Chimeric antigen receptor targeting Fc receptor-like 5 and its use

Abstract

To provide chimeric antigen receptors targeting Fc receptor-like 5 and uses thereof.SOLUTION: The presently disclosed subject matter provides methods and compositions for treating a neoplasia (e.g., multiple myeloma). The presently disclosed subject matter relates to chimeric antigen receptors (CARs) that specifically target Fc receptor-like 5 (FcRL5), e.g., domain 9 of FcRL5, and immunoresponsive cells comprising such CARs. The presently disclosed FcRL5-targeted CARs have enhanced immune-activating properties, including anti-tumor activity. The chimeric antigen receptors (CARs) may comprise an extracellular antigen-binding domain, a transmembrane domain and an intracellular domain.SELECTED DRAWING: None

Description

[Technical Field]

[0001] (Cross-reference to related applications) This application claims priority to U.S. Provisional Patent Application No. 62 / 088,164, filed on December 5, 2014, the contents of which this Provisional Patent Application is incorporated in its entirety by reference, and priority to this Provisional Patent Application is claimed.

[0002] (Introduction) The subject matter of this disclosure provides methods and compositions for treating cancer. The subject matter of this disclosure relates to Fc receptor-like 5 (FcRL5), for example, a chimeric antigen receptor (CAR) that specifically targets domain 9 of FcRL5, immune response cells containing such CARs, and methods for using such cells to treat cancer (e.g., multiple myeloma). [Background technology]

[0003] (Background of the invention) Cell-based immunotherapy is a treatment with curative potential for treating cancer. T cells and other immune cells can be modified to target tumor antigens through the introduction of genetic material that encodes artificial or synthetic receptors for antigens, called chimeric antigen receptors (CARs), which are specific to selected antigens. T-cell targeted therapy using CARs has shown recent clinical success in treating hematological malignancies.

[0004] Multiple myeloma (MM) is the second leading cause of death from hematological malignancies (Siegel et al., CA: a cancer journal for clinicians, Vol. 63, pp. 11-30 (2013)). Approximately 25% of patients have high-risk cytogenetic abnormalities that foreshadow a median survival of less than two years (Boyd et al., Genes, chromosomes & cancer, Vol. 50, pp. 765-774). 2011; Shaughnessy et al., Blood Vol. 109, pp. 2276-2284 (2007). Although recent research has been conducted, regardless of cytogenetics, this disease is still considered incurable except through the immunotherapeutic graft-versus-myeloma (GvM) effect of allogeneic transplantation. However, allogeneic transplantation is limited by ineligibility and high transplant-related morbidity and mortality rates (Gahrton et al., The New England Journal of Medicine Vol. 325, pp. 1267-1284). Page 273 (1991). Similar to the GvM effect, potentially therapeutic T-cell effects can be achieved with minimal toxicity through autologous adoptive T-cell therapy. Myeloma is predicted to be an ideal disease for testing adoptive T-cell therapy. Firstly, allogeneic transplantation has demonstrated that T cells can be a curative treatment, with or without minimal chemotherapy, after non-myeloablative transplantation or post-transplant donor lymphocyte infusion. Secondly, pre-conditioning chemotherapy may thus enhance the efficacy of adoptive T-cell therapy, possibly through a mechanism that depletes regulatory T cells (Tregs) (Brentjens et al., Blood vol. 118, pp. 4817-4828 (2011) and Pegram et al., Blood vol. 119, pp. 4828). (pp. 133-4141 (2012)) The period immediately following autologous transplantation may be the optimal time for T cell administration, and myeloma is one of the few diseases for which autologous stem cell transplantation is the standard treatment. Thirdly, the immunomodulatory agent lenalidomide can improve CAR-based treatment as shown in mice (Bertilaccio et al., Blood Vol. 122, p. 4171 (2013)), and lenalidomide is commonly used to treat MM. Fourthly, adoptive T cell therapy is superior to myeloma when compared to solid tumors or extramedullary CLL (Brentjens et al. (2011)). It works best in cerebrospinal diseases, such as ALL (Brentjens et al., Science Translational Medicine Vol. 5, 177ra p. 138 (2013); Davila et al., Science Translational Medicine Vol. 6, 224ra p. 225 (2014)), and like ALL, myeloma is It is a disease of the bone marrow. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Siegel et al., CA: A Cancer Journal for Clinicians, Vol. 63, pp. 11-30 (2013) [Non-Patent Document 2] Boyd et al., Genes, Chromosomes & Cancer, Vol. 50, pp. 765-774 (2011) [Non-Patent Document 3] Shaughnessy et al., Blood Vol. 109, pp. 2276-2284 (2007) [Non-Patent Document 4] Gahrton et al., The New England Journal of Medicine, Vol. 325, pp. 1267-1273 (1991). [Non-Patent Document 5] Brentjens et al., Blood, Vol. 118, pp. 4817-4828 (2011) [Non-Patent Document 6] Pegram et al., Blood Vol. 119, pp. 4133-4141 (2012) [Non-Patent Document 7] Bertilaccio et al., Blood, Vol. 122, p. 4171 (2013) [Non-Patent Document 8] Brentjens et al., Science Translational Medicine, Vol. 5, 177ra, p. 138 (2013) [Non-Patent Document 9] Davila et al., Science Translational Medicine, Vol. 6, 224ra, p. 225 (2014) [Overview of the Initiative] [Means for solving the problem]

[0006] While there are several reasons to predict that adoptive T-cell therapy may work well in myeloma, extending adoptive T-cell therapy to myeloma presents its own unique challenges. Unlike other B-cell malignancies, CD19 expression is found in only 2% of myeloma patients (Bataille et al., Haematologica vol. 91, pp. 1234-1240 (2006)). Furthermore, C Unlike D19, all common extracellular immunophenotypic markers in myeloma (CD138, CD38, and CD56) are co-expressed on other essential cell types, and CARs against any of these targets can result in unacceptable "off-tumor, on-target" toxicity that can be lethal even at targets where the antibody is well tolerated, similar to CARs targeting HER2 (Morgan et al., Molecular therapy: the journal of the American Society of Gene Therapy, Vol. 18, pp. 843-851 (2010)) (Brentjens et al. (2013)). Therefore, treating multiple myeloma requires careful consideration. Novel therapeutic strategies are needed to design CARs that target antigens highly expressed in MM cells but limitedly expressed in normal tissues, enabling potent tumor eradication with minimal toxicity and immunogenicity.

[0007] (Summary of the invention) The subject matter of this disclosure generally provides chimeric antigen receptors (CARs) that specifically target Fc receptor-like 5 (FcRL5), immune response cells containing such CARs, and the use of these CARs and immune response cells for treating multiple myeloma.

[0008] The subject of this disclosure is to provide a CAR. In a non-limiting example, this CAR comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular domain, the extracellular antigen-binding domain specifically binding to FcRL5. In a particular embodiment, this extracellular antigen-binding domain binds to domain 9 of FcRL5.

[0009] In certain non-limiting embodiments, this extracellular antigen-binding domain is a single-chain variable fragment (scFv). In certain embodiments, this extracellular antigen-binding domain is a murine scFv. In certain embodiments, this extracellular antigen-binding domain is a human scFv. In certain non-limiting embodiments, this extracellular antigen-binding domain is an optionally cross-linked Fab. In certain non-limiting embodiments, this extracellular binding domain is an F(ab)2. In certain non-limiting embodiments, any of the molecules described above may be included within a fusion protein, together with a heterologous sequence, to form an extracellular antigen-binding domain. In certain embodiments, this extracellular antigen-binding domain has a binding affinity (K -11 ) of from about 1×10 -6 M to about 3×10 -10 M, from 1×10 -6 M to about 3×10 -9 M or from 1×10 -6 M to about 3×10 d M and specifically binds to FcRL5. In certain embodiments, this extracellular antigen-binding domain has a K -9 of from about 1×10 -6 M to about 3×10 d and specifically binds to domain 8 or 9 of FcRL5.

[0010] In certain embodiments, this extracellular antigen-binding domain is associated with SEQ ID NOs: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, 87, 91, 95, 99, 103, 107, 111, 115, 119, 123, 127, 131, 135, 139, 143, 147, 151, 155, 159, 163, 167, 171, 175, 179, 183, 187, 191, 195 , Sequence ID 199, Sequence ID 203, Sequence ID 207, Sequence ID 211, Sequence ID 215, Sequence ID 219, Sequence ID 223, Sequence ID 227, Sequence ID 231, Sequence ID 235, Sequence ID 239, Sequence ID 243, Sequence ID 247, Sequence ID 251, Sequence ID 255, Sequence ID 259, Sequence ID 263, Sequence ID 267, Sequence ID 271, Sequence ID 275, Sequence ID 279, Sequence ID 283, Sequence ID 287, Sequence ID 291, Array The light chain variable region contains an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to an amino acid sequence selected from the group consisting of number 295, SEQ ID NO. 299, SEQ ID NO. 303, SEQ ID NO. 917, and SEQ ID NO. 921, and this extracellular antigen-binding domain binds to FcRL5.

[0011] In certain embodiments, this extracellular antigen-binding domain is used for SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, 172, 176, 180, 184, 188, 192, 196 , Sequence ID 200, Sequence ID 204, Sequence ID 208, Sequence ID 212, Sequence ID 216, Sequence ID 220, Sequence ID 224, Sequence ID 228, Sequence ID 232, Sequence ID 236, Sequence ID 240, Sequence ID 244, Sequence ID 248, Sequence ID 252, Sequence ID 256, Sequence ID 260, Sequence ID 264, Sequence ID 268, Sequence ID 272, Sequence ID 276, Sequence ID 280, Sequence ID 284, Sequence ID 288, Sequence ID 292, Array The extracellular antigen-binding domain contains a heavy chain variable region that includes an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to an amino acid sequence selected from the group consisting of number 296, SEQ ID NO. 300, SEQ ID NO. 304, SEQ ID NO. 915, and SEQ ID NO. 919, and this extracellular antigen-binding domain binds to FcRL5.

[0012] In certain embodiments, this extracellular antigen-binding domain is (a) SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NOs: 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, 87, 91, 95, 99, 103, 107, 111, 115, 119, 123, 127, 131, 135, 139, 143, 147, 151, 155, 159, 163, 167, 171, 175, 179, 183, 187, 191, 195, 199 , a light chain variable region and (b) sequence number 203, 207, 211, 215, 219, 223, 227, 231, 235, 239, 243, 247, 251, 255, 259, 263, 267, 271, 275, 279, 283, 287, 291, 295, 299, 303, 917 and 921, containing an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs. Sequence No. 4, Sequence No. 8, Sequence No. 12, Sequence No. 16, Sequence No. 20, Sequence No. 24, Sequence No. 28, Sequence No. 32, Sequence No. 36, Sequence No. 40, Sequence No. 44, Sequence No. 48, Sequence No. 52, Sequence No. 56, Sequence No. 60, Sequence No. 64, Sequence No. 68, Sequence No. 72, Sequence No. 76, Sequence No. 80, Sequence No. 84, Sequence No. 88, Sequence No. 92, Sequence No. 96, Sequence No. 100, Sequence No. 104, Sequence No. 108, Sequence No. 112, Sequence No. 116, Sequence No. 120, Sequence No. 124, Sequence No. 128, Sequence No. 132, Sequence No. 136, Sequence No. 140, Sequence No. 144, Sequence No. 148, Sequence No. 152, Sequence No. 156, Sequence No. 160, Sequence No. 164, Sequence No. 168, Sequence No. 172, Sequence No. 176, Sequence No. 180, Sequence No. 184, Sequence No. 188, Sequence No. 192, Sequence No. 196, Sequence No. 200, Sequence No. 2 04, SEQ ID NO: 208, SEQ ID NO: 212, SEQ ID NO: 216, SEQ ID NO: 220, SEQ ID NO: 224, SEQ ID NO: 228, SEQ ID NO: 232, SEQ ID NO: 236, SEQ ID NO: 240, SEQ ID NO: 244, SEQ ID NO: 248, SEQ ID NO: 252, SEQ ID NO: 256, SEQ ID NO: 260, SEQ ID NO: 264, SEQ ID NO: 268, SEQ ID NO: 272, SEQ ID NO: 276, SEQ ID NO: 280, SEQ ID NO: 284, SEQ ID NO: 288, SEQ ID NO: 292, SEQ ID NO: 296, Distribution The extracellular antigen-binding domain contains a heavy chain variable region that includes an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to an amino acid sequence selected from the group consisting of sequence number 300, sequence number 304, sequence number 915, and sequence number 919, and this extracellular antigen-binding domain binds to FcRL5.

[0013] In certain embodiments, this extracellular antigen-binding domain is associated with SEQ ID NOs: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, 87, 91, 95, 99, 103, 107, 111, 115, 119, 123, 127, 131, 135, 139, 143, 147, 151, 155, 159, 163, 16 7. Includes a light chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 171, 175, 179, 183, 187, 191, 195, 199, 203, 207, 211, 215, 219, 223, 227, 231, 235, 239, 243, 247, 251, 255, 259, 263, 267, 271, 275, 279, 283, 287, 291, 295, 299, 303, 917, 921 and their conservative modifications.

[0014] In certain embodiments, this extracellular antigen-binding domain is associated with SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, and 1 It includes a heavy chain variable region containing an amino acid sequence selected from the group consisting of 68, SEQ ID NO: 172, SEQ ID NO: 176, SEQ ID NO: 180, SEQ ID NO: 184, SEQ ID NO: 188, SEQ ID NO: 192, SEQ ID NO: 196, SEQ ID NO: 200, SEQ ID NO: 204, SEQ ID NO: 208, SEQ ID NO: 212, SEQ ID NO: 216, SEQ ID NO: 220, SEQ ID NO: 224, SEQ ID NO: 228, SEQ ID NO: 232, SEQ ID NO: 236, SEQ ID NO: 240, SEQ ID NO: 244, SEQ ID NO: 248, SEQ ID NO: 252, SEQ ID NO: 256, SEQ ID NO: 260, SEQ ID NO: 264, SEQ ID NO: 268, SEQ ID NO: 272, SEQ ID NO: 276, SEQ ID NO: 280, SEQ ID NO: 284, SEQ ID NO: 288, SEQ ID NO: 292, SEQ ID NO: 296, SEQ ID NO: 300, SEQ ID NO: 304, SEQ ID NO: 915, SEQ ID NO: 919 and their conservative modifications.

[0015] In certain embodiments, the extracellular antigen-binding domain includes a heavy chain variable region containing amino acids having the sequence described in SEQ ID NO: 915. In certain embodiments, the extracellular antigen-binding domain includes a light chain variable region containing amino acids having the sequence described in SEQ ID NO: 917. In certain embodiments, the extracellular antigen-binding domain includes a heavy chain variable region containing amino acids having the sequence described in SEQ ID NO: 919. In certain embodiments, the extracellular antigen-binding domain includes a light chain variable region containing amino acids having the sequence described in SEQ ID NO: 921. In certain embodiments, the extracellular antigen-binding domain includes a heavy chain variable region containing amino acids having the sequence described in SEQ ID NO: 144. In certain embodiments, the extracellular antigen-binding domain includes a light chain variable region containing amino acids having the sequence described in SEQ ID NO: 143. In certain embodiments, the extracellular antigen-binding domain includes a heavy chain variable region containing amino acids having the sequence described in SEQ ID NO: 216. In certain embodiments, the extracellular antigen-binding domain includes a light chain variable region containing amino acids having the sequence described in SEQ ID NO: 215. In certain embodiments, the extracellular antigen-binding domain includes a heavy chain variable region containing amino acids having the sequence described in SEQ ID NO: 220. In certain embodiments, the extracellular antigen-binding domain includes a light chain variable region containing amino acids having the sequence described in SEQ ID NO: 219. In certain embodiments, the extracellular antigen-binding domain includes a heavy chain variable region containing amino acids having the sequence described in SEQ ID NO: 236. In certain embodiments, the extracellular antigen-binding domain includes a light chain variable region containing amino acids having the sequence described in SEQ ID NO: 235. In certain embodiments, the extracellular antigen-binding domain includes a heavy chain variable region containing amino acids having the sequence described in SEQ ID NO: 268. In certain embodiments, the extracellular antigen-binding domain includes a light chain variable region containing amino acids having the sequence described in SEQ ID NO: 267. In certain embodiments, the extracellular antigen-binding domain includes a heavy chain variable region containing amino acids having the sequence described in SEQ ID NO: 116. In certain embodiments, the extracellular antigen-binding domain includes a light chain variable region containing amino acids having the sequence described in SEQ ID NO: 115.In certain embodiments, the extracellular antigen-binding domain includes a heavy chain variable region containing amino acids having the sequence described in SEQ ID NO: 172. In certain embodiments, the extracellular antigen-binding domain includes a light chain variable region containing amino acids having the sequence described in SEQ ID NO: 171.

[0016] In certain embodiments, this extracellular antigen-binding domain is (a) SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NOs: 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, 87, 91, 95, 99, 103, 107, 111, 115, 119, 123, 127, 131, 135, 139, 143, 147, 151, 155, 159, 163, 167, 171, 175, 179 (b) a light chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 183, 187, 191, 195, 199, 203, 207, 211, 215, 219, 223, 227, 231, 235, 239, 243, 247, 251, 255, 259, 263, 267, 271, 275, 279, 283, 287, 291, 295, 299, 303, 917, 921 and their conservative modifications, and (b) SEQ ID NOs: 4, 8, Column number 12, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO: 56, SEQ ID NO: 60, SEQ ID NO: 64, SEQ ID NO: 68, SEQ ID NO: 72, SEQ ID NO: 76, SEQ ID NO: 80, SEQ ID NO: 84, SEQ ID NO: 88, SEQ ID NO: 92, SEQ ID NO: 96, SEQ ID NO: 100, SEQ ID NO: 104, SEQ ID NO: 108, SEQ ID NO: 112, SEQ ID NO: 116, SEQ ID NO: 120, SEQ ID NO: 124, SEQ ID NO: 128, SEQ ID NO: 132, SEQ ID NO: 136, SEQ ID NO: 140, SEQ ID NO: 144, SEQ ID NO: 148, SEQ ID NO: 152, SEQ ID NO: 156, SEQ ID NO: 160, SEQ ID NO: 164, SEQ ID NO: 168, SEQ ID NO: 172, SEQ ID NO: 176 The sequence includes a heavy chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 224, 228, 232, 236, 240, 244, 248, 252, 256, 260, 264, 268, 272, 276, 280, 284, 288, 292, 296, 300, 304, 915, 919 and their conservative modifications.

