFv fragment fused to an Fc constant region.

[0084] In some embodiments, the anti-Claudin 18.2 binding agent comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, 2 or 3, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 4 or 5, a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6, a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9.

[0085] In some embodiments, the anti-Claudin 18.2 binding agent comprises the amino acid sequence set forth in SEQ ID NO:10, and the VL region comprises the amino acid sequence set forth in SEQ ID NO:11.

[0086] In some embodiments, the anti-Claudin 18.2 binding agent comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 16, 17 or 18, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 19 or 20, a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 21, a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 22, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 23, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 24.

[0087] In some embodiments, the anti-Claudin 18.2 binding agent comprises the amino acid sequence set forth in SEQ ID NO:25, and the VL region comprises the amino acid sequence set forth in SEQ ID NO:26.

[0088] In some embodiments, the anti-Claudin 18.2 binding agent comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 31, 32 or 33, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34 or 35, a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 36, a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 37, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 38, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 39.

[0089] In some embodiments, the anti-Claudin 18.2 binding agent comprises the amino acid sequence set forth in SEQ ID NO:40, and the VL region comprises the amino acid sequence set forth in SEQ ID NO:41.

[0090] In some embodiments, the anti-Claudin 18.2 binding agent comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 46, 47 or 48, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 49 or 50, a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 51, a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 52, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 53, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 54.

[0091] In some embodiments, the anti-Claudin 18.2 binding agent comprises the amino acid sequence set forth in SEQ ID NO:55, and the VL region comprises the amino acid sequence set forth in SEQ ID NO:56.

[0092] In some embodiments, the anti-Claudin 18.2 binding agent comprises 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, a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 66, a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 67, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 68, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 69.

[0093] In some embodiments, the anti-Claudin 18.2 binding agent comprises the amino acid sequence set forth in SEQ ID NO:70, and the VL region comprises the amino acid sequence set forth in SEQ ID NO:71.

[0094] In some embodiments, the anti-Claudin 18.2 binding agent comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 76, 77 or 78, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 79 or 80, a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 81, a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 82, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 83, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 84.

[0095] In some embodiments, the anti-Claudin 18.2 binding agent comprises the amino acid sequence set forth in SEQ ID NO:85, and the VL region comprises the amino acid sequence set forth in SEQ ID NO:86.

[0096] In some embodiments, the anti-Claudin 18.2 binding agent comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 89, 90 or 91, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 92 or 93, a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 94, a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 95, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 96, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 97.

[0097] In some embodiments, the anti-Claudin 18.2 binding agent comprises the amino acid sequence set forth in SEQ ID NO:98, and the VL region comprises the amino acid sequence set forth in SEQ ID NO:99.

[0098] In some embodiments, the anti-Claudin 18.2 binding agent comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 102, 103 or 104, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 105 or 106, a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 107, a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 108, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 109, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 110.

[0099] In some embodiments, the anti-Claudin 18.2 binding agent comprises the amino acid sequence set forth in SEQ ID NO:111, and the VL region comprises the amino acid sequence set forth in SEQ ID NO:112.

[0100] In some embodiments, the anti-Claudin 18.2 binding agent comprises 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, a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 120, a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 121, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 122, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 123.

[0101] In some embodiments, the anti-Claudin 18.2 binding agent comprises the amino acid sequence set forth in SEQ ID NO:124, and the VL region comprises the amino acid sequence set forth in SEQ ID NO:125.

[0102] In some embodiments, the anti-Claudin 18.2 binding agent comprises a variable heavy (VH) chain sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 10, and a variable light (VL) chain sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to SEQ ID NO: 11.

[0103] In some embodiments, the anti-Claudin 18.2 binding agent comprises a variable heavy (VH) chain sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 25, and a variable light (VL) chain sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to SEQ ID NO: 26.

[0104] In some embodiments, the anti-Claudin 18.2 binding agent comprises a variable heavy (VH) chain sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 40, and a variable light (VL) chain sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 41.

[0105] In some embodiments, the anti-Claudin 18.2 binding agent comprises a variable heavy (VH) chain sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 55, and a variable light (VL) chain sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to SEQ ID NO: 56.

[0106] In some embodiments, the anti-Claudin 18.2 binding agent comprises a variable heavy (VH) chain sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 70, and a variable light (VL) chain sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 71.

[0107] In some embodiments, the anti-Claudin 18.2 binding agent comprises a variable heavy (VH) chain sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 85, and a variable light (VL) chain sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to SEQ ID NO: 86.

[0108] In some embodiments, the anti-Claudin 18.2 binding agent comprises a variable heavy (VH) chain sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:98, and a variable light (VL) chain sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to SEQ ID NO:99.

[0109] In some embodiments, the anti-Claudin 18.2 binding agent comprises a variable heavy (VH) chain sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:111, and a variable light (VL) chain sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:112.

[0110] In some embodiments, the anti-Claudin 18.2 binding agent comprises a variable heavy (VH) chain sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 124, and a variable light (VL) chain sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 125.

[0111] In some embodiments, the anti-Claudin 18.2 binding agent is a monospecific antibody. In some embodiments, the anti-Claudin 18.2 binding agent is a bispecific antibody. In some embodiments, the bispecific antibody also binds to CD3.

[0112] In some aspects, the present disclosure provides a pharmaceutical composition comprising an anti-Claudin 18.2 binding agent disclosed herein and a pharmaceutically acceptable excipient. In some aspects, the present disclosure provides a method for treating a disease or disorder in a subject in need thereof, comprising administering to the subject an anti-Claudin 18.2 binding agent or a pharmaceutical composition comprising an anti-Claudin 18.2 binding agent as disclosed herein. In some embodiments, the disease or disorder is cancer. In some embodiments, the disease or disorder is gastric cancer, gastroesophageal junction (GEJ) cancer, or pancreatic cancer. In some embodiments, the disease or disorder is cancer, an autoimmune disease, or an infectious disease. [Brief explanation of the drawings]

[0113] [Figure 1] Figure 1 is a series of plots showing that purified anti-Claudin 18.2 antibodies described herein bind to HEK-293T cells expressing human or mouse Claudin 18.2, but not to parental HEK-293T cells. Solid and dashed lines represent staining with anti-Claudin 18.2 antibodies or isotype controls, respectively. [Figure 2]Figure 2 is a series of plots showing antigen-specific killing of target cells using anti-Claudin 18.2 CAR T cells in a 3-day cytotoxicity assay. Non-transduced (NTD) T cells were used as a negative control. [Figure 3] Figures 3A-3C are a series of plots and tables showing the transduction efficiency and phenotype of anti-Claudin 18.2 CARs with safety switches. Figure 3A shows representative FACS plots demonstrating efficient transduction of anti-Claudin 18.2 CARs in different rituximab off-switch formats. Figure 3B summarizes the transduction efficiency in two different donors. Figure 3C shows the memory phenotype of CAR T cells at the end of production, as determined by FACS analysis of the expression of CD62L and CD45RO markers. [Figure 4] Figures 4A-4B are a series of plots showing the continuous killing of target cells using anti-Claudin 18.2 CAR T cells with and without a safety switch (Figure 4A only). Figure 4A shows long-term cytotoxicity against one gastric cancer cell line overexpressing Claudin 18.2 (MKN45 / hClaudin 18.2) and pancreatic cancer cell lines expressing endogenous Claudin 18.2 (PATU8988s, Panc05.04). Figure 4B shows long-term cytotoxicity against gastric cancer cell lines expressing endogenous Claudin 18.2 (SNU-601, SNU-620, NUGC-4, GSU). [Figure 5] Figure 5 is a series of bar graphs showing cytokine release by anti-Claudin 18.2 CAR T cells after co-culture with Claudin 18.2-positive gastric (SNU-601) and pancreatic (PATU8988s) cell lines at a 1:1 effector:target ratio for 24 hours. Supernatants were collected and IFN-γ, IL-2, and TNF-α levels were measured using MSD's Human ProInflammatory 9-Plex Kit. Dotted lines indicate the limit of detection for individual cytokines. [Figure 6]Figures 6A and 6B are plots showing tumor volume (Figure 6A) and body weight (Figure 6B) of mice treated with different anti-Claudin 18.2 CARs at 1 x 106 cell doses in a subcutaneous xenograft model (N = 5 per group). Figures 6C and 6D are plots showing tumor volume (Figure 6C) and body weight (Figure 6D) of mice treated with different anti-Claudin 18.2 CARs at 3 x 106 and 10 x 106 cell doses in the same subcutaneous xenograft model (N = 8 per group). Individual mouse body weight changes are plotted against the number of days after CAR T treatment in Figures 6E-6I. [Figure 7] Figures 7A-7B are plots showing tumor volume (Figure 7A) and body weight (Figure 7B) from an in vivo experiment using an SNU-601 intraperitoneal xenograft model. Representative bioluminescence imaging of the same mice as in Figure 7C. [Figure 8] Figures 8A-8D are plots showing tumor volume and body weight of mice treated with different anti-Claudin 18.2 CARs at 3 x 106 cell doses (Figures 8A and 8C) and 1 x 106 cell doses (Figures 8B and 8D) in the NUGC-4 subcutaneous model (N = 5 per group). Individual mouse body weight changes are plotted against days after CAR T treatment in Figures 8E-8I. Figure 8J shows CAR T cell proliferation in blood collected from mice treated with 3 x 106 CAR+ cells. Results represent the mean ± SEM. [Figure 9] Figure 9 shows the results of the off-target or on-target risk analysis of anti-Claudin 18.2 CAR T. [Figure 10] Figures 10A-C show the results of an analysis of Claudin 18.2 clone 2A4 CAR T cells or Claudin 18.2 clone 2A4 / CD70 tandem or dual CAR T cells. [Figure 11]Figures 11A-11B show data from an MLR assay. Claudin 18.2 CAR T cells expressing a CD70 CAR depleted alloreactive T cells (right panel) and resisted T cell-mediated rejection (left panel). Alloreactive T cell MLR was performed using TRACKO graft donor T cells co-expressing Claudin 18.2 CAR and CD70 CAR. Data are representative of two graft-host donor pairs (Figures 11A and 11B). Data represent the mean ± SEM. [Figure 12] Figures 12A-B show the results of comparing the activity of Claudin 18.2 CAR with the CD8 hinge domain and transmembrane domain and the CD28 hinge domain and transmembrane domain. DETAILED DESCRIPTION OF THE INVENTION

[0114] Provided herein are Claudin 18.2-specific antibodies and chimeric antigen receptors (CARs).The Claudin 18.2-specific CARs described herein comprise an extracellular domain, a transmembrane domain, and an intracellular domain, and the extracellular domain comprises a Claudin 18.2 antigen binding domain that specifically binds to Claudin 18.2, and a polynucleotide encoding these CARs.Also provided are immune cells, such as CAR-T cells, that comprise these Claudin 18.2-specific CARs, and pharmaceutical compositions that comprise these immune cells.Methods for producing and using these Claudin 18.2-specific CARs and immune cells that comprise these Claudin 18.2-specific CARs are also disclosed, for example, for the treatment of cancer.

[0115] I. Claudin 18.2 Binder The present disclosure provides a Claudin 18.2 binding agent (e.g., a molecule comprising a Claudin 18.2 antigen-binding domain, a Claudin 18.2 antibody, or a fragment thereof) that specifically binds to Claudin 18.2. As used herein, the term "antibody" refers to a polypeptide that contains sufficient standard immunoglobulin sequence elements to confer specific binding to a particular target antigen (e.g., Claudin 18.2). As is known in the art, naturally occurring intact antibodies are approximately 150 kD tetrameric agents consisting of two identical heavy chain polypeptides (about 50 kD each) and two identical light chain polypeptides (about 25 kD each) that associate with each other and bind to what is commonly referred to as a "Y-shaped" structure. Each heavy chain consists of at least four domains (each about 110 amino acids long)—an amino-terminal variable (VH) domain (located at the tip of the Y structure), followed by three constant domains: a CHI, a CH2, and a carboxy-terminal CH3 domain (located at the base of the stem of the Y). A short region known as the "switch" connects the variable and constant regions of the heavy chain. A "hinge" connects the CH2 and CH3 domains to the rest of the antibody. Two disulfide bonds in this hinge region bind the two heavy chain polypeptides to each other in an intact antibody. Each light chain consists of two domains: an amino-terminal variable (VL) domain followed by a carboxy-terminal constant (CL) domain, separated from each other by another "switch." Those skilled in the art are familiar with antibody structure and sequence elements and will recognize "variable" and "constant" regions in the provided sequences, and will understand that there can be some flexibility in the definition of the "boundary" between such domains, such that different presentations of the same antibody chain sequence can exhibit such boundaries at positions that are shifted by, for example, one or a few residues.

[0116] The assignment of amino acids to each of the framework, CDR, and variable domains is typically based on Kabat numbering (see, e.g., Kabat et al. in Sequences of Proteins of Immunological Interest, 5th Ed., NIH Publication 91-3242, Bethesda, Md. 1991), Chothia numbering (see, e.g., Chothia & Lesk, (1987), J Mol Biol 196:901-917; Al-Lazikani et al., (1997) J Mol Biol 273:927-948; Chothia et al., (1992) J Mol Biol 227:799-817; Tramontano et al., (1990) J Mol Biol 215(1):175-82; and U.S. Pat. No. 7,709,226), Contact numbering, or the AbM scheme (Antibody Modeling program, Oxford Molecular).

[0117] Therefore, in some embodiments, the CDRs of the Claudin 18.2 binding agents provided herein are numbered according to the Kabat numbering scheme. In other embodiments, the CDRs of the Claudin 18.2 binding agents provided herein are numbered according to the Chothia numbering scheme. In other embodiments, the CDRs of the Claudin 18.2 binding agents provided herein are numbered according to the Contact numbering scheme. In other embodiments, the CDRs of the Claudin 18.2 binding agents provided herein are numbered according to the AbM numbering scheme.

[0118] An intact antibody tetramer consists of two heavy-light chain dimers, where the heavy and light chains are linked to each other by one disulfide bond and two other disulfide bonds link the heavy chain hinge regions together, linking the dimers to form a tetramer. Naturally occurring antibodies are also glycosylated, typically on the CH2 domain. Each domain in a natural antibody has a structure characterized by an "immunoglobulin fold" formed from two beta sheets (e.g., three-, four-, or five-stranded sheets) packed together into a compressed antiparallel beta barrel. Each variable domain contains three hypervariable loops known as "complement-determining regions" (CDR1, CDR2, and CDR3) and four relatively invariant "framework" regions (FR1, FR2, FR3, and FR4). When a natural antibody folds, the FR regions form a beta sheet that provides the structural framework for the domain, and the CDR loop regions from both the heavy and light chains assemble in three-dimensional space to generate a single hypervariable antigen-binding site located at the tip of a Y-structure. The Fc region of a naturally occurring antibody binds to elements of the complement system and also to receptors on effector cells, including those that mediate cytotoxicity. As is known in the art, the affinity and / or other binding properties of the Fc region for Fc receptors can be modulated through glycosylation or other modifications. In some embodiments, antibodies produced and / or utilized according to the present invention comprise a glycosylated Fc domain, including Fc domains with modified or engineered glycosylation.

[0119] For purposes of the present invention, in certain embodiments, a polypeptide or complex of polypeptides comprising sufficient immunoglobulin domain sequences found in a natural antibody may be referred to and / or used as an "antibody," regardless of whether such polypeptide is produced naturally (e.g., produced by an organism in response to an antigen) or produced by recombinant engineering, chemical synthesis, or other artificial system or methodology. In some embodiments, antibodies are polyclonal, and in some embodiments, antibodies are monoclonal. In some embodiments, antibodies have constant region sequences characteristic of murine, rabbit, primate, or human antibodies. In some embodiments, antibody sequence elements are humanized, primatized, chimeric, etc., as known in the art.

[0120] Furthermore, as used herein, the term "antibody" can refer, in appropriate embodiments, to any of the constructs or formats known or developed in the art for utilizing the structural and functional characteristics of antibodies in alternative presentations (unless otherwise stated or clear from the context). For example, in some embodiments, antibodies utilized in accordance with the present invention include, but are not limited to, intact IgA, IgG, IgE, or IgM antibodies; bispecific or multispecific antibodies (e.g., Zybodies®, etc.); antibody fragments such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated CDRs or sets thereof, single chain Fvs; polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); camelid antibodies; masked antibodies (e.g., Probodies®); Small Modular ImmunoPharmaceuticals ("SMIPs™"); single chain or tandem diabodies (TandAb®); VHHs; Anticalins®; Nanobodies® minibodies; BiTEs®; ankyrin repeat proteins or DARPINs®; Avimers®; DARTs; TCR-like antibodies; Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®, Centyrins®; and KALBITOR®. In some embodiments, the antibody may lack a covalent modification (e.g., attachment of a glycan) that it has when produced naturally. In some embodiments, the antibody may contain a covalent modification (e.g., attachment of a glycan, a payload (e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.), or other pendant group (e.g., polyethylene glycol, etc.)).

[0121] Antibodies include antibody fragments. Antibodies also include, but are not limited to, polyclonal, monoclonal, chimeric dAb (domain antibody), single chain, Fab , F a , F (ab ) 2 fragments, scFv, and F ab The antibody may be a whole antibody, or an immunoglobulin, or an antibody fragment.

[0122] As detailed above, a whole antibody consists of two pairs of "light chains" (LC) and "heavy chains" (HC) (such light chain (LC) / heavy chain pairs are abbreviated herein as LC / HC). The light and heavy chains of such antibodies are polypeptides consisting of several domains. In a whole antibody, each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises heavy chain constant domains CH1, CH2, and CH3 (antibody classes IgA, IgD, and IgG), and optionally a heavy chain constant domain CH4 (antibody classes IgE and IgM). Each light chain comprises a light chain variable domain VL and a light chain constant domain CL. The variable domains VH and VL can be further subdivided into regions of hypervariability, called complementarity-determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (Janeway, CA, Jr, et al, (2001). Immunobiology., 5th ed., Garland Publishing, and Woof, J., Burton, D., Nat Rev Immunol 4 (2004) 89-99). Two pairs of heavy and light chains (HC / LC) can specifically bind to the same antigen. Therefore, the whole antibody is a bivalent monospecific antibody. Such "antibodies" include, for example, murine antibodies, human antibodies, chimeric antibodies, humanized antibodies, and genetically engineered antibodies (variant antibodies or mutated antibodies), so long as their characteristic properties are retained. In some embodiments, the antibody or binding agent is a humanized antibody, particularly a recombinant human antibody or humanized antibody.

[0123] In some embodiments, an antibody or binding agent can be "symmetric." By "symmetric," we mean that the antibody or binding agent has the same type of Fv region (e.g., an antibody has two Fab regions). In some embodiments, an antibody or binding agent can be "asymmetric." By "asymmetric," we mean that the antibody or binding agent has at least two different types of Fv regions (e.g., an antibody has Fab and scFv regions, Fab and scFv2 regions, or Fab-VHH regions). Various asymmetric antibody or binding agent architectures are known in the art (Brinkman and Kontermann et al., 2017 Mabs(9)(2):182-212).

[0124] As used herein, the term "antibody agent" refers to an agent that specifically binds to a particular antigen. In some embodiments, the term encompasses any polypeptide or polypeptide complex that contains sufficient immunoglobulin structural elements to confer specific binding. Exemplary antibody agents include, but are not limited to, monoclonal or polyclonal antibodies. In some embodiments, an antibody agent may include one or more constant region sequences characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, an antibody agent may include one or more sequence elements that are humanized, primatized, chimerized, etc., as known in the art. In many embodiments, the term "antibody agent" is used to refer to one or more of the constructs or formats known or developed in the art for utilizing the structural and functional characteristics of antibodies in alternative presentations. For example, antibody agents utilized in accordance with the present invention include, but are not limited to, intact IgA, IgG, IgE, or IgM antibodies; bispecific or multispecific antibodies (e.g., Zybodies®, etc.); antibody fragments such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated CDRs or sets thereof, and single chain Fvs; polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); camelid antibodies; masked antibodies (e.g., Probodies®); Small Modular ImmunoPharmaceuticals ("SMIPs™"); single chain or tandem diabodies (TandAbs®); VHHs; Anticalins®; Nanobodies®; minibodies; BiTEs®; ankyrin repeat proteins or DARPINs®; Avimers®; DARTs; TCR-like antibodies; Adnectins®; Affilins®; Trans-bodies®; Afffibodies®; TrimerX®; MicroProteins; Fynomers®, Centyrins®; and KALBITOR®.

[0125] In some embodiments, an antibody may lack covalent modifications (e.g., glycan attachment) that it has when produced naturally. In some embodiments, an antibody may contain covalent modifications (e.g., the attachment of a glycan, a payload (e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.), or other pendant groups (e.g., polyethylene glycol, etc.). In many embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence includes one or more structural elements recognized by those skilled in the art as complementarity-determining regions (CDRs), and in some embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence includes at least one CDR (e.g., at least one heavy chain CDR and / or at least one light chain CDR) that is substantially identical to that found in a reference antibody. In some embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the art as an immunoglobulin variable domain. In some embodiments, an antibody agent is a polypeptide protein having a binding domain that is homologous or largely homologous to an immunoglobulin binding domain.

[0126] The encoded antibody or antigen-binding molecule of the present invention can be single-chain or double-chain. In some embodiments, the antibody or antigen-binding molecule is single-chain. In certain embodiments, the antigen-binding molecule is selected from the group consisting of scFv, Fab, Fab', Fv, F(ab')2, dAb, and any combination thereof.

[0127] In some embodiments, the anti-Claudin 18.2 antibody agent is isolated. In some embodiments, the antibody agent can be purified to greater than 95% or 99% purity, as determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC) (see, for example, Flatman et al., J. Chromatogr., B 848:79-87 (2007)). In some aspects, the present disclosure provides a composition comprising a Claudin 18.2 binding agent (e.g., a Claudin 18.2-specific antibody) and a pharmaceutically acceptable carrier or excipient.

[0128] In some embodiments, the anti-Claudin 18.2 antibody agent comprises an Fc domain. The Fc domain can interact with cell surface receptors, which can enable the antibody to activate the immune system. In IgG, IgA, and IgD antibody isotypes, the Fc region is composed of two identical protein fragments derived from the second and third constant domains of the antibody's two heavy chains, while IgM and IgE Fc regions contain three heavy chain constant domains (C) in each polypeptide chain. H The Fc region of IgG contains domains 2-4. The Fc region of IgG may have a highly conserved N-glycosylation site (N297). Glycosylation of the Fc fragment may be essential for Fc receptor-mediated activity. The N-glycans attached to this site may be primarily complex-type, core-fucosylated, biantennary structures.

[0129] The constant regions of the light and heavy chains may not be directly involved in binding the antibody to the antigen, but may influence the orientation of the variable region. The constant regions may also exhibit various effector functions, such as participation in antibody-dependent complement-mediated lysis or antibody-dependent cellular cytotoxicity, through interactions with effector molecules and cells.

[0130] The disclosed anti-Claudin 18.2 antibody agents can be antibodies of any isotype, including isotype IgA, isotype IgD, isotype IgE, isotype IgG, or isotype IgM. In some embodiments, the anti-Claudin 18.2 antibody contains an IgG1, IgG2, IgG3, or IgG4 constant domain.

[0131] Provided herein are Claudin 18.2 binding agents (e.g., antibodies) that can bind to various regions or domains of Claudin 18.2 targets. Epitopes can be, for example, consecutive amino acids of Claudin 18.2 targets (linear or continuous epitopes), or can be combined from two or more non-contiguous regions of Claudin 18.2 targets (conformational, non-linear, discontinuous, or discontinuous epitopes). The epitopes that Claudin 18.2 antigen-binding domains bind to can be determined by various assays, such as NMR spectroscopy, X-ray diffraction crystallography, ELISA assays, hydrogen / deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligo-peptide scanning assays, flow cytometry, and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping).

[0132] In some embodiments, the Claudin 18.2 binding agent comprises a variable heavy chain (VH), wherein the amino acid sequence of the VH is selected from the VH sequences presented in Table 1a. In some embodiments, the anti-Claudin 18.2 binding agent comprises an immunoglobulin variable heavy chain having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence presented in Table 1a. Kabat CDR definitions are shown in bold, and Chothia CDR definitions are underlined.

[0133] [Table 1]

[0134] In some embodiments, the Claudin 18.2 binding agent comprises a variable light chain (VL), wherein the amino acid sequence of the VL is selected from the VL sequences presented in Table lb. In some embodiments, the anti-Claudin 18.2 binding agent comprises an immunoglobulin light chain variable region having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence presented in Table lb.

[0135] [Table 2]

[0136] Provided herein are Claudin 18.2 binding agents, wherein the Claudin 18.2 antigen-binding domain comprises a variable heavy chain (VH) and a variable light chain (VL), wherein the amino acid sequence of the VH is selected from the VH sequences presented in Table 1a, and the amino acid sequence of the VL is selected from the VL sequences presented in Table 1b.

[0137] In some embodiments, the Claudin 18.2 binding agent comprises heavy chain CDR1, CDR2, and CDR3. In some embodiments, the heavy chain CDR1, CDR2, and CDR3 sequences are selected from the heavy chain CDRs presented in Table 1c. In Table 1d, Kabat CDR definitions are shown in bold and Chothia CDR definitions are underlined.

[0138] [Table 3-1] [Table 3-2]

[0139] In some embodiments, the Claudin 18.2 binding agent comprises a light chain CDR1, CDR2, and CDR3. In some embodiments, the light chain CDR1, CDR2, and CDR3 sequences are selected from the light chain CDRs presented in Table 1d. In Table 1e, Kabat CDR definitions are shown in bold and Chothia CDR definitions are underlined.

[0140] [Table 4]

[0141] The present disclosure encompasses modifications to Claudin 18.2 antibody agents containing the sequences shown in Tables 1a, 1b, 1c, 1d, and 1e, including functionally equivalent Claudin 18.2 antibody agents with modifications that do not significantly affect their properties, as well as variants with enhanced or reduced activity and / or affinity. For example, amino acid sequences can be mutated to obtain Claudin 18.2 antigen binding agents with desired binding affinity to Claudin 18.2. Modification of polypeptides is routine practice 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 using chemical analogs.

[0142] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue or the antibody fused to an epitope tag. Other insertional variants of antibody molecules include the fusion to the N- or C-terminus of the antibody of an enzyme or a polypeptide which increases the half-life of the antibody in the blood circulation.

[0143] Substitutional variants have at least one amino acid residue in the antigen-binding domain removed and a different residue inserted in its place. In some embodiments, sites of interest for substitutional mutagenesis include hypervariable regions / CDRs, although FR changes are also contemplated. Conservative substitutions are shown under the heading "Conservative Substitutions" in Table 2. If such substitutions result in altered biological activity, more substantial changes, designated "exemplary substitutions" in Table 2 or further described below for amino acid classes, can be introduced and the products screened.

[0144] [Table 5]

[0145] i.Antibody fragment In one aspect, the anti-Claudin 18.2 antibody agent of any of the above embodiments may be an antibody fragment. Antibody fragments include a portion of an intact antibody, such as the antigen-binding or variable region of the intact antibody. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab'), Fv, diabodies, linear antibodies, multispecific antibodies formed from antibody and scFv fragments, and other fragments described below. In some embodiments, the antibody is a full-length antibody, such as an intact IgG1 antibody, or other antibody classes or isotypes described herein. (See, e.g., Hudson et al., Nat. Med., 9:129-134 (2003); Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, pp. 269-315 (1994); Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993); WO 93 / 01161; and U.S. Pat. Nos. 5,571,894, 5,869,046, 6,248,516, and 5,587,458.) A full-length, intact, or whole antibody is an antibody having a structure substantially similar to that of a native antibody or an antibody having a heavy chain containing an Fc region as defined herein. Antibody fragments can be produced by a variety of techniques, as known in the art, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., E. coli or phage).

[0146] An Fv antibody fragment contains a complete antigen recognition and binding site. This fragment may comprise a dimer of one heavy chain variable region domain and one light chain variable region domain in tight, non-covalent association. The folding of these two domains produces six hypervariable loops (three loops from each H chain and L chain) that contribute amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable region (or half of an Fv containing only three antigen-specific CDRs) has the ability to recognize and bind to an antigen, although with lower affinity than the entire binding site.

[0147] Diabodies are V domains that are fused together so that interchain pairing of V domains is achieved but intrachain pairing is not achieved. H and V L A small antibody fragment prepared by constructing an sFv fragment with a short linker (e.g., about 5-10 residues) between the domains, resulting in a bivalent fragment. Bispecific diabodies are small antibody fragments prepared by constructing an sFv fragment with a short linker (e.g., about 5-10 residues) between the domains, resulting in a bivalent fragment. H and V L It is a heterodimer of two crossover sFv fragments in which the domains are present on different polypeptide chains (see, e.g., EP 404,097, WO 93 / 11161, and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)).

[0148] Domain antibodies (dAbs), which can be produced in fully human form, are the smallest known antigen-binding fragments of antibodies, ranging from about 11 kDa to about 15 kDa. Dabs are the robust variable regions of the heavy and light chains of immunoglobulins (V and V, respectively). H and V L). They are highly expressed in microbial cell culture, exhibit favorable biophysical properties (e.g., including but not limited to, solubility and temperature stability), and are well suited to selection and affinity maturation by in vitro selection systems (e.g., phage display, etc.). dAbs are biologically active as monomers, and because of their small size and inherent stability, they can be formatted into larger molecules to generate drugs with extended serum half-lives or other pharmacological activities. (See, e.g., WO94 / 25591 and US20030130496).

[0149] Fv and scFv are species with intact binding sites that lack constant regions. Therefore, they may be suitable for reducing non-specific binding during in vivo use. Single-chain Fv (sFv or scFv) consists of VFs linked in a single polypeptide chain. H and V L An sFv polypeptide is an antibody fragment that contains an antibody domain. The V domain enables the sFv to form the desired structure for antigen binding. H and V L A polypeptide linker may further be included between the domains (see, e.g., Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); Borrebaeck 1995 (infra)). scFv fusion proteins can be constructed to result in fusion of an effector protein at either the amino or carboxy terminus of the sFv. Antibody fragments may also be "linear antibodies" (see, e.g., U.S. Pat. No. 5,641,870). Such linear antibody fragments may be monospecific or bispecific. The amino acid sequence of an exemplary Claudin 18.2-specific scFv is provided in Table 1e, with Kabat CDR definitions in bold and Chothia CDR definitions underlined.

[0150] [Table 6]

[0151] [Table 7] JPEG2025539380000009.jpg243170 JPEG2025539380000010.jpg236170

[0152] In some embodiments, the Claudin 18.2 antigen-binding domain comprises an scFv containing the light chain variable (VL) and heavy chain variable (VH) regions of a Claudin 18.2-specific monoclonal antibody connected by a flexible linker. Single-chain variable region fragments may be generated by linking the light and / or heavy chain variable regions using a linking peptide. An example of a linking peptide is the amino acid sequence (GGGGS). xwhere x is 1, 2, 3, 4, or 5 (SEQ ID NO: 215) (GGGGS(GS sequence (1)) is SEQ ID NO: 163). 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: 135). Generally, linkers can be short, flexible polypeptides, generally consisting of about 20 or fewer amino acid residues. Furthermore, linkers can be modified for additional functionality, such as, for example, attachment of a drug or attachment to a solid support. Single-chain variants can be produced recombinantly or synthetically. For synthetic production of scFvs, an automated synthesizer can be used. For recombinant production of scFv, a suitable plasmid containing a polynucleotide encoding the scFv can be introduced into a suitable host cell, either 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. A polynucleotide encoding the desired scFv 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.

[0153] In exemplary embodiments, provided herein is a Claudin 18.2 antigen-binding domain comprising a VH region comprising the VH CDR1, VH CDR2, and VH CDR3 of the VH sequence shown in Table 1a, and / or a VL region comprising the VL CDR1, VL CDR2, and VL CDR3 of the VL sequence shown in Table 1b. In some embodiments, the VH and VL are linked together by a linker, e.g., a linker listed in Table 7a, e.g., a "GS" linker comprising only G (glycine) and S (serine) residues. In some embodiments, the linker comprises the amino acid sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 135) ("(GGGGS)4"). In some embodiments, the linker may be encoded by a DNA sequence comprising GGCGGTGGAGGCTCCGGAGGGGGGGGCTCTGGCGGAGGGGGCTCC (SEQ ID NO: 151). In some embodiments, the linker may be encoded by a DNA sequence comprising GGCGGCGGCGGCTCTGGAGGAGGAGGCAGCGGCGGAGGAGGCTCCGGAGGCGGCGGCTCT (SEQ ID NO: 152). In some embodiments, the linker comprises the amino acid sequence GGGGSGGGSGGGGGS (SEQ ID NO: 162). In some embodiments, the linker is an scFv Whitlow linker, which may comprise the amino acid sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO: 164). The scFv Whitlow linker may be encoded by a DNA sequence comprising GGGTCTACATCCGGCTCCGGGAAGCCCGGAAGTGGCGAAGGTAGTACAAAGGGG (SEQ ID NO: 165). In some embodiments, the VH and VL sequences of the disclosed scFvs may be oriented with the VH sequence located at the N-terminus of the scFv, followed by the linker, followed by the VL sequence, while in other embodiments, the scFv may be oriented with the VL sequence located at the N-terminus, followed by the linker, followed by the VH sequence.

[0154] ii. Chimeric and humanized antibodies In some embodiments, the anti-Claudin 18.2 antibody agent is or comprises a monoclonal antibody, including a chimeric antibody, a humanized antibody, or a human antibody.

[0155] In some embodiments, the anti-Claudin 18.2 antibody agents provided herein may be chimeric antibodies (see, e.g., U.S. Pat. No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). A chimeric antibody may be an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species. In one example, a chimeric antibody may contain a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In a further example, a chimeric antibody may be a "class-switched" antibody in which the class or subclass has been changed from that of the parent antibody. A chimeric antibody includes an antigen-binding fragment thereof.

[0156] In some embodiments, a chimeric antibody can be a humanized antibody (see, e.g., Almagro and Fransson, Front. Biosci., 13:1619-1633 (2008); Riechmann et al., Nature, 332:323-329 (1988); Queen et al., Proc. Natl. Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005); Padlan, Mol. Immunol. 28:489-498 (1991); Dall'Acqua et al., Methods. 36:43-60 (2005); Osbourn et al., Methods. 36:61-68 (2005); and Klimka et al., Br. J. Cancer. 83:252-260 (2000)). Humanized antibodies are chimeric antibodies containing amino acid residues derived from non-human hypervariable regions and human FRs. In certain embodiments, humanized antibodies comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable regions (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all of the FRs correspond to those of a human antibody. Humanized antibodies may optionally contain at least a portion of an antibody constant region derived from a human antibody.

[0157] Non-human antibodies may be humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. A humanized antibody may contain one or more variable domains comprising one or more CDRs, or portions thereof, derived from a non-human antibody. A humanized antibody may contain one or more variable domains comprising one or more FRs, or portions thereof, derived from a human antibody sequence. A humanized antibody may optionally contain at least a portion of a human constant region. In some embodiments, one or more FR residues in a humanized antibody are substituted with the corresponding residue from a non-human antibody (e.g., the antibody from which the CDR residues are derived) to restore or improve antibody specificity or affinity.

[0158] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best fit" method, framework regions derived from the consensus sequence of human antibodies of a particular subpopulation of light chain variable regions or heavy chain variable regions, human mature (somatically mutated) framework regions or human germline framework regions, and framework regions derived from screening FR libraries (e.g., Sims et al., J. Immunol, 151:2296 (1993); Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol, 151:2623 (1993); Baca et al., J. Biol. Chem., 272:10678-10684 (1997); and Rosok et al., J. Immunol, 151:2623 (1993)). al., J. Biol. Chem., 271:22611-22618 (1996).

[0159] iii. Human antibodies In some embodiments, the anti-Claudin 18.2 antibody agent provided herein is a human antibody.Human antibodies can be produced using various techniques known in the art (see, for example, van Dijk and van de Winkel, Curr. Opin. Pharmacol, 5:368-74 (2001); and Lonberg, Curr. Opin. Immunol, 20:450-459 (2008)).Human antibodies may have amino acid sequences that correspond to the amino acid sequences of antibodies produced by humans or human cells, or derived from non-human sources that utilize human antibody repertoires or other human antibody coding sequences.This definition of human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies can be prepared by administering an immunogen (e.g., Claudin 18.2 protein) to transgenic animals that have been engineered to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge (see, e.g., Lonberg, Nat. Biotech., 23:1117-1125 (2005); U.S. Patent Nos. 6,075,181, 6,150,584, 5,770,429, and 7,041,870, and U.S. Patent Application Publication No. US 2007 / 0061900). The human variable regions from intact antibodies produced by such animals may be further engineered, for example, by combining them with different human constant regions.

[0160] Human antibodies may also be made by hybridoma-based methods. For example, human antibodies can be produced from human myeloma and mouse-human heteromyeloma cell lines using human B cell hybridoma technology, and other methods (e.g., Kozbor, J. Immunol, 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (1987); Boerner et al., J. Immunol, 147:86 (1991); Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006); U.S. Patent No. 7,189,826; Ni, Xiandai Mianyixue, 26(4):265-268 (2006); Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005); and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005). Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences can then be combined with desired human constant regions.

[0161] Modification of the oligosaccharides in antibodies can be made, for example, to create antibody variants with specific improved properties. For example, antibody glycosylation variants can have improved CDC function. In some embodiments, the present disclosure contemplates antibody variants that retain some, but not all, effector functions, making them desirable candidates for applications in which the in vivo half-life of the antibody is important but certain effector functions (such as complement) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / depletion of CDC activity. iv. Antibody derivative

[0162] In some embodiments, the antibody agents provided herein may be further modified to contain additional nonproteinaceous moieties that are known in the art and readily available. Moieties suitable for derivatization of antibodies may include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone), polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have manufacturing advantages due to its stability in water.

[0163] The polymers may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they may be the same or different molecules.

[0164] In some embodiments, a conjugate of an antibody and a non-proteinaceous moiety is provided that can be selectively heated by exposure to radiation. In some embodiments, the non-proteinaceous moiety can be a carbon nanotube (see, for example, Kam et al., Proc. Natl. Acad. Sci. USA, 102:11600-11605 (2005)). The radiation can be of any wavelength, including but not limited to, a wavelength that is not harmful to normal cells but heats the non-proteinaceous moiety to a temperature that kills cells in the vicinity of the antibody-non-proteinaceous moiety.

[0165] A Claudin 18.2 binder (e.g., a molecule comprising an antigen-binding domain) is said to "specifically bind" to its target antigen (e.g., human, cynomolgus monkey, or mouse Claudin 18.2) if the dissociation constant (Kd) is about 1 nM. An antigen-binding domain specifically binds to an antigen with "high affinity" when the Kd is 1-5 nM, and with "ultra-high affinity" when the Kd is 0.1-0.5 nM. In one embodiment, the antigen-binding domain has a Kd of about 1 nM. In one embodiment, the off-rate is <1 x 10 -5 In other embodiments, the antigen-binding domain is about 1 x 10 -7 M~1×10 -12 M, and in yet another embodiment, the antigen-binding domain binds with a Kd of about 1 x 10 -5 M~1×10 -12 It binds with a Kd of M.

[0166] As provided herein, the antigen-binding domain of the present disclosure specifically binds to mammalian Claudin 18.2 (e.g., human Claudin 18.2, cynomolgus monkey Claudin 18.2, or mouse Claudin 18.2). In certain embodiments, the Claudin 18.2 antigen-binding domain of the present disclosure specifically binds to mammalian Claudin 18.2 at a concentration of 1×10 -6 Less than M, 1 x 10 -7 Less than M, 1 x 10 -8 Less than M or 1 x 10 -9 In one particular embodiment, the Claudin 18.2 antigen-binding domain binds to mammalian Claudin 18.2 (e.g., human Claudin 18.2, cynomolgus monkey Claudin 18.2, or mouse Claudin 18.2) with a Kd of less than 1×10 -7 In another embodiment, the Claudin 18.2 antigen-binding domain binds to mammalian Claudin 18.2 (e.g., human Claudin 18.2, cynomolgus monkey Claudin 18.2, or mouse Claudin 18.2) with a Kd of less than 1 x 10 -8In some embodiments, the Claudin 18.2 antigen-binding domain binds to mammalian Claudin 18.2 (e.g., human Claudin 18.2, cynomolgus Claudin 18.2) with a Kd of less than about 1 x 10 -7 M, approx. 2 x 10 -7 M, about 3 x 10 -7 M, approx. 4 x 10 -7 M, about 5 x 10 -7 M, about 6 x 10 -7 M, about 7 x 10 -7 M, about 8 x 10 -7 M, about 9 x 10 -7 M, about 1 x 10 -8 M, approx. 2 x 10 -8 M, about 3 x 10 -8 M, approx. 4 x 10 -8 M, about 5 x 10 -8 M, about 6 x 10 -8 M, about 7 x 10 -8 M, about 8 x 10 -8 M, about 9 x 10 -8 M, about 1 x 10 -9 M, approx. 2 x 10 -9 M, about 3 x 10 -9 M, approx. 4 x 10 -9 M, about 5 x 10 -9 M, about 6 x 10 -9 M, about 7 x 10 -9 M, about 8 x 10 -9 M, about 9 x 10 -9 M, about 1 x 10 ~10 M, or approximately 5 x 10 -10 In certain embodiments, the Kd is off / K on It is calculated as the quotient of and K on and K. off is determined using a monovalent antibody, such as a Fab fragment, as measured, for example, by BIAcore® surface plasmon resonance technology. In other embodiments, Kd is off / K on It is calculated as the quotient of K on and K. off is determined using a bivalent antibody, such as a Fab fragment, measured, for example, by BIAcore® surface plasmon resonance technology.

[0167] In some embodiments, the Claudin 18.2 antigen-binding domain is administered to mammalian Claudin 18.2 (e.g., human Claudin 18.2, cynomolgus monkey Claudin 18.2, or mouse Claudin 18.2) at 1×10 -4 M -1 s -1 Less than 2 x 10 -4 M -1 s -1 Less than 3 x 10 -4 M -1 s -1 Less than 4 x 10 -4 M -1 s -1 Less than 5 x 10 -4 M -1 s -1 Less than 7 x 10 -4 M -1 s -1 Less than 8 x 10 -4 M -1 s -1 Less than 9 x 10 -4 M -1 s -1 Less than 1×10 -5 M -1 s -1 Less than 2 x 10 -5 M -1 s -1 Less than 3 x 10 -5 M -1 s -1 Less than 4 x 10 -5 M -1 s -1 Less than 5 x 10 -5 M -1 s -1 Less than 6 x 10 -5 M -1 s -1 Less than 7 x 10 -5 M -1 s -1 Less than 8 x 10 -5 M -1 s -1 Less than 9 x 10 -5 M -1 s -1 Less than 1×10 -6 M -1 s -1 Less than 2 x 10-6 M -1 s -1 Less than 3 x 10 -6 M -1 s -1 Less than 4 x 10 -6 M -1 s -1 Less than 5 x 10 -6 M -1 s -1 Less than 6 x 10 -6 M -1 s -1 Less than 7 x 10 -6 M -1 s -1 Less than 8 x 10 -6 M -1 s -1 Less than 9 x 10 -6 M -1 s -1 Less than or equal to 1 x 10 -7 M -1 s -1 The binding rate (k on In certain embodiments, k on is determined using a monovalent antibody, such as a Fab fragment, measured, for example, by BIAcore® surface plasmon resonance technology. on is determined, for example, using bivalent antibodies measured by BIAcore® surface plasmon resonance technology.