[0017] In certain embodiments, the extracellular antigen-binding domain includes (a) a heavy chain variable region having the sequence described in SEQ ID NO: 915 and (b) a light chain variable region having the sequence described in SEQ ID NO: 917. In certain embodiments, the extracellular antigen-binding domain includes (a) a heavy chain variable region having the sequence described in SEQ ID NO: 919 and (b) a light chain variable region having the sequence described in SEQ ID NO: 921. In certain embodiments, the extracellular antigen-binding domain includes (a) a heavy chain variable region having the sequence described in SEQ ID NO: 144 and (b) a light chain variable region having the sequence described in SEQ ID NO: 143. In certain embodiments, the extracellular antigen-binding domain includes (a) a heavy chain variable region having the sequence described in SEQ ID NO: 216 and (b) a light chain variable region having the sequence described in SEQ ID NO: 215. In certain embodiments, the extracellular antigen-binding domain includes (a) a heavy chain variable region having the sequence described in SEQ ID NO: 220 and (b) a light chain variable region having the sequence described in SEQ ID NO: 219. In certain embodiments, the extracellular antigen-binding domain includes (a) a heavy chain variable region having the sequence described in SEQ ID NO: 236 and (b) a light chain variable region having the sequence described in SEQ ID NO: 235. In certain embodiments, the extracellular antigen-binding domain includes (a) a heavy chain variable region having the sequence described in SEQ ID NO: 268 and (b) a light chain variable region having the sequence described in SEQ ID NO: 267. In certain embodiments, the extracellular antigen-binding domain includes (a) a heavy chain variable region having the sequence described in SEQ ID NO: 116 and (b) a light chain variable region having the sequence described in SEQ ID NO: 115. In certain embodiments, the extracellular antigen-binding domain includes (a) a heavy chain variable region having the sequence described in SEQ ID NO: 172 and (b) a light chain variable region having the sequence described in SEQ ID NO: 171.

[0018] In certain non-limiting embodiments, the extracellular antigen-binding domain comprises both a heavy chain and a light chain, and optionally includes a linker sequence, e.g., a linker peptide, between the heavy chain variable region and the light chain variable region. For example, in certain non-limiting embodiments, the extracellular antigen-binding domain comprises (a) a heavy chain variable region having the sequence described in SEQ ID NO: 915 and (b) a light chain variable region having the sequence described in SEQ ID NO: 917, and optionally includes (c) a linker sequence, e.g., a linker peptide, between the heavy chain variable region and the light chain variable region. In certain embodiments, the extracellular antigen-binding domain comprises (a) a heavy chain variable region having the sequence described in SEQ ID NO: 919 and (b) a light chain variable region having the sequence described in SEQ ID NO: 92, and optionally includes (c) a linker sequence, e.g., a linker peptide, between the heavy chain variable region and the light chain variable region. In certain embodiments, the extracellular antigen-binding domain includes (a) a heavy chain variable region having the sequence described in SEQ ID NO: 144 and (b) a light chain variable region having the sequence described in SEQ ID NO: 143, and optionally (c) a linker sequence between the heavy chain variable region and the light chain variable region, such as a linker peptide. In certain embodiments, the extracellular antigen-binding domain includes (a) a heavy chain variable region having the sequence described in SEQ ID NO: 216 and (b) a light chain variable region having the sequence described in SEQ ID NO: 215, and optionally (c) a linker sequence between the heavy chain variable region and the light chain variable region, such as a linker peptide. In certain embodiments, the extracellular antigen-binding domain includes (a) a heavy chain variable region having the sequence described in SEQ ID NO: 220 and (b) a light chain variable region having the sequence described in SEQ ID NO: 219, and optionally (c) a linker sequence between the heavy chain variable region and the light chain variable region, such as a linker peptide. In certain embodiments, the extracellular antigen-binding domain includes (a) a heavy chain variable region having the sequence described in SEQ ID NO: 236 and (b) a light chain variable region having the sequence described in SEQ ID NO: 235, and optionally (c) a linker sequence between the heavy chain variable region and the light chain variable region, such as a linker peptide.In certain embodiments, the extracellular antigen-binding domain includes (a) a heavy chain variable region having the sequence described in SEQ ID NO: 268 and (b) a light chain variable region having the sequence described in SEQ ID NO: 267, optionally, and (c) a linker sequence between the heavy chain variable region and the light chain variable region, such as a linker peptide. In certain embodiments, the extracellular antigen-binding domain includes (a) a heavy chain variable region having the sequence described in SEQ ID NO: 116 and (b) a light chain variable region having the sequence described in SEQ ID NO: 115, optionally, and (c) a linker sequence between the heavy chain variable region and the light chain variable region, such as a linker peptide. In certain embodiments, the extracellular antigen-binding domain includes (a) a heavy chain variable region having the sequence described in SEQ ID NO: 172 and (b) a light chain variable region having the sequence described in SEQ ID NO: 171, optionally, and (c) a linker sequence between the heavy chain variable region and the light chain variable region, such as a linker peptide.

[0019] In certain embodiments, this extracellular antigen-binding domain is (a) SEQ ID NOs: 311, 317, 323, 328, 334, 337, 342, 347, 351, 356, 362, 368, 374, 376, 380, 384, 389, 394, 396, 400, 405, 408, 412, 415, 422, 427, 432, 437, 442, 446, 451, 453, 456, 458 The group consists of 459, 463, 464, 467, 473, 476, 482, 486, 489, 492, 494, 497, 502, 507, 512, 517, 522, 527, 529, 532, 536, 539, 543, 546, 550, 553, 555, 561, 567, 570, 574, 577, 578, 579, 584, 578, 587, 591, 925, and 931. Heavy chain variable region CDR3 containing an amino acid sequence selected from; and (b) SEQ ID NOs: 314, 320, 325, 331, 339, 345, 350, 353, 359, 365, 371, 377, 383, 386, 392, 395, 399, 402, 407, 410, 414, 418, 419, 424, 430, 435, 439, 443, 449, 452, 455, 457, 4 It contains a light chain variable region CDR3 comprising an amino acid sequence selected from the group consisting of 62, 465, 470, 479, 485, 488, 491, 493, 495, 499, 505, 509, 514, 519, 524, 528, 530, 531, 535, 541, 542, 545, 549, 554, 558, 564, 569, 573, 576, 581, 592, 928, and 934.

[0020] In certain embodiments, this extracellular antigen-binding domain includes: (a) a heavy chain variable region CDR1 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 309, 315, 321, 326, 332, 335, 340, 346, 354, 360, 366, 372, 378, 387, 393, 403, 411, 420, 425, 436, 440, 444, 471, 480, 500, 510, 515, 520, 525, 537, 551, 559, 565, 582, 589, 923 and 929; (b) SEQ ID NOs: 310, 316, 322, 327, Heavy chain variable region CDR2 containing an amino acid sequence selected from the group consisting of 333, 336, 341, 355, 361, 367, 373, 379, 388, 404, 412, 421, 426, 431, 441, 445, 450, 466, 472, 475, 481, 496, 501, 506, 511, 516, 521, 526, 538, 552, 560, 566, 583, 590, 924, and 930; (c) SEQ ID NOs: 311, 317, 323, 328, 334, 337, 342, 347, 351, 356, 362, 368, 374, 376, 380 The group consists of 384, 389, 394, 396, 400, 405, 408, 412, 415, 422, 427, 432, 437, 442, 446, 451, 453, 456, 458, 459, 463, 464, 467, 473, 476, 482, 486, 489, 492, 494, 497, 502, 507, 512, 517, 522, 527, 529, 532, 536, 539, 543, 546, 550, 553, 555, 561, 567, 570, 574, 577, 578, 579, 584, 578, 587, 591, 925 and 931. (d) Heavy chain variable region CDR3 containing an amino acid sequence selected from; (d) Light chain variable region CDR1 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 312, 318, 324, 329, 338, 343, 348, 352, 357, 363, 369, 381, 390, 397, 401, 406, 416, 423, 428, 433, 447, 460, 468, 474, 477, 483, 490, 498, 503, 508, 518, 533, 540, 544, 547, 556, 562, 568, 571, 580, 585, 588, 926 and 932;(e) Light chain variable region CDR2 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 313, 319, 330, 344, 349, 358, 364, 370, 382, ​​385, 391, 398, 409, 417, 429, 434, 438, 448, 454, 461, 469, 478, 484, 487, 504, 513, 523, 534, 429, 448, 548, 557, 563, 572, 575, 586, 927 and 933; and (f) SEQ ID NOs: 314, 320, 325, 331, 339, 345, 35; 0, 353, 359, 365, 371, 377, 383, 386, 392, 395, 399, 402, 407, 410, 414, 418, 419, 424, 430, 435, 439, 443, 449, 452, 455, 457, 462, 465, 470, 479, 485, 488, 491, 493 Light chain variable region CDR3 containing an amino acid sequence selected from the group consisting of 495, 499, 505, 509, 514, 519, 524, 528, 530, 531, 535, 541, 542, 545, 549, 554, 558, 564, 569, 573, 576, 581, 592, 928, and 934.

[0021] In certain embodiments, the extracellular antigen-binding domain includes (a) a heavy chain variable region CDR1 containing an amino acid having the sequence or a conserved modification thereof described in SEQ ID NO: 923, (b) a heavy chain variable region CDR2 containing an amino acid having the sequence or a conserved modification thereof described in SEQ ID NO: 924, and (c) a heavy chain variable region CDR3 containing an amino acid having the sequence or a conserved modification thereof described in SEQ ID NO: 925. In certain embodiments, the extracellular antigen-binding domain includes (a) a light chain variable region CDR1 containing an amino acid having the sequence or a conserved modification thereof described in SEQ ID NO: 926, (b) a light chain variable region CDR2 containing an amino acid having the sequence or a conserved modification thereof described in SEQ ID NO: 927, and (c) a light chain variable region CDR3 containing an amino acid having the sequence or a conserved modification thereof described in SEQ ID NO: 928.

[0022] In certain embodiments, this extracellular antigen-binding domain is (a) the sequence described in Sequence ID No. 929 (b) SEQ ID NO: 1, a heavy chain variable region CDR1 containing an amino acid sequence having the above, or a conservative modification thereof. (c) Heavy chain variable region CDR2 containing the amino acid sequence having the sequence described in 930 or a conservative modification thereof, and heavy chain variable region C containing the amino acid sequence having the sequence described in SEQ ID NO: 931. This includes DR3 or a conservative modification thereof. In certain embodiments, this extracellular antigen-binding domain is (a) a light chain variable region CDR1 comprising an amino acid sequence having the sequence described in SEQ ID NO: 932. or a conservative modification thereof, (b) a light amino acid sequence having the sequence described in Sequence ID No. 933 (c) Having the chain variable region CDR2 or its conservative modification and the sequence described in Sequence ID No. 934 This includes the light chain variable region CDR3 containing the amino acid sequence or a conservative modification thereof.

[0023] In certain embodiments, the extracellular antigen-binding domain is: (a) a heavy chain variable region CDR1 or a conserved modification thereof, comprising an amino acid sequence having the sequence described in SEQ ID NO: 923; (b) SEQ ID NO: (c) Heavy chain variable region CDR2 containing an amino acid sequence having the sequence described in 924 or a conservative modification thereof, (c) Heavy chain variable region CD containing an amino acid sequence having the sequence described in SEQ ID NO: 925 R3 or its conservative modification, (d) an amino acid sequence having the sequence described in SEQ ID NO: 926 (e) having the sequence described in Sequence ID No. 927, a light chain variable region CDR1 or a conservative modification thereof. Light chain variable region CDR2 containing the amino acid sequence or its conservative modification, and (f) Sequence ID This includes a light chain variable region CDR3 containing an amino acid sequence having the sequence described in 928, or a conservative modification thereof.

[0024] In certain embodiments, the extracellular antigen-binding domain is: (a) a heavy chain variable region CDR1 or a conserved modification thereof, comprising an amino acid sequence having the sequence described in SEQ ID NO: 929; (b) SEQ ID NO: (c) Heavy chain variable region CDR2 containing an amino acid sequence having the sequence described in 930 or a conservative modification thereof, (c) Heavy chain variable region CDR containing an amino acid sequence having the sequence described in SEQ ID NO: 931 3. A conservative modification thereof, (d) comprising an amino acid sequence having the sequence described in Sequence ID No. 932 Light chain variable region CDR1 or a conservative modification thereof, having the sequence described in (e) Sequence ID No. 933 (f)SEQ ID NO: 9 This includes a light chain variable region CDR3 or a conservative modification thereof, which contains an amino acid sequence having the sequence described in 34.

[0025] In certain embodiments, the extracellular antigen-binding domain includes (a) a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 411 or a conserved modification thereof; (b) a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 412 or a conserved modification thereof; (c) a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 463 or a conserved modification thereof; (d) a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 318 or a conserved modification thereof; (e) a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 319 or a conserved modification thereof; and (f) a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 419 or a conserved modification thereof.

[0026] In certain embodiments, the extracellular antigen-binding domain includes: (a) heavy chain variable region CDR1 or a conserved modification thereof containing the amino acid sequence of SEQ ID NO: 515; (b) heavy chain variable region CDR2 or a conserved modification thereof containing the amino acid sequence of SEQ ID NO: 516; (c) heavy chain variable region CDR3 or a conserved modification thereof containing the amino acid sequence of SEQ ID NO: 517; (d) light chain variable region CDR1 or a conserved modification thereof containing the amino acid sequence of SEQ ID NO: 318; (e) light chain variable region CDR2 or a conserved modification thereof containing the amino acid sequence of SEQ ID NO: 319; and (f) light chain variable region CDR3 or a conserved modification thereof containing the amino acid sequence of SEQ ID NO: 531.

[0027] In certain embodiments, this extracellular antigen binding includes (a) the amino acid sequence of SEQ ID NO: 403 (b) heavy chain variable region CDR1 or its conservative modification; (b) including the amino acid sequence of SEQ ID NO: 404 (c) Heavy chain variable region CDR2 or a conservative modification thereof; (d) Light chain variable region CDR1 or a conservative modification thereof including the amino acid sequence of SEQ ID NO: 532; (e) Light chain variable region CDR2 or a conservative modification thereof including the amino acid sequence of SEQ ID NO: 533; and (f) Light chain variable region CDR3 including the amino acid sequence of SEQ ID NO: 535.

[0028] In certain embodiments, this extracellular antigen-binding domain is: (a) the heavy chain variable region CDR1 or a conserved modification thereof, comprising the amino acid sequence of SEQ ID NO: 411; (b) the amino acid sequence of SEQ ID NO: 412 (c) Heavy chain variable region CDR2 or a conservative modification thereof containing the sequence; (d) Light chain variable region CDR1 or a conservative modification thereof containing the amino acid sequence of SEQ ID NO: 543; (e) Light chain variable region CDR2 or a conservative modification thereof containing the amino acid sequence of SEQ ID NO: 448; and (f) Light chain variable region CDR3 or a conservative modification thereof containing the amino acid sequence of SEQ ID NO: 545.

[0029] In certain embodiments, this extracellular antigen-binding domain is: (a) the heavy chain variable region CDR1 or a conserved modification thereof, comprising the amino acid sequence of SEQ ID NO: 372; (b) the amino acid sequence of SEQ ID NO: 475 (c) Heavy chain variable region CDR2 containing the sequence or a conservative modification thereof; (d) Light chain variable region CDR1 containing the amino acid sequence of SEQ ID NO: 571; (e) Light chain variable region CDR2 containing the amino acid sequence of SEQ ID NO: 572 or a conservative modification thereof; and (f) Light chain variable region CDR3 containing the amino acid sequence of SEQ ID NO: 573 or a conservative modification thereof.

[0030] In certain embodiments, this extracellular antigen-binding domain is: (a) the heavy chain variable region CDR1 or a conservative modification thereof, comprising the amino acid sequence of SEQ ID NO: 440; (b) the amino acid sequence of SEQ ID NO: 441 (c) Heavy chain variable region CDR2 or a conservative modification thereof containing the sequence; (d) Light chain variable region CDR1 or a conservative modification thereof containing the amino acid sequence of SEQ ID NO: 442; (e) Light chain variable region CDR2 or a conservative modification thereof containing the amino acid sequence of SEQ ID NO: 329; and (f) Light chain variable region CDR3 or a conservative modification thereof containing the amino acid sequence of SEQ ID NO: 443.

[0031] In certain embodiments, this extracellular antigen-binding domain is: (a) the heavy chain variable region CDR1 or a conserved modification thereof, comprising the amino acid sequence of SEQ ID NO: 309; (b) the amino acid sequence of SEQ ID NO: 310 (c) Heavy chain variable region CDR2 or a conservative modification thereof containing the sequence; (d) Light chain variable region CDR1 or a conservative modification thereof containing the amino acid sequence of SEQ ID NO: 489; (e) Light chain variable region CDR2 or a conservative modification thereof containing the amino acid sequence of SEQ ID NO: 490; and (f) Light chain variable region CDR3 or a conservative modification thereof containing the amino acid sequence of SEQ ID NO: 491.