[0168] In some embodiments, the Claudin 18.2 antigen-binding domain is administered to mammalian Claudin 18.2 (e.g., human Claudin 18.2, cynomolgus monkey Claudin 18.2, or mouse Claudin 18.2) at 1×10 -2 s -1 Less than 2 x 10 -2 s -1 Less than 3 x 10 -2 s -1 Less than 4 x 10 -2 s -1 Less than 5 x 10 -2 s -1 Less than 6 x 10 -2 s -1 Less than 7 x 10 -2 s -1Less than 8 x 10 -2 s -1 Less than 9 x 10 -2 s -1 Less than 1×10 -3 s -1 Less than 2 x 10 -3 s -1 Less than 3 x 10 -3 s -1 Less than 4 x 10 -3 s -1 Less than 5 x 10 -3 s -1 Less than 6 x 10 -3 s -1 Less than 7 x 10 -3 s -1 Less than 8 x 10 -3 s -1 Less than 9 x 10 -3 s -1 Less than 1×10 -4 s -1 Less than 2 x 10 -4 s -1 Less than 3 x 10 -4 s -1 Less than 4 x 10 -4 s -1 Less than 5 x 10 -4 s -1 Less than 6 x 10 -4 s -1 Less than 7 x 10 -4 s -1 Less than 8 x 10 -4 s -1 Less than 9 x 10 -4 s -1 Less than 1×10 -5 s -1 Less than or 5 x 10 -4 s -1 Dissociation rate (k off In certain embodiments, k off is determined using a monovalent antibody, such as a Fab fragment, measured, for example, by BIAcore® surface plasmon resonance technology. off is determined, for example, using bivalent antibodies measured by BIAcore® surface plasmon resonance technology.

[0169] II. Chimeric Antigen Receptors As used herein, a chimeric antigen receptor (CAR) is a protein that specifically recognizes a target antigen (e.g., a target antigen on a cancer cell). When bound to a target antigen, the CAR can activate immune cells to attack and destroy cells bearing that antigen (e.g., cancer cells). CARs can also incorporate costimulatory or signal transduction domains to increase their efficacy. See Krause et al., J. Exp. Med., Volume 188, No. 4, 1998 (619-626); Finney et al., Journal of Immunology, 1998, 161:2791-2797; Song et al., Blood 119:696-706 (2012); Kalos et al., Sci. Transl. Med. 3:95 (2011); Porter et al., N. Engl. J. Med. 365:725-33 (2011); and Gross et al., Annu. Rev. Pharmacol. Toxicol. 56:59-83 (2016); U.S. Patent Nos. 7,741,465 and 6,319,494.

[0170] The chimeric antigen receptors described herein comprise an extracellular domain, a transmembrane domain, and an intracellular domain, and the extracellular domain comprises a Claudin 18.2 antigen-binding domain that specifically binds to Claudin 18.2. In some embodiments, a Claudin 18.2-specific CAR comprises the following elements from 5' to 3': a signal sequence, a Claudin 18.2 antigen-binding domain (e.g., an anti-Claudin 18.2 scFv), a hinge and transmembrane region, and one or more continuous signaling domains. In certain embodiments, a Claudin 18.2-specific CAR disclosed herein comprises the following elements from 5' to 3': a CD8α signal sequence, a Claudin 18.2 scFv comprising the Claudin 18.2 variable heavy chain and / or variable light chain described herein, a CD8α hinge and transmembrane region, a 41BB cytoplasmic signaling domain, and a CD3ζ cytoplasmic signaling domain. In some embodiments, a Claudin 18.2-specific CAR comprises the following elements from 5' to 3': a Claudin 18.2 antigen-binding domain (e.g., an anti-Claudin 18.2 scFv), a hinge region and transmembrane region, and one or more continuous signaling domains. In certain embodiments, a Claudin 18.2-specific CAR disclosed herein comprises the following elements from 5' to 3': a Claudin 18.2 scFv comprising a Claudin 18.2 variable heavy chain and / or variable light chain described herein, a CD8α hinge region and transmembrane region, a 41BB cytoplasmic signaling domain, and a CD3ζ cytoplasmic signaling domain. Table 7a lists exemplary CAR component amino acid sequences.

[0171] In some embodiments, the Claudin 18.2-specific CAR further comprises a safety switch and / or one or more monoclonal antibody-specific epitopes.

[0172] a. Antigen-binding domain As described above, the Claudin 18.2 CAR described herein comprises an antigen-binding domain. As used herein, "antigen-binding domain" refers to any polypeptide that binds to a specific target antigen, for example, the specific target antigen may be a Claudin 18.2 (Claudin 18.2) protein or a fragment thereof (interchangeably referred to herein as a "Claudin 18.2 antigen," "Claudin 18.2 target antigen," or "Claudin 18.2 target"). In some embodiments, the antigen-binding domain binds to a Claudin 18.2 antigen on tumor cells. In some embodiments, the antigen-binding domain binds to a Claudin 18.2 antigen on cells involved in hyperproliferative diseases.

[0173] In some embodiments, the antigen-binding domain comprises a variable heavy chain, a variable light chain, and / or one or more CDRs described herein. In some embodiments, the antigen-binding domain is a single-chain variable fragment (scFv) comprising CDR1, CDR2, and CDR3 of the light chain CDRs, and CDR1, CDR2, and CDR3 of the heavy chain CDRs.

[0174] In some embodiments, the Claudin 18.2-specific CAR comprises the VH amino acid sequence shown in Table 1a. In some embodiments, the Claudin 18.2-specific CAR comprises the VL amino acid sequence shown in Table 1b. In some embodiments, the Claudin 18.2-specific CAR comprises the heavy chain CDR1, CDR2, CDR3 amino acid sequences shown in Table 1d. In some embodiments, the Claudin 18.2-specific CAR comprises the light chain CDR1, CDR2, CDR3 amino acid sequences shown in Table 1e.

[0175] Variants of antigen-binding domains (e.g., CDR, VH, and / or VL variants) are also within the scope of the present disclosure, including variable light chains and / or variable heavy chains, each of which has at least 70-80%, 80-85%, 85-90%, 90-95%, 95-97%, 97-99%, or greater than 99% identity to the amino acid sequence of an antigen-binding domain sequence described herein. In some examples, such molecules contain at least one heavy chain and one light chain, while in other examples, variant forms contain two variable light chains and two variable heavy chains (or subportions thereof). Those of skill in the art can determine suitable variants of the antigen-binding domains described herein using well-known techniques. In certain embodiments, those skilled in the art can identify appropriate regions of the molecule that can be altered without destroying activity by targeting regions not believed to be important for activity.

[0176] In certain embodiments, the polypeptide structure of the antigen-binding domain is based on an antibody, including, but not limited to, a monoclonal antibody, a bispecific antibody, a minibody, a domain antibody, a synthetic antibody (sometimes referred to herein as an "antibody mimetic"), a chimeric antibody, a humanized antibody, a human antibody, an antibody fusion (sometimes referred to herein as an "antibody conjugate"), and fragments thereof, respectively. In some embodiments, the antigen-binding domain comprises or consists of an avimer.

[0177] A Claudin 18.2 antigen-binding domain is said to be "selective" if it binds more strongly to one target than it binds to a second target.

[0178] In some embodiments, the Claudin 18.2 antigen-binding domain is an scFv. In some embodiments, the Claudin 18.2-specific CAR comprises an scFv provided in Table 1c.

[0179] In some embodiments, the Claudin 18.2-specific CAR comprises a leader or signal peptide, and in some embodiments, the leader peptide comprises an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the amino acid sequence MALPVTALLLPLALLLHAARP (SEQ ID NO: 134). In some embodiments, the leader (signal) peptide comprises the amino acid sequence of SEQ ID NO: 134. In some embodiments, the leader (signal) peptide is encoded by a nucleic acid sequence comprising ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGCCGCACGCCCG (SEQ ID NO: 166).

[0180] In other embodiments, the disclosure relates to isolated polynucleotides encoding any one of the Claudin 18.2 antigen-binding domains described herein. In some embodiments, the disclosure relates to isolated polynucleotides encoding a Claudin 18.2 CAR described in Table 10. Also provided herein are vectors comprising the polynucleotides, and methods of making the polynucleotides.

[0181] [Table 8] JPEG2025539380000012.jpg251157 JPEG2025539380000013.jpg250157 JPEG2025539380000014.jpg250157 JPEG2025539380000015.jpg249157 JPEG2025539380000016.jpg57168

[0182] b. Safety switch and monoclonal antibody specific epitope safety switch It will be appreciated that adverse events can be minimized by transducing immune cells (containing one or more CARs) with a suicide gene. It may also be desirable to incorporate an inducible "on" or "facilitator" switch into immune cells. Suitable techniques include the use of inducible caspase-9 (U.S. Application No. 2011 / 0286980) or thymidine kinase before, after, or simultaneously with transduction of cells with the CAR constructs of the present disclosure. Additional methods for introducing suicide genes and / or "on" switches include TALENS, zinc fingers, RNAi, siRNA, shRNA, antisense technology, and other techniques known in the art.

[0183] According to the present disclosure, additional on-off or other types of control switch technologies can be incorporated herein. These technologies can employ the use of dimerization domains and optional activators of such domain dimerization. These technologies include, for example, those described by Wu et al., Science 2014 350(6258), which utilizes the FKBP / rapalog dimerization system in certain cells, the contents of which are incorporated herein by reference in their entirety. Additional dimerization technologies are described, for example, in Fegan et al. Chem. Rev. 2010, 110, 3315-3336, and U.S. Patent Nos. 5,830,462, 5,834,266, 5,869,337, and 6,165,787, the contents of which are also incorporated herein by reference in their entirety. Additional dimerization pairs may include cyclosporin-A / cyclophilin receptor, estrogen / estrogen receptor (optionally using tamoxifen), glucocorticoid / glucocorticoid receptor, tetracycline / tetracycline receptor, vitamin D / vitamin D receptor. Further examples of dimerization techniques are described, for example, in WO 2014 / 127261, WO 2015 / 090229, US 2014 / 0286987, US2015 / 0266973, US2016 / 0046700, U.S. Patent No. 8,486,693, US 2014 / 0171649, and US 2012 / 0130076, the contents of which are incorporated herein by reference in their entireties.

[0184] In some embodiments, the CAR immune cells (e.g., CAR-T cells) of the present disclosure comprise a polynucleotide encoding a suicide polypeptide or safety switch, such as, for example, RQP8. See, for example, WO2013153391A, which is incorporated herein by reference in its entirety. In the CAR immune cells (e.g., CAR-T cells) comprising the polynucleotide, the suicide polypeptide is expressed on the surface of the CAR immune cells (e.g., CAR-T cells). In some embodiments, the suicide polypeptide is SEQ ID NO: 167: It contains the amino acid sequence shown in CPYSNPSLCSGGGGSELPTQGTFSNVSTNVSPAKPTTTACPYSNPSLCSGGGGSPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVV (SEQ ID NO: 167).

[0185] The suicide polypeptide may also contain a signal peptide at the amino terminus, such as MGTSLLCWMALCLLGADHADA (SEQ ID NO: 169). In some embodiments, the suicide polypeptide contains the amino acid sequence set forth in SEQ ID NO: 168, which includes the signal sequence of SEQ ID NO: 169: MGTSLLCWMALCLLGADHADACPYSNPSLCSGGGGSELPTQGTFSNVSTNVSPAKPTTTACPYSNPSLCSGGGGSPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVV (SEQ ID NO: 168).

[0186] When the suicide polypeptide is expressed on the surface of CAR immune cells (e.g., CAR-T cells), rituximab binds to the R epitope of the polypeptide, resulting in cell lysis. Two or more molecules of rituximab can bind to each polypeptide expressed on the cell surface. Each R epitope of the polypeptide may bind to a separate rituximab molecule. Deletion of Claudin 18.2-specific CAR immune cells (e.g., CAR-T cells) can occur in vivo, for example, by administering rituximab to a patient. The decision to delete the introduced cells can arise from undesirable effects detected in patients due to the introduced cells, for example, when unacceptable levels of toxicity are detected.

[0187] In some embodiments, the suicide polypeptide is expressed on the surface of a cell. In some embodiments, the suicide polypeptide is included in a CAR construct. In some embodiments, the suicide polypeptide is not part of a Claudin 18.2 CAR construct.

[0188] In some embodiments, the extracellular domain of any one of the Claudin 18.2-specific CARs disclosed herein can comprise 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. Exemplary mAb-specific epitopes are disclosed in International Patent Publication No. 2016 / 120216, which is incorporated herein in its entirety. In these embodiments, the extracellular domain of the CAR comprises an antigen-binding domain that specifically binds to Claudin 18.2 and one or more epitopes that bind to one or more monoclonal antibodies (mAbs). CARs that comprise mAb-specific epitopes can be single-chain or multi-chain.

[0189] The inclusion of an epitope specific to a monoclonal antibody in the extracellular domain of the CAR described herein allows for the separation and depletion of engineered immune cells expressing the CAR. In some embodiments, this function also promotes the recovery of endogenous Claudin 18.2-expressing cells that are depleted by the administration of engineered immune cells expressing the CAR. In some embodiments, allowing depletion provides a safety switch in the event of adverse effects, for example, when administered to a subject.

[0190] Thus, in some embodiments, the present disclosure relates to methods for sorting and / or depleting engineered immune cells endowed with CARs containing mAb-specific epitopes, and methods for promoting recovery of endogenous Claudin 18.2-expressing cells.

[0191] Some epitope-monoclonal antibody conjugates can be used to generate CARs containing monoclonal antibody-specific epitopes, particularly CARs already approved for medical use, such as, but not limited to, CD20 epitope / rituximab.

[0192] The present disclosure also encompasses methods for sorting engineered immune cells equipped with Claudin 18.2-specific CARs that express mAb-specific epitopes, and therapeutic methods in which the activation of these CAR-equipped engineered immune cells is modulated by depleting the cells using antibodies that target the external ligand-binding domain of the CAR. Table 4 provides exemplary mimotope sequences that can be inserted into the extracellular domain of any one of the CARs of the present disclosure.

[0193] [Table 9] In some embodiments, the extracellular binding domain of the CAR comprises 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 -; -V 1- (L) x -Epitope 1-(L) x -V2; -V 1- (L) x -Epitope 1-(L) x -V 2- (L) x -Epitope 2-(L) x ; -V 1- (L) x -Epitope 1-(L) x -V 2- (L) x-Epitope 2-(L) x -Epitope 3-(L) x ; -V 1- (L) x -Epitope 1-(L) x -V 2- (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 and - L1, V H Chain V L is a linker suitable for joining to a chain, - L is a linker comprising glycine and serine residues, and each occurrence of L in the extracellular binding domain can be identical to or different from other occurrences of L in the same extracellular binding domain, such as SGGGG (SEQ ID NO: 177), GGGGS (SEQ ID NO: 163), or SGGGGS (SEQ ID NO: 178) (all of which occur, for example, in SEQ ID NO: 52); - x is 0 or 1 or 2, and each occurrence of x is selected independently of the others; epitope 1, epitope 2, epitope 3, and epitope 4 are mAb-specific epitopes, which may be identical or different, and V H is the heavy chain variable fragment, and VL is a light chain variable fragment. In some embodiments, epitope 1, epitope 2, epitope 3, and epitope 4 each comprise the amino acid sequence of SEQ ID NO: 140. In some embodiments, epitope 1, epitope 2, epitope 3, and epitope 4 may each comprise the amino acid sequence of SEQ ID NO: 148 or 149. In some embodiments, epitope 1, epitope 2, and epitope 4 are mAb-specific epitopes having the amino acid sequence of SEQ ID NO: 140, and epitope 3 is a mAb-specific epitope having the amino acid sequence of SEQ ID NO: 148. In some embodiments, epitope 1, epitope 2, and epitope 4 are mAb-specific epitopes having the amino acid sequence of SEQ ID NO: 140, and epitope 3 is a mAb-specific epitope having the amino acid sequence of SEQ ID NO: 149.

[0194] C hinge domain The extracellular domain of the CAR of the present disclosure can include a "hinge" domain (or hinge region). This term generally refers to any polypeptide that functions to connect the transmembrane domain in the CAR to the extracellular antigen-binding domain in the CAR. In particular, the hinge domain can be used to provide more flexibility and accessibility to the extracellular antigen-binding domain.

[0195] The hinge domain can comprise up to 300 amino acids, and in some embodiments, 10-100 amino acids, or in some embodiments, 25-50 amino acids. The hinge domain can originate from all or part of a naturally occurring molecule derived from the extracellular region of CD8, CD4, CD28, 4-1BB, or IgG (particularly, it is understood that this may include all or part of the hinge region of IgG, i.e., some or all of the immunoglobulin members, such as IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, etc., or fragments thereof), or all or part of an antibody heavy chain constant region. Alternatively, the A domain may be a synthetic sequence corresponding to a naturally occurring A sequence or may be a completely synthetic A sequence. In some embodiments, the A domain is a portion of the human CD8 α chain (e.g., NP_001139345.1). In another specific embodiment, the hinge domain and transmembrane domain comprise a portion of the human CD8 α chain. In some embodiments, the hinge domain of a CAR described herein comprises a subsequence of CD8α, CD28, IgG1, IgG4, PD-1, or FcγRIIIα, particularly the hinge region of any of CD8α, CD28, IgG1, IgG4, PD-1, or FcγRIIIα. In some embodiments, the hinge domain comprises a human CD8α hinge, a human CD28 hinge domain, a human IgG1 hinge, a human IgG4, a human PD-1, or a human FcγRIIIα hinge. In some embodiments, a CAR disclosed herein comprises an scFv, a human CD8α hinge domain and transmembrane domain, a CD3ζ signaling domain, and a 4-1BB signaling domain. In some embodiments, a CAR disclosed herein comprises an scFv, a human CD28 hinge domain and transmembrane domain, a CD3ζ signaling domain, and a 4-1BB signaling domain. Table 5 provides the amino acid sequences of exemplary hinges provided herein.

[0196] [Table 10]

[0197] In certain embodiments, the hinge region comprises an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to an extracellular domain amino acid sequence set forth herein in Table 5.

[0198] D transmembrane domain The CAR of the present disclosure is designed with a transmembrane domain fused to the extracellular domain of the CAR. This can also be fused to the intracellular domain of the CAR. In some examples, the transmembrane domain can be selected or modified by amino acid substitution to avoid the binding of this domain to the transmembrane domain of the same or different surface membrane protein, and minimize the interaction with other members of the receptor complex. In some embodiments, a short linker can form a bond between any or part of the extracellular domain, transmembrane domain, and intracellular domain of the CAR.

[0199] Suitable transmembrane domains of the CARs disclosed herein include (a) transmembrane domains that are transmembrane-specific, e.g., T helper (T h ) cells, cytotoxic T (T c ) cells, T regulatory (T reg ) the ability to be expressed on the surface of immune cells, such as immune cells, or lymphocytes, such as natural killer (NK) cells, and / or (b) the ability to interact with an extracellular antigen-binding domain and an intracellular signaling domain to direct a cellular response of the immune cell against a target cell.

[0200] Transmembrane domains can be derived from either natural or synthetic sources. If the source is natural, the domain can be derived from any membrane-bound or transmembrane protein.

[0201] Transmembrane regions of particular use in the present disclosure include CD8α, CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD1-1a / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class 1 molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor , integrin, signaling lymphocyte activation molecule (SLAM protein), activating NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE, CD103, ITGAL, CD1 1a, LFA-1, ITGAM, CD1 1b, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT and ligands that specifically bind to AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83, or any combination thereof.

[0202] As non-limiting examples, the transmembrane region may be derived from, for example, the α, β, γ, or δ polypeptides constituting the CD3 complex, the IL-2 receptor p55 (α chain), p75 (β chain), or γ chain, a subunit chain of an Fc receptor, or may be part of a T cell receptor, particularly in the case of Fcγ receptor III or CD proteins. Alternatively, the transmembrane domain may be synthetic and comprise 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).

[0203] In some embodiments, the transmembrane domain in a CAR of the present disclosure is a CD8α transmembrane domain. In some embodiments, the transmembrane domain in a CAR of the present disclosure is a CD8α transmembrane domain comprising the amino acid sequence IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 154). In some embodiments, the hinge and transmembrane domain in a CAR of the present disclosure is a CD8α hinge and transmembrane domain comprising the amino acid sequence of SEQ ID NO: 136.

[0204] In some embodiments, the transmembrane domain in a CAR of the present disclosure is a CD28 transmembrane domain. In some embodiments, the transmembrane domain in a CAR of the present disclosure is a CD28 transmembrane domain comprising the amino acid sequence FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 157).

[0205] e. intracellular domain The intracellular (cytoplasmic) domain of a CAR of the present disclosure can provide for activation of at least one normal effector function of an immune cell involving the CAR, e.g., signal 1 / activation, and / or signal 2 / costimulation. Effector function of a T cell can refer to, for example, cytolytic activity or helper activity, including cytokine secretion.

[0206] In some embodiments, activating intracellular signaling domains for use in CARs may be, for example, but not limited to, the cytoplasmic sequences of T cell receptors and co-receptors that act in concert to initiate signaling following antigen receptor engagement, as well as any derivatives or variants of these sequences, and any synthetic sequences that have the same function.

[0207] Suitable (e.g., activation) intracellular domains include, but are not limited to, CD3 zeta, CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD1-1a / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class 1 molecule, TNF receptor protein, immunoglobulin protein, cytokines, and the like. Kine receptor, integrin, signaling lymphocyte activation molecule (SLAM protein), activating NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE, CD103, ITGAL, CD1 1a, LFA-1, ITGAM, CD1 1b, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT The signaling domains comprise a ligand that specifically binds to AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83, or any combination thereof.

[0208] In addition to the activation domain described above, the intracellular domain of the CAR of the present disclosure can incorporate a costimulatory signaling domain (interchangeably referred to herein as a costimulatory molecule) to increase its efficacy. The costimulatory domain can provide a signal in addition to the primary signal provided by the activation molecule described herein.