[0032] In certain non-limiting embodiments, this extracellular antigen-binding domain includes an amino acid having the sequence described in SEQ ID NO: 664 or a conserved modification thereof. In certain embodiments, this extracellular antigen-binding domain includes an amino acid having the sequence described in SEQ ID NO: 700 or a conserved modification thereof. In certain embodiments, this extracellular antigen-binding domain includes an amino acid having the sequence described in SEQ ID NO: 702 or a conserved modification thereof. In certain embodiments, this extracellular antigen-binding domain includes an amino acid having the sequence described in SEQ ID NO: 710 or a conserved modification thereof. In certain embodiments, this extracellular antigen-binding domain includes an amino acid having the sequence described in SEQ ID NO: 726: or a conserved modification thereof. In certain embodiments, this detailed The extracellular antigen-binding domain includes an amino acid having the sequence described in SEQ ID NO: 650 or a conserved modification thereof. In certain embodiments, this extracellular antigen-binding domain includes an amino acid having the sequence described in SEQ ID NO: 678 or a conserved modification thereof.

[0033] In certain non-limiting embodiments, this extracellular antigen-binding domain is (a) Sequence ID No. 923 (ii) a heavy chain variable region CDR1 or a conserved modification thereof containing an amino acid sequence having the sequence described in (ii), a heavy chain variable region CDR2 or a conserved modification thereof containing an amino acid sequence having the sequence described in SEQ ID NO: 924, and a heavy chain variable region CDR3 or a conserved modification thereof containing an amino acid sequence having the sequence described in SEQ ID NO: 925, and (ii) a light chain variable region CDR1 or a conserved modification thereof containing an amino acid sequence having the sequence described in SEQ ID NO: 926, a light chain variable region CDR2 or a conserved modification thereof containing an amino acid sequence having the sequence described in SEQ ID NO: 927, and a light chain variable region CDR3 or a conserved modification thereof containing an amino acid sequence having the sequence described in SEQ ID NO: 928, optionally, (iii) a linker sequence between the heavy chain variable region and the light chain variable region, e.g. For example, it contains linker peptides.

[0034] In another non-limiting embodiment, this extracellular antigen-binding domain is (i) linked to Sequence ID No. 929. (ii) a heavy chain variable region CDR1 or a conserved modification thereof containing an amino acid sequence having the sequence described, a heavy chain variable region CDR2 or a conserved modification thereof containing an amino acid sequence having the sequence described in SEQ ID NO: 930, and a heavy chain variable region CDR3 or a conserved modification thereof containing an amino acid sequence having the sequence described in SEQ ID NO: 931, and (ii) a light chain variable region CDR1 or a conserved modification thereof containing an amino acid sequence having the sequence described in SEQ ID NO: 932, a light chain variable region CDR2 or a conserved modification thereof containing an amino acid sequence having the sequence described in SEQ ID NO: 933, and a light chain variable region CDR3 or a conserved modification thereof containing an amino acid sequence having the sequence described in SEQ ID NO: 934, optionally comprising (iii) a linker sequence between the heavy chain variable region and the light chain variable region, such as a linker peptide.

[0035] In certain embodiments, this extracellular antigen-binding domain is (a) the amino acid of SEQ ID NO: 411 (b) the heavy chain variable region CDR1 containing the sequence or its conservative modification; (b) the amino acid sequence of SEQ ID NO: 412 (c) Heavy chain variable region CDR2 or a conserved modification thereof containing the sequence; (d) Light chain variable region CDR1 or a conserved modification thereof containing the amino acid sequence of SEQ ID NO: 463; (e) Light chain variable region CDR2 or a conserved modification thereof containing the amino acid sequence of SEQ ID NO: 318; and (f) Light chain variable region CDR3 or a conserved modification thereof containing the amino acid sequence of SEQ ID NO: 419, optionally accompanied by (g) a linker sequence between the heavy chain variable region and the light chain variable region, e.g., a linker peptide.

[0036] In certain embodiments, this extracellular antigen-binding domain is (a) the amino acid of SEQ ID NO: 515 (b) the heavy chain variable region CDR1 containing the sequence or its conservative modification; (b) the amino acid sequence of SEQ ID NO: 516 (c) Heavy chain variable region CDR2 or a conserved modification thereof containing the sequence; (d) Light chain variable region CDR1 or a conserved modification thereof containing the amino acid sequence of SEQ ID NO: 517; (e) Light chain variable region CDR2 or a conserved modification thereof containing the amino acid sequence of SEQ ID NO: 319; and (f) Light chain variable region CDR3 or a conserved modification thereof containing the amino acid sequence of SEQ ID NO: 531, optionally, (g) Heavy chain variable region and Light chain variable region It includes a linker sequence between the chain variable region, such as a linker peptide.

[0037] In certain embodiments, this extracellular antigen binding includes (a) the amino acid sequence of SEQ ID NO: 403 (b) heavy chain variable region CDR1 or its conservative modification; (b) including the amino acid sequence of SEQ ID NO: 404 (c) Heavy chain variable region CDR2 or its conserved modification; (d) Light chain variable region CDR1 or its conserved modification containing the amino acid sequence of SEQ ID NO: 532; (e) Light chain variable region CDR2 or its conserved modification containing the amino acid sequence of SEQ ID NO: 534; and (f) Light chain variable region CDR3 containing the amino acid sequence of SEQ ID NO: 535, optionally, (g) Linker arrangement between the heavy chain variable region and the light chain variable region A column, for example, containing a linker peptide.

[0038] In certain embodiments, this extracellular antigen-binding domain is: (a) the heavy chain variable region CDR1 or a conserved modification thereof, comprising the amino acid sequence of SEQ ID NO: 411; (b) the amino acid sequence of SEQ ID NO: 412 (c) Heavy chain variable region CDR2 or a conserved modification thereof containing the amino acid sequence of SEQ ID NO: 543; (d) Light chain variable region CDR1 or a conserved modification thereof containing the amino acid sequence of SEQ ID NO: 544; (e) Light chain variable region CDR2 or a conserved modification thereof containing the amino acid sequence of SEQ ID NO: 448; and (f) Light chain variable region CDR3 or a conserved modification thereof containing the amino acid sequence of SEQ ID NO: 545, optionally, (g) Heavy chain variable region and Light chain It includes a linker sequence between the chain variable region, such as a linker peptide.

[0039] In certain embodiments, this extracellular antigen-binding domain is: (a) the heavy chain variable region CDR1 or a conserved modification thereof, comprising the amino acid sequence of SEQ ID NO: 372; (b) the amino acid sequence of SEQ ID NO: 475 (c) Heavy chain variable region CDR2 or a conserved modification thereof containing the sequence; (d) Light chain variable region CDR1 or a conserved modification thereof containing the amino acid sequence of SEQ ID NO: 570; (e) Light chain variable region CDR2 or a conserved modification thereof containing the amino acid sequence of SEQ ID NO: 572; and (f) Light chain variable region CDR3 or a conserved modification thereof containing the amino acid sequence of SEQ ID NO: 573, optionally, (g) Heavy chain variable region and Light chain variable region It includes a linker sequence between the chain variable region, such as a linker peptide.

[0040] In certain embodiments, this extracellular antigen-binding domain is: (a) the heavy chain variable region CDR1 or a conservative modification thereof, comprising the amino acid sequence of SEQ ID NO: 440; (b) the amino acid sequence of SEQ ID NO: 441 (c) Heavy chain variable region CDR2 or a conserved modification thereof containing the sequence; (d) Light chain variable region CDR1 or a conserved modification thereof containing the amino acid sequence of SEQ ID NO: 442; (e) Light chain variable region CDR2 or a conserved modification thereof containing the amino acid sequence of SEQ ID NO: 330; and (f) Light chain variable region CDR3 or a conserved modification thereof containing the amino acid sequence of SEQ ID NO: 443, optionally, (g) Heavy chain variable region and Light chain variable region It includes a linker sequence between the chain variable region, such as a linker peptide.

[0041] In certain embodiments, this extracellular antigen-binding domain is: (a) the heavy chain variable region CDR1 or a conserved modification thereof, comprising the amino acid sequence of SEQ ID NO: 309; (b) the amino acid sequence of SEQ ID NO: 310 (c) Heavy chain variable region CDR2 or a conserved modification thereof containing the amino acid sequence of SEQ ID NO: 489; (d) Light chain variable region CDR1 or a conserved modification thereof containing the amino acid sequence of SEQ ID NO: 490; (e) Light chain variable region CDR2 or a conserved modification thereof containing the amino acid sequence of SEQ ID NO: 313; and (f) Light chain variable region CDR3 or a conserved modification thereof containing the amino acid sequence of SEQ ID NO: 491, optionally, (g) Heavy chain variable region and Light chain It includes a linker sequence between the chain variable region, such as a linker peptide.

[0042] In certain embodiments, the linker peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 307 and SEQ ID NO: 897.

[0043] In certain embodiments, the extracellular antigen-binding domain binds to an FcRL5 containing the amino acid sequence described in SEQ ID NO: 899. In certain embodiments, this extracellular antigen-binding domain binds to an epitope containing the amino acid sequence described in SEQ ID NO: 964. In certain embodiments, this extracellular antigen-binding domain binds to an epitope containing the amino acid sequence described in SEQ ID NO: 965.

[0044] According to the subject matter of this disclosure, the extracellular antigen-binding domain is covalently conjugated to the transmembrane domain. This extracellular antigen-binding domain may include a signal peptide covalently conjugated to the 5' end of the extracellular antigen-binding domain. In certain embodiments, the transmembrane domain of CAR includes CD8 polypeptide, CD28 polypeptide, CD3ζ polypeptide, CD4 polypeptide, 4-1BB polypeptide, OX40 polypeptide, ICOS polypeptide, CTLA-4 polypeptide, PD-1 polypeptide, LAG-3 polypeptide, 2B4 polypeptide, BTLA polypeptide, synthetic peptide (not based on a protein associated with an immune response), or a combination thereof. In one non-limiting embodiment, this transmembrane domain includes CD8 polypeptide. In certain embodiments, this transmembrane domain includes CD28 polypeptide.

[0045] According to the subject matter of this disclosure, in certain embodiments, the intracellular domain comprises a CD3ζ polypeptide. In certain embodiments, this intracellular domain further comprises at least one signaling region. In certain embodiments, this at least one signaling region comprises a CD28 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a DAP-10 polypeptide, a PD-1 polypeptide, a CTLA-4 polypeptide, a LAG-3 polypeptide, a 2B4 polypeptide, a BTLA polypeptide, a synthetic peptide (not based on a protein associated with an immune response), or a combination thereof. In certain embodiments, the signaling region is a co-stimulatory signaling region. In certain embodiments, this co-stimulatory signaling region comprises a CD28 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a DAP-10 polypeptide, or a combination thereof. In certain embodiments, this at least one co-stimulatory signaling region comprises a CD28 polypeptide. In certain non-limiting embodiments, the transmembrane domain comprises a CD28 polypeptide, the intracellular domain comprises a CD3ζ polypeptide, and the co-stimulatory signaling domain comprises a CD28 polypeptide. In certain non-limiting embodiments, the transmembrane domain comprises a CD8 polypeptide, the intracellular domain comprises a CD3ζ polypeptide, and the co-stimulatory signaling domain comprises a 4-1BB polypeptide.

[0046] In certain embodiments, the above-mentioned CAR is expressed by recombination. This CAR can be expressed from a vector. In certain embodiments, this vector is a gamma-retroviral vector.

[0047] The subject matter of this disclosure also provides isolated immune response cells containing the above-mentioned CAR. In certain embodiments, these isolated immune response cells are transduced with the CAR, for example, constitutively expressed on the surface of the immune response cells. In certain embodiments, these isolated immune response cells are further transduced with at least one costimulatory ligand so that the immune response cells express at least one costimulatory ligand. In certain embodiments, this at least one costimulatory ligand is selected from the group consisting of 4-1BBL, CD80, CD86, CD70, OX40L, CD48, TNFRSF14, and combinations thereof. In certain embodiments, these isolated immune response cells are further transduced with at least one cytokine so that the immune response cells secrete at least one cytokine. In certain embodiments, this at least one cytokine is selected from the group consisting of IL-2, IL-3, IL-6, IL-7, IL-11, IL-12, IL-15, IL-17, IL-21, and combinations thereof. In certain embodiments, the isolated immune response cells are selected from a group consisting of T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, human embryonic stem cells, lymphocyte precursor cells, T cell progenitor cells, and pluripotent stem cells that can differentiate into lymphoid cells. In certain embodiments, the immune response cells are T cells.

[0048] The subject matter of this disclosure further provides nucleic acid molecules encoding the CAR of this disclosure, vectors comprising such nucleic acid molecules, and host cells expressing such nucleic acid molecules. In certain embodiments, the nucleic acid molecule comprises a nucleic acid having the sequence described in SEQ ID NO: 951. In certain embodiments, the nucleic acid molecule comprises a nucleic acid having the sequence described in SEQ ID NO: 952. In certain embodiments, the vector is a γ-retroviral vector. In certain embodiments, the host cell is a T cell.

[0049] Furthermore, the subject matter of this disclosure provides a method for using the above-mentioned immune response cells to reduce tumor burden in a subject. For example, the subject matter of this disclosure provides a method for reducing tumor burden in a subject, comprising the steps of administering an effective amount of the immune response cells of this disclosure to the subject, thereby inducing tumor cell death in the subject. In certain embodiments, the method reduces the number of tumor cells. In another embodiment, the method reduces the tumor size. In yet another embodiment, the method eradicates the tumor in the subject. In certain embodiments, the tumor is selected from the group consisting of multiple myeloma, non-Hodgkin lymphoma (particularly mantle cell), Hodgkin lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, Burkitts lymphoma, and Waldenström macroglobulinemia. In certain embodiments, the tumor is multiple myeloma. In certain embodiments, the subject is human. In certain embodiments, the immune response cells are T cells.

[0050] Furthermore, the subject matter of this disclosure provides a method for using the above-mentioned immune response cells to increase or prolong the survival of a subject having a neoplasm. For example, the subject matter of this disclosure provides a method for increasing or prolonging the survival of a subject having a neoplasm, comprising the step of administering an effective amount of the immune response cells of this disclosure to the subject, thereby increasing or prolonging the survival of the subject. In certain embodiments, the neoplasm is selected from the group consisting of multiple myeloma, non-Hodgkin lymphoma (particularly mantle cell), Hodgkin lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, Burkitt lymphoma, and Waldenström macroglobulinemia. In certain embodiments, the neoplasm is multiple myeloma. In certain embodiments, this method reduces or eradicates the tumor burden in the subject.

[0051] The subject matter of this disclosure also provides a method for producing immune response cells that bind to Fc receptor-like 5 (FcRL5), for example, domain 9 of FcRL5. In a non-limiting embodiment, the method includes the step of introducing a nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular domain into immune response cells, wherein the extracellular antigen-binding domain specifically binds to Fc receptor-like 5 (FcRL5). In a specific non-limiting embodiment, the extracellular antigen-binding domain is scFv.

[0052] The subject matter of this disclosure further provides pharmaceutical compositions comprising an effective amount of immune response cells of this disclosure and pharmaceutically acceptable excipients. In certain embodiments, these pharmaceutical compositions are for treating neoplasms. In certain embodiments, the neoplasm is selected from the group consisting of multiple myeloma, non-Hodgkin lymphoma (particularly mantle cell), Hodgkin lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, Burkitt lymphoma, and Waldenström macroglobulinemia. In certain embodiments, the neoplasm is multiple myeloma.

[0053] The subject matter of this disclosure further provides kits for treating neoplasms, including immune response cells of this disclosure. In certain embodiments, the kit further includes instructions for the use of immune response cells to treat neoplasms. In certain embodiments, the neoplasm is selected from the group consisting of multiple myeloma, non-Hodgkin lymphoma (particularly mantle cell), Hodgkin lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, Burkitt lymphoma, and Waldenström macroglobulinemia. In certain embodiments, the neoplasm is multiple myeloma.

[0054] The following detailed description, which is illustrated by illustrative methods but is not intended to limit the present invention to the specific embodiments described, can be understood in conjunction with the accompanying drawings. [Brief explanation of the drawing]

[0055] [Figure 1-1] Figure 1 shows FcRL5 expression in various normal tissues and human cancer cell lines. [Figure 1-2] Figure 1 shows FcRL5 expression in various normal tissues and human cancer cell lines. [Figure 1-3] Figure 1 shows FcRL5 expression in various normal tissues and human cancer cell lines. [Figure 1-4] Figure 1 shows FcRL5 expression in various normal tissues and human cancer cell lines.

[0056] [Figure 2] Figure 2 shows the screening of anti-FcRL5 scFv using 3T3 cells expressing FcRL5 or FcRL1, 2, 3, 4, or 6.

[0057] [Figure 3A] (A) Presentation of the domains and solubility of FcRL5, glycosylphosphatidylinositol (GPI) anchored and transmembrane morphology. (B) Presentation of the vector used to express the mutant morph of FcRL5 lacking domain 9 (also referred to herein as FcRL5Δdom9). (C) Nucleotide sequences of full-length FcRL5 and the morph of FcRL5 lacking domain 9. (D) Presentation of differences in the nucleotide sequences of full-length FcRL5 and the mutant morph of FcRL5 with domain 9 deletion (referred to herein as "FcRL5Δdom9"). [Figure 3B] (A) Presentation of the domains and solubility of FcRL5, glycosylphosphatidylinositol (GPI) anchored and transmembrane morphology. (B) Presentation of the vector used to express the mutant morph of FcRL5 lacking domain 9 (also referred to herein as FcRL5Δdom9). (C) Nucleotide sequences of full-length FcRL5 and the morph of FcRL5 lacking domain 9. (D) Presentation of differences in the nucleotide sequences of full-length FcRL5 and the mutant morph of FcRL5 with domain 9 deletion (referred to herein as "FcRL5Δdom9"). [Figure 3C](A) Presentation of the domains and solubility of FcRL5, glycosylphosphatidylinositol (GPI) anchored and transmembrane morphology. (B) Presentation of the vector used to express the mutant morph of FcRL5 lacking domain 9 (also referred to herein as FcRL5Δdom9). (C) Nucleotide sequences of full-length FcRL5 and the morph of FcRL5 lacking domain 9. (D) Presentation of differences in the nucleotide sequences of full-length FcRL5 and the mutant morph of FcRL5 with domain 9 deletion (referred to herein as "FcRL5Δdom9"). [Figure 3D-1] (A) Presentation of the domains and solubility of FcRL5, glycosylphosphatidylinositol (GPI) anchored and transmembrane morphology. (B) Presentation of the vector used to express the mutant morph of FcRL5 lacking domain 9 (also referred to herein as FcRL5Δdom9). (C) Nucleotide sequences of full-length FcRL5 and the morph of FcRL5 lacking domain 9. (D) Presentation of differences in the nucleotide sequences of full-length FcRL5 and the mutant morph of FcRL5 with domain 9 deletion (referred to herein as "FcRL5Δdom9"). [Figure 3D-2] (A) Presentation of the domains and solubility of FcRL5, glycosylphosphatidylinositol (GPI) anchored and transmembrane morphology. (B) Presentation of the vector used to express the mutant morph of FcRL5 lacking domain 9 (also referred to herein as FcRL5Δdom9). (C) Nucleotide sequences of full-length FcRL5 and the morph of FcRL5 lacking domain 9. (D) Presentation of differences in the nucleotide sequences of full-length FcRL5 and the mutant morph of FcRL5 with domain 9 deletion (referred to herein as "FcRL5Δdom9").