[0209] Suitable costimulatory domains within the scope of the present disclosure include, for example, CD28, OX40, 4-1BB / CD137, CD2, CD3 (alpha, beta, delta, epsilon, gamma, zeta), CD4, CD5, CD7, CD9, CD16, CD22, CD27, CD30, CD33, CD37, CD40, CD45, CD64, CD80, CD86, CD134, CD137, CD154, PD-1, ICOS, lymphocyte function-associated antigen-1 (LF A-1 (CD1 1a / CD18), CD247, CD276 (B7-H3), LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class I molecule, TNFR, integrin, signaling lymphocyte activation molecule, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD 8beta, IL-2Rbeta, IL-2Rgamma, IL-7Ralpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1-1d, ITGAE, CD103, ITGAL, CD1-1a, LFA-1, ITGAM, CD1 -1b, ITGAX, CD1-1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT It is understood that the co-stimulatory molecules may be derived from AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83 ligand, or fragments or combinations thereof. It is understood that additional co-stimulatory molecules or fragments thereof not listed above are within the scope of the present disclosure.

[0210] In some embodiments, the intracellular / cytoplasmic domain of the CAR may be designed to include the 41BB / CD137 domain by itself, or may be combined with any other desired intracellular domain useful in the context of the CARs of the present disclosure. The complete native amino acid sequence of 41BB / CD137 is set forth in NCBI Reference Sequence: NP_ 001552.2. The complete native 41BB / CD137 nucleic acid sequence is set forth in NCBI Reference Sequence: NM_ 001561.5.

[0211] In some embodiments, the intracellular / cytoplasmic domain of the CAR may be designed to include the CD28 domain by itself, or may be combined with any other desired intracellular domain useful in the context of the CAR of the present disclosure. The complete native amino acid sequence of CD28 is set forth in NCBI Reference Sequence: NP_006130.1. The complete native CD28 nucleic acid sequence is set forth in NCBI Reference Sequence: NM_006139.1.

[0212] In some embodiments, the intracellular / cytoplasmic domain of a CAR may be designed to include a CD3 zeta domain by itself or may be combined with any other desired intracellular domain useful in the context of the CAR of the present disclosure. In some embodiments, the intracellular signaling domain of a CAR may include a CD3ζ signaling domain having an amino acid sequence with 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: 138 or SEQ ID NO: 139 (see Table 7a). For example, the intracellular domain of a CAR may include a CD3 zeta chain portion and a portion of a costimulatory signaling molecule. The intracellular signaling sequences within the intracellular signaling portion of a CAR of the present disclosure can be linked to each other randomly or in a specific order. In some embodiments, the intracellular domain is designed to include the activation domain of CD3 zeta and the signaling domain of CD28. In some embodiments, the intracellular domain is designed to include the activation domain of CD3 zeta and the costimulatory / signaling domain of 4-1BB.

[0213] In some embodiments, 4-1BB (intracellular domain) has the amino acid sequence In some embodiments, the 4-1BB (intracellular domain) is encoded by the nucleic acid sequence: AAGCGCGGCAGGAAGAAGCTCCTCTACATTTTTAAGCAGCCTTTTATGAGGCCCGTACAGACAACACAGGAGGAAGATGGCTGTAGCTGCAGATTTCCCGAGGAGGAGGAAGGTGGGTGCGAGCTG (SEQ ID NO: 172).

[0214] In some embodiments, the intracellular domain in the CAR is designed to include portions of CD28 and CD3 zeta, where the intracellular CD28 is encoded by the nucleic acid sequence set forth in SEQ ID NO: 173. AGATCCAAAAGAAGCCGCCTGCTCCATAGCGATTACATGAATATGACTCC ACGCCGCCCTGGCCCCACAAGGAAACACTACCAGCCTTACGCACCACCTAGAGATTTCGCTGCCTATCGGAGC (SEQ ID NO: 173).

[0215] In some embodiments, the intracellular domain in the CAR is designed to comprise the amino acid sequence RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (CD28-YMNM intracellular domain ("YMNM" disclosed as SEQ ID NO: 217), SEQ ID NO: 174). The CD3 zeta amino acid sequence may comprise SEQ ID NO: 138 or 139, and the nucleic acid sequence encoding the CD3 zeta amino acid sequence may comprise SEQ ID NO: 175: AGGGTGAAGTTTTCCAGATCTGCAGATGCACCAGCGTATCAGCAGGGCCAGAACCAACTGTATAACGAGCTCAACCTGGGACGCAGGGAAGAGTATGACGTTTTGGACAAGCGCAGAGGACGGGACCCTGAGATGGGTGGCAAACCAAGACGAAAAAACCCCAGGAGGGTCT CTATAATGAGCTGCAGAAGGATAAGATGGCTGAAGCCTATTCTGAAATAGGCATGAAAGGAGAGCGGAGAAGGGGAAAAGGGCACGACGGTTTGTACCAGGGACTCAGCACTGCTACGAAGGATACTTATGACGCTCTCCACATGCAAGCCCTGCCACCTAGG (SEQ ID NO: 175).

[0216] In some embodiments, the intracellular signaling domain of a CAR of the present disclosure comprises a domain of a costimulatory molecule. In some embodiments, the intracellular signaling domain of a CAR of the present disclosure comprises a portion of a costimulatory molecule selected from the group consisting of a fragment of 4-1BB (GenBank: AAA53133) and CD28 (NP_006130.1). In some embodiments, the intracellular signaling domain of a CAR of the present disclosure comprises an amino acid sequence comprising 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: 137 and SEQ ID NO: 174. In some embodiments, the intracellular signaling domain of a CAR of the present disclosure comprises an amino acid sequence comprising 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: 137 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: 174.

[0217] In an exemplary embodiment, a CAR of the present disclosure comprises, from N- to C-terminus: a CD8α signal sequence, Claudin 18.2 scFv, a CD8α hinge and transmembrane region, a 4-1BB cytoplasmic (co-stimulatory) signaling domain, and a CD3ζ cytoplasmic (stimulatory) signaling domain.

[0218] III. CAR-containing immune cells a. Immune cells Provided herein are engineered immune cells that express the CARs of the present disclosure (e.g., CAR-T cells).

[0219] In some embodiments, the engineered immune cells comprise a population of CARs, each CAR comprising a different extracellular antigen-binding domain. In some embodiments, the immune cells comprise a population of CARs, each CAR comprising an extracellular antigen-binding domain.

[0220] The engineered immune cells can be allogeneic or autologous.

[0221] In some embodiments, the engineered immune cells are T cells (e.g., inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes (Tregs), helper T lymphocytes, tumor-infiltrating lymphocytes (TILs)), natural killer T cells (NKTs), TCR-expressing cells, dendritic cells, killer dendritic cells, mast cells, or B cells. In some embodiments, the cells can arise from the group consisting of CD4+ T-lymphocytes and CD8+ T-lymphocytes. In some exemplary embodiments, the engineered immune cells are T cells. In some exemplary embodiments, the engineered immune cells are gamma delta T cells. In some exemplary embodiments, the engineered immune cells are macrophages. In some exemplary embodiments, the engineered immune cells are natural killer (NK) cells.

[0222] In some embodiments, the engineered immune cells may be derived from, for example, but not limited to, stem cells, which may be adult stem cells, non-human embryonic stem cells, more particularly 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.

[0223] In some embodiments, the cells are obtained or prepared from peripheral blood. In some embodiments, the cells are obtained or prepared from peripheral blood mononuclear cells (PBMCs). In some embodiments, the cells are obtained or prepared from bone marrow. In some embodiments, the cells are obtained or prepared from umbilical cord blood. In some embodiments, the cells are human cells.

[0224] In some embodiments, cells are transfected or transduced with a nucleic acid vector using a method selected from the group consisting of electroporation, sonoporation, biolistics (e.g., Gene Gun), lipid transfection, polymer transfection, nanoparticles, viral transfection (e.g., retrovirus, lentivirus, AAV), or polyplexes.

[0225] In some embodiments, engineered immune cells expressing a Claudin 18.2-specific CAR of the present disclosure on their cell surface membrane comprise a proportion of stem cell memory and central memory cells that is greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. In some embodiments, engineered immune cells expressing a Claudin 18.2-specific CAR of the present disclosure on their cell surface membrane comprise a proportion of stem cell memory and central memory cells that is greater than about 10% to about 100%, 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 10% to about 30%, about 10% to about 20%, about 15% to about 100%, about 15% to about 90%, about 15% to about 80%, Approximately 15% to approximately 70%, approximately 15% to approximately 60%, approximately 15% to approximately 50%, approximately 15% to approximately 40%, approximately 15% to approximately 30%, approximately 20% to approximately 100%, approximately 20% to approximately 90%, approximately 20% to approximately 80%, approximately 20% to approximately 70%, approximately 20% to approximately 60%, approximately 20% to approximately 50%, approximately 20% to approximately 40%, approximately 20% to approximately 30%, approximately 30% to approximately 100%, approximately 30% to approximately 90%, approximately 30% to Approximately 80%, approximately 30% to approximately 70%, approximately 30% to approximately 60%, approximately 30% to approximately 50%, approximately 30% to approximately 40%, approximately 40% to approximately 100%, approximately 40% to approximately 90%, approximately 40% to approximately 80%, approximately 40% to approximately 70%, approximately 40% to approximately 60%, approximately 40% to approximately 50%, approximately 50% to approximately 100%, approximately 50% to approximately 90%, approximately 50% to approximately 80%, approximately 50% to approximately 70%, approximately 50% to approximately 60%, The proportion of stem cell memory cells and central memory cells is about 60% to about 100%, about 60% to about 90%, about 60% to about 80%, about 60% to about 70%, about 70% to about 90%, about 70% to about 80%, about 80% to about 100%, about 80% to about 90%, about 90% to about 100%, about 25% to about 50%, about 75% to about 100%, or about 50% to about 75%.

[0226] In some embodiments, the immune cells are inflammatory T lymphocytes expressing any one of the CARs described herein. In some embodiments, the immune cells are cytotoxic T lymphocytes expressing any one of the CARs described herein. In some embodiments, the immune cells are regulatory T lymphocytes expressing any one of the CARs described herein. In some embodiments, the immune cells are helper T lymphocytes expressing any one of the CARs described herein.

[0227] Prior to expansion and genetic modification, cell sources can be obtained from a subject through a variety of non-limiting methods. Cells can be obtained from numerous sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, any number of T cell lines available and known to those skilled in the art can be used. In some embodiments, cells can be derived from a healthy donor, or a patient (e.g., a patient diagnosed with cancer), or a patient diagnosed with an infectious disease. In some embodiments, cells can be part of a mixed population of cells exhibiting different phenotypic characteristics.

[0228] Also provided herein are cell lines obtained from transformed immune cells (e.g., T cells) according to any of the above-described methods. Also provided herein are modified cells that are resistant to immunosuppressive treatment. In some embodiments, the isolated cells according to the present disclosure comprise a polynucleotide encoding a CAR.

[0229] The immune cells of the present disclosure can be activated and proliferated by generally known methods before or after genetic modification of immune cells.Generally, the engineered immune cells of the present disclosure can be proliferated by, for example, contacting with an agent that stimulates the CD3 TCR complex and costimulatory molecules on the surface of T cells, thereby generating an activation signal for T cells.For example, chemical substances 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 T cells.

[0230] In some embodiments, a population of T cells may be stimulated in vitro by contacting them with, for example, an anti-CD3 antibody, such as an OKT3 antibody, or an antigen-binding fragment thereof, or a surface-immobilized anti-CD2 antibody, or by contacting them with a protein kinase C activator (e.g., bryostatin) in combination with a calcium ion phore. Co-stimulation of accessory molecules on the surface of T cells is achieved using a ligand that binds to the accessory molecule. For example, a population of T cells can be contacted with an anti-CD3 antibody (e.g., an OKT3 antibody) and an anti-CD28 antibody under conditions appropriate for stimulating T cell proliferation. The anti-CD3 antibody and anti-CD28 antibody can be disposed on beads, such as plastic or magnetic beads, or on a plate or other substrate. Suitable conditions for T cell culture include an appropriate medium (e.g., Minimum Essential Medium or RPMI Medium 1640, or X-vivo 15, (Lonza)), which may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-2, IL-15, TGF-beta, 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 can include RPMI 1640, A1M-V, DMEM, MEM, a-MEM, F-12, X-Vivo15, and X-Vivo20, or Optimizer with additional amino acids, sodium pyruvate, and vitamins, and can be serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones and / or cytokines sufficient for T cell proliferation (e.g., IL-7 and / or IL-15). Antibiotics, such as penicillin and streptomycin, are included only in experimental cultures and not in cultures of cells infused into subjects. Target cells are maintained under conditions necessary to support growth, such as an appropriate temperature (e.g., 37°C) and atmosphere (e.g., air + 5% CO2).T cells exposed to different stimulation times may exhibit different characteristics.In some embodiments, the cells of the present disclosure can be grown by co-culturing with tissue or cells.Cells can also be grown in vivo, for example, in the blood of subject after administering cells to subject.

[0231] In some embodiments, engineered immune cells according to the present disclosure may comprise one or more disrupted or inactivated genes. In some embodiments, engineered immune cells according to the present disclosure comprise one disrupted or inactivated gene selected from the group consisting of CD52, Claudin 18.2, 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, isolated cells comprise a polynucleotide encoding a polypeptide comprising a multi-chain CAR. In some embodiments, 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, Claudin 18.2 and CD52, Claudin 18.2 and TCRα, Claudin 18.2 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, 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. In some embodiments, the methods include disrupting or inactivating one or more genes by introducing an endonuclease into the cell, which can selectively inactivate genes by selective DNA cleavage.In some embodiments, the endonuclease may be, for example, a zinc finger nuclease (ZFN), megaTAL nuclease, meganuclease, transcription activator-like effector nuclease (TALE nuclease / TALEN), or CRISPR (e.g., Cas9 or Cas12) endonuclease.

[0232] In some embodiments, the TCR does not function in a cell according to the present disclosure by disrupting or inactivating the TCR alpha and / or TCR beta genes. In some embodiments, a method of obtaining modified cells from an individual is provided, 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 being obtained by the present methods and are therefore encompassed within the scope of the present disclosure. The modified cells disclosed herein can be used to treat a patient in need thereof for host-versus-graft (HvG) rejection and graft-versus-host disease (GvHD); therefore, within the scope of the present disclosure, a method of treating a patient in need thereof for host-versus-graft (HvG) rejection and graft-versus-host disease (GvHD) comprises treating the patient by administering to the patient an effective amount of modified cells comprising a disrupted or inactivated TCR alpha and / or TCR beta gene.

[0233] 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), thereby creating immune cells suitable for cancer treatment combining adoptive immunotherapy and chemotherapy. Exemplary PNAs include, for example, clofarabine, fludarabine, cyclophosphamide, and cytarabine, alone or in combination. PNAs are metabolized by deoxycytidine kinase (dCK) to monophosphate PNA, diphosphate PNA, and triphosphate PNA. Their triphosphate forms compete with ATP for DNA synthesis, act as proapoptotic agents, and are potent inhibitors of ribonucleotide reductase (RNR), which is involved in trinucleotide production. Claudin 18.2-specific CAR-T cells containing a disrupted or inactivated dCK gene are provided herein. In some embodiments, dCK knockout cells are generated by transfection of T cells with a polynucleotide encoding a TAL nuclease specific for the dCK gene, for example, by electroporation of mRNA. dCK knockout Claudin 18.2-specific CAR-T cells are resistant to PNAs, including, for example, chlorofarabine and / or fludarabine, and maintain T cell cytotoxic activity against Claudin 18.2-expressing cells.

[0234] In some embodiments, the isolated cells or cell lines of the present disclosure may comprise 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.

[0235] The present disclosure also provides engineered immune cells comprising any of the CAR polynucleotides described herein.In some embodiments, 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 provide identification and / or selection of cells that have received the vector.

[0236] The CAR polypeptide can be synthesized in situ within the cell after the introduction of a polynucleotide encoding the CAR polypeptide into the cell. Alternatively, the CAR polypeptide can 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 (e.g., using a lentiviral vector) can be used to integrate the polynucleotide construct into the genome of the cell. The polynucleotide construct can be integrated into the genome of the cell, for example, by random integration mediated by a lentiviral vector, or by site-specific integration mediated by homologous recombination via an adeno-associated viral vector. The polynucleotide construct can be integrated into the genome of the subject by homologous recombination, for example, at one or more genomic loci where disruption (e.g., knockout) occurs in one or more endogenous genes. Examples of endogenous genes include, but are not limited to, TCRα, TCRβ, CD52, glucocorticoid receptor (GR), deoxycytidine kinase (dCK), CD70, or immune checkpoint proteins such as programmed death-1 (PD-1).

[0237] In other embodiments, transient transformation methods can be used to transiently express polynucleotide constructs, and polynucleotide constructs that are not integrated into the genome of a cell. In other embodiments, viral-mediated methods can be used. Polynucleotides can be introduced into cells by any suitable means, such as, for example, recombinant viral vectors (e.g., retroviruses, adenoviruses), liposomes, etc. Transient transformation methods include, but are not limited to, microinjection, electroporation, or particle bombardment. Polynucleotides can be contained in vectors, such as, for example, plasmid vectors or viral vectors.

[0238] In some embodiments, an isolated nucleic acid is provided, comprising a promoter operably linked to a first polynucleotide encoding a Claudin 18.2 antigen-binding domain, at least one costimulatory molecule, and an activation domain. In some embodiments, the nucleic acid construct is contained within a viral vector. In some embodiments, the viral vector is selected from the group consisting of a retroviral vector, a murine leukemia virus vector, an SFG vector, an adenoviral vector, a lentiviral vector, an adeno-associated virus (AAV) vector, a herpes virus vector, and a vaccinia virus vector. In some embodiments, the nucleic acid is contained within a plasmid.

[0239] b. Immune cells with increased resistance to immune rejection In certain aspects, the present disclosure provides engineered immune cells or populations of engineered immune cells that comprise or express a Claudin 18.2-specific CAR and (1) further comprise or express an immune rejection evasion protein, and / or (2) further comprise one or more genomic modifications that functionally impair or reduce expression of one or more of CD58, NLRC5, RFX5, ICAM-1, TAP2, β2M, CIITA, RFXAP, RFXANK, and CD48. In some embodiments, the engineered immune cells or populations of engineered immune cells are allogeneic engineered immune cells. In some embodiments, the engineered immune cells or populations of engineered immune cells exhibit enhanced or increased resistance to host alloreactive immune cell rejection. In some embodiments, the increased resistance to alloreactive immune cell rejection is determinable and / or determined by a mixed lymphocyte reaction (MLR) assay, for example, an MLR assay as described herein.

[0240] In some embodiments, the engineered immune cell or population of engineered immune cells comprises one or more polynucleotides encoding a Claudin 18.2 CAR and an immune rejection evasion protein. In some embodiments, the polynucleotide encoding the Claudin 18.2 CAR and the polynucleotide encoding the immune rejection evasion protein may be part of the same polynucleotide or different polynucleotides. In some embodiments, the immune rejection evasion protein comprises a CD70 binding protein. In some embodiments, the CD70 binding protein comprises or is a CD70 chimeric antigen receptor (CAR). In some embodiments, the CD70 binding protein comprises a CD70 binding domain and a transmembrane domain. In some embodiments, the CD70 binding domain comprises a CD70 antibody or antigen-binding fragment thereof, or a receptor for CD70 or a CD70-binding fragment thereof. In certain embodiments, the CD70 antibody comprises the amino acid sequence of SEQ ID NO: 204, 205, 206, and / or 207. In other embodiments, the CD70 binding domain comprises an anti-CD70 antibody, optionally an scFv.

[0241] In some embodiments, the CD70 binding protein comprises a CD8 transmembrane domain or a CD28 transmembrane domain. In a further embodiment, the CD70 binding protein further comprises a hinge domain, optionally comprising a CD8 hinge domain or a CD28 hinge domain. In other embodiments, the CD70 binding protein further comprises one or more intracellular signaling domains selected from the group consisting of a CD3z signaling domain, a CD3d signaling domain, a CD3g signaling domain, a CD3e signaling domain, a CD28 signaling domain, a CD2 signaling domain, an OX40 signaling domain, and a 4-1BB signaling domain, or variants thereof. In a further embodiment, the CD70 binding protein comprises a CD3z or CD3g signaling domain and does not comprise a costimulatory domain, such as a CD28 signaling domain or a 4-1BB signaling domain, or variants thereof. In other embodiments, the CD70 binding protein comprises a 4-1BB signaling domain and does not comprise a CD3z signaling domain. In another embodiment, the CD70 binding protein comprises a 4-1BB signaling domain and a CD3z signaling domain. In other embodiments, the one or more intracellular domains comprise the amino acid sequence of one or more of SEQ ID NOs: 137, 138, 139, 158, 159, or 174. In some embodiments, the CD70 binding protein comprises: In another embodiment, the CD70 binding protein does not comprise an intracellular signaling domain. In some embodiments, the engineered immune cell further comprises one or more genomic modifications that functionally impair or reduce expression of CD70.

[0242] In some embodiments, genomic modifications may be introduced by zinc finger nucleases (ZFNs), megaTAL nucleases, meganucleases, transcription activator-like effector nucleases (TALE nucleases / TALEN®), or CRISPR (e.g., Cas9 or Cas12) endonucleases.

[0243] c. Manufacturing method Provided herein are methods of making the CARs and CAR-containing immune cells of the present disclosure.

[0244] Various known techniques can be used to produce polynucleotides, polypeptides, vectors, antigen-binding domains, immune cells, compositions, and the like according to the present disclosure.

[0245] Prior to the in vitro manipulation or genetic modification of immune cells described herein, the cells can be obtained from a subject. The cells expressing the Claudin 18.2 CAR can be derived from an allogeneic or autologous process.

[0246] i. Raw materials In some embodiments, the immune cells include T cells. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, T cells can be obtained from a unit of blood drawn from a subject using any number of techniques known to those skilled in the art, such as FICOLL™ separation.

[0247] Cells can be obtained from an individual's circulating blood by apheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In certain embodiments, the cells collected by apheresis can be washed to remove the plasma fraction and placed in a suitable buffer or medium for subsequent processing.

[0248] In certain embodiments, T cells are isolated from PBMCs by lysing red blood cells and depleting monocytes, for example, by centrifugation through a PERCOLL™ gradient. Specific subpopulations of T cells (e.g., CD28+, CD4+, CD5+, CD45RA-, CD45RO+, CD5+, CD62-, CD95-, CD95+, IL2Rβ+, IL2Rβ-, CCR7+, CCR7-, CDL-, CD62L+, and combinations thereof) can be further isolated by positive or negative selection techniques known in the art. In one example, the subpopulation of T cells is CD45RA+, CD95+, IL-2Rβ-, CCR7+, CD62L+. In one example, the subpopulation of T cells is CD45RA+, CD95+, IL-2Rβ+, CCR7+, CD62L+. In one example, a subpopulation of T cells is CD45RO+, CD95+, IL-2Rβ+, CCR7+, and CD62L+. In one example, a subpopulation of T cells is CD45RO+, CD95+, IL-2Rβ+, CCR7-, and CD62L-. In one example, a subpopulation of T cells is CD45RA+, CD95+, IL-2Rβ+, CCR7-, and CD62L-. For example, enrichment of a T cell population by negative selection can be achieved using a combination of antibodies directed against surface markers unique to the negatively selected cells. One method for use herein is cell sorting and / or selection via negative magnetic immunoadherence or flow cytometry using a cocktail of monoclonal antibodies directed against cell surface markers present on the negatively selected cells. For example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. Flow cytometry and cell sorting can also be used to isolate cell populations of interest for use in the present disclosure.

[0249] PBMCs can be used directly for genetic modification with immune cells (such as CARs or TCRs) using the methods described herein. In certain embodiments, after isolating PBMCs, T lymphocytes can be further isolated, and both cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subpopulations, either before or after genetic modification and / or expansion.

[0250] In some embodiments, CD8+ cells are further sorted into naive, stem cell memory, central memory, and effector cells by identifying characteristic cell surface antigens associated with each of these types of CD8+ cells. In some embodiments, the expression of phenotypic markers of central memory T cells includes CD45RO, CD62L, CCR7, CD28, CD3, and CD127, and is negative for granzyme B. In some embodiments, stem cell memory T cells are CD45RO-, CD62L+, CD8+ T cells. In some embodiments, central memory T cells are CD45RO+, CD62L+, CD8+ T cells. In some embodiments, effector T cells are negative for CD62L, CCR7, CD28, and CD127, and positive for granzyme B and perforin.

[0251] In certain embodiments, CD4+ T cells are further sorted into subpopulations. For example, CD4+ T helper cells can be sorted into naive, central memory, and effector cells by identifying cell populations with distinctive cell surface antigens.

[0252] ii. Stem cell-derived immune cells In some embodiments, immune cells may be derived from stem cells, such as progenitor cells, bone marrow stem cells, induced pluripotent stem cells, iPSCs, hematopoietic stem cells, and mesenchymal stem cells. iPS cells and other types of stem cells may be cultured as immortal cell lines or isolated directly from patients. Various methods for isolating, expressing, and / or culturing stem cells are known in the art and can be used in the practice of the present invention.

[0253] In some embodiments, the immune cells are induced pluripotent stem cells (iPSCs) derived from reprogrammed T cells. In some embodiments, the source material may be induced pluripotent stem cells (iPSCs) derived from T cells or non-T cells. The source material may alternatively be B cells, or peripheral blood mononuclear cell isolates, hematopoietic progenitor cells, hematopoietic stem cells, mesenchymal stem cells, adipose stem cells, or any other cells from any other somatic cell type.

[0254] iii. Genetic modification of isolated cells Immune cells, such as T cells, can be genetically modified after isolation using known methods, or the immune cells can be activated and expanded (or differentiated in the case of progenitor cells) in vitro before being genetically modified. In some embodiments, isolated immune cells are genetically modified to reduce or eliminate expression of endogenous TCRα and / or CD52. In some embodiments, the cells are genetically modified using gene editing techniques (e.g., CRISPR / Cas9, CRISPR / CAS12, zinc finger nucleases (ZFNs), TALENs, MegaTALs, meganucleases) to reduce or eliminate expression of endogenous proteins (e.g., TCRα and / or CD52). In another embodiment, optionally, immune cells, such as T cells, are further genetically modified with a chimeric antigen receptor described herein (e.g., further transduced with a viral vector comprising one or more nucleotide sequences encoding a CAR) and then activated and / or expanded in vitro.

[0255] Methods for activating and expanding T cells are known in the art and are described, for example, in U.S. Patent Nos. 6,905,874, 6,867,041, 6,797,514, and PCT WO2012 / 079000, the contents of which are incorporated herein by reference in their entireties. Generally, such methods involve contacting PBMCs or isolated T cells with stimulatory and costimulatory molecules, such as anti-CD3 and anti-CD28 antibodies, typically attached to plastic or magnetic beads or other surfaces, in a culture medium containing appropriate cytokines, such as IL-2. The anti-CD3 and anti-CD28 antibodies bound to the beads serve as "surrogate" antigen-presenting cells (APCs). One example is the Dynabeads® system, which is a CD3 / CD28 activator / stimulator system for the physiological activation of human T cells. In other embodiments, T cells can be activated and stimulated to expand with feeder cells and appropriate antibodies and cytokines using the methods described in U.S. Pat. No. 6,040,177, U.S. Pat. No. 5,827,642, and WO2012129514, the contents of which are incorporated by reference in their entireties.

[0256] Certain methods for generating the constructs and engineered immune cells of the present disclosure are described in PCT application PCT / US15 / 14520, the contents of which are incorporated herein by reference in their entirety.

[0257] It will be understood that PBMCs may further contain other cytotoxic lymphocytes, such as NK cells or NKT cells. An expression vector carrying the coding sequence of the chimeric receptor disclosed herein can be introduced into a population of human donor T cells, NK cells, or NKT cells. Successfully transduced T cells carrying the expression vector can be sorted using flow cytometry to isolate CD3-positive T cells, which can then be further expanded to increase the number of these CAR-expressing T cells in addition to cell activation using anti-CD3 antibodies and IL-2 or other methods known in the art as described elsewhere herein. Standard procedures are used to cryopreserve CAR-expressing T cells for storage and / or preparation for use in human subjects. In one embodiment, in vitro transduction, culture, and / or expansion of T cells is performed in the absence of non-human animal-derived products, such as fetal calf serum and fetal bovine serum. In one embodiment, cryopreservation may involve freezing in an appropriate medium such as CryoStor® CS10, CryoStor® CS2, or CryoStor® CS5 (BioLife Solutions).

[0258] For cloning a polynucleotide, a vector can be introduced into a host cell (isolated host cell) to allow the vector itself to replicate, thereby amplifying copies of the polynucleotide contained therein. Cloning vectors generally contain sequence components, including but not limited to, an origin of replication, a promoter sequence, a transcription initiation sequence, an enhancer sequence, and a selection marker. These elements can be selected appropriately by those skilled in the art. For example, an origin of replication can be selected to promote autonomous replication of the vector in the host cell.

[0259] In certain embodiments, the present disclosure provides an isolated host cell containing the vector provided herein. The host cell containing the vector can be useful for expressing or cloning the polynucleotide contained in the vector. Suitable host cells can include, but are not limited to, prokaryotic cells, fungal cells, yeast cells, or higher eukaryotic cells such as mammalian cells, more particularly human cells.

[0260] Vectors can be introduced into host cells using any suitable method known in the art, including, but not limited to, DEAE-dextran-mediated delivery, calcium phosphate precipitation, cationic lipid-mediated delivery, liposome-mediated transfection, electroporation, biolistics, receptor-mediated gene delivery, polylysine, histones, chitosan, and peptide-mediated delivery. Standard methods for viral transfection and transformation of cells for expression of a vector of interest are well known in the art. In a further embodiment, a mixture of different expression vectors can be used to genetically modify a donor population of immune effector cells, with each vector encoding a different CAR disclosed herein. The resulting transduced immune effector cells form a mixed population of engineered cells, with a proportion of the engineered cells expressing one or more different CARs.

[0261] In one embodiment, the present disclosure provides a method for preserving engineered cells expressing a CAR that targets the Claudin 18.2 protein. In one embodiment, this involves cryopreserving the immune cells so that the cells remain viable upon thawing. In one embodiment, cryopreservation can involve freezing in an appropriate medium, such as CryoStor® CS10, CryoStor® CS2, or CryoStor® CS5 (BioLife Solutions). A fraction of the immune cells expressing the CAR can be cryopreserved by methods known in the art to provide a permanent source of such cells for future treatment of patients with malignant tumors. If needed, the cryopreserved transformed immune cells can be thawed, grown, and expanded to obtain more such cells.

[0262] In some embodiments, cells are formulated by first harvesting the cells from their culture medium, then washing and concentrating them in a medium and container system suitable for administration in therapeutically effective amounts (a "pharmaceutically acceptable" carrier). Suitable infusion media are any isotonic media formulation, typically saline, Normosol™ R (Abbott), or Plasma-Lyte™ A (Baxter), although 5% dextrose or Ringer's lactate in water can also be utilized. Infusion media can be supplemented with human serum albumin.

[0263] iv. Allogeneic CAR T cells Briefly, the process for manufacturing allogeneic CAR T therapy involves harvesting healthy, selected, screened, and tested PBMCs or T cells from healthy donors. The allogeneic T cells are then gene-edited to reduce the risk of graft-versus-host disease (GvHD) and prevent allogeneic rejection. Selected T cell receptor genes (e.g., TCRα, TCRβ) are knocked out to avoid GvHD. The CD52 gene can be knocked out to render the CAR T product resistant to anti-CD52 antibody treatment. Anti-CD52 antibody treatment can therefore be used to lymphodeplete the host immune system, allowing the CAR T cells to achieve full therapeutic efficacy while remaining engrafted. Next, the T cells are engineered to express a CAR that recognizes a specific cell surface protein (e.g., Claudin 18.2) expressed on hematological or solid tumors. The engineered T cells then undergo a purification process and are ultimately cryopreserved in vials for delivery to patients.

[0264] v. Autologous CAR T cells Autologous chimeric antigen receptor (CAR) T cell therapy involves collecting a patient's own cells (e.g., white blood cells, including T cells) and genetically engineering the T cells to express a CAR that recognizes a target antigen expressed on the cell surface of one or more specific cancer cells and kills the cancer cells. The engineered cells are then cryopreserved and subsequently administered to the patient, whose cells have been removed for engineering.

[0265] IV. Treatment method The present disclosure includes methods of treating or preventing conditions associated with Claudin 18.2 or undesirable and / or elevated Claudin 18.2 levels in a patient, comprising administering to a patient in need thereof an effective amount of at least one CAR, or immune cells comprising a CAR disclosed herein.

[0266] Methods for treating diseases or disorders, including cancer, are provided. In some embodiments, the present disclosure relates to generating a T cell-mediated immune response in a subject, comprising administering to the subject an effective amount of engineered immune cells of the present application. In some embodiments, the T cell-mediated immune response is directed against a target cell(s). In some embodiments, the engineered immune cells comprise a chimeric antigen receptor (CAR). In some embodiments, the target cell is a tumor cell. In some aspects, the present disclosure includes a method for treating or preventing a malignancy, the method comprising administering to a subject in need thereof an effective amount of at least one isolated antigen-binding domain described herein. In some aspects, the present disclosure includes a method for treating or preventing a malignancy, the method comprising administering to a subject in need thereof an effective amount of at least one immune cell, the immune cell comprising at least one chimeric antigen receptor and / or isolated antigen-binding domain described herein. The CAR-containing immune cells of the present disclosure can be used to treat malignancies associated with aberrant expression of Claudin 18.2. In some embodiments, the CAR-containing immune cells of the present disclosure can be used to treat malignancies such as gastric cancer, gastroesophageal junction (GEJ) cancer, pancreatic cancer, small cell lung cancer, melanoma, low-grade glioma, glioblastoma, medullary thyroid carcinoma, carcinoid, dispersed neuroendocrine tumors in the pancreas, bladder, and prostate, testicular cancer, and lung adenocarcinoma with neuroendocrine features. In an exemplary embodiment, the CAR-containing immune cells, such as the anti-Claudin 18.2 CAR-T cells of the present disclosure, are used to treat small cell lung cancer.

[0267] Also provided is a method of reducing the size of a tumor in a subject, comprising administering to the subject engineered cells of the present disclosure, wherein the cells comprise a chimeric antigen receptor comprising a Claudin 18.2 antigen-binding domain and bind to the Claudin 18.2 antigen on the tumor.

[0268] In some embodiments, the subject has a solid tumor or a hematological malignancy such as lymphoma or leukemia. In some embodiments, the engineered cells are delivered to a tumor bed, such as that found in small cell lung cancer. In some embodiments, the cancer is present in the subject's bone marrow. In some embodiments, the engineered cells are autoimmune cells, e.g., autologous T cells. In some embodiments, the engineered cells are allogeneic immune cells, e.g., allogeneic T cells. In some embodiments, the engineered cells are xenogeneic immune cells, e.g., xenogeneic T cells. In some embodiments, the engineered cells are transfected or transduced ex vivo. As used herein, the term "in vitro cells" refers to any cells cultured ex vivo.

[0269] An "effective amount" is any amount that, when used alone or in combination with another agent, provides a desired or beneficial result. A "therapeutically effective amount," "effective dose," or "therapeutically effective dose" of a therapeutic agent, such as an engineered CAR T cell, when used alone or in combination with another therapeutic agent, is any amount that protects a subject against the onset of disease, or promotes disease regression as indicated by a decrease in the severity of disease symptoms, increases the frequency and duration of disease-free periods, or prevents functional impairment or disability due to disease. The ability of a therapeutic agent to promote disease regression can be evaluated using various methods known to skilled practitioners (e.g., physicians or clinicians), for example, by assaying the activity of the agent in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or in in vitro assays.

[0270] The terms "patient" and "subject" are used interchangeably and include human and non-human animal subjects, as well as those with a formally diagnosed disorder, those without a formally recognized disorder, those under medical attention, those at risk of developing a disorder, and the like.

[0271] The terms "treat" and "treatment" include therapeutic treatment, prophylactic treatment, and uses that reduce a subject's risk of developing a disorder or other risk factors. Treatment does not require a complete cure of the disorder, but encompasses embodiments in which symptoms or underlying risk factors are reduced. The term "prevent" does not require 100% elimination of the likelihood of an event. Rather, it indicates a reduced likelihood of an event occurring in the presence of a compound or method.

[0272] The desired total therapeutic amount of cells in the composition is at least 2 cells (e.g., at least one CD8+ T cell and at least one CD4+ T cell, or two CD8+ T cells, or two CD4+ T cells), or more typically 10 2 More than 10 cells, and up to 10 6 pcs, up to 10 including that number 8 pieces or 10 9 cells, and 10 10 or 10 12 The number of cells will depend on the desired use for which the composition is intended and the type of cells contained therein. Desired cell densities are typically between 10 and 10 6 cells / ml, generally above 10 7 cells / ml, typically >10 8 cells / ml. Clinically significant numbers of immune cells are greater than 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , or 10 12 The cells can be distributed over multiple injections that cumulatively equal or exceed 10 cells. In some embodiments of the present disclosure, particularly because all injected cells are reoriented to a specific target antigen (e.g., Claudin 18.2), 6 / kilogram (10 per patient) 6 ~10 11) can be administered. CAR therapy can be administered multiple times at doses within these ranges. Cells can be autologous, allogeneic, or xenogeneic to the patient undergoing therapy.

[0273] In some embodiments, the therapeutically effective amount of CAR T cells is about 1×10 5 cells / kg, approximately 2×10 5 cells / kg, approximately 3×10 5 cells / kg, approximately 4×10 5 cells / kg, approximately 5×10 5 cells / kg, approximately 6×10 5 cells / kg, approximately 7×10 5 cells / kg, approximately 8×10 5 cells / kg, approximately 9×10 5 cells / kg, 2×10 6 cells / kg, approximately 3×10 6 cells / kg, approximately 4×10 6 cells / kg, approximately 5×10 6 cells / kg, approximately 6×10 6 cells / kg, approximately 7×10 6 cells / kg, approximately 8×10 6 cells / kg, approximately 9×10 6 cells / kg, approximately 1×10 7 cells / kg, approximately 2×10 7 cells / kg, approximately 3×10 7 cells / kg, approximately 4×10 7 cells / kg, approximately 5×10 7 cells / kg, approximately 6×10 7 cells / kg, approximately 7×10 7 cells / kg, approximately 8×10 7 cells / kg, or approximately 9 × 10 7 cells / kg.

[0274] In some embodiments, the target dose for CAR+ / CAR-T+ cells is about 1 x 10 6 ~Approx. 1×10 10 cells / kg, e.g., about 1 x 10 6 cells / kg, approximately 1×10 7 cells / kg, approximately 1×10 8cells / kg, approximately 1×10 9 cells / kg, or approximately 1 x 10 10 The range is 1000mg / kg of cells.Doses above and below this range may be appropriate for a particular subject, and appropriate dose levels can be determined by medical professionals as needed.Furthermore, multiple doses of cells can be provided according to the present disclosure.

[0275] In some aspects, the present disclosure includes pharmaceutical compositions comprising at least one antigen-binding domain described herein and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition further comprises an additional active agent.

[0276] The CAR-expressing cell population of the present disclosure can be administered as a pharmaceutical composition, alone or in combination with a diluent and / or in combination with other components such as IL-2 or other cytokines or cell populations. The pharmaceutical composition of the present disclosure may comprise a CAR-expressing cell population, such as a T cell described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include a buffer, such as neutral buffered saline or phosphate buffered saline; a carbohydrate, such as glucose, mannose, sucrose, or dextran; an amino acid, such as mannitol, a protein, polypeptide, or glycine; an antioxidant; a chelating agent, such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide); and a preservative. The composition of the present disclosure may be formulated for intravenous administration.

[0277] Pharmaceutical compositions (solutions, suspensions, etc.) may contain one or more of the following: a sterile diluent such as water for injection; saline, preferably saline, Ringer's solution, isotonic sodium chloride; fixed oils such as synthetic mono- or diglycerides that may serve as solvents or suspending media; polyethylene glycol, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium sulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates; agents for adjusting tonicity such as sodium chloride or dextrose. Parenteral preparations may be enclosed in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic. For therapeutic use, injectable pharmaceutical compositions are preferably sterile.

[0278] In some embodiments, when administered to a patient, engineered immune cells expressing any one of the Claudin 18.2-specific CARs described herein on their cell surface can reduce, kill, or lyse the patient's endogenous Claudin 18.2-expressing cells. In one embodiment, the rate of reduction or lysis of Claudin 18.2-expressing endogenous cells or cells of a Claudin 18.2-expressing cell line by engineered immune cells expressing any one of the Claudin 18.2-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 greater than 95%. In one embodiment, the rate of reduction or lysis of Claudin 18.2-expressing endogenous cells or cells of a Claudin 18.2-expressing cell line by engineered immune cells expressing any one of the Claudin 18.2-specific CARs described herein is between about 5% and about 95%, between about 10% and about 95%, between about 10% and about 90%, between about 10% and about 80%, between about 10% and about 70%, between about 10% and about 60%, between about 10% and about 50%, between about 10% and about 40%, between about 20% and about 90%, between about 20% and about 80%, between about 20% and about 70%, between about 20% and about 60%, between about 20% and about 50%, between about 25% and about 75%, or between about 25% and about 60%.

[0279] In one embodiment, the rate of reduction or lysis of target cells, e.g., cell lines expressing Claudin 18.2, by engineered immune cells that express a Claudin 18.2-specific CAR of the present disclosure on their cell surface membrane can be measured using the assays disclosed herein.

[0280] In certain embodiments, compositions comprising the CAR-expressing immune effector cells disclosed herein may be administered to a patient systemically, e.g., by intravenous injection, or locally in proximity to a site of disease, e.g., by intraperitoneal injection. In some embodiments, compositions comprising anti-Claudin 18.2 CAR T cells may be administered intraperitoneally. In some embodiments, local, non-systemic delivery of the anti-Claudin 18.2 CAR T cells described herein may achieve efficacy at lower doses and / or reduce adverse events compared to systemic delivery, thereby improving the overall therapeutic index of the CAR T therapy.

[0281] The methods may further include administering one or more chemotherapeutic agents to the patient prior to administering the engineered cells provided herein. In certain embodiments, the chemotherapeutic agent is a lymphodepleting (conditioning) chemotherapy. For example, certain beneficial doses of cyclophosphamide (200 mg / m 2 / day~2000 mg / m 2 / day, approximately 100 mg / m 2 / day ~ approx. 2000 mg / m 2 / day, e.g., about 100 mg / m 2 / day, approximately 200 mg / m 2 / day, approximately 300 mg / m 2 / day, approximately 400 mg / m 2 / day, approximately 500 mg / m 2 / day, approximately 600 mg / m 2 / day, approximately 700 mg / m 2 / day, approximately 800 mg / m 2 / day, approximately 900 mg / m 2 / day, approximately 1000 mg / m 2 / day, approximately 1500 mg / m 2 / day or approximately 2000 mg / m 2 / day) and a specific dose of fludarabine (20 mg / m 2 / day~900 mg / m 2 / day, approximately 10 mg / m 2 / day ~ approx. 900 mg / m 2 / day; e.g., about 10 mg / m 2 / day, approximately 20 mg / m 2 / day, approximately 30 mg / m 2 / day, approximately 40 mg / m 2 / day, approximately 40 mg / m 2 / day, approximately 50 mg / m 2 / day, approximately 60 mg / m 2 / day, approximately 70 mg / m 2 / day, approximately 80 mg / m 2 / day, approximately 90 mg / m 2 / day, approximately 100 mg / m 2 / day, approximately 500 mg / m 2 / day or approximately 900 mg / m 2 / day) to the patient. An exemplary dosing regimen involves treating a patient with daily administration of about 300 mg / m2 / day of cyclophosphamide to the patient in combination with, before or after, administration of about 30 mg / m2 / day of fludarabine for three days prior to administration of a therapeutically effective amount of engineered T cells to the patient.