[0058] [Figure 4] Figure 4 shows the screening of anti-FcRL5 scFv ET200-39 on 3T3 cells expressing FcRL5Δdom9.

[0059] [Figure 5]Figure 5 shows the screening of anti-FcRL5 scFv ET200-104 on 3T3 cells expressing FcRL5Δdom9.

[0060] [Figure 6-1] Figure 6 shows the screening of anti-FcRL5 scFv ET200-105 on 3T3 cells expressing FcRL5Δdom9. [Figure 6-2] Figure 6 shows the screening of anti-FcRL5 scFv ET200-105 on 3T3 cells expressing FcRL5Δdom9.

[0061] [Figure 7] Figure 7 shows the screening of anti-FcRL5 scFv ET200-109 on 3T3 cells expressing FcRL5Δdom9.

[0062] [Figure 8] Figure 8 shows the screening of anti-FcRL5 scFv ET200-117 on 3T3 cells expressing FcRL5Δdom9.

[0063] [Figure 9] These are schematic diagrams of FcRL5-targeting chimeric antigen receptors according to non-limiting embodiments of the subject matter of this disclosure. (A) Schematic diagram of an FcRL5-targeting CAR having a CD28 costimulatory domain and a CD3 zeta. (B) Schematic diagram of an FcRL5-targeting CAR having a 4-1BB costimulatory domain and a CD3 zeta.

[0064] [Figure 10] Figure 10 shows a vector map of a chimeric antigen receptor targeting FcRL5 using scFV ET200-31 according to one non-limiting embodiment of the subject matter of this disclosure.

[0065] [Figure 11]Figure 11 shows a vector map of a chimeric antigen receptor targeting FcRL5 using scFV ET200-39 according to one non-limiting embodiment of the subject matter of this disclosure.

[0066] [Figure 12] Figure 12 shows a vector map of a chimeric antigen receptor targeting FcRL5 using scFV ET200-69 according to one non-limiting embodiment of the subject matter of this disclosure.

[0067] [Figure 13] Figure 13 shows a vector map of a chimeric antigen receptor targeting FcRL5 using scFV ET200-104 according to one non-limiting embodiment of the subject matter of this disclosure.

[0068] [Figure 14] Figure 14 shows a vector map of a chimeric antigen receptor targeting FcRL5 using scFV ET200-105 according to one non-limiting embodiment of the subject matter of this disclosure.

[0069] [Figure 15] Figure 15 shows a vector map of a chimeric antigen receptor targeting FcRL5 using scFV ET200-109 according to one non-limiting embodiment of the subject matter of this disclosure.

[0070] [Figure 16] Figure 16 shows a vector map of a chimeric antigen receptor targeting FcRL5 using scFV ET200-117 according to one non-limiting embodiment of the subject matter of this disclosure.

[0071] [Figure 17] Figure 17 shows the expression of chimeric antigen receptors targeting FcRL5 on the surface of transduced T cells.

[0072] [Figure 18] Figure 18 shows the cytotoxicity of FcRL5-targeted chimeric antigen receptor T cells against FcRL5-expressing cells.

[0073] [Figure 19] Figure 19 shows the induction of cytokine secretion by chimeric antigen receptor T cells targeted by FcRL5.

[0074] [Figure 20] Figure 20 shows the proliferation of chimeric antigen receptor T cells that target FcRL5 upon antigen stimulation.

[0075] [Figure 21] Figure 21 illustrates the CLIPS technique. The CLIPS reaction occurs between the bromine group of the CLIPS scaffold and the thiol side chain of cysteine. The reaction is rapid and specific under mild conditions. Using this sophisticated chemistry, native protein sequences are transformed into CLIPS constructs with various structures. From left to right: two different single T2 loops, a T3 double loop, a conjugated T2+T3 loop, a stabilized beta sheet, and a stabilized alpha helix (Timmerman et al., J Mol Recognit 2007; Vol. 20: pp. 283-29).

[0076] [Figure 22] Figure 22 illustrates combinatorial clip library screening. Target proteins containing discontinuous conformational epitopes (left) are converted into a matrix library (center). Combinatorial peptides are synthesized on a dedicated minicard and chemically converted into spatially defined CLIPS constructs (right).

[0077] [Figure 23] Figure 23 shows the T3 looped CLIPSTM construct.

[0078] [Figure 24]Figures 24A-D illustrate the heatmap technique. (A) A table of combination peptides having two subsequences labeled "Loop 1" and "Loop 2". (B) Data from A shown as a matrix. (C) Color bar display of the heatmap presentation. (D) Heatmap visualization of the data from A.

[0079] [Figure 25] Figure 25 shows a heatmap analysis of the data recorded for Herceptin.

[0080] [Figure 26] Figure 26 shows a heatmap analysis of the data recorded for ET200-104.

[0081] [Figure 27] Figure 27 illustrates a 3D model of amino acid residues 380–731 of FcRL55 with the highlighted peptide stretch 657SRPILTFRAPR667. [Modes for carrying out the invention]

[0082] (Detailed description of the invention) The subject matter of this disclosure generally provides a chimeric antigen receptor (CAR) that targets FcRL5. In a non-limiting example, the CAR comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular domain, the extracellular antigen-binding domain specifically binding to FcRL5. In certain embodiments, the extracellular antigen-binding domain specifically binds to domains 7, 8, or 9 of FcRL5. The subject matter of this disclosure also provides immune response cells expressing FcRL5-targeted CARs (e.g., T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, human embryonic stem cells, lymphocyte precursor cells, T cell-progenitor cells, and pluripotent stem cells into which lymphoid cells can differentiate), and methods of using such immune response cells to treat tumors, such as multiple myeloma. I. Definition

[0083] Unless otherwise specified, all technical and scientific terms used herein have meanings that are generally understood by those skilled in the art to which this invention pertains. The following references provide general definitions of many of the terms used herein: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd edition, 1994); The Cambridge Dictionary of Science and Technology (Walker, ed., 1988); The Glossary of Genetics, 5th edition, R. Rieger et al. (eds.), Springer Verlag (1991); and Hale and Marham, The Harper Collins Dictionary of Biology (1994). (1991). As used herein, the following terms have the meanings set forth below unless otherwise specified.

[0084] As used herein, the terms “about” or “approximately” mean within an acceptable margin of error for a particular value, as determined by a person skilled in the art, and depending in part to the limits of the measuring system, how the value was measured or determined. For example, “about” may mean within or greater than 3 standard deviations in practice in the art. Alternatively, “about” may mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and still more preferably up to 1% of a given value. Or, particularly with respect to biological systems or processes, the term may mean within a certain order of magnitude of a given value, preferably up to 5 times, and more preferably up to 2 times.

[0085] As used herein, the term “cell population” means a group of at least two cells expressing similar or different phenotypes. In non-limiting examples, a cell population may include at least about 10, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, or at least about 1000 cells expressing similar or different phenotypes.

[0086] As used herein, the term “antibody” means not only intact antibody molecules but also fragments of antibody molecules that retain immunogenicity. Such fragments are also well known in the art and are commonly used both in vitro and in vivo. Therefore, as used herein, the term “antibody” means not only intact immunoglobulin molecules but also the well known active fragments F(ab')2 and Fab. F(ab')2 and Fab fragments lacking the Fe fragment of the intact antibody evaporate more rapidly from circulation and may have less nonspecific tissue binding to the intact antibody (Wahl et al., J Nucl Med vol. 24: pp. 316-325 (1983)). The antibodies of the present invention include whole native antibodies, bispecific antibodies; chimeric antibodies; Fab, Fab', single-chain V-region fragments (scFv), fusion polypeptides, and atypical antibodies.

[0087] As used herein, the terms “single-chain variable fragment” or “scFv” mean V H ::VL heterodimers are covalently linked (V) regions of the heavy chain of immunoglobulins (e.g., mouse or human). H ) and the variable region of the light chain (V L It is a fusion protein with ). Heavy chain (V H ) and light chain (V L ) means that they are directly joined or V H The N-terminus of V L Connect to the C terminal or V H The C-terminus of V LIt is either attached to the N-terminus or joined by a peptide code linker (e.g., 10, 15, 20, 25 amino acids). This linker is typically rich in glycine for flexibility and serine or threonine for solubility. This linker can link the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain. Non-limiting examples of linkers are disclosed in Shen et al., Anal Chem vol. 80(no. 6): pp. 1910-1917 (2008) and WO2014 / 087010, the contents of which are incorporated herein by reference in their entirety. In certain embodiments, this linker is a G4S linker.

[0088] In a non-limiting example, this linker includes amino acids having the sequence described in Sequence ID No. 897 provided below. GGGGSGGGGSGGGGS[SEQ ID NO: 897]. In a particular embodiment, the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 897 is described in SEQ ID NO: 898, provided below: GGTGGAGGTGGATCAGGTGGAGGTGGATCTGGTGGAGGTGGATCT[SEQ ID NO: 898].

[0089] In another non-limiting example, the linker includes amino acids having the sequence described in Sequence ID No. 307 provided below. SRGGGGSGGGGSGGGGSLEMA [SEQ ID NO: 307] In a particular embodiment, the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 307 is described in SEQ ID NO: 305, which is provided below: TCTAGAGGTGGTGGTGGTAGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCCTCGAGATGGCC[Sequence No. 305]

[0090] Despite the removal of the constant region and the introduction of a linker, the scFv protein retains the specificity of the original immunoglobulin. Single-chain Fv polypeptide antibodies were found in Huston et al. (Proc Nat). As described in Acad Sci USA, Vol. 85: pp. 5879-5883, 1988, V H Code array and V L It can be expressed from nucleic acids containing coding sequences. See also U.S. Patents 5,091,513, 5,132,405 and 4,956,778; and U.S. Patent Publications 20050196754 and 20050196754. Inhibitory antagonist scFvs have been described (e.g., Zhao et al., Hyrbidoma (Larchmt) 2008, Vol. 27 (No. 6): pp. 455-451; Peter et al., J Cachexia Sarcopenia Muscle, August 12, 2012; Shieh et al., J Imunol 2009, Vol. 183 (No. 4): pp. 2277-2285; Giomarelli et al., Thromb Haemost 2007, Vol. 97 (No. 6): pp. 955-963; Fife et al., J Clin Invst See Brocks et al., Immunotechnology 1997 Vol. 3 (No. 3): pp. 2252-2261; Moosmayer et al., Ther Immunol 1995 Vol. 2 (No. 10): pp. 31-40). Agonist scFv with stimulating activity has been described (for example, Peter et al., J Bioi Chern 2003 Vol. 25278 (No. 38): pp. 36740-77; Xie et al., Nat Biotech, Vol. 15 (No. 8), 1997: pp. 768-71. Ledbetter et al., Crit Rev Immunol 1997, Vol. 17 (Nos. 5-6): pp. 427-425. (See Ho et al., BioChim Biophys Acta 2003, Vol. 1638 (No. 3): pp. 257-256).

[0091] As used herein, "F(ab)" refers to a fragment of an antibody structure that binds to an antigen, is monovalent, and lacks an Fc region. For example, an antibody digested by the enzyme papain produces two F(ab) fragments and one Fc fragment (e.g., a heavy (H) chain constant region; an Fc region that does not bind to the antigen).

[0092] As used herein, "F(ab')2" refers to an antibody fragment produced by pepsin digestion of a whole IgG antibody, which has two antigen-binding (ab') (bivalent) regions, each (ab') region containing two separate amino acid chains, a portion of the H chain and a light (L) chain linked by a disulfide bond to bind the antigen, with the remaining H chain portions linked together. The "F(ab')2" fragment can be split into two individual Fab' fragments.

[0093] As used herein, the term “vector” refers to any genetic element, such as plasmids, phages, transposons, cosmids, chromosomes, viruses, virions, etc., that can be replicated in association with appropriate regulatory elements and into which a gene sequence can be transferred into a cell. Therefore, the term includes cloning and expression vehicles, as well as viral vectors and plasmid vectors.

[0094] As used herein, the term “expression vector” refers to a recombinant nucleic acid sequence, i.e., a recombinant DNA molecule, that contains a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism. Nucleic acid sequences required for expression in prokaryotes typically include promoters, operators (optional), and ribosome-binding sites, often in combination with other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.

[0095] As used herein, "CDR" is defined as the complementarity-determining region amino acid sequence of an antibody, which is the hypervariable region of the immunoglobulin heavy and light chains. See, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, US Department of Health and Human Services, National Institutes of Health (1987). Regarding illumination. Generally, antibodies contain three heavy chains and three light chain CDRs or CDR regions within their variable region. The CDRs provide the majority of contact residues for the antibody's binding to the antigen or epitope. In certain embodiments, these CDR regions are Kabat-system (Kabat, E A et al. (1991) Sequences of Proteins of Immunological Interest, 5th edition. This is described using the US Department of Health and Human Services (NIH Publication No. 91-3242).

[0096] As used herein, the term “affinity” refers to a measure of binding strength. While not theoretically bound, affinity depends on the closeness of stereochemical fit between the antibody binding site and the antigenic determinant, the size of the contact area between them, and the distribution of charged and hydrophobic groups. Affinity also includes the term “avidity,” which refers to the strength of antigen-antibody binding after the formation of a reversible complex. Methods for calculating the affinity of an antibody to an antigen, including the use of binding experiments for affinity calculation, are known in the art. Antibody activity in functional assays (e.g., flow cytometry assays) also reflects antibody affinity. Antibodies and affinity can be phenotypically characterized and compared using functional assays (e.g., flow cytometry assays).

[0097] Nucleic acid molecules useful in the methods of the present invention include any nucleic acid molecule or fragment thereof that encodes the polypeptide of the present invention. Such nucleic acid molecules do not need to be 100% identical to the endogenous nucleic acid sequence, but typically exhibit substantial identity. Polynucleotides having “substantial identity” with respect to the endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. “Hybridizing” means pairing complementary polynucleotide sequences (e.g., genes described herein) or portions thereof to form a double-stranded molecule under various stringency conditions (see, e.g., Wahl, GM and SL Berger (1987) Methods Enzymol Vol. 152: p. 399; Kimmel, AR (1987) Methods Enzymol Vol. 152: p. 507).

[0098] For example, stringent salt concentrations are typically less than about 750 mM NaCl and less than 75 mM trisodium citrate, preferably less than about 500 mM NaCl and less than 50 mM trisodium citrate, more preferably less than about 250 mM NaCl and less than 25 mM trisodium citrate. Low-stringency hybridization can be obtained in the absence of organic solvents, such as formamide, while high-stringency hybridization can be obtained in the presence of at least about 35% formamide, more preferably at least about 50% formamide. Stringent temperature conditions typically include a temperature of at least about 30°C, more preferably at least about 37°C, and most preferably at least about 42°C. Various further parameters, such as hybridization time, the concentration of surfactants, such as sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are achieved by combining these various conditions as needed. In one preferred embodiment, hybridization occurs at 30°C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In one more preferred embodiment, hybridization occurs at 37°C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg / ml denatured salmon sperm DNA (ssDNA). In one most preferred embodiment, hybridization occurs at 42°C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA. Useful variations of these conditions are readily apparent to those skilled in the art.

[0099] For most applications, the washing step after hybridization also varies in stringency. The stringency conditions for washing can be defined by salt concentration and temperature. As described above, the stringency of washing can be increased by decreasing the salt concentration or by increasing the temperature. For example, a stringent salt concentration for the washing step is preferably less than about 30 mM NaCl and less than 3 mM trisodium citrate, most preferably less than about 15 mM NaCl and less than 1.5 mM trisodium citrate. A stringent temperature condition for the washing step typically includes a temperature of at least about 25°C, more preferably at least about 42°C, and even more preferably at least about 68°C. In one preferred embodiment, the washing step takes place at 25°C in 30 mM NaCl, 3 mM trisodium citrate and 0.1% SDS. In one more preferred embodiment, the washing step takes place at 42°C in 15 mM NaCl, 1.5 mM trisodium citrate and 0.1% SDS. In a more preferred embodiment, the washing step takes place at 68°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Further variations of these conditions are readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, by Benton and Davis (Science vol. 196: p. 180, 1977); Grunstein and Rogness (Proc Natl Acad Sci, USA vol. 72: p. 3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

[0100] "Substantially identical" means a polypeptide or nucleic acid molecule that exhibits at least 50% identity with respect to a reference amino acid sequence (e.g., any one of the amino acid sequences described herein) or a nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein). Preferably, such sequence is at least 60%, more preferably 80% or 85%, more preferably 90%, 95%, or even 99% identical at the amino acid level or nucleic acid level to the sequence used for comparison.

[0101] Sequence identity is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning a degree of homology to various substitutions, deletions, and / or other modifications. In an exemplary approach to determining the degree of identity, the BLAST program may be used, with probability scores between e-3 and e-100 indicating closely related sequences.

[0102] As used herein, the term “analog” refers to a structurally related polypeptide or nucleic acid molecule that has the function of a reference polypeptide or nucleic acid molecule.

[0103] As used herein, the term “ligand” refers to a molecule that binds to a receptor. In particular, ligands bind to receptors on other cells, enabling cell-to-cell recognition and / or interaction.

[0104] As used herein, the term “disease” means any condition or disorder that impairs or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include neoplasms or cellular pathogen infections.

[0105] As used herein, the term “effective dose” means an amount sufficient to have a therapeutic effect. In certain embodiments, the “effective dose” is an amount sufficient to halt, induce remission, or inhibit the continued proliferation, growth, or metastasis (e.g., invasion or migration) of a neoplasm.

[0106] As used herein, the term “heterogeneic nucleic acid molecule or polypeptide” means a nucleic acid molecule (e.g., a cDNA, DNA, or RNA molecule) or polypeptide that is not normally present in cells or samples obtained from cells. This nucleic acid may be of another biological origin or may be, for example, an mRNA molecule that is not normally expressed in cells or samples.

[0107] As used herein, the term "immune response cells" refers to cells that function in an immune response, or their precursors or offspring.

[0108] As used herein, the term “modulate” means to change something positively or negatively. Exemplary modulations include changes of approximately 1%, 2%, 5%, 10%, 25%, 50%, 75%, or 100%.

[0109] As used herein, the term “increase” means a change of at least about 5% positive, including but not limited to a change of about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, or about 100% positive.

[0110] As used herein, the term “reduce” means to change by at least about 5%, including but not limited to changing by about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, or about 100%.

[0111] As used herein, the term “isolated cell” refers to a cell separated from the molecules and / or cellular components naturally associated with it.

[0112] As used herein, the terms “isolated,” “purified,” or “biologically pure” refer to material that has been freed to a variable degree from the components that would normally accompany it if found in its native state. “Isolated” indicates a degree of separation from its original source or surroundings. “Purified” indicates a higher degree of separation than isolation. A “purified” or “biologically pure” protein is sufficiently free of other materials that any impurities do not substantially affect the biological properties of the protein or cause other harmful consequences. That is, the nucleic acids or peptides of the present invention are purified if, when produced by recombinant DNA technology, they substantially do not contain cellular material, viral material, or culture medium, or if chemically synthesized, they substantially do not contain chemical precursors or other chemicals. Purity and homogeneity are typically determined using analytical chemistry techniques, e.g., polyacrylamide gel electrophoresis or high-performance liquid chromatography. The term “purified” may indicate that the nucleic acid or protein produces essentially one band in an electrophoretic gel. For proteins that can be modified, for example, phosphorylation or glycosylation, different modifications may result in different isolated proteins that can be purified separately.

[0113] As used herein, the term “secreted” means polypeptides released from a cell as vesicles that transiently fuse in the cellular plasma membrane and release proteins to the outside of the cell via secretory pathways through the endoplasmic reticulum and Golgi apparatus.

[0114] As used herein, the terms “specifically bind to,” “specifically bind to,” or “specifically target” mean a polypeptide or fragment thereof that recognizes and binds to a biological molecule of interest (e.g., a polypeptide), but does not substantially recognize and bind to other molecules in a sample naturally containing the polypeptide of the present invention, such as a biological sample.

[0115] As used herein, the terms “to treat” or “treatment” refer to a clinical intervention in an attempt to alter the disease process of the individual or cell being treated, which may be performed for preventive purposes or during the course of clinicopathological disease. The therapeutic effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, reducing symptoms, diminishing any direct or indirect pathological consequences of disease, preventing metastasis, slowing the rate of disease progression, achieving remission or mitigation of the disease state, and achieving remission or an improved prognosis. By preventing the progression of disease or disability, treatment can prevent exacerbations caused by the disability in a diseased or diagnosed subject or a subject suspected of having the disability, but treatment can also prevent the onset of disability or symptoms of disability in a subject at risk of disability or a subject suspected of having the disability.

[0116] As used herein, the term “subject” means any animal (e.g., mammal) including, but not limited to, humans, non-human primates, rodents, etc. (e.g., the recipient of a particular procedure, or from which cells are recovered). II. Fc receptor-like 5 (FcRL5)

[0117] Fc receptor-like 5 (FcRL5) (also known as "CD307e" or "IRTA2") is expressed on B cells and plasma cells, making it a reasonable target for treating multiple myeloma. FcRL5 binds to the Fc portion of IgG and contributes to B cell receptor signaling and B cell proliferation (Franco et al., Journal of Immunology, Vol. 190, 5739). ~5746 pages (2013); Dement-Brown et al., Journal of leukocyte biology 9 (Vol. 1, pp. 59-67 (2012)). FcRL5 was found to be an alternative to CD138 as a FACS marker for malignant plasma cells in fresh or frozen patient samples (n=23) with an average relative MFI between 10 and 55 (Ise et al., Leukemia Vol. 21, pp. 169-174 (2007)). Another study confirmed cell surface expression of FcRL5 by FACS on primary patient samples from most cases of chronic lymphocytic leukemia (CLL) and mantle cell lymphoma tested, as well as all cases of multiple myeloma (MM) (n=8) (Ise et al. (2007)). The third group studied normal bone High surface staining was observed on plasma cells in MGUS (n=16) and MM (n=16) derived from bone marrow (n=7) (similar MFI in all three groups, approximately 1000-fold increase compared to isotype controls) (Elkins et al., Molecular Cancer Therapeutics Vol. 11, 2 pp. 222-2232 (2012). FcRL5 is located on 1q21 and has been found to be involved in 1q21 abnormalities in B-cell malignancies (Hatzivassiliou et al., Immunity). Volume 14, pp. 277-289 (2001). Amplification of 1q21 was found in 48% of MM patients at diagnosis and in 67% of patients at relapse, and correlated with a worse prognosis (An et al., Haematologica, Volume 99, pp. 353-359 (2014)). FcRL5 Targeted antibody-drug conjugates were effective in treating an in vivo mouse model of MM (Elkins et al. (2012)).

[0118] Non-restrictive examples of human FcRL5 amino acid sequences can be found under GenBank protein accession numbers: AAI01070.1;XP_011508332.1;XP_011508334.1;XP_011508333.1;XP_011508332.1; and NP_001182317.1.

[0119]

[0120] In certain embodiments, FcRL5 comprises nine immunoglobulin (Ig)-like domains, e.g., domain 1, domain 2, domain 3, domain 4, domain 5, domain 6, domain 7, domain 8, and domain 9 (see Figures 3A and 3C). In certain embodiments, domain 9 of FcRL5 comprises the amino acid sequence described in Sequence ID No. 900. Sequence ID No. 900 is provided below. RPVLTLRAPGTHAAVGDLLELHCEALRGSPLILYRFFHEDVTLGNRSSPSGGASLNLSLTAEHSGNYSCEADNGLGAQRSETVTLYI[Sequence ID 900].

[0121] In certain embodiments, domain 9 of FcRL5 may have the amino acid sequence or a fragment thereof described in SEQ ID NO: 963. SEQ ID NO: 963 is provided below: GTHAAVGDLLELHCEALRGSPLILYRFFHEDVTLGNRSSPSGGASLNLSLTAEHSGNYSCEADNGLGAQRSETVTLYI[Sequence ID 963].

[0122] In certain embodiments, domain 1 may contain amino acids 23-100 of SEQ ID NO: 899; domain 2 may contain amino acids 105-185 of SEQ ID NO: 899; domain 3 may contain amino acids 191-273 of SEQ ID NO: 899; domain 4 may contain amino acids 287-373 of SEQ ID NO: 899; domain 5 may contain amino acids 380-466 of SEQ ID NO: 899; domain 6 may contain amino acids 490-555 of SEQ ID NO: 899; domain 7 may contain amino acids 565-638 of SEQ ID NO: 899; domain 8 may contain amino acids 658-731 of SEQ ID NO: 899; and domain 9 may contain amino acids 754-835 of SEQ ID NO: 899.

[0123] In certain embodiments, domain 9 of FcRL5 contains an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the amino acid sequence of sequence number 900 or 963. III. Chimeric Antigen Receptors (CARs)

[0124] Chimeric antigen receptors (CARs) are engineered receptors that transfer or confer desired specificity onto immune effector cells. CARs can be used to transfer the specificity of monoclonal antibodies onto T cells; the transfer of their coding sequences is facilitated by retroviral vectors.

[0125] Three generations of CARs exist. "First-generation" CARs typically consist of an extracellular antigen-binding domain (e.g., a single-chain variable fragment (scFv)) fused to a transmembrane domain fused to the cytoplasmic / intracellular domain of the T cell receptor chain. "First-generation" CARs typically have an intracellular domain derived from the CD3ξ chain, which is the primary signaling factor from the endogenous TCR. "First-generation" CARs can provide de novo antigen recognition and, independently of HLA-mediated antigen presentation, transmit signals to CD4 via their CD3ζ chain signaling domain in a single fusion molecule. + T cells and CD8 +Both types of T cell activation can be induced. "Second-generation" CARs add intracellular domains derived from various co-stimulatory molecules (e.g., CD28, 4-1BB, ICOS, OX40) to the cytoplasmic tail of the CAR to provide further signaling to T cells. "Second-generation" CARs include those that provide both co-stimulation (e.g., CD28 or 4-1BB) and activation (CD3ζ). Preclinical studies have shown that "second-generation" CARs can improve the antitumor activity of T cells. For example, the robust efficacy of "second-generation" CAR-modified T cells has been demonstrated in clinical trials targeting the CD19 molecule in patients with chronic lymphoblastic leukemia (CLL) and acute lymphoblastic leukemia (ALL). "Third-generation" CARs include those that provide multiple co-stimulatory (e.g., CD28 and 4-1BB) and activation (CD3ζ).

[0126] According to the subject matter of this disclosure, these CARs comprise an extracellular antigen-binding domain, a transmembrane domain, and an intracellular domain, the extracellular antigen-binding domain binding to FcRL5. In one specific, non-limiting embodiment, the extracellular antigen-binding domain is scFv. In one specific, non-limiting embodiment, the extracellular antigen-binding domain is optionally crosslinked Fab. In one specific, non-limiting embodiment, the extracellular binding domain is F(ab)2. In one specific, non-limiting embodiment, any of the above molecules may be included within a fusion protein with a heterologous sequence to form an extracellular antigen-binding domain.

[0127] In certain non-limiting embodiments, the extracellular antigen-binding domain of the CAR of the Disclosure has high binding specificity and high binding affinity to FcRL5 or domain 9 of FcRL5. In certain non-limiting embodiments, the extracellular antigen-binding domain of the CAR of the Disclosure has high binding specificity and high binding affinity to domain 8 of FcRL5. In certain non-limiting embodiments, the extracellular antigen-binding domain of the CAR of the Disclosure has high binding specificity and high binding affinity to domain 7 of FcRL5. For example, in such embodiments, the extracellular antigen-binding domain of the CAR (embodied, for example, scFv or its analogue) has approximately 3 × 10⁻¹⁶ -6 M or a dissociation constant less than (K) d ) and bind to FcRL5 (or domain 8 or domain 9 of FcRL5). In certain embodiments, this K d It is approximately 1 x 10 -6 M or less, approximately 1 x 10 -7 M or less, approximately 1 x 10 -8 M or less, approximately 1 x 10 -9 M or less, approximately 1 x 10 -10 M or less or approximately 1 x 10 -11 M or less. In certain embodiments, this K d It is approximately 1 x 10 -11 From M to approximately 3 x 10 -6 Up to M, 1 x 10 -10 From M to approximately 3 x 10 -6 Up to M, or approximately 1 x 10 -9 From M to approximately 3 x 10 -6 Up to M, for example, approximately 1 × 10 -9 From M to approximately 1 x 10 -8 Up to M, approximately 1 x 10 -8 From M to approximately 1 x 10 -7 Up to M, or approximately 1 x 10 -7 From M to approximately 1 x 10 -6 Up to M, or approximately 1 x 10 -6 From M to approximately 3 x 10 -6 It goes up to M.

[0128] The binding of the extracellular antigen-binding domain of the CAR of this disclosure (e.g., embodied by scFv or its analogues) to FcRL5 (or domain 8 or domain 9 of FcRL5) can be confirmed, for example, by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), or Western blot assay. Each of these assays generally detects the presence of a particular protein-antibody complex of interest by using a reagent (e.g., antibody or scFv) specifically labeled for the complex of interest. For example, scFv may be radioactively labeled and used in radioimmunoassay (RIA) (see, for example, Weintraub, B, Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March 1986, incorporated herein by reference). Radioactive isotopes can be used with a γ counter or It can be detected by means such as the use of a scintillation counter or by autoradiography. In certain embodiments, the extracellular antigen-binding domain targeted by FcRL5 is labeled with a fluorescent marker. Non-limiting examples of fluorescent markers include green fluorescent protein (GFP), blue fluorescent protein (e.g., EBFP, EBFP2, Azurite, and mKalama1), cyan fluorescent protein (e.g., ECFP, Cerulean, and CyPet), and yellow fluorescent protein (e.g., YFP, Citrine, Venus, and YPet). In certain embodiments, the human scFv targeted by FcRL5 is labeled with GFP.

[0129] In certain embodiments, the extracellular antigen-binding domain of the CAR of the Disclosure includes a single-chain variable fragment (scFv). In one specific embodiment, the extracellular antigen-binding domain of the CAR of the Disclosure includes a human scFv that specifically binds to human FcRL5. In another specific embodiment, the extracellular antigen-binding domain of the CAR of the Disclosure includes a mouse scFv that specifically binds to human FcRL5. In certain embodiments, the extracellular antigen-binding domain of the CAR of the Disclosure includes an scFv that specifically binds to at least a portion of domain 7 of FcRL5. In certain embodiments, the extracellular antigen-binding domain of the CAR of the Disclosure includes an scFv that specifically binds to at least a portion of domain 8 of FcRL5. In certain embodiments, the extracellular antigen-binding domain of the CAR of the Disclosure includes an scFv that specifically binds to at least a portion of domain 9 of FcRL5. For example, but not limited to, domain 9 of FcRL5 includes the amino acid sequence described in SEQ ID NO: 900 or 963, or a fragment thereof.

[0130] In certain embodiments, these extracellular antigen-binding domains are incorporated herein by reference, each of them being Franco et al., Journal of Immunology (2013). The mouse scFv is a mouse obtained from two commercially available mouse hybridomas that bind different extracellular epitopes on human FcRL5, characterized in: Vol. 190: pp. 5739-5746; Ise et al., Clinical cancer research: an official fournal of the American Association for Cancer Research (2005); Vol. 11: pp. 87-96; and Ise et al., Clinical chemistry and laboratory medicine: CCLM / FESCC (2006); Vol. 44: pp. 594-602. In certain embodiments, this extracellular antigen-binding domain is the heavy chain variable region of antibodies that bind to human FcRL5, for example, antibodies F56 and F119 disclosed by Ise et al. (2005), whose entirety is incorporated herein by reference. And it is a mouse scFv derived from the light chain variable region. CAR's extracellular antigen-binding domain

[0131] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is used in SEQ ID NOs: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, 87, 91, 95, 99, 103, 107, 111, 115, 119, 123, 127, 131, 135, 139, 143, 147, 151, 155, 159, 163, 167, This scFv antibody contains a light chain variable region containing an amino acid sequence selected from the group consisting of sequence number 171, SEQ ID NO: 175, SEQ ID NO: 179, SEQ ID NO: 183, SEQ ID NO: 187, SEQ ID NO: 191, SEQ ID NO: 195, SEQ ID NO: 199, SEQ ID NO: 203, SEQ ID NO: 207, SEQ ID NO: 211, SEQ ID NO: 215, SEQ ID NO: 219, SEQ ID NO: 223, SEQ ID NO: 227, SEQ ID NO: 231, SEQ ID NO: 235, SEQ ID NO: 239, SEQ ID NO: 243, SEQ ID NO: 247, SEQ ID NO: 251, SEQ ID NO: 255, SEQ ID NO: 259, SEQ ID NO: 263, SEQ ID NO: 267, SEQ ID NO: 271, SEQ ID NO: 275, SEQ ID NO: 279, SEQ ID NO: 283, SEQ ID NO: 287, SEQ ID NO: 291, SEQ ID NO: 295, SEQ ID NO: 299, SEQ ID NO: 303, SEQ ID NO: 917, and SEQ ID NO: 921, and binds to the FcRL5 polypeptide.

[0132] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is used in sequence numbers 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, sequence number It includes a heavy chain variable region containing an amino acid sequence selected from the group consisting of number 164, SEQ ID NO: 168, SEQ ID NO: 172, SEQ ID NO: 176, SEQ ID NO: 180, SEQ ID NO: 184, SEQ ID NO: 188, SEQ ID NO: 192, SEQ ID NO: 196, SEQ ID NO: 200, SEQ ID NO: 204, SEQ ID NO: 208, SEQ ID NO: 212, SEQ ID NO: 216, SEQ ID NO: 220, SEQ ID NO: 224, SEQ ID NO: 228, SEQ ID NO: 232, SEQ ID NO: 236, SEQ ID NO: 240, SEQ ID NO: 244, SEQ ID NO: 248, SEQ ID NO: 252, SEQ ID NO: 256, SEQ ID NO: 260, SEQ ID NO: 264, SEQ ID NO: 268, SEQ ID NO: 272, SEQ ID NO: 276, SEQ ID NO: 280, SEQ ID NO: 284, SEQ ID NO: 288, SEQ ID NO: 292, SEQ ID NO: 296, SEQ ID NO: 300, SEQ ID NO: 304, SEQ ID NO: 915, and SEQ ID NO: 919.