[0282] In some embodiments, particularly when the engineered cells provided herein have been gene-edited to eliminate or minimize surface expression of CD52, lymphodepletion further comprises administration of an anti-CD52 antibody, such as alemtuzumab. In some embodiments, the CD52 antibody is administered at a dose of about 1-20 mg / day IV, e.g., about 13 mg / day IV, e.g., about 20 mg / day IV, e.g., about 30 mg / day IV, for 1, 2, 3, or more days. The antibody may be administered in combination with, before, or after the administration of other components of the lymphodepletion regimen (e.g., cyclophosphamide and / or fludarabine).

[0283] In other embodiments, the antigen binding domain, transduced (or otherwise engineered) cells, and chemotherapeutic agent are each administered in an amount effective to treat the disease or condition of the subject.

[0284] In certain embodiments, compositions comprising the CAR-expressing immune effector cells disclosed herein can be administered in conjunction with any number of chemotherapeutic agents. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN™); alkylsulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meteuredopa, and uredopa; ethylenimines and methylameramines, including altretamine, triethylenemelamine, triethylenephosphamide, triethylenethiophosphamide, and trimethylolmelamine; nitrogen mustards, such as methylameramine, ... Nitrosoureas, such as chlorambucil, chlornaphazine, colofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembicine, phenesterine, prednimustine, trofosfamide, uracil mustard, carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine, and other nitrosoureas; antibiotics, such as arachnidin, actinomycin, anthramycin, azaserine, bleomycin, sactinomycin, camomycin, Lithiamycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfilomycin, puromycin, queramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubeni mexamex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU;Androgens, e.g., calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testosterone; antiadrenal agents, e.g., aminoglutethimide, mitotane, trilostane; folic acid supplements, e.g., furoic acid, aceglatone; aldophosphamide glycoside; aminolevulinic acid, amsacrine; bestravcil; bisantrene; edatraxate, defafamine; demecolcine; diaziconazole; elformitin; elliptinium acetate, etoglucide; gallium nitrate, hydroxyurea; lentinan; lonidamine; mitoguazone, mitoxantrone; mogamulizumab Pidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllic acid, 2-ethylhydrazide; procarbazine; PSK®, razoxane; sizofiran; spirogermanium; tenuazonic acid, triazicon; 2,2',2'-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mytalactol; pipobroman; gacytosine; arabinoside (Ara-C), cyclophosphamide; thiotepa; taxoids, such as paclitaxel (TAXOL®, Bristol-Myers Squibb) and docetaxel (TAXOTERE®, Rhone-Poulenc Rorer); chlorambucil; gemcitabine; 6-thioguanine; platinum analogues, such as mercaptopurine, methotrexate, cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16), ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RF S2000; difluoromethylomitin (DMFO); retinoic acid derivatives such as Targretin™ (bexarotene), Panretin™, (alitretinoin); ONTAK™ (denileukin diftitox); esperamycin, capecitabine;and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing. Also included within this definition are anti-estrogens (e.g., tamoxifen, raloxifene, aromatase-inhibiting 4(5)-imidazole, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston)), and anti-hormonal agents that regulate or inhibit hormone action on tumors, such as anti-androgens (e.g., flutamide, nilutamide, bicalutamide, leuprolide, and goserelin), as well as pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing. Chemotherapeutic combinations may also be administered where appropriate, including, but not limited to, CHOP, i.e., cyclophosphamide (Cytoxan®), doxorubicin (hydroxydoxorubicin), vincristine (Oncovin®), and prednisone.

[0285] In some embodiments, the chemotherapeutic agent is administered simultaneously with, or within one week of, administration of the engineered cells, polypeptide, or nucleic acid. In other embodiments, the chemotherapeutic agent is administered about 1 to 7 days, about 1 to about 4 weeks, about 1 week to about 1 month, about 1 week to about 2 months, about 1 week to about 3 months, about 1 week to about 6 months, about 1 week to about 9 months, or about 1 week to about 12 months after administration of the engineered cells, polypeptide, or nucleic acid. In other embodiments, the chemotherapeutic agent is administered at least one month before administration of the cells, polypeptide, or nucleic acid. In some embodiments, the method further comprises administering two or more chemotherapeutic agents.

[0286] A variety of additional therapeutic agents can be used in conjunction with the compositions described herein. For example, potentially useful additional therapeutic agents include PD-1 inhibitors such as nivolumab (Opdivo®), pembrolizumab (Keytruda®), pembrolizumab, pidilizumab, and atezolizumab.

[0287] Additional therapeutic agents suitable for use in combination with the present disclosure include, but are not limited to, ibrutinib (Imbruvica®), ofatumumab (Arzera®), rituximab (Rituxan®), bevacizumab (Avastin®), trastuzumab (Herceptin®), trastuzumab emtansine (Kadcyla®), imatinib ( Gleevec (registered trademark), cetuximab (Erbitux (registered trademark), panitumumab) (Vectibix (registered trademark), catumaxomab, ibritumomab, ofatumumab, tositumomab, brentuximab, alemtuzumab, gemtuzumab, erlotinib, gefitinib, vandetanib, afatinib, lapatinib, neratinib, axitinib, masitinib, pazopanib, sucralose, Nitinib, sorafenib, toceranib, lestaurtinib, axitinib, cediranib, lenvatinib, nintedanib, pazopanib, regorafenib, semaxanib, sorafenib, sunitinib, tivozanib, toceranib, vandetanib, entrectinib, cabozantinib, imatinib, dasatinib, nilotinib, ponatinib, radotinib, bosutinib, lestaurtinib, ruxoli mTOR inhibitors such as tinib, pacritinib, cobimetinib, selumetinib, trametinib, binimetinib, alectinib, ceritinib, crizotinib, aflibercept, adipotide, denileukin diftitox, everolimus and temsirolimus, hedgehog inhibitors such as sonidegib and vismodegib, and CDK inhibitors such as palbociclib.

[0288] In some embodiments, compositions comprising CAR-containing immune cells can be administered with a therapeutic regimen to prevent or reduce cytokine release syndrome (CRS) or neurotoxicity. Therapeutic regimens to prevent cytokine release syndrome (CRS) or neurotoxicity can include lenzilumab, tocilizumab, atrial natriuretic peptide (ANP), anakinra, and iNOS inhibitors (e.g., L-NIL or 1400W). In additional embodiments, compositions comprising CAR-containing immune cells can be administered with an anti-inflammatory agent. Anti-inflammatory agents or drugs include, but are not limited to, steroids and glucocorticoids (betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone), nonsteroidal anti-inflammatory drugs (NSAIDS) (including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF drugs, cyclophosphamide and mycophenolic acid).Exemplary NSAIDs include ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors and sialic acid.Exemplary analgesics include acetaminophen, oxycodone, proporoxifene hydrochloride, tramadol. Exemplary glucocorticoids include cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Exemplary biological response modifiers include molecules directed against cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors, such as TNF antagonists (e.g., etanercept (ENBREL®), adalimumab (HUMIRA®), and infliximab (REMICADE®), chemokine inhibitors, and adhesion molecule inhibitors. Biological response modifiers include monoclonal antibodies and recombinant molecules. Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, gold (oral (auranofin) and intramuscular), and minocycline.

[0289] In certain embodiments, the compositions described herein are administered in conjunction with cytokines. Examples of cytokines include lymphokines, monokines, and conventional polypeptide hormones. Cytokines include growth hormones (such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone), parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, prorelaxin, glycoprotein hormones (such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH)), hepatocyte growth factor (HGF), fibroblast growth factor (FGF), prolactin, placental lactogen, Mullerian inhibitory substance, mouse gonadotropin-related peptide, inhibin, activin, vascular endothelial growth factor, integrins, thrombopoietin (TPO), nerve growth factor (NGF) (such as NGF-beta), platelet growth factor, transforming growth factor (TGF) (TGF-alpha and TG). F-beta), insulin-like growth factor-I and -II, erythropoietin (EPO), osteoinductive factor, interferons (such as interferon-alpha, beta, and -gamma), colony-stimulating factors (CSFs) (such as macrophage-CSF (M-CSF)), granulocyte-macrophage-CSF (GM-CSF), and granulocyte-CSF (G-CSF), interleukins (IL) (such as IL-1, IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15, IL-21), tumor necrosis factors (such as TNF-alpha or TNF-beta), and other polypeptide factors, including LIF and Kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or recombinant cell culture, as well as biologically active equivalents of the native sequence cytokines.

[0290] V. Sorting and Depletion Methods In some embodiments, a method for in vitro sorting of an immune cell population is provided, wherein a subset of the immune cell population comprises engineered immune cells that express any one of Claudin 18.2-specific CARs that comprise an epitope (e.g., an exemplary mimotope sequence) specific for a monoclonal antibody. 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 Claudin 18.2-specific CAR.

[0291] In some embodiments, the monoclonal antibody specific for the epitope is optionally conjugated to a fluorophore, in which case the step of selecting cells that bind the monoclonal antibody can be performed by fluorescence-activated cell sorting (FACS).

[0292] In some embodiments, the monoclonal antibody specific for the epitope is optionally conjugated to a magnetic particle, in which case the step of selecting cells that bind to the monoclonal antibody can be performed by magnetic-activated cell sorting (MACS).

[0293] In some embodiments, the mAb used in the method of sorting immune cells expressing a CAR is selected from alemtuzumab, ibritumomab tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, bevacizumab, certolizumab pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab, palivizumab, ranibizumab, tocilizumab, trastuzumab, 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.

[0294] In some embodiments, the population of CAR-expressing immune cells obtained when using the above-described methods for in vitro sorting of CAR-expressing immune cells comprises at least 70%, 75%, 80%, 85%, 90%, 95% CAR-expressing immune cells. In some embodiments, the population of CAR-expressing immune cells obtained when using the in vitro methods for sorting CAR-expressing immune cells comprises at least 85% CAR-expressing immune cells.

[0295] In some embodiments, the population of CAR-expressing immune cells obtained when using the above-described in vitro selection method for CAR-expressing immune cells exhibits increased in vitro cytotoxic activity compared to the initial (unsorted) cell population. In some embodiments, said cytotoxic activity is increased by 10%, 20%, 30%, or 50% in vitro. In some embodiments, the immune cells are T cells.

[0296] In some embodiments, the mAb is previously bound to a support or surface. Non-limiting examples of solid supports can include beads, agarose beads, plastic beads, magnetic beads, plastic bonded plates, glass bonded plates, ceramic bonded plates, columns, or cell culture bags.

[0297] The CAR-expressing immune cells administered to the recipient can be enriched in vitro from a source population. Methods for expanding the source population can include selecting cells that express an antigen, such as the CD34 antigen, using a combination of density centrifugation, immunomagnetic bead purification, affinity chromatography, and fluorescence-activated cell sorting.

[0298] Flow cytometry can be used to quantify specific cell types in a cell population.Generally, flow cytometry is a method for quantifying cellular components or structural features mainly by optical means.By quantifying structural features, different cell types can be distinguished, so that flow cytometry and cell sorting can be used to count and sort cells of different phenotypes in a mixture.

[0299] Flow cytometry analysis involves two major steps: 1) labeling a selected cell type with one or more markers, and 2) determining the number of labeled cells relative to the total number of cells in a population. In some embodiments, the method of labeling a cell type involves binding a labeled antibody to a marker expressed by a particular cell type. The antibody can be directly labeled with a fluorescent compound or indirectly labeled, for example, with a fluorescently labeled secondary antibody that recognizes the first antibody.

[0300] In some embodiments, the method used to sort CAR-expressing T cells is magnetically activated cell sorting (MACS). Magnetically activated cell sorting (MACS) is a method for separating various cell populations according to their surface antigens (CD molecules) using superparamagnetic nanoparticles and columns. Using MACS, pure cell populations can be obtained. Cells in a single-cell suspension can be magnetically labeled with microbeads. The sample is then loaded onto a column made of ferromagnetic spheres, coated with a cell-friendly coating, allowing for rapid and gentle cell separation. Unlabeled cells pass through, while magnetically labeled cells are retained within the column. The flow-through can be collected as the unlabeled cell fraction. After a washing step, the column is removed from the separator, and the magnetically labeled cells are eluted from the column.

[0301] Detailed protocols for the purification of specific cell populations, such as T cells, can be found in Basu S et al. (2010). (Basu S, Campbell HM, Dittel BN, Ray A. Purification of specific cell populations by fluorescence activated cell sorting (FACS). J Vis Exp. (41): 1546).

[0302] In some embodiments, the present disclosure provides a method for depleting Claudin 18.2-specific CAR-expressing immune cells by in vivo depletion. In vivo depletion can include administering a therapeutic agent (e.g., a molecule that binds to an epitope on the CAR) to a mammalian organism, with the aim of stopping the proliferation of CAR-expressing immune cells by inhibition or elimination.

[0303] One aspect of the present invention relates to a method for in vivo depletion of engineered immune cells expressing a Claudin 18.2 CAR comprising a mAb-specific epitope, comprising contacting the engineered immune cells or the CAR-expressing immune cells with at least one epitope-specific mAb. Another aspect of the present invention relates to a method for in vivo depletion of CAR-expressing immune cells comprising a chimeric scFv (e.g., formed by insertion of a mAb-specific epitope) by contacting the engineered immune cells with an epitope-specific antibody. In some embodiments, the immune cells are T cells and / or the antibody is a monoclonal antibody.

[0304] According to one embodiment, in vivo depletion of immune-engineered cells is performed on engineered immune cells previously sorted using the in vitro method of the present invention. In this case, an infused mAb can be used. In some embodiments, the mAb-specific antigen is CD20 antigen and the epitope-specific mAb is rituximab. In some embodiments, the present invention relates to a method for in vivo depletion of engineered immune cells expressing a CAR containing a mAb-specific epitope (CAR-expressing immune cells) in a patient, comprising contacting the CAR-expressing immune cells with at least one epitope-specific mAb.

[0305] In some embodiments, contacting the engineered immune cells or the CAR-expressing immune cells with at least one epitope-specific mAb comprises injecting the epitope-specific mAb (e.g., rituximab) into the patient. In some embodiments, the amount of epitope-specific mAb administered to the patient is sufficient to eliminate at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the CAR-expressing immune cells in the patient.

[0306] In some embodiments, contacting the engineered immune cells or the CAR-expressing immune cells with at least one epitope-specific mAb comprises administering to the patient about 375 mg / m 2 In some embodiments, the mAb (e.g., rituximab) is administered once a week.

[0307] In some embodiments, when immune cells expressing a CAR containing a mAb-specific epitope (CAR-expressing immune cells) are depleted in a complement-dependent cytotoxicity (CDC) assay using an epitope-specific mAb, the amount of viable CAR-expressing immune cells is reduced. In some embodiments, the amount of viable CAR-expressing immune cells is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In some embodiments, the mAb-specific epitope is a CD20 epitope or mimotope, and / or the epitope-specific mAb is rituximab.

[0308] In certain embodiments, in vivo depletion of CAR-engineered immune cells is performed by injecting bispecific antibodies. By definition, bispecific monoclonal antibodies (BsAbs) are engineered proteins composed of fragments of two different monoclonal antibodies, which consequently bind to two different types of antigens. These BsAbs and their use in immunotherapy have been reviewed in Muller D and Kontermann RE (2010) Bispecific Antibodies for Cancer Immunotherapy, BioDrugs 24(2):89-98.

[0309] According to another specific embodiment, the injected bispecific mAb can bind both the mAb-specific epitope carried by the engineered immune cells expressing the chimeric scFv and the surface antigen of effector and cytotoxic cells (e.g., immune cells such as lymphocytes, macrophages, dendritic cells, natural killer cells (NK cells), and cytotoxic T lymphocytes (CTLs)). In this way, depletion of the engineered immune cells induced by the BsAb can occur via antibody-dependent cellular cytotoxicity (ADCC). (Deo YM, Sundarapandiyan K, Keler T, Wallace PK, and Graziano RF, (2000), Journal of Immunology, 165(10):5954-5961).

[0310] In some embodiments, cytotoxic agents are linked to epitope-specific mAbs, which can be used to deplete CAR-expressing immune cells.By combining the targeting ability of monoclonal antibodies with the cancer-killing ability of cytotoxic agents, antibody-drug conjugates (ADCs) allow for the differentiation of sensitivity between healthy tissue and diseased tissue, compared with the use of drugs alone.Some ADCs have been approved for the market, and the technology for producing them, especially on linkers, has been described (Payne, G. (2003) Cancer Cell 3:207-212; Trail et al. (2003) Cancer Immunol. Immunother. 52:328-337; Syrigos and Epeneto (1999) Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drug Del. Rev. 26:151-172, U.S. Patent No. 4,975,278).

[0311] In some embodiments, the injected epitope-specific mAb is pre-conjugated with a molecule that can promote complement-dependent cytotoxicity (CDC). Thus, the complement system aids or complements the antibody's ability to eliminate pathogens from the organism. When stimulated, an activation cascade is initiated, resulting in a massive amplification of the response and activation of the cell-killing membrane attack complex. Different molecules can be used to bind mAbs, such as glycans (Courtois, A, Gac-Breton, S., Berthou, C, Guezennec, J., Bordron, A. and Boisset, C. (2012). Complement-dependent cytotoxicity activity of therapeutic antibody fragments can be acquired by immunogenic glycan coupling, Electronic Journal of Biotechnology ISSN: 0717-3458; http: / / www.ejbiotechnology.info DOI: 10.2225 / voll5-issue5).

[0312] VI. Kits and Articles of Manufacture The present application provides kits comprising any one of the Claudin 18.2 containing CAR or Claudin 18.2 CAR-containing immune cells described herein, as well as pharmaceutical compositions thereof. In one embodiment, the engineered CAR cells are frozen in an appropriate medium, such as CryoStor® CS10, CryoStor® CS2, or CryoStor® CS5 (BioLife Solutions).

[0313] In some exemplary embodiments, the kits of the present disclosure comprise allogeneic Claudin 18.2 CAR-containing T cells and a CD52 antibody for administration to a subject for use in lymphodepleting and CAR-T regimens.

[0314] The present application also provides an article of manufacture comprising any one of the therapeutic compositions or kits described herein. An example of an article of manufacture is a vial (e.g., a sealed vial). [Example]

[0315] Example 1. Characterization of Claudin 18.2-targeting antibodies To test the binding capacity and specificity of Claudin 18.2 antibodies, HEK-293T, a Claudin 18-negative cell line, was engineered to overexpress human or mouse Claudin 18.1 or 18.2. Cells were stained with purified anti-Claudin 18.2 scFv-hIgG2Fc or control scFv-hIgG Fc at 5 μg / mL in PBS for 30 minutes at 4°C. Bound Claudin 18.2 antibodies were then detected with a PE-labeled anti-human IgG Fcγ antibody (Jackson ImmunoResearch, catalog no. 109-0116-098) at a 1:200 dilution. Stained samples were analyzed by flow cytometry. Representative images showing binding of Claudin 18.2 antibodies to 293T parental and engineered cells are included in Figure 1. Solid and dashed lines represent staining with anti-Claudin 18.2 antibodies or isotype controls, respectively. Due to the high sequence homology between human and mouse Claudin 18.2, all anti-human Claudin 18.2 antibodies cross-react and bind to mouse Claudin 18.2. Clones that specifically bind to human Claudin 18.2 but not human Claudin 18.1 are considered optimal clones for reformatting into CARs for higher specificity.

[0316] Example 2. Generation of Claudin 18.2 CAR T cells Claudin 18.2 CAR T cells were prepared using lentiviral transduction. To generate lentivirus encoding the Claudin 18.2 CAR, HEK-293T cells were transduced at 0.8 × 10 per well of a 6-well plate in 2 mL of DMEM (Gibco) supplemented with 10% FBS, 1 × non-essential amino acids, 1 mM sodium pyruvate, and 25 mM HEPES. 6The cells were seeded at 1000 cells / mL. The next day, lentiviral packaging vectors containing 1.5 μg of psPAX2 and 0.5 μg of pMD2.G were mixed with 0.5 μg of a plasmid expressing a CAR construct in 250 μL of Opti-MEM. 10 μL of Lipofectamine 2000 in 250 μL of Opti-MEM was incubated at room temperature for 5 minutes and then added to the DNA mixture. A total of 500 μL of the DNA / Lipofectamine mixture was incubated at room temperature for 20 minutes before being added to the wells containing the HEK-293T cells. The cells were then returned to a 37°C incubator containing 5% CO2 overnight. One day after transfection, the medium from each well of the 6-well plate was replaced with 2 mL of X-VIVO™ 15 supplemented with 10% FBS. Supernatants were collected 24 hours later, passed through a 0.45 μm filter (EMD Millipore), and then concentrated to lentivirus using Lenti-X (Takara). The concentrated virus was resuspended in 200 μL of X-VIVO™ 15 containing 10% FBS and used directly for T cell transduction.