[0133] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) is a heavy chain variable region comprising amino acids having the sequence described in Sequence ID No. 915 provided below. VKLQESGGGLVQPGGSRKLSCAASGFTFSIFGLHWVRQAPEKGLEWVAYISGDSNTIYYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCARNSYYALDYWGQGTTVTVSS[Sequence ID 915]

[0134] The nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 915 is described in SEQ ID NO: 916, provided below: GTGAAGCTGCAGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGGTCCCGGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTATCTTTGGATTGCACTGGGTTCGTCAGGCTCCAGAGAAGGGGCTGGAGTGGGTCGCATACATTAGTGGTGACAGTAATACCATCTACTATG CAGACACAGTGAAGGGCCGATTCACCATCTCCAGAGACAATCCCAAGAACACCCTGTTCCTGCAAATGACCAGTCTAAGGTCTGAGGACACGGCCATGTATTACTGTGCAAGAAATAGCTACTATGCTCTGGACTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA [SEQ ID NO: 916]

[0135] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) includes a light chain variable region comprising amino acids having the sequence described in Sequence ID No. 917 provided below. DIELTQSPAIMSVSPGEKVTMTCRASSSVSSSYLHWYQQRSGASPKIWIYSTSNLASGVPARFSGSGTGTSYSLTISSVEAEDAATYYCQQYSGYPWTFGGGTKLEI[Sequence ID 917]

[0136] The nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 917 is described in SEQ ID NO: 918, provided below: GACATTGAGCTCACCCAGTCTCCAGCAATCATGTCTGTATCTCCAGGTGAAAAGGTCACCATGACCTGCAGGGCCAGCTCAAGTGTCAGTTCCAGTTACTTGCACTGGTACCAGCAGAGGTCAGGTGCCTCCCCCAAAATCTGGATTTATAGCACATCCAACTTG GCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGACTGGGACCTCTTACTCTCTCACAATCAGCAGTTGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGTACAGTGGTTACCCGTGGACGTTCGGTGGAGGGACCAAGCTGGAGATC [SEQ ID NO: 918]

[0137] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) includes a heavy chain variable region comprising amino acids having the sequence described in Sequence ID No. 919 provided below. VQLQESGGGLVQPGGSRKLSCTASGFTFSSFGMHWVRQAPEKGLEWVAYISSGSNNIYFADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCARSEYYGSSHMDYWGQGTTVTVSS[Sequence ID 919]

[0138] The nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 3 is described in SEQ ID NO: 920, provided below:

[0139] GTCCAACTGCAGGAGTCAGGGGAGGCTTAGTGCAGCCTGGAGGGTCCCGGAAACTCTCCTGTACAGCCTCTGGATTCACTTTCAGTAGCTTTGGAATGCACTGGGTTCGTCAGGCTCCAGAGAAGGGGCTGGAGTGGGTCGCATACATTAGTAGTGGCAGTAATAACATCTACTTTGCGGAC ACAGTGAAGGGCCGATTCACCATCTCCAGAGACAATCCCAAGAACACCCTGTTCCTGCAAATGACCAGTCTAAGGTCTGAGGACACGGCCATGTATTACTGTGCAAGATCGGAATACTACGGTAGTAGCCATATGGACTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA [SEQ ID NO: 920]

[0140] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) includes a light chain variable region comprising amino acids having the sequence described in Sequence ID No. 921 provided below. DIELTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKPLIYSATYRNSGVPDRFTGSGSGTDFTLTITNVQSKDLADYFCQQYNRYPYTSGGGTKLEI[Sequence ID 921]

[0141] The nucleic acid sequence encoding the amino acid sequence of Sequence ID No. 921 is described in Sequence ID No. 922, provided below:

[0142] GACATTGAGCTCACCCAGTCTCCAAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCGTCACCTGCAAGGCCAGTCAGAATGTGGGTACTAATGTAGCCTGGTATCAACAGAAACCAGGACAATCTCCTAACCACTGATTTACTCGGCAACCTAC CGGAACAGTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCACTAACGTGCAGTCTAAAGACTTGGCAGACTATTTCTGTCAACAATATAACAGGTATCGGTACACGTCCGGAGGGGGGACCAAGCTGGAGATC [SEQ ID NO. 922]

[0143] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) is a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 144, which includes a heavy chain variable region containing amino acids having the sequence described in SEQ ID NO: 144, as described in SEQ ID NO: 142.

[0144] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) includes a light chain variable region containing amino acids having the sequence described in SEQ ID NO: 143. The nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 143 is described in SEQ ID NO: 141.

[0145] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) includes a heavy chain variable region containing amino acids having the sequence described in SEQ ID NO: 216. The nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 216 is described in SEQ ID NO: 214.

[0146] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) includes a light chain variable region containing amino acids having the sequence described in SEQ ID NO: 215. The nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 215 is described in SEQ ID NO: 213.

[0147] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) includes a heavy chain variable region containing amino acids having the sequence described in SEQ ID NO: 220. The nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 220 is described in SEQ ID NO: 218.

[0148] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) includes a light chain variable region containing amino acids having the sequence described in SEQ ID NO: 219. The nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 219 is described in SEQ ID NO: 217.

[0149] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) includes a heavy chain variable region containing amino acids having the sequence described in SEQ ID NO: 236. The nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 236 is described in SEQ ID NO: 234.

[0150] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) includes a light chain variable region containing amino acids having the sequence described in SEQ ID NO: 235. The nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 235 is described in SEQ ID NO: 232.

[0151] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) includes a heavy chain variable region containing amino acids having the sequence described in SEQ ID NO: 268. The nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 268 is described in SEQ ID NO: 266.

[0152] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) includes a light chain variable region containing amino acids having the sequence described in SEQ ID NO: 267. The nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 267 is described in SEQ ID NO: 265.

[0153] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) includes a heavy chain variable region containing amino acids having the sequence described in SEQ ID NO: 172. The nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 172 is described in SEQ ID NO: 170.

[0154] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) includes a light chain variable region containing amino acids having the sequence described in SEQ ID NO: 171. The nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 171 is described in SEQ ID NO: 169.

[0155] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) includes a heavy chain variable region containing amino acids having the sequence described in SEQ ID NO: 116. The nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 116 is described in SEQ ID NO: 114.

[0156] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) includes a light chain variable region containing amino acids having the sequence described in SEQ ID NO: 115. The nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 115 is described in SEQ ID NO: 113.

[0157] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence Number 3, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 55, SEQ ID NO: 59, SEQ ID NO: 63, SEQ ID NO: 67, SEQ ID NO: 71, SEQ ID NO: 75, SEQ ID NO: 79, SEQ ID NO: 83, SEQ ID NO: 87, SEQ ID NO: 91, SEQ ID NO: 95, SEQ ID NO: 99, SEQ ID NO: 103, SEQ ID NO: 107, SEQ ID NO: 111, SEQ ID NO: 115, SEQ ID NO: 119, SEQ ID NO: 123, SEQ ID NO: 127, SEQ ID NO: 131, SEQ ID NO: 135, SEQ ID NO: 139, SEQ ID NO: 143, SEQ ID NO: 147, SEQ ID NO: 151, SEQ ID NO: 155, SEQ ID NO: 159, SEQ ID NO: 163, SEQ ID NO: 167, SEQ ID NO: 171, SEQ ID NO: 1 (b) a light chain variable region containing an amino acid sequence selected from the group consisting of 75, SEQ ID NO: 179, SEQ ID NO: 183, SEQ ID NO: 187, SEQ ID NO: 191, SEQ ID NO: 195, SEQ ID NO: 199, SEQ ID NO: 203, SEQ ID NO: 207, SEQ ID NO: 211, SEQ ID NO: 215, SEQ ID NO: 219, SEQ ID NO: 223, SEQ ID NO: 227, SEQ ID NO: 231, SEQ ID NO: 235, SEQ ID NO: 239, SEQ ID NO: 243, SEQ ID NO: 247, SEQ ID NO: 251, SEQ ID NO: 255, SEQ ID NO: 259, SEQ ID NO: 263, SEQ ID NO: 267, SEQ ID NO: 271, SEQ ID NO: 275, SEQ ID NO: 279, SEQ ID NO: 283, SEQ ID NO: 287, SEQ ID NO: 291, SEQ ID NO: 295, SEQ ID NO: 299, SEQ ID NO: 303, SEQ ID NO: 917, SEQ ID NO: 921, and (b) SEQ ID NO: 4, SEQ ID NO: 8, sequence Number 12, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO: 56, SEQ ID NO: 60, SEQ ID NO: 64, SEQ ID NO: 68, SEQ ID NO: 72, SEQ ID NO: 76, SEQ ID NO: 80, SEQ ID NO: 84, SEQ ID NO: 88, SEQ ID NO: 92, SEQ ID NO: 96, SEQ ID NO: 100, SEQ ID NO: 104, SEQ ID NO: 108, SEQ ID NO: 112, SEQ ID NO: 116, SEQ ID NO: 120, SEQ ID NO: 124, SEQ ID NO: 128, SEQ ID NO: 132, SEQ ID NO: 136, SEQ ID NO: 140, SEQ ID NO: 144, SEQ ID NO: 148, SEQ ID NO: 152, SEQ ID NO: 156, SEQ ID NO: 160, SEQ ID NO: 164, SEQ ID NO: 168, SEQ ID NO: 172, SEQ ID NO: 176, SEQ ID NO: 180, SEQ ID NO: 184, SEQ ID NO: 188, SEQ ID NO: 192, SEQ ID NO: 196, SEQ ID NO: 200, SEQ ID NO: 204 The extracellular binding domain includes a heavy chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 208, 212, 216, 220, 224, 228, 232, 236, 240, 244, 248, 252, 256, 260, 264, 268, 272, 276, 280, 284, 288, 292, 296, 300, 304, 917, and 921, and this extracellular binding domain binds to the FcRL5 polypeptide.

[0158] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) includes (a) a light chain variable region comprising an amino acid having the sequence described in SEQ ID NO: 3, and (b) a heavy chain variable region comprising an amino acid having the sequence described in SEQ ID NO: 4.

[0159] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b) The light chain variable region containing the amino acid sequence having the sequence described in number 7 and the sequence described in (b) Sequence ID 8 It includes a heavy chain variable region containing an amino acid sequence having the sequence shown.

[0160] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b) The light chain variable region includes an amino acid sequence having the sequence described in (b) Sequence ID No. 12.

[0161] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b) Light chain variable region containing an amino acid sequence having the sequence described in (b) Sequence ID No. 16 It includes a heavy chain variable region containing an amino acid sequence having the sequence described above.

[0162] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b) Light chain variable region containing an amino acid sequence having the sequence described in number 19 and (b) Sequence ID 20 It includes a heavy chain variable region containing an amino acid sequence having the sequence described above.

[0163] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b) Light chain variable region containing an amino acid sequence having the sequence described in (b) Sequence ID No. 24 It includes a heavy chain variable region containing an amino acid sequence having the sequence described above.

[0164] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence Light chain variable region containing an amino acid sequence having the sequence described in (b) number 27: and heavy chain variable region containing an amino acid sequence having the sequence described in (b) sequence number 28.

[0165] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b) Light chain variable region containing an amino acid sequence having the sequence described in (b) Sequence ID No. 32 It includes a heavy chain variable region containing an amino acid sequence having the sequence described above.

[0166] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b) Light chain variable region containing an amino acid sequence having the sequence described in (b) Sequence ID No. 36 It includes a heavy chain variable region containing an amino acid sequence having the sequence described above.

[0167] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b) Light chain variable region containing the amino acid sequence having the sequence described in number 39 and (b) Sequence ID 40 It includes a heavy chain variable region containing an amino acid sequence having the sequence described above.

[0168] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b)Sequence ID 44 It includes a heavy chain variable region containing an amino acid sequence having the sequence described above.

[0169] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b) Light chain variable region containing an amino acid sequence having the sequence described in number 47 and (b) Sequence ID 48 It includes a heavy chain variable region containing an amino acid sequence having the sequence described above.

[0170] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b) Light chain variable region containing an amino acid sequence having the sequence described in number 51 and (b) Sequence ID 52 It includes a heavy chain variable region containing an amino acid sequence having the sequence described above.

[0171] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b)Sequence ID 56 It includes a heavy chain variable region containing an amino acid sequence having the sequence described above.

[0172] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b) Light chain variable region containing an amino acid sequence having the sequence described in number 59 and (b) Sequence ID 60 It includes a heavy chain variable region containing an amino acid sequence having the sequence described above.

[0173] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b)Sequence ID 64 It includes a heavy chain variable region containing an amino acid sequence having the sequence described above.

[0174] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b)Sequence ID 68 It includes a heavy chain variable region containing an amino acid sequence having the sequence described above.

[0175] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b) Light chain variable region containing an amino acid sequence having the sequence described in number 71 and (b) Sequence ID 72 It includes a heavy chain variable region containing an amino acid sequence having the sequence described above.

[0176] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b) Light chain variable region containing an amino acid sequence having the sequence described in number 75 and (b) Sequence ID 76 It includes a heavy chain variable region containing an amino acid sequence having the sequence described above.

[0177] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b) Light chain variable region containing an amino acid sequence having the sequence described in number 79 and (b) Sequence ID 80 It includes a heavy chain variable region containing an amino acid sequence having the sequence described above.

[0178] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b)Sequence ID 84 It includes a heavy chain variable region containing an amino acid sequence having the sequence described above.

[0179] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b)Sequence ID 88 It includes a heavy chain variable region containing an amino acid sequence having the sequence described above.

[0180] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b) Light chain variable region containing an amino acid sequence having the sequence described in number 91 and (b) Sequence ID 92 It includes a heavy chain variable region containing an amino acid sequence having the sequence described above.

[0181] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b) Light chain variable region containing an amino acid sequence having the sequence described in number 95 and (b) Sequence ID 96 It includes a heavy chain variable region containing an amino acid sequence having the sequence described above.

[0182] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b) Light chain variable region containing an amino acid sequence having the sequence described in number 99 and (b) Sequence ID 10 It includes a heavy chain variable region containing an amino acid sequence having the array described in 0.

[0183] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) an array a light chain variable region containing an amino acid sequence having the sequence described in SEQ ID NO: 103 and (b) a heavy chain variable region containing an amino acid sequence having the sequence described in SEQ ID NO: 1 04.

[0184] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) an array a light chain variable region containing an amino acid sequence having the sequence described in SEQ ID NO: 107 and (b) a heavy chain variable region containing an amino acid sequence having the sequence described in SEQ ID NO: - 1 08.

[0185] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) an array a light chain variable region containing an amino acid sequence having the sequence described in SEQ ID NO: 111 and (b) a heavy chain variable region containing an amino acid sequence having the sequence described in SEQ ID NO: 112.

[0186] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) an array a light chain variable region containing an amino acid sequence having the sequence described in SEQ ID NO: 115 and (b) a heavy chain variable region containing an amino acid sequence having the sequence described in SEQ ID NO: 1 16.

[0187] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) an array a light chain variable region containing an amino acid sequence having the sequence described in SEQ ID NO: 119 and (b)' a heavy chain variable region containing an amino acid sequence having the sequence described in SEQ ID NO: 1 20.

[0188] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) an array (b)Sequence ID 1 It includes a heavy chain variable region containing an amino acid sequence having the sequence described in 24.

[0189] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b) Light chain variable region containing an amino acid sequence having the sequence described in number 127 and (b) Sequence ID 1 It includes a heavy chain variable region containing an amino acid sequence having the sequence described in 28.

[0190] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b)Sequence ID 1 It includes a heavy chain variable region containing an amino acid sequence having the sequence described in 32.

[0191] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b)Sequence ID 1 It includes a heavy chain variable region containing an amino acid sequence having the sequence described in 36.

[0192] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b)Sequence ID 1 It includes a heavy chain variable region containing an amino acid sequence having the sequence described in 40.

[0193] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b)Sequence ID 1 It includes a heavy chain variable region containing an amino acid sequence having the sequence described in 44.

[0194] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) comprises (a) a light chain variable region comprising an amino acid sequence having the sequence set forth in SEQ ID NO: 147 and (b) a heavy chain variable region comprising an amino acid sequence having the sequence set forth in SEQ ID NO: 148.

[0195] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) comprises (a) a light chain variable region comprising an amino acid sequence having the sequence set forth in SEQ ID NO: 151 and (b) a heavy chain variable region comprising an amino acid sequence having the sequence set forth in SEQ ID NO: 152.

[0196] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) comprises (a) a light chain variable region comprising an amino acid sequence having the sequence set forth in SEQ ID NO: 155 and (b) a heavy chain variable region comprising an amino acid sequence having the sequence set forth in SEQ ID NO: 156. <000096I>

[0197] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) comprises (a) a light chain variable region comprising an amino acid sequence having the sequence set forth in SEQ ID NO: 159 and (b) a heavy chain variable region comprising an amino acid sequence having the sequence set forth in SEQ ID NO: 160.

[0198] [[ID=e2]] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) comprises (a) a light chain variable region comprising an amino acid sequence having the sequence set forth in SEQ ID NO: 163 and (b) a heavy chain variable region comprising an amino acid sequence having the sequence set forth in SEQ ID NO: 164.

[0199] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) comprises (a) a light chain variable region comprising an amino acid sequence having the sequence set forth in SEQ ID NO: 167 and (b) a heavy chain variable region comprising an amino acid sequence having the sequence set forth in SEQ ID NO: 168. <00009TS>It includes a heavy chain variable region containing an amino acid sequence having the sequence described in 68.

[0200] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b)Sequence ID 1 It includes a heavy chain variable region containing an amino acid sequence having the sequence described in 72.

[0201] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b)Sequence ID 1 It includes a heavy chain variable region containing an amino acid sequence having the sequence described in 76.

[0202] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b)Sequence ID 1 It includes a heavy chain variable region containing an amino acid sequence having the sequence described in 80.

[0203] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b)Sequence ID 1 It includes a heavy chain variable region containing an amino acid sequence having the sequence described in 84.

[0204] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b)Sequence ID 1 It includes a heavy chain variable region containing an amino acid sequence having the sequence described in 88.

[0205] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b)Sequence ID 1 It includes a heavy chain variable region containing an amino acid sequence having the sequence described in 92.

[0206] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b)Sequence ID 1 It includes a heavy chain variable region containing an amino acid sequence having the sequence described in 96.

[0207] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b)Sequence ID 2 It includes a heavy chain variable region containing an amino acid sequence having the sequence described in 00.

[0208] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b)Sequence ID 2 It includes a heavy chain variable region containing an amino acid sequence having the sequence described in 04.

[0209] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b)Sequence ID 2 It includes a heavy chain variable region containing an amino acid sequence having the sequence described in 08.

[0210] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b) A light chain variable region containing an amino acid sequence having the sequence described in (b) SEQ ID NO: 212 and a heavy chain variable region containing an amino acid sequence having the sequence described in SEQ ID NO: 212.

[0211] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b)Sequence ID 2 It includes a heavy chain variable region containing an amino acid sequence having the sequence described in 16.

[0212] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b)Sequence ID 2 It includes a heavy chain variable region containing an amino acid sequence having the sequence described in 20.

[0213] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b)Sequence ID 2 It includes a heavy chain variable region containing an amino acid sequence having the sequence described in 24.

[0214] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b)Sequence ID 2 It includes a heavy chain variable region containing an amino acid sequence having the sequence described in 28.

[0215] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b)Sequence ID 2 It includes a heavy chain variable region containing an amino acid sequence having the sequence described in 32.

[0216] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b)Sequence ID 2 It includes a heavy chain variable region containing an amino acid sequence having the sequence described in 36.

[0217] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b)Sequence ID 2 It includes a heavy chain variable region containing an amino acid sequence having the sequence described in 40.

[0218] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b)Sequence ID 2 It includes a heavy chain variable region containing an amino acid sequence having the sequence described in 44.

[0219] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b)Sequence ID 2 It includes a heavy chain variable region containing an amino acid sequence having the sequence described in 48.

[0220] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b)Sequence ID 2 It includes a heavy chain variable region containing an amino acid sequence having the sequence described in 52.

[0221] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b)Sequence ID 2 It includes a heavy chain variable region containing an amino acid sequence having the sequence described in 56.

[0222] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b)Sequence ID 2 It includes a heavy chain variable region containing an amino acid sequence having the sequence described in 60.

[0223] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b)Sequence ID 2 It includes a heavy chain variable region containing an amino acid sequence having the sequence described in 64.

[0224] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b)Sequence ID 2 It includes a heavy chain variable region containing an amino acid sequence having the sequence described in 68.

[0225] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b)Sequence ID 2 It includes a heavy chain variable region containing an amino acid sequence having the sequence described in 72.

[0226] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b)Sequence ID 2 It includes a heavy chain variable region containing an amino acid sequence having the sequence described in 76.

[0227] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b)Sequence ID 2 It includes a heavy chain variable region containing an amino acid sequence having the sequence described in 80.

[0228] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b)Sequence ID 2 It includes a heavy chain variable region containing an amino acid sequence having the sequence described in 84.

[0229] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b)Sequence ID 2 It includes a heavy chain variable region containing an amino acid sequence having the sequence described in 88.

[0230] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b)Sequence ID 2 It includes a heavy chain variable region containing an amino acid sequence having the sequence described in 92.

[0231] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b)Sequence ID 2 It includes a heavy chain variable region containing an amino acid sequence having the sequence described in 80.

[0232] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b)Sequence ID 2 It includes a heavy chain variable region containing an amino acid sequence having the sequence described in 84.

[0233] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b)Sequence ID 2 It includes a heavy chain variable region containing an amino acid sequence having the sequence described in 88.

[0234] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b)Sequence ID 2 It includes a heavy chain variable region containing an amino acid sequence having the sequence described in 92.

[0235] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b) A light chain variable region containing an amino acid sequence having the sequence described in (b) SEQ ID NO: 296 and a heavy chain variable region containing an amino acid sequence having the sequence described in (b) SEQ ID NO: 296.

[0236] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b)Sequence ID 3 It includes a heavy chain variable region containing an amino acid sequence having the sequence described in 00.

[0237] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b)Sequence ID 3 It includes a heavy chain variable region containing an amino acid sequence having the sequence described in 04.

[0238] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) includes (a) a heavy chain variable region having the sequence described in SEQ ID NO: 915 and (b) a light chain variable region having the sequence described in SEQ ID NO: 917.

[0239] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) includes (a) a heavy chain variable region having the sequence described in SEQ ID NO: 919 and (b) a light chain variable region having the sequence described in SEQ ID NO: 921.

[0240] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) includes heavy and light chain variable regions containing amino acid sequences homologous to the amino acid sequences described herein and disclosed in Tables 1-76. For example, but not limited to, this extracellular antigen-binding domain (e.g., scFv) is: SEQ ID NOs: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, 87, 91, 95, 99, 103, 107, 111, 115, 119, 123, 127, 131, 135, 139, 143, 147, 151, 155, 159, 163, 167, 171, 175, 179, 183, 187, 191, 195 The light chain variable region includes an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs: 199, 203, 207, 211, 215, 219, 223, 227, 231, 235, 239, 243, 247, 251, 255, 259, 263, 267, 271, 275, 279, 283, 287, 291, 295, 299, 303, 917, and 921.

[0241] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is used in SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, 172, 176, 180, 184, Sequence ID 188, Sequence ID 192, Sequence ID 196, Sequence ID 200, Sequence ID 204, Sequence ID 208, Sequence ID 212, Sequence ID 216, Sequence ID 220, Sequence ID 224, Sequence ID 228, Sequence ID 232, Sequence ID 236, Sequence ID 240, Sequence ID 244, Sequence ID 248, Sequence ID 252, The heavy chain variable region includes an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs. 256, 260, 264, 268, 272, 276, 280, 284, 288, 292, 296, 300, 304, 915, and 919.

[0242] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) distributed Column number 3, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 55, SEQ ID NO: 59, SEQ ID NO: 63, SEQ ID NO: 67, SEQ ID NO: 71, SEQ ID NO: 75, SEQ ID NO: 79, SEQ ID NO: 83, SEQ ID NO: 87, SEQ ID NO: 91, SEQ ID NO: 95, SEQ ID NO: 99, SEQ ID NO: 103, SEQ ID NO: 107, SEQ ID NO: 111, SEQ ID NO: 115, SEQ ID NO: 119, SEQ ID NO: 123, SEQ ID NO: 127, SEQ ID NO: 131, SEQ ID NO: 135, SEQ ID NO: 139, SEQ ID NO: 143, SEQ ID NO: 147, SEQ ID NO: 151, SEQ ID NO: 155, SEQ ID NO: 159, SEQ ID NO: 163, SEQ ID NO: 167, SEQ ID NO: 171, SEQ ID NO: 175, SEQ ID NO: 179, SEQ ID NO: 183, SEQ ID NO: 187, SEQ ID NO: 191, SEQ ID NO: 195, SEQ ID NO: 199, SEQ ID NO: 203, SEQ ID NOs: 207, 211, 215, 219, 223, 227, 231, 235, 239, 243, 247, 251, 255, 259, 263, 267, 271, 275, 279, 283, 287, 291, 295, 29 9. A light chain variable region containing an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs. 90, SEQ ID NOs. 917, SEQ ID NOs. 921, and (b) SEQ ID NOs. 90, 0, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO: 56, SEQ ID NO: 60, SEQ ID NO: 64, SEQ ID NO: 68, SEQ ID NO: 72, SEQ ID NO: 76, SEQ ID NO: 80, SEQ ID NO: 84, SEQ ID NO: 88, SEQ ID NO: 92, SEQ ID NO: 96, SEQ ID NO: 100, SEQ ID NO: 104, SEQ ID NO: 108, SEQ ID NO: 112, SEQ ID NO: 116, SEQ ID NO: 120, SEQ ID NO: 124, SEQ ID NO: 128, SEQ ID NO: 132, SEQ ID NO: 136, SEQ ID NO: 140, SEQ ID NO: 144, SEQ ID NO: 148, SEQ ID NO: 152, SEQ ID NO: 156, SEQ ID NO: 160, SEQ ID NO: 164, SEQ ID NO: 168, SEQ ID NO: 172, SEQ ID NO: 176, SEQ ID NO: 180, SEQ ID NO: 184, Sequence ID 188, Sequence ID 192, Sequence ID 196, Sequence ID 200, Sequence ID 204, Sequence ID 208, Sequence ID 212, Sequence ID 216, Sequence ID 220, Sequence ID 224, Sequence ID 228, Sequence ID 232, Sequence ID 236, Sequence ID 240, Sequence ID 244, Sequence ID 248, Sequence ID 252, The heavy chain variable region includes an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs. 256, 260, 264, 268, 272, 276, 280, 284, 288, 292, 296, 300, 304, 915, and 919.

[0243] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises (a) a light chain variable region containing an amino acid sequence homologous to at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence described in SEQ ID NO: 143, and (b) a heavy chain variable region containing an amino acid sequence homologous to at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence described in SEQ ID NO: 144, wherein the extracellular antigen-binding domain binds to the FcRL5 polypeptide.

[0244] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) distributed (b) Light chain variable regions containing amino acid sequences homologous to at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence described in sequence number 215, and (b) light chain variable regions containing amino acid sequences homologous to at least 80%, It contains a heavy chain variable region with 1%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous amino acid sequences, and this extracellular antigen-binding domain binds to the FcRL5 polypeptide.

[0245] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) distributed (b) The extracellular antigen-binding domain comprises a light chain variable region containing an amino acid sequence homologous to at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence described in (b) Sequence ID No. 220, and a heavy chain variable region containing an amino acid sequence homologous to at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence described in Sequence ID No. 220, wherein the extracellular antigen-binding domain binds to the FcRL5 polypeptide.

[0246] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises (a) a light chain variable region containing an amino acid sequence homologous to at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence described in SEQ ID NO: 235, and (b) at least 80% homologous to the amino acid sequence described in SEQ ID NO: 236. It contains a heavy chain variable region with 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous amino acid sequences, and this extracellular antigen-binding domain binds to the FcRL5 polypeptide.

[0247] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) distributed (b) Light chain variable regions containing amino acid sequences homologous to at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence described in sequence number 268, and (b) light chain variable regions containing amino acid sequences homologous to at least 80%, 8 It contains a heavy chain variable region with 1%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous amino acid sequences, and this extracellular antigen-binding domain binds to the FcRL5 polypeptide.

[0248] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) distributed (b) Heavy chain variable regions containing amino acid sequences homologous to at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence described in sequence number 915, and (b) at least 80%, 8 It contains a light chain variable region with 1%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous amino acid sequences, and this extracellular antigen-binding domain binds to the FcRL5 polypeptide.

[0249] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) distributed (b) A heavy chain variable region containing an amino acid sequence homologous to at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence described in sequence number 919, and (b) at least 80%, 8 It contains a light chain variable region with 1%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous amino acid sequences, and this extracellular antigen-binding domain binds to the FcRL5 polypeptide.

[0250] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) is a light chain variable comprising (a) an amino acid sequence homologous to at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence homologous to (b) an amino acid sequence homologous to at least 80%, 81%, of the amino acid sequence homologous to (a) an amino acid sequence homologous to (b) an amino acid sequence homologous to (a It contains a heavy chain variable region with 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous amino acid sequences, and this extracellular antigen-binding domain binds to the FcRL5 polypeptide.

[0251] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) is a light chain variable comprising (a) an amino acid sequence homologous to at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence homologous to (b) an amino acid sequence homologous to at least 80%, 81%, of the amino acid sequence homologous to (a) the amino acid sequence homologous to (b) the amino acid sequence homologous to (a It contains a heavy chain variable region with 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous amino acid sequences, and this extracellular antigen-binding domain binds to the FcRL5 polypeptide.

[0252] V of the above array H Region and V L V has high homology (i.e., 80% or more) to the region. H Region and / or V LAn extracellular antigen-binding domain (e.g., scFv) containing a region can be obtained by mutagenesis (e.g., site-directed or PCR-mediated mutagenesis) followed by testing of the encoded modified scFv for retained function (i.e., binding affinity) using the binding assays described herein. In certain embodiments, a V having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity may be obtained. L The sequence contains substitutions (e.g., conservative substitutions that produce a conservative modification of the sequence), insertions, or deletions compared to the reference sequence, but the extracellular antigen-binding domain (e.g., scFv) containing the sequence retains the ability to bind to FcRL5. In certain embodiments, V has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. H The sequence contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the extracellular antigen-binding domain (e.g., scFv) containing the sequence retains its ability to bind to FcRL5. In certain embodiments, a total of about 1 to about 10 amino acids are substituted, inserted, and / or deleted in the disclosed sequence. For example, V, not as an limitation. H Array or V L The sequence may have up to approximately 1, up to approximately 2, up to approximately 3, up to approximately 4, up to approximately 5, up to approximately 6, up to approximately 7, up to approximately 8, up to approximately 9, or up to approximately 10 modified and / or substituted amino acid residues. Non-limiting examples of conservative modifications are provided below, for example, in Table 231.

[0253] The subject matter of this disclosure further provides extracellular antigen-binding domains (e.g., scFv) including heavy chain variable regions and light chain variable regions CDRs, e.g., CDR1, CDR2, and CDR3, as disclosed herein in Tables 229 and 230. These CDR regions are described using the Kabat system (Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, 5th edition, US Department of Health and Human Services, NIH Publication No. 91-3242). This disclosure further provides extracellular antigen-binding domains (e.g., scFv) including conserved modifications of antibody sequences disclosed herein. For example, but not as an limitation, the extracellular antigen-binding domain (e.g., scFv) of the subject matter of this disclosure comprises a heavy chain variable region containing CDR1, CDR2, and CDR3 sequences and a light chain variable region containing CDR1, CDR2, and CDR3 sequences, one or more of these CDR sequences comprising the identified amino acid sequences disclosed herein or their conserved modifications, and these extracellular antigen-binding domains retain the desired functional properties. See Tables 229 and 230.

[0254] In certain embodiments, the subject of this disclosure provides an extracellular antigen-binding domain (e.g., scFv) comprising a light chain variable region, which includes (a) SEQ ID NOs: 312, 3118, 324, 329, 338, 343, 348, 352, 357, 363, 369, 381, 390, 397, 401, 406, 416, 423, 428, 433, 447, 460, 468, 474, 477, 483, 490, 498, 503, 508, 518, 533, 5 CDR1 containing amino acid sequences selected from the group consisting of 40, 544, 547, 556, 562, 568, 571, 580, 585, 588, 926 and 932, and their conservative modifications; (b) SEQ ID NOs: 313, 319, 330, 344, 349, 358, 364, 370, 382, ​​385, 391, 398, 409, 417, 429, 434, 438, 448, 454, 461, 469, 478, 484, 487, 504, 513, 52 CDR2 containing amino acid sequences selected from the group consisting of 3, 534, 429, 448, 548, 557, 563, 572, 575, 586, 927 and 933, and their conservative modifications; and (c) SEQ ID NOs: 314, 320, 325, 331, 339, 345, 350, 353, 359, 365, 371, 377, 383, 386, 392, 395, 399, 402, 407, 410, 414, 418, 419, 424, 430, 435 The CDR3 includes an amino acid sequence selected from the group consisting of 439, 443, 449, 452, 455, 457, 462, 465, 470, 479, 485, 488, 491, 493, 495, 499, 505, 509, 514, 519, 524, 528, 530, 531, 535, 541, 542, 545, 549, 554, 558, 564, 569, 573, 576, 581, 592, 928, and 934, as well as their conservative modifications.

[0255] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) is a heavy chain variable region, the heavy chain variable region comprising (a) CDR1 amino acid sequences selected from the group consisting of SEQ ID NOs: 309, 315, 321, 326, 332, 335, 340, 346, 354, 360, 366, 372, 378, 387, 393, 403, 411, 420, 425, 436, 440, 444, 471, 480, 500, 510, 515, 520, 525, 537, 551, 559, 565, 582, 589, 923 and 929 and their conserved modifications; (b) SEQ ID NO: 310 Select from the group consisting of 316, 322, 327, 333, 336, 341, 355, 361, 367, 373, 379, 388, 404, 412, 421, 426, 431, 441, 445, 450, 466, 472, 475, 481, 496, 501, 506, 511, 516, 521, 526, 538, 552, 560, 566, 583, 590, 924 and 930 and their conservative modifications. CDR2 containing the amino acid sequence; and (c) SEQ ID NOs: 311, 317, 323, 328, 334, 337, 342, 347, 351, 356, 362, 368, 374, 376, 380, 384, 389, 394, 396, 400, 405, 408, 412, 415, 422, 427, 432, 437, 442, 446, 451, 453, 456, 458, 459, 463, 464, 467, 47 The CDR3 comprises an amino acid sequence selected from the group consisting of 3, 476, 482, 486, 489, 492, 494, 497, 502, 507, 512, 517, 522, 527, 529, 532, 536, 539, 543, 546, 550, 553, 555, 561, 567, 570, 574, 577, 578, 579, 584, 578, 587, 591, 925, and 931, and their conservative modifications.

[0256] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) distributed (b) Sequence of the heavy chain variable region CDR1 containing the amino acid sequence of column number 411 or a conservative modification thereof; (c) a heavy chain variable region CDR2 or a conserved modification thereof containing the amino acid sequence of number 412; and (c) a heavy chain variable region CDR3 or a conserved modification thereof containing the amino acid sequence of sequence number 463. In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b) Light chain variable region CDR1 containing the amino acid sequence of number 318 or a conservative modification thereof; (b) Light chain variable region CDR2 containing the amino acid sequence of sequence number 319 or a conservative modification thereof; and (c) Light chain variable region CDR3 containing the amino acid sequence of sequence number 419 or a conservative modification thereof.

[0257] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) distributed (b) Sequence of the heavy chain variable region CDR1 containing the amino acid sequence of column number 515 or a conservative modification thereof; (c) a heavy chain variable region CDR2 or a conserved modification thereof containing the amino acid sequence of number 516; and (c) a heavy chain variable region CDR3 or a conserved modification thereof containing the amino acid sequence of sequence number 517. In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b) Light chain variable region CDR1 containing the amino acid sequence of number 318 or a conservative modification thereof; (b) Light chain variable region CDR2 containing the amino acid sequence of sequence number 319 or a conservative modification thereof; and (c) Light chain variable region CDR3 containing the amino acid sequence of sequence number 531 or a conservative modification thereof.