[0317] To generate T cells expressing the Claudin 18.2 CAR, primary human T cells were purified directly from LeukoPak (StemCell Technologies) using the EasySep™ Human T Cell Isolation Kit (StemCell Technologies, Catalog No. 17951) and cryopreserved. T cells were activated immediately after recovery from cryopreservation with human T cell TransAct (Miltenyi Biotec, Catalog No. 130-111-160, 1:100 dilution) in X-VIVO™ 15 (Lonza) supplemented with 10% FBS and 100 IU / mL human IL-2 (Miltenyi Biotec). After 2 days, activated T cells were harvested and resuspended at 1 x 10 in 1 mL of fresh medium containing IL-2. 6The cells were resuspended at 1000 cells / mL. The prepared virus was then added for CAR transduction. On day 5 after activation, the medium was replaced with X-VIVO™ 15 T cell growth medium supplemented with 5% human AB serum (Gemini Bio) along with 100 IU / mL human IL-2 to feed the cells. On day 6, the TCRa constant (TRAC) and CD52 genes were knocked out by transcription activator-like effector nuclease (TALEN)-mediated gene editing. Cells were expanded into larger flasks or G-Rex vessels (Wilson Wolf) as needed using T cell growth medium with IL-2. On day 14, TCRα / β-negative cells were purified using the EasySep Human TCRa / b Depletion Kit (StemCell Technologies) and placed overnight in T cell growth medium containing 100 IU / mL human IL-2 before being cryopreserved on day 15. The percentage of CAR+ cells across samples was normalized to the sample with the lowest transduction efficiency by adding non-transduced (NTD) T cells immediately before cryopreservation. CAR transduction efficiency and phenotype were assessed by flow cytometry on days 9 and 14. To determine the percentage of CAR transduction, T cells were first stained with 50 μg / mL biotinylated recombinant protein L (Thermo Scientific, Cat. No. 29997) in PBS for 30 minutes at 4°C, followed by staining with PE-streptavidin (Biolegend, Cat. No. 405204) at a 1:200 dilution for 30 minutes at 4°C. Memory phenotypes were assigned according to CD62L and CD45RO expression within the CAR+ cell population (gating based on PE-conjugated rituximab detection) as follows: stem cell memory (TSCM, CD45RO- / CD62L+), central memory (TCM, CD45RO+ / CD62L+), effector memory (TEM, CD45RO+ / CD62L-), effector cells (TEFF, CD45RO- / CD62L-).

[0318] Examples of Claudin 18.2 CAR T cells with safety switches are shown in Figures 3A-3C. Figure 3A shows representative FACS plots demonstrating varying levels of transduction of anti-Claudin 18.2 CARs in different rituximab off-switch formats. Constructs with lower transduction efficiencies were deemed less desirable for large-scale manufacturing. Figure 3B summarizes the transduction efficiencies in two different donors. Figure 3C shows comparable distribution of T cell differentiation subsets among different Claudin 18.2 CAR T cells.

[0319] Example 3. In vitro cytotoxicity of Claudin 18.2 CAR T cells To test Claudin 18.2-specific killing, we used multiple human cancer cell lines, including Claudin 18.2-positive cells (PATU8988s, MKN45 / hClaudin 18.2), Claudin 18.1-positive cells (MKN45 / hClaudin 18.1), and cells negative for both Claudin 18.1 and 18.2 (22rv1). All cell lines were engineered to express firefly luciferase. On day 0 of the assay, 1 x 10 cells were cultured. 4 Target cells were seeded in 100 μL of RPMI supplemented with 10% FBS per well in a white, flat-bottom 96-well tissue culture plate. After target cells adhered to the bottom of the plate, Claudin 18.2 CAR T cells were thawed and added to the seeded target cells at different effector:target (E:T) ratios ranging from 1:9 to 3:1 in 100 μL of RPMI supplemented with 10% FBS. Cell viability was measured after 72 hours using the One-glo luciferase reagent kit (Promega). Each condition was assayed in duplicate, and the rate of target cell lysis was calculated by normalizing the luciferase activity for each CAR treatment to the target-only control.

[0320] Figure 2 shows that the majority of Claudin 18.2 CAR T cells specifically killed Claudin 18.2-expressing target cells. Clones that showed no cytotoxicity against Claudin 18.2 (4A5, 17F11) or cross-reactivity with Claudin 18.1 (10D11) were excluded from the following screening.

[0321] In addition to the short-term killing assay, a serial killing assay involving repeated antigen exposure was also used to further evaluate Claudin 18.2 CARs with different safety switches. Briefly, on day 0 of the assay, 1 × 10 4 Target cells were seeded in 100 μL of RPMI supplemented with 10% FBS per well in a white, flat-bottom 96-well tissue culture plate. After the target cells adhered to the bottom of the plate, Claudin 18.2 CAR T cells were thawed and added to the seeded target cells at a 3:1 effector:target (E:T) ratio in 100 μL of RPMI supplemented with 10% FBS. Every 2–3 days, 100 μL of medium containing T cells was transferred to freshly seeded target cells, and the percentage of target cell lysis was determined at each time point using the 1-glo luciferase assay kit (Promega). Each condition was assayed in triplicate.

[0322] The mean percentage of lysis and standard error of the mean are plotted in Figure 4A-B. Figure 4A shows the long-term cytotoxicity against one gastric cancer cell line overexpressing Claudin 18.2 (MKN45 / hClaudin 18.2) and one pancreatic cancer cell line expressing endogenous Claudin 18.2 (PATU8988s, Panc05.04). The optimal clone with the highest target cell lysis throughout the assay period was selected and further tested in a separate experiment against four gastric cancer cell lines expressing endogenous Claudin 18.2 (SNU-601, SNU-620, NUGC-4, GSU). As shown in Figure 4B, 2A4.R2S appeared to be more potent than the other two clones in two different donors. Example 4. In vitro cytokine secretion of Claudin 18.2 CAR T cells

[0323] Cytokines secreted from T cells produced according to the method of Example 2 were measured using a Human ProInflammatory 9-Plex Tissue Culture Kit (Meso Scale Discovery, 15007B). Briefly, 100 μL of RPMI containing 10% FBS was added to 1×10 4 100 μL of target cells (SNU-601, PATU8988s) were added to a 96-well plate. Claudin 18.2 CAR T cells were thawed and added at a 1:1 effector:target (E:T) ratio in 100 μL of RPMI medium containing 10% FBS. After 24 hours, the media from the co-culture was collected from each well and spun down to pellet T cells. The supernatant was stored at -80°C and then thawed for cytokine analysis using the Meso Scale Discovery assay according to the manufacturer's protocol.

[0324] Figure 5 shows experimental data demonstrating that Claudin 18.2 CAR T cells secreted cytokines in a Claudin 18.2-dependent manner. While low levels of spontaneous cytokines were detected in the absence of target, coculture with SNU-601 or PATU8988s induced significant release of IFN-γ, IL-2, and TNF-α. The dotted lines in each graph indicate the detection limit for each individual cytokine.

[0325] Example 5. In vivo antitumor efficacy of Claudin 18.2 CAR T cells To test the in vivo antitumor activity of Claudin 18.2 CAR T cells, an SNU-601 gastric cancer xenograft model was used. On day 0, immunodeficient NSG mice were inoculated with 5 × 10 6 SNU-601 cells were implanted subcutaneously, and tumor growth was monitored using a digital caliper. Tumor size was calculated using the following formula: tumor volume = (width × width) × (mass × mass). 2 × length / 2) was used to calculate the tumor size. 3Once the tumor reached a volume of 1 × 10, mice were randomized and treated intravenously with freshly thawed Claudin 18.2 CAR T or non-transduced (NTD) T cells. Cells were resuspended in PBS and injected via tail vein injection in a volume of 200 μL. Tumor and body weight were monitored every 3–4 days until the end of the study. Figures 6A–6B show tumors from 1 × 10 tumors. 6 Experimental data are presented showing that a CAR+ cell dose of anti-Claudin 18.2 CAR T cells can control and eliminate established gastric cancer tumors in an in vivo SNU-601 tumor model (Figure 6A) without causing weight loss (Figure 6B). 6 For CAR T cells, weight loss was observed in some mice receiving Claudin 18.2 clone 2A4 CAR T cells, followed by weight regain at later time points. See Figure 6D (mean) and Figure 6H (individual plots). 6 Mice receiving Claudin 18.2 clone 2A4 CAR T cells showed significant weight loss, and studies with these mice were terminated at an early time point in accordance with established animal care protocols. Figure 6C shows that both anti-Claudin 18.2 clone 1E7 CAR and clone 2A4 CAR effectively controlled tumor growth in the SNU-601 SC mouse model at all doses tested, except for 10 × 10 of 2A4. 6 Note that CAR T dosing data was only available up to day 15. 1E7 CAR T cells of the anti-Claudin 18.2 clone were administered at a high dose of 10 × 10 6 Mice administered with CAR T cells showed initial weight loss but regained weight at later time points (Figures 6D and 6G). Thus, a therapeutic index can be established for both clone 2A4 and 1E7 CARs.

[0326] Further experiments were performed using an intraperitoneal xenograft model that mimics peritoneal metastasis. To establish this model, mice were inoculated with 3 × 10 6SNU-601 cells were injected intraperitoneally, and tumor cell engraftment was confirmed using luminescence imaging. Mice were then injected with two dose levels (1 × 10) via different injection routes (IP: intraperitoneal or IV: intravenous). 6 or 3×10 6 CAR + cells) were treated with Claudin 18.2 clone 1E7 CAR T cells. Tumor regression was measured by bioluminescence spectroscopy. Data in Figure 7B show that a low dose of 1 x 10 cells administered intravenously in an SNU-601 intraperitoneal xenograft model significantly reduced tumor regression. 6 This figure shows that CAR T cells did not effectively eliminate tumors in an SNU-601 intraperitoneal xenograft model. The same dose administered intraperitoneally effectively eliminated tumors in the same model (Figure 7A). While not intending to be bound by any particular mechanism, this suggests that local, regional delivery of CAR T cells may be superior to systemic delivery, at least in this model. For local or regional delivery, lower doses may be used to further ensure safety. None of the animals showed significant loss of body weight (Figure 7B). Results represent the mean ± SEM. Representative bioluminescence images of the same mice as in Figures 7A-B at days 1, 6, 10, and 31 after CAR T treatment are shown in Figure 7C.

[0327] The in vivo antitumor efficacy and safety of Claudin 18.2 CAR T cells were again tested in a different animal model, the NUGC-4 subcutaneous model, in comparison with two tool CARs: Tool CAR 1 contains the anti-Claudin 18.2 antibody described in Shah et al., Nat Med, 2133-2141 (2023), and Tool CAR 2 is described in Jiang et al., 2019, JNCI Natl Cancer Inst, 111(4):djy134. On day 0, immunodeficient NSG mice were inoculated with 3 × 10 6 NUGC-4 cells were implanted subcutaneously, and tumor growth was monitored by digital calipers as previously described. At day 20, tumors grew to approximately 150–200 mm 3A volume of 1000 cells was achieved, and mice were randomized and treated intravenously with freshly thawed Claudin 18.2 CAR T or non-transduced (NTD) T cells. Cells were resuspended in PBS and administered intravenously at 1 x 10 cells per mouse via tail vein injection in a volume of 200 μL. 6 CAR+ cells or 3 × 10 6 Tumors and body weights were monitored weekly from day 4 after CAR T administration until the end of the study.

[0328] Figures 8A and 8B show that both anti-Claudin 18.2 clone 1E7 and 2A4 CAR T cells were positive for 1 × 10 6 These results show that CAR T cell doses controlled established gastric cancer tumors more effectively than tool CAR 1 and tool CAR 2. Anti-Claudin 18.2 clone 2A4 CAR T cells performed even better than clone 1E7 in controlling tumor volume, but at a higher dose of 3 × 10 6 For CAR T cells, some mice receiving anti-Claudin 18.2 clone 2A4 CAR T showed significant weight loss, which led to the termination of the study in these mice according to established animal care protocols (Figure 8C). Individual mouse weight changes were calculated based on a 3 × 10 6 CAR T cell dose was plotted against days after CAR T treatment.

[0329] As shown in Figures 8F-8I, with the exception of mice receiving tool CAR 1 (Figure 8H), which was the most effective CAR in controlling tumor volume (Figure 8A), early weight loss was observed in most mice injected with anti-Claudin 18.2 CAR T cells (compare Figure 8F, 8G with Figure 8I). Mice receiving tool CAR 2 CAR T cells showed similar initial weight loss and tumor volume reduction compared to mice receiving anti-Claudin 18.2 clone 1E7 and 2A4 CAR T cells. Compare Figure 8I with Figures 8F and 8G and the activity data in Figure 8A. Thus, early weight loss may be associated with CAR T efficacy, and anti-Claudin 18.2 clone 1E7 and 2A4 CARs achieved better efficacy than tool CAR 1 and tool CAR 2, while tool CAR 2 showed comparable initial weight loss. Of the two most effective clones, 1E7 and 2A4, all mice treated with clone 1E7 CAR T cells and all mice treated with clone 2A4 CAR T cells maintained in the study regained weight at later time points (see Figures 8F and 8G). The data demonstrated that, at the selected doses, anti-Claudin 18.2 clone 1E7 and 2A4 CAR T cells can effectively reduce established gastric cancer tumors in mice without causing irreversible weight loss. Finally, CAR T cell proliferation in the blood was analyzed. 3 × 10 6 The number of CAR T cells harvested from mice treated with CAR+ cells was plotted against the number of days after CAR T treatment (Figure 8J). Claudin 18.2 clone 2A4 CAR T expanded best, while Claudin 18.2 clone 1E7 CAR T cells did not expand as well as Claudin 18.2 clone 2A4 CAR T cells, although 1E7 showed comparable tumor-killing activity to 2A4 in this experiment (Figure 8A).

[0330] Example 6. Generation of Claudin 18.2-specific CAR T cells This example describes the construction of an anti-Claudin 18.2 chimeric antigen receptor (CAR).

[0331] The anti-Claudin 18.2 antibody clones 1E7, 2A4, 9G2, 2A10, 12H6, 10D11, 17F11, 6B2, and 4A5 were reformatted as CARs. The amino acid sequences of the heavy and light chain variable regions of these antibodies (Tables 1a and 1b) were used to design single-chain variable fragments (scFvs) with the general structure of heavy chain variable region-linker-light chain variable region (exemplary amino acid sequences are listed in Table 1c). The linker had the following amino acid sequence: GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 135). The scFv amino acid sequence was combined with hinge, transmembrane, and cytoplasmic amino acid sequences to generate CARs, as described below.

[0332] An exemplary chimeric antigen receptor was designed to include the following elements from 5' to 3' (see Tables 7a and 7b): the CD8α signal sequence of the human CD8α molecule (SEQ ID NO: 134), the anti-Claudin 18.2 scFv, the hinge region and transmembrane region (SEQ ID NO: 136), the cytoplasmic portion of the 41BB molecule (SEQ ID NO: 137), and the cytoplasmic portion of the CD3ζ molecule (SEQ ID NO: 138 or 139).

[0333] [Table 11-1] [Table 11-2] [Table 11-3]

[0334] [Table 12] JPEG2025539380000023.jpg250159 JPEG2025539380000024.jpg255166

[0335] [Table 13] JPEG2025539380000026.jpg249154

[0336] [Table 14]

[0337] Example 7: Anti-Claudin 18.2 CAR construct with a safety switch This example describes the construction of an anti-Claudin 18.2 CAR with a safety switch. The anti-Claudin 18.2 CARs disclosed herein may be formatted to include different safety switch structures, such as any of the safety switch structures listed below (Table 8). In Table 8, "R" refers to the rituximab recognition site, also known as the CD20 mimotope, "Q" refers to the QBEND-10 epitope, and "S" refers to an scFv, such as an anti-Claudin scFv disclosed herein.

[0338] [Table 15]

[0339] Exemplary protein sequences of anti-Claudin 18.2 CAR constructs comprising a safety switch are shown in Table 9a. Exemplary safety switch constructs may comprise a CD8α signal sequence (SEQ ID NO: 134), an anti-Claudin 18.2 scFv described herein, a CD20 mimotope (SEQ ID NO: 140), a QBEND-10 epitope (SEQ ID NO: 148 or SEQ ID NO: 149), the hinge and transmembrane regions of a human CD8α molecule (SEQ ID NO: 136), the cytoplasmic portion of a 4-1BB molecule (SEQ ID NO: 137), and the cytoplasmic portion of a CD3ζ molecule (SEQ ID NO: 138 or SEQ ID NO: 139). An exemplary safety switch construct may comprise the anti-Claudin 18.2 scFv described herein, a CD20 mimotope (SEQ ID NO: 140), a QBEND-10 epitope (SEQ ID NO: 148 or SEQ ID NO: 149), the hinge and transmembrane regions of the human CD8α molecule (SEQ ID NO: 136), the cytoplasmic portion of the 4-1BB molecule (SEQ ID NO: 137), and the cytoplasmic portion of the CD3ζ molecule (SEQ ID NO: 138 or SEQ ID NO: 139) (without the CD8α signal sequence).

[0340] [Table 16]

[0341] [Table 17] JPEG2025539380000031.jpg224170

[0342] [Table 18] JPEG2025539380000033.jpg225168

[0343] Example 8: In vitro detection and depletion of Claudin 18.2 CAR T cells using a rituximab-based safety switch To deplete or turn off CAR T cells in the event of unwanted activity, a rituximab off-switch is developed by inserting a rituximab mimotope into one or more of the various positions in the extracellular domain of the CAR described herein. A complement-dependent cytotoxicity assay is used to evaluate rituximab-dependent in vitro depletion of Claudin 18.2 CAR T cells. In this assay, frozen CAR-T cells are thawed and 1 x 10 5 Cells are incubated in RPMI 1640 medium supplemented with 10% FBS in 96-well plates. Cells are incubated for 3 hours in the absence or presence of 25% baby rabbit complement (Cedarlane, CL3441-S) and rituximab antibody (produced in-house; 100 mg / mL). Cells are stained with recombinant Claudin 18.2 (adipogen), and cytotoxicity is analyzed by flow cytometry. Anti-Claudin 18.2 CAR T cells can be detected by both recombinant Claudin 18.2 and rituximab staining. Claudin 18.2 CAR T cells are depleted in vitro in a rituximab- and complement-dependent manner.

[0344] Example 9: Analysis of potential off-target and on-target risks of Claudin 18.2 CAR T cells Next, we investigated whether the anti-Claudin 18.2 antibody might cross-react with any other membrane proteins. Using a membrane proteome array, we assessed the binding profile of the Claudin 18.2 CAR and identified potential off-target hits for both clones 1E7 and 2A4. The membrane protein GPRC5D was identified as a potential off-target hit for clone 1E7 but not for clone 2A4. We tested the off-target toxicity of the anti-Claudin 8.2 clones 1E7 and A4 CAR T in a short-term killing assay. 293T cells or 293T cells expressing GPRC5D, Claudin 18.1, or Claudin 18.2 were co-cultured with Claudin 18.2 CAR for 72 hours at different E:T ratios in the short-term killing assay described herein. The data in Figure 9 show that Claudin 18.2 CAR T cells exhibit an acceptable safety profile, as cytotoxicity was specific to Claudin 18.2. Results represent the mean ± SEM and represent technical replicates of n = 3. Experiments were performed in triplicate using CAR T cells from two different donors, and representative data are shown in Figure 9.

[0345] Claudin 18.2 has been shown to be expressed on normal stomach tissue. Histopathological analysis was then performed to assess the potential on-target toxicity of Claudin 18.2 CAR T cells. An in vivo NUGC4 subcutaneous tumor model was used for the experiment. Briefly, mice were inoculated subcutaneously with tumor cells on day -20 and intravenously administered with CAR T cells on day 0. Tissues were collected on day 40 or earlier if rapid weight loss was observed. Histopathological analysis showed infiltration and tissue damage in the stomach, which is expected and consistent with normal tissue expression of Claudin 18.2. The histopathological scores of both the 1E7 and 2A4 Claudin 18.2 CARs were comparable to those of two tool CARs in clinical development as CAR or antibody therapeutics. Findings are summarized in Table 11 below.

[0346] [Table 19]

[0347] Example 10: Claudin 18.2 CAR T cells with enhanced resistance to host cell rejection It has previously been demonstrated that co-expressing a rejection evasion protein, such as a CD70 binding protein or CD70 CAR, in CAR T cells can improve the resistance of CAR T cells to alloreactive immune cells. See WO2022 / 266203, which is incorporated herein in its entirety for all purposes. An expression cassette encoding a Claudin 18.2 CAR and a CD70 CAR (anti-CD70 scFv-CD8 hinge domain and transmembrane domain-CD3z) linked by P2A was constructed (Claudin 18.2 / CD70 dual CAR). Alternatively, the anti-CD70 scFv coding sequence was added to the 5' end of the Claudin 18.2 scFv coding sequence to generate a CD70 / Claudin18.2 tandem CAR construct. Clone 2A4 was tested in this experiment. CAR T cells expressing either Claudin 18.2 CAR alone, dual CARs, or tandem CARs were generated, and the expression of CD70 scFv and Claudin 18.2 scFv on the cell surface from each CAR T cell was verified by flow cytometry (data not shown).

[0348] The characteristics of Claudin 18.2 / CD70 dual and tandem CAR T cells, including levels of activation markers (Figure 10A) and T cell phenotype (Figure 10B), were analyzed in comparison with Claudin 18.2 CAR T cells. Expression of CD70 CAR did not significantly affect the cytotoxic activity of Claudin 18.2 CAR T cells (Figure 10C).

[0349] To evaluate the activity of Claudin 18.2 CAR T cells co-expressing allorejection evasion proteins such as CD70-binding proteins or CD70 CARs, we performed an alloreactive T cell mixed lymphocyte reaction (MLR) assay. To prime alloreactive T cells, unedited graft donor T cells were irradiated at 30 Gy and co-cultured with host PBMCs at a 1:1 ratio in RPMI supplemented with 10% FBS and 20 IU / mL of recombinant human IL-2, IL-7, and IL-15. On day 4 of co-culture, half of the medium was replaced with fresh RPMI supplemented with 10% FBS. On day 7, T cells were isolated using a human Pan T cell isolation kit (Miltenyi Biotech) as directed by the manufacturer's protocol. Primed alloreactive T cells were then co-cultured with graft T cells at a 1:1 ratio in 200 μL of RPMI medium supplemented with 10% FBS and 20 U / mL of recombinant human IL-2 in round-bottom 96-well plates. If the MLR co-culture lasted more than 4 days, half of the medium was replaced on day 4. Cells were analyzed by flow cytometry at the indicated time points.

[0350] As shown in Figure 11A, Claudin 18.2 CAR T cells expressing the CD70 CAR resisted alloreactive T cell-mediated rejection, while CAR T cells expressing neither the NTD nor the CD70 CAR were completely rejected (left panel). Both tandem and dual CARs showed improved survival to varying degrees and significantly reduced the absolute number of host T cells, demonstrating effective immune rejection evasion activity. Conversely, host T cells proliferated when co-cultured with the NTD and Claudin 18.2 CAR T cells without expressing the CD70 CAR (right panel). The same experiment was repeated with different graft-donor pairs (Figure 11B).

[0351] Example 11: Claudin 18.2 CAR with CD28 hinge and transmembrane domains Next, we constructed an anti-Claudin 18.2 CAR construct with a CD28 hinge and transmembrane domain (CD28 HTM) and compared CAR T cells with anti-Claudin 18.2 CAR T cells with a CD8a hinge and transmembrane domain (8HTM). Figure 12A shows that CAR T cells exhibit similar cytotoxicity in short-term killing assays, with CAR T cells expressing CD28 HTM displaying slightly fewer activation markers and more stem-center memory cells (Figure 12B).

[0352] [Table 20]

[0353] All references cited herein, including patents, patent applications, articles, textbooks, etc., and the references cited therein, to the extent not already cited, are hereby incorporated by reference in their entirety.

[0354] While the teachings of the present disclosure 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 set forth in the claims that follow. 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.

Claims

1. A chimeric antigen receptor comprising an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises a Claudin 18.2 antigen-binding domain that specifically binds to Claudin 18.2, and the antigen-binding domain comprises: (a) a variable heavy chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3, 16-18, 31-33, 46-48, 61-63, 76-78, 89-91, 102-104, 115, 116, and 117; (b) a variable heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-5, 19-20, 34-35, 49-50, 64-65, 79-80, 92-93, 105-106, 118, and 119; and (c) a variable heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 21, 36, 51, 66, 81, 94, 107 and 120; and (d) a variable light chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 22, 37, 52, 67, 82, 95, 108 and 121; and (e) a variable light chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 23, 38, 53, 68, 83, 96, 109, and 122; and (f) a variable light chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 24, 39, 54, 69, 84, 97, 110, and 123.

2. A chimeric antigen receptor comprising an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises a Claudin 18.2 antigen-binding domain that specifically binds to Claudin 18.2, and the antigen-binding domain comprises: (a) a variable heavy chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3, 16-18, 31-33, 46-48, 61-63, 76-78, 89-91, 102-104, and 115-117; (b) a variable heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-5, 19-20, 34-35, 49-50, 64-65, 79-80, 92-93, 105-106, and 118-119; and (c) a variable heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 21, 36, 51, 66, 81, 94, 107, and 120.

3. A chimeric antigen receptor comprising an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises a Claudin 18.2 antigen-binding domain that specifically binds to Claudin 18.2, and the antigen-binding domain comprises: (a) a variable light chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 22, 37, 52, 67, 82, 95, 108, and 121; (b) a variable light chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 23, 38, 53, 68, 83, 96, 109, and 122; and (c) a variable light chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 24, 39, 54, 69, 84, 97, 110, and 123. The chimeric antigen receptor of any one of claims 1 to 2, comprising at least one of the following:

4. the antigen-binding domain (a) a variable heavy chain CDR1 (CDRH1) comprising an amino acid sequence selected from SEQ ID NOs: 1-3, a CDRH2 comprising an amino acid sequence selected from SEQ ID NOs: 4-5, a CDRH3 comprising the amino acid sequence of SEQ ID NO: 6, a variable light chain CDR1 (CDRL1) comprising the amino acid sequence of SEQ ID NO: 7, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 8, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 9; or (b) a CDRH1 comprising an amino acid sequence selected from SEQ ID NOs: 16-18, a CDRH2 comprising an amino acid sequence selected from SEQ ID NOs: 19-20, a CDRH3 comprising the amino acid sequence of SEQ ID NO: 21, a CDRL1 comprising the amino acid sequence of SEQ ID NO: 22, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 23, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 24; or (c) a CDRH1 comprising an amino acid sequence selected from SEQ ID NOs: 31-33, a CDRH2 comprising an amino acid sequence selected from SEQ ID NOs: 34-35, a CDRH3 comprising the amino acid sequence of SEQ ID NO: 36, a CDRL1 comprising the amino acid sequence of SEQ ID NO: 37, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 38, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 39; or (d) a CDRH1 comprising an amino acid sequence selected from SEQ ID NOs: 46-48, a CDRH2 comprising an amino acid sequence selected from SEQ ID NOs: 49-50, a CDRH3 comprising the amino acid sequence of SEQ ID NO: 51, a CDRL1 comprising the amino acid sequence of SEQ ID NO: 52, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 53, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 54; or (e) a CDRH1 comprising an amino acid sequence selected from SEQ ID NOs: 61-63, a CDRH2 comprising an amino acid sequence selected from SEQ ID NOs: 64-65, a CDRH3 comprising the amino acid sequence of SEQ ID NO: 66, a CDRL1 comprising the amino acid sequence of SEQ ID NO: 67, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 68, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 69; or (f) a CDRH1 comprising an amino acid sequence selected from SEQ ID NOs: 76-78, a CDRH2 comprising an amino acid sequence selected from SEQ ID NOs: 79-80, a CDRH3 comprising the amino acid sequence of SEQ ID NO: 81, a CDRL1 comprising the amino acid sequence of SEQ ID NO: 82, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 83, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 84; or (g) a CDRH1 comprising an amino acid sequence selected from SEQ ID NOs: 89-91, a CDRH2 comprising an amino acid sequence selected from SEQ ID NOs: 92-93, a CDRH3 comprising the amino acid sequence of SEQ ID NO: 94, a CDRL1 comprising the amino acid sequence of SEQ ID NO: 95, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 96, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 97; or (h) a CDRH1 comprising an amino acid sequence selected from SEQ ID NOs: 102-104, a CDRH2 comprising an amino acid sequence selected from SEQ ID NOs: 105-106, a CDRH3 comprising the amino acid sequence of SEQ ID NO: 107, a CDRL1 comprising the amino acid sequence of SEQ ID NO: 108, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 109, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 110; or (i) the chimeric antigen receptor of any one of claims 1 to 3, comprising: a CDRH1 comprising an amino acid sequence selected from SEQ ID NOs: 115 to 117; a CDRH2 comprising an amino acid sequence selected from SEQ ID NOs: 118 to 119; a CDRH3 comprising the amino acid sequence of SEQ ID NO: 120; a CDRL1 comprising the amino acid sequence of SEQ ID NO: 121; a CDRL2 comprising the amino acid sequence of SEQ ID NO: 122; and a CDRL3 comprising SEQ ID NO:

123.

5. A chimeric antigen receptor comprising an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises a Claudin 18.2 antigen-binding domain that specifically binds to Claudin 18.2, and the antigen-binding domain comprises: (a) a variable heavy chain comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 25, 40, 55, 70, 85, 98, 111, and 124; and (b) a variable light chain comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 26, 41, 56, 71, 86, 99, 112, and 125; The chimeric antigen receptor according to any one of claims 1 to 4, wherein the variable heavy chain and the variable light chain are linked by at least one linker.

6. A chimeric antigen receptor comprising an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises a Claudin 18.2 antigen-binding domain that specifically binds to Claudin 18.2, and the antigen-binding domain comprises: (a) a variable heavy chain comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 25, 40, 55, 70, 85, 98, 111, and 124; and (b) a variable light chain comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 26, 41, 56, 71, 86, 99, 112, and 125; The chimeric antigen receptor according to any one of claims 1 to 5, wherein the variable heavy chain and the variable light chain are linked by at least one linker.

7. the antigen-binding domain (a) a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 10, and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 11; or (b) a VH comprising the amino acid sequence of SEQ ID NO: 25, and a VL comprising the amino acid sequence of SEQ ID NO: 26; or (c) a VH comprising the amino acid sequence of SEQ ID NO: 40, and a VL comprising the amino acid sequence of SEQ ID NO: 41; or (d) a VH comprising the amino acid sequence of SEQ ID NO: 55, and a VL comprising the amino acid sequence of SEQ ID NO: 56; or (e) a VH comprising the amino acid sequence of SEQ ID NO: 70, and a VL comprising the amino acid sequence of SEQ ID NO: 71; or (f) a VH comprising the amino acid sequence of SEQ ID NO: 85, and a VL comprising the amino acid sequence of SEQ ID NO: 86; or (g) a VH comprising the amino acid sequence of SEQ ID NO: 98, and a VL comprising the amino acid sequence of SEQ ID NO: 99; or (h) a VH comprising the amino acid sequence of SEQ ID NO: 111, and a VL comprising the amino acid sequence of SEQ ID NO: 112; or The chimeric antigen receptor of any one of claims 1 to 6, comprising: (i) a VH comprising the amino acid sequence of SEQ ID NO: 124, and a VL comprising the amino acid sequence of SEQ ID NO:

125.

8. The chimeric antigen receptor of any one of claims 1 to 7, comprising an scFv comprising an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises a Claudin 18.2 antigen-binding domain that specifically binds to Claudin 18.2, and the antigen-binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 27, 42, 57, 72, 187, 189, 191, and 193.

9. The chimeric antigen receptor of any one of claims 1 to 8, wherein the chimeric antigen receptor comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 13, 28, 43, 58, 73, 87, 100, 113, 126, 128, 130, 132, 179-186, 195-198, 200-201, and 208-211.

10. The chimeric antigen receptor according to any one of claims 1 to 9, wherein the transmembrane domain comprises the transmembrane domain of human CD8a, CD28, or CD2.

11. The chimeric antigen receptor of any one of claims 1 to 10, further comprising a hinge domain.

12. The chimeric antigen receptor of claim 11, wherein the hinge domain comprises human CD8a, CD28, or CD2.

13. The chimeric antigen receptor of claim 11 or 12, wherein the hinge domain and transmembrane domain are those of human CD8a, CD28, or CD2.

14. The chimeric antigen receptor of any one of claims 1 to 13, wherein the intracellular domain comprises at least one costimulatory domain.

15. Co-stimulatory domains include CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell co-stimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1 (CD1 la / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class I molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, signaling lymphocyte activation molecule (SLAM protein), activating NK cell receptor, BTLA, and Toll Ligand Receptor. Body, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE, CD103, ITGAL, CD1 1a, LFA-1, ITGAM, CD1 1b, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL , DNAMI (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT The chimeric antigen receptor of claim 14, which is a signaling region of a ligand that specifically binds to AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83, or any combination thereof.

16. The chimeric antigen receptor of claim 15, wherein the costimulatory domain comprises one or more signaling regions selected from the group consisting of a 4-1BB / CD137 signaling region, a CD28 signaling region, and variants thereof.

17. The chimeric antigen receptor of claim 16, wherein the costimulatory domain comprises a signaling region comprising one or more amino acid sequences of SEQ ID NOs: 137, 158, 159, and 174.

18. The chimeric antigen receptor of any one of claims 1 to 17, wherein the intracellular domain comprises at least one activation domain.

19. 19. The chimeric antigen receptor of claim 18, wherein the activation domain comprises a CD3 signaling domain.

20. 20. The chimeric antigen receptor of claim 19, wherein the CD3 signaling domain comprises a CD3 zeta signaling domain.

21. 21. The chimeric antigen receptor of claim 20, wherein the CD3 zeta signaling domain comprises the amino acid sequence of SEQ ID NO: 138 or a fragment thereof, or the amino acid sequence of SEQ ID NO: 139 or a fragment thereof.

22. The chimeric antigen receptor of any one of claims 1 to 20, wherein the chimeric antigen receptor is encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 15, 30, 45, 60, 75, 88, 101, 114, and 127.

23. The chimeric antigen receptor of any one of claims 1 to 22, wherein the extracellular domain further comprises an anti-CD70 scFv that specifically binds to CD70.

24. The chimeric antigen receptor of claim 23, wherein the anti-CD70 scFv comprises the amino acid sequence of SEQ ID NO: 204, 205, and / or 206.

25. The chimeric antigen receptor of any one of claims 1 to 24, further comprising a safety switch.

26. The chimeric antigen receptor of claim 25, wherein the safety switch comprises a CD20 mimotope or a QBEND-10 epitope.

27. The chimeric antigen receptor of claim 26, wherein the safety switch comprises one or more CD20 mimotopes or one or more QBEND-10 epitopes, or a combination thereof.

28. The chimeric antigen receptor of any one of claims 1 to 27, wherein the chimeric antigen receptor comprises one or more safety switches in the form of QR3, SR2, RSR, or R2S.

29. The chimeric antigen receptor of any one of claims 1 to 28, wherein the chimeric antigen receptor comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 128, 130, 132, 184-186, and 208-211.

30. An isolated polynucleotide encoding the chimeric antigen receptor of any one of claims 1 to 29.

31. A vector comprising the polynucleotide of claim 30.

32. 32. The vector of claim 31, wherein the vector is a retroviral vector, a DNA vector, a plasmid, an RNA vector, an adenoviral vector, an adeno-associated viral vector, a lentiviral vector, or any combination thereof.

33. An engineered immune cell expressing the chimeric antigen receptor of any one of claims 1 to 29.

34. 33. An engineered immune cell comprising or expressing the polynucleotide of claim 30 or the vector of claim 31 or 32.

35. 35. The engineered immune cell of claim 33 or 34, wherein the immune cell is a T cell, a tumor infiltrating lymphocyte (TIL), an NK cell, a TCR-expressing cell, a dendritic cell, or an NK-T cell.

36. 36. The engineered immune cell of claim 35, wherein the cell is an autologous T cell.

37. 36. The engineered immune cell of claim 35, wherein the cell is an allogeneic T cell.

38. 38. The engineered immune cell of any one of claims 33 to 37, wherein the engineered immune cell further comprises or expresses a CD70 binding protein.

39. 39. The engineered immune cell of claim 38, wherein the CD70 binding protein comprises an anti-CD70 antibody or an antigen-binding fragment thereof, and a transmembrane domain.

40. 40. The engineered immune cell of claim 39, wherein the anti-CD70 antibody comprises the amino acid sequence of SEQ ID NO: 204, 205, and / or 206.

41. 41. The engineered immune cell of any one of claims 38-40, wherein the CD70 binding protein further comprises one intracellular domain selected from the group consisting of a CD3z signaling domain, a CD3d signaling domain, a CD3g signaling domain, a CD3e signaling domain, a CD28 signaling domain, a CD2 signaling domain, an OX40 signaling domain, and a 4-1BB signaling domain, or a variant thereof.

42. 42. The engineered immune cell of any one of claims 38-41, wherein the CD70 binding protein comprises a CD3z signaling domain and does not comprise a costimulatory domain.

43. 43. The engineered immune cell of any one of claims 38-42, wherein the CD70 binding protein further comprises a hinge domain, optionally wherein the hinge domain comprises a CD8 hinge domain.

44. 44. The engineered immune cell of any one of claims 38 to 43, wherein the CD70 binding protein comprises the amino acid sequence of SEQ ID NO:

207.

45. A pharmaceutical composition comprising the engineered immune cells of any one of claims 33 to 44 and at least one pharmaceutically acceptable excipient.

46. 46. ​​A method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject an effective amount of the engineered immune cell of any one of claims 33-44 or the pharmaceutical composition of claim 45.

47. 47. The method of claim 46, wherein the engineered immune cells or the pharmaceutical composition are administered to the subject intravenously, subcutaneously, or intraperitoneally.

48. 48. The method of claim 47, wherein the engineered immune cells or the pharmaceutical composition are administered to the subject by intravenous injection, intraperitoneal injection or subcutaneous injection.

49. The method of any one of claims 46 to 48, wherein the disease or disorder is cancer.

50. 50. The method of any one of claims 46 to 49, wherein the disease or disorder is gastric cancer, gastroesophageal junction (GEJ) cancer, or pancreatic cancer.

51. An article of manufacture comprising an engineered immune cell according to any one of claims 33 to 44, or a pharmaceutical composition according to claim 45.

52. An anti-Claudin 18.2 binding agent, comprising: (a) a variable heavy chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3, 16-18, 31-33, 46-48, 61-63, 76-78, 89-91, 102-104, and 115-117; (b) a variable heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-5, 19-20, 34-35, 49-50, 64-65, 79-80, 92-93, 105-106, and 118-119; (c) a variable heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 21, 36, 51, 66, 81, 94, 107, 120; (d) a variable light chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 22, 37, 52, 67, 82, 95, 108, and 121; (e) a variable light chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 23, 38, 53, 68, 83, 96, 109, 122; and (f) An anti-Claudin 18.2 binding agent comprising a variable light chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 24, 39, 54, 69, 84, 97, 110, 123.

53. (a) a variable heavy chain CDR1 (CDRH1) comprising an amino acid sequence selected from SEQ ID NOs: 1-3, a CDRH2 comprising an amino acid sequence selected from SEQ ID NOs: 4-5, a CDRH3 comprising the amino acid sequence of SEQ ID NO: 6, a variable light chain CDR1 (CDRL1) comprising the amino acid sequence of SEQ ID NO: 7, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 8, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 9; or (b) a CDRH1 comprising an amino acid sequence selected from SEQ ID NOs: 16-18, a CDRH2 comprising an amino acid sequence selected from SEQ ID NOs: 19-20, a CDRH3 comprising the amino acid sequence of SEQ ID NO: 21, a CDRL1 comprising the amino acid sequence of SEQ ID NO: 22, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 23, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 24; or (c) a CDRH1 comprising an amino acid sequence selected from SEQ ID NOs: 31-33, a CDRH2 comprising an amino acid sequence selected from SEQ ID NOs: 34-35, a CDRH3 comprising the amino acid sequence of SEQ ID NO: 36, a CDRL1 comprising the amino acid sequence of SEQ ID NO: 37, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 38, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 39; or (d) a CDRH1 comprising an amino acid sequence selected from SEQ ID NOs: 46-48, a CDRH2 comprising an amino acid sequence selected from SEQ ID NOs: 49-50, a CDRH3 comprising the amino acid sequence of SEQ ID NO: 51, a CDRL1 comprising the amino acid sequence of SEQ ID NO: 52, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 53, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 54; or (e) a CDRH1 comprising an amino acid sequence selected from SEQ ID NOs: 61-63, a CDRH2 comprising an amino acid sequence selected from SEQ ID NOs: 64-65, a CDRH3 comprising the amino acid sequence of SEQ ID NO: 66, a CDRL1 comprising the amino acid sequence of SEQ ID NO: 67, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 68, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 69; or (f) a CDRH1 comprising an amino acid sequence selected from SEQ ID NOs: 76-78, a CDRH2 comprising an amino acid sequence selected from SEQ ID NOs: 79-80, a CDRH3 comprising the amino acid sequence of SEQ ID NO: 81, a CDRL1 comprising the amino acid sequence of SEQ ID NO: 82, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 83, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 84; or (g) a CDRH1 comprising an amino acid sequence selected from SEQ ID NOs: 89-91, a CDRH2 comprising an amino acid sequence selected from SEQ ID NOs: 92-93, a CDRH3 comprising the amino acid sequence of SEQ ID NO: 94, a CDRL1 comprising the amino acid sequence of SEQ ID NO: 95, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 96, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 97; or (h) a CDRH1 comprising an amino acid sequence selected from SEQ ID NOs: 102-104, a CDRH2 comprising an amino acid sequence selected from SEQ ID NOs: 105-106, a CDRH3 comprising the amino acid sequence of SEQ ID NO: 107, a CDRL1 comprising the amino acid sequence of SEQ ID NO: 108, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 109, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 110; or 53. The anti-Claudin 18.2 binding agent of claim 52, comprising: (i) a CDRH1 comprising an amino acid sequence selected from SEQ ID NOs: 115-117, a CDRH2 comprising an amino acid sequence selected from SEQ ID NOs: 118-119, a CDRH3 comprising the amino acid sequence of SEQ ID NO: 120, a CDRL1 comprising the amino acid sequence of SEQ ID NO: 121, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 122, and a CDRL3 SEQ ID NO:

123.

54. The binding agent is an antibody, an antibody conjugate, or an antigen-binding fragment thereof, optionally an F(ab') 2 54. The Claudin 18.2 binding agent of claim 52 or 53, which is a Fab' fragment, a Fab fragment, an Fv fragment, a scFv fragment, a dsFv fragment, or a dAb fragment.

55. The anti-Claudin 18.2 binding agent of any one of claims 52 to 54, wherein the binding agent is a monoclonal antibody comprising an IgG constant region.

56. 56. The anti-Claudin 18.2 binding agent of any one of claims 52-55, comprising a variable heavy (VH) chain sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 25, 40, 55, 70, 85, 98, 111, and 124.

57. 57. The anti-Claudin 18.2 binding agent of any one of claims 52-56, comprising a variable light (VL) chain sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 26, 41, 56, 71, 86, 99, 112, and 125.

58. 58. The anti-Claudin 18.2 binding agent of any one of claims 52-57, wherein the binding agent comprises a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 27, 42, 57, 72, 187, 189, 191, and 193.

59. The anti-Claudin 18.2 binding agent of any one of claims 52 to 58, wherein the binding agent is a fusion protein comprising an scFv fragment fused to an Fc constant region.

60. The anti-Claudin 18.2 binding agent of any one of claims 52 to 59, wherein the binding agent is a bispecific antibody.

61. 61. The anti-Claudin 18.2 binding agent of claim 60, wherein the bispecific antibody binds to CD3.

62. 62. A pharmaceutical composition comprising the anti-Claudin 18.2 binding agent of any one of claims 52 to 61 and a pharmaceutically acceptable excipient.

63. 62. A method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject the anti-Claudin 18.2 binding agent of any one of claims 52 to 61, or the pharmaceutical composition of claim 62.

64. 64. The method of claim 63, wherein the disease or disorder is cancer.

65. 65. The method of claim 63 or 64, wherein the disease or disorder is gastric cancer, gastroesophageal junction (GEJ) cancer, or pancreatic cancer.

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