[0258] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) distributed (b) Sequence of the heavy chain variable region CDR1 containing the amino acid sequence of column number 403 or a conservative modification thereof; (c) a heavy chain variable region CDR2 or a conserved modification thereof containing the amino acid sequence of number 404; and (c) a heavy chain variable region CDR3 or a conserved modification thereof containing the amino acid sequence of sequence number 532. In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b) Light chain variable region CDR1 containing the amino acid sequence of number 533 or a conservative modification thereof; (b) Light chain variable region CDR2 containing the amino acid sequence of sequence number 534 or a conservative modification thereof; and (c) Light chain variable region CDR3 containing the amino acid sequence of sequence number 535 or a conservative modification thereof.

[0259] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) distributed (b) Sequence of the heavy chain variable region CDR1 containing the amino acid sequence of column number 411 or a conservative modification thereof; (c) a heavy chain variable region CDR2 or a conserved modification thereof containing the amino acid sequence of number 412; and (c) a heavy chain variable region CDR3 or a conserved modification thereof containing the amino acid sequence of sequence number 543. In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b) Light chain variable region CDR1 containing the amino acid sequence of number 544 or a conservative modification thereof; (b) Light chain variable region CDR2 containing the amino acid sequence of sequence number 448 or a conservative modification thereof; and (c) Light chain variable region CDR3 containing the amino acid sequence of sequence number 545 or a conservative modification thereof.

[0260] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) a heavy chain variable region CDR1 or a conserved modification thereof, comprising the amino acid sequence of SEQ ID NO: 372; (b) sequence (c) Heavy chain variable region CDR2 or a conserved modification thereof containing the amino acid sequence of number 475; and (c) Heavy chain variable region CDR3 or a conserved modification thereof containing the amino acid sequence of SEQ ID NO: 570. In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b) Light chain variable region CDR1 containing the amino acid sequence of number 571 or a conservative modification thereof; (b) Light chain variable region CDR2 containing the amino acid sequence of sequence number 572 or a conservative modification thereof; and (c) Light chain variable region CDR3 containing the amino acid sequence of sequence number 573 or a conservative modification thereof.

[0261] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b) Sequence number 440, heavy chain variable region CDR1 or its conservative modification; (a) a heavy chain variable region CDR2 or a conserved modification thereof containing the amino acid sequence of sequence number 441; and (c) a heavy chain variable region CDR3 or a conserved modification thereof containing the amino acid sequence of sequence number 442. In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence number (b) Light chain variable region CDR1 containing the amino acid sequence of SEQ ID NO. 329 or a conservative modification thereof; (b) Light chain variable region CDR2 containing the amino acid sequence of SEQ ID NO. 330 or a conservative modification thereof; and (c) Light chain variable region CDR3 containing the amino acid sequence of SEQ ID NO. 443 or a conservative modification thereof.

[0262] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b) Sequence number 309, heavy chain variable region CDR1 or a conservative modification thereof; (a) a heavy chain variable region CDR2 or a conserved modification thereof containing the amino acid sequence of sequence number 310; and (c) a heavy chain variable region CDR3 or a conserved modification thereof containing the amino acid sequence of sequence number 489. In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence number (b) Light chain variable region CDR1 containing the amino acid sequence of SEQ ID NO. 490 or a conservative modification thereof; (b) Light chain variable region CDR2 containing the amino acid sequence of SEQ ID NO. 313 or a conservative modification thereof; and (c) Light chain variable region CDR3 containing the amino acid sequence of SEQ ID NO. 491 or a conservative modification thereof.

[0263] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b) Heavy chain variable region CDR1 or a conserved modification thereof, comprising an amino acid sequence having the sequence described in SEQ ID NO. 923; (b) Heavy chain variable region CDR2 or a conserved modification thereof, comprising an amino acid sequence having the sequence described in SEQ ID NO. 924; and (c) Heavy chain variable region CDR3 or a conserved modification thereof, comprising an amino acid sequence having the sequence described in SEQ ID NO. 925. In certain embodiments, this extracellular antigen-binding domain comprises (a) an amino acid sequence having the sequence described in SEQ ID NO. 926. (b) Light chain variable region CDR1 or a conservative modification thereof; (b) Light chain variable region CDR2 or a conservative modification thereof comprising an amino acid sequence having the sequence described in SEQ ID NO: 927; and (c) Light chain variable region CDR3 or a conservative modification thereof comprising an amino acid sequence having the sequence described in SEQ ID NO: 928.

[0264] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b) Heavy chain variable region CDR1 or a conserved modification thereof, comprising an amino acid sequence having the sequence described in number 929; (b) Heavy chain variable region CDR2 or a conserved modification thereof, comprising an amino acid sequence having the sequence described in SEQ ID NO: 930; and (c) Heavy chain variable region CDR3 or a conserved modification thereof, comprising an amino acid sequence having the sequence described in SEQ ID NO: 931. In certain embodiments, this extracellular antigen-binding domain comprises (a) an amino acid sequence having the sequence described in SEQ ID NO: 932 (b) Light chain variable region CDR1 or a conservative modification thereof; (b) Light chain variable region CDR2 or a conservative modification thereof comprising an amino acid sequence having the sequence described in SEQ ID NO: 933; and (c) Light chain variable region CDR3 or a conservative modification thereof comprising an amino acid sequence having the sequence described in SEQ ID NO: 934.

[0265] The subject matter of this disclosure provides an extracellular antigen-binding domain (e.g., scFv) comprising a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences and a light chain variable region comprising CDR1, CDR2, and CDR3 sequences, (a) the heavy chain variable region CDR3 is sequence numbers 311, 317, 323, 328, 334, 337, 342, 347, 351, 356, 362, 368, 374, 376, 380, 384, 389, 394, 396, 400, 405, 408, 412, 415, 422, 427, A selected from the group consisting of 432, 437, 442, 446, 451, 453, 456, 458, 459, 463, 464, 467, 473, 476, 482, 486, 489, 492, 494, 497, 502, 507, 512, 517, 522, 527, 529, 532, 536, 539, 543, 546, 550, 553, 555, 561, 567, 570, 574, 577, 578, 579, 584, 578, 587, 591, 925 and 931, and their conservative modifications. (b) Light chain variable region CDR3 contains a mino acid sequence, and the sequence numbers are 314, 320, 325, 331, 339, 345, 350, 353, 359, 365, 371, 377, 383, 386, 392, 395, 399, 402, 407, 410, 414, 418, 419, 424, 430, 435, 439, 443, 449, 452, 455, 457, 4 The extracellular antigen-binding domain comprises an amino acid sequence selected from the group consisting of 62, 465, 470, 479, 485, 488, 491, 493, 495, 499, 505, 509, 514, 519, 524, 528, 530, 531, 535, 541, 542, 545, 549, 554, 558, 564, 569, 573, 576, 581, 592, 928, and 934 and their conserved modifications, and this extracellular antigen-binding domain specifically binds to human FcRL5.

[0266] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b) comprising a heavy chain variable region CDR3 or a conserved modification thereof containing the amino acid sequence of number 463; and (b) a light chain variable region CDR3 or a conserved modification thereof containing the amino acid sequence of sequence number 419; this extracellular binding domain specifically binds to FcRL5.

[0267] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b) comprising a heavy chain variable region CDR3 or a conserved modification thereof containing the amino acid sequence of number 517; and (b) a light chain variable region CDR3 or a conserved modification thereof containing the amino acid sequence of sequence number 531; this extracellular binding domain specifically binds to FcRL5.

[0268] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b) comprising a heavy chain variable region CDR3 or a conserved modification thereof containing the amino acid sequence of number 532; and (b) a light chain variable region CDR3 or a conserved modification thereof containing the amino acid sequence of SEQ ID NO: 535; the antibody or its antigen-binding fragment specifically binds to FcRL5.

[0269] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b) comprising a heavy chain variable region CDR3 or a conserved modification thereof containing the amino acid sequence of number 543; and (b) a light chain variable region CDR3 or a conserved modification thereof containing the amino acid sequence of sequence number 545; this extracellular binding domain specifically binds to FcRL5.

[0270] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b) comprising a heavy chain variable region CDR3 or a conserved modification thereof containing the amino acid sequence of number 570; and (b) a light chain variable region CDR3 or a conserved modification thereof containing the amino acid sequence of SEQ ID NO: 573; the antibody or its antigen-binding fragment specifically binds to FcRL5.

[0271] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) distributed (b) a heavy chain variable region CDR3 or a conserved modification thereof containing an amino acid sequence having the sequence described in column number 925; and (b) a light chain variable region CDR3 or a conserved modification thereof containing an amino acid sequence having the sequence described in sequence number 928; this extracellular binding domain specifically binds to FcRL5.

[0272] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b) a heavy chain variable region CDR3 or a conservative modification thereof comprising an amino acid sequence having the sequence described in number 931; and (b) a light chain variable region CDR3 or a conservative modification thereof comprising an amino acid sequence having the sequence described in sequence number 934.

[0273] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b) comprising a heavy chain variable region CDR3 or a conserved modification thereof containing the amino acid sequence of number 442; and (b) a light chain variable region CDR3 or a conserved modification thereof containing the amino acid sequence of sequence number 443; this extracellular binding domain specifically binds to FcRL5.

[0274] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b) comprising a heavy chain variable region CDR3 or a conserved modification thereof containing the amino acid sequence of number 489; and (b) a light chain variable region CDR3 or a conserved modification thereof containing the amino acid sequence of sequence number 491; this extracellular binding domain specifically binds to FcRL5.

[0275] In certain embodiments, the subject matter of this disclosure provides an extracellular antigen-binding domain (e.g., scFv) comprising a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences and a light chain variable region comprising CDR1, CDR2, and CDR3 sequences, wherein (a) the heavy chain variable region CDR1 is sequence numbers 309, 315, 321, 326, 332, 335, 340, 346, 354, 360, 366, 372, 378, 387, 393, 403, 411, 420, 425, 436, 440, 444, 471, 480 (b) The heavy chain variable region CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 310, 316, 322, 327, 333, 336, 341, 355, 361, 367, 373, 379, 388, 404, 412, 421, 426, 431, 441, 445, 450, 466, 472, 475, 481, 496, 501, 50 (c) The heavy chain variable region CDR3 comprises amino acid sequences selected from the group consisting of SEQ ID NOs: 6, 511, 516, 521, 526, 538, 552, 560, 566, 583, 590, 924 and 930, and their conservative modifications; (c) The heavy chain variable region CDR3 comprises SEQ ID NOs: 311, 317, 323, 328, 334, 337, 342, 347, 351, 356, 362, 368, 374, 376, 380, 384, 389, 394, 396, 400, 405, 408, 412, 415, 422, 427, 432, 437, 4 It comprises amino acid sequences selected from the group consisting of 42, 446, 451, 453, 456, 458, 459, 463, 464, 467, 473, 476, 482, 486, 489, 492, 494, 497, 502, 507, 512, 517, 522, 527, 529, 532, 536, 539, 543, 546, 550, 553, 555, 561, 567, 570, 574, 577, 578, 579, 584, 578, 587, 591, 925 and 931, as well as their conservative modifications;(d) Light chain variable region CDR1 is sequence numbers 312, 3118, 324, 329, 338, 343, 348, 352, 357, 363, 369, 381, 390, 397, 401, 406, 416, 423, 428, 433, 447, 460, 468, 474, 477, 483, 490, 498, 503, 508, 518, 533, 540, 544, 547, 556, 562, 568, 571, 580, 585, 588, 926 and 93 2, and amino acid sequences selected from the group consisting of their conservative modifications; (e) light chain variable region CDR2 is sequence numbers 313, 319, 330, 344, 349, 358, 364, 370, 382, ​​385, 391, 398, 409, 417, 429, 434, 438, 448, 454, 461, 469, 478, 484, 487, 504, 513, 523, 534, 429, 448, 548, 557, 563, 572, 575, 5 (f) The light chain variable region CDR3 includes amino acid sequences selected from the group consisting of 86, 927 and 933, and their conservative modifications; (f) the light chain variable region CDR3 includes SEQ ID NOs: 314, 320, 325, 331, 339, 345, 350, 353, 359, 365, 371, 377, 383, 386, 392, 395, 399, 402, 407, 410, 414, 418, 419, 424, 430, 435, 439, 443, 449, 452, 455, 457, 46 The extracellular antigen-binding domain comprises amino acid sequences selected from the group consisting of 2, 465, 470, 479, 485, 488, 491, 493, 495, 499, 505, 509, 514, 519, 524, 528, 530, 531, 535, 541, 542, 545, 549, 554, 558, 564, 569, 573, 576, 581, 592, 928, and 934, as well as their conserved modifications; this extracellular antigen-binding domain specifically binds FcRL5.

[0276] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b) Sequence number 411; (b) Heavy chain variable region CDR1 or a conservative modification thereof; (c) Heavy chain variable region CDR2 containing the amino acid sequence of SEQ ID NO. 412 or a conservative modification thereof; (d) Light chain variable region CDR1 containing the amino acid sequence of SEQ ID NO. 318 or a conservative modification thereof; (e) Light chain variable region CDR2 containing the amino acid sequence of SEQ ID NO. 319 or a conservative modification thereof; and (f) Light chain variable region CDR3 containing the amino acid sequence of SEQ ID NO. 419 or a conservative modification thereof.

[0277] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b) Sequence number 515, heavy chain variable region CDR1 or its conservative modification; (c) Heavy chain variable region CDR2 containing the amino acid sequence of SEQ ID NO. 516 or a conservative modification thereof; (d) Light chain variable region CDR1 containing the amino acid sequence of SEQ ID NO. 318 or a conservative modification thereof; (e) Light chain variable region CDR2 containing the amino acid sequence of SEQ ID NO. 319 or a conservative modification thereof; and (f) Light chain variable region CDR3 containing the amino acid sequence of SEQ ID NO. 531 or a conservative modification thereof.

[0278] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b) Sequence number 403: Heavy chain variable region CDR1 or a conservative modification thereof; (c) Heavy chain variable region CDR2 containing the amino acid sequence of SEQ ID NO. 404 or a conservative modification thereof; (d) Light chain variable region CDR1 containing the amino acid sequence of SEQ ID NO. 533 or a conservative modification thereof; (e) Light chain variable region CDR2 containing the amino acid sequence of SEQ ID NO. 534 or a conservative modification thereof; and (f) Light chain variable region CDR3 containing the amino acid sequence of SEQ ID NO. 535 or a conservative modification thereof.

[0279] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b) Sequence number 411; (b) Heavy chain variable region CDR1 or a conservative modification thereof; (c) Heavy chain variable region CDR2 containing the amino acid sequence of SEQ ID NO. 412 or a conservative modification thereof; (d) Light chain variable region CDR1 containing the amino acid sequence of SEQ ID NO. 544 or a conservative modification thereof; (e) Light chain variable region CDR2 containing the amino acid sequence of SEQ ID NO. 448 or a conservative modification thereof; and (f) Light chain variable region CDR3 containing the amino acid sequence of SEQ ID NO. 545 or a conservative modification thereof.

[0280] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b) Sequence number 372, heavy chain variable region CDR1 or its conservative modification; (c) Heavy chain variable region CDR2 containing the amino acid sequence of SEQ ID NO. 475 or a conservative modification thereof; (d) Light chain variable region CDR1 containing the amino acid sequence of SEQ ID NO. 571 or a conservative modification thereof; (e) Light chain variable region CDR2 containing the amino acid sequence of SEQ ID NO. 572 or a conservative modification thereof; and (f) Light chain variable region CDR3 containing the amino acid sequence of SEQ ID NO. 573 or a conservative modification thereof.

[0281] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b) Heavy chain variable region CDR1 or a conservative modification thereof containing an amino acid sequence having the sequence described in number 923; (c) Heavy chain variable region CDR2 or a conservative modification thereof containing an amino acid sequence having the sequence described in sequence number 924; (d) Light chain variable region CDR1 or a conservative modification thereof containing an amino acid sequence having the sequence described in sequence number 925; (e) Light chain variable region CDR2 or a conservative modification thereof containing an amino acid sequence having the sequence described in sequence number 927; and (f) Light chain variable region CDR3 or a conservative modification thereof containing an amino acid sequence having the sequence described in sequence number 928.

[0282] In certain embodiments, this extracellular antigen-binding domain (e.g., scFv) is (a) sequence (b) Heavy chain variable region CDR1 or a conservative modification thereof containing an amino acid sequence having the sequence described in Sequence ID No. 929; (c) Heavy chain variable region CDR2 or a conservative modification thereof containing an amino acid sequence having the sequence described in Sequence ID No. 930; (d) Light chain variable region CDR1 or a conservative modification thereof containing an amino acid sequence having the sequence described in Sequence ID No. 932; (e) Light chain variable region CDR2 or a conservative modification thereof containing an amino acid sequence having the sequence described in Sequence ID No. 933; and (f) Light chain variable region CDR3 containing an amino acid sequence having the sequence described in Sequence ID No. 934. This includes preservative modifications.

[0283] In certain embodiments, the anti-FcRL5 antibody or its antigen-binding fragment of the present disclosure is (a) sequence (b) Sequence number 440, heavy chain variable region CDR1 or its conservative modification; (c) Heavy chain variable region CDR2 containing the amino acid sequence of SEQ ID NO. 441 or a conservative modification thereof; (d) Light chain variable region CDR1 containing the amino acid sequence of SEQ ID NO. 329 or a conservative modification thereof; (e) Light chain variable region CDR2 containing the amino acid sequence of SEQ ID NO. 330 or a conservative modification thereof; and (f) Light chain variable region CDR3 containing the amino acid sequence of SEQ ID NO. 443 or a conservative modification thereof.

[0284] In certain embodiments, the anti-FcRL5 antibody or its antigen-binding fragment of the present disclosure is (a) sequence (b) Sequence number 309, heavy chain variable region CDR1 or a conservative modification thereof; (c) Heavy chain variable region CDR2 containing the amino acid sequence of SEQ ID NO. 310 or a conservative modification thereof; (d) Light chain variable region CDR1 containing the amino acid sequence of SEQ ID NO. 490 or a conservative modification thereof; (e) Light chain variable region CDR2 containing the amino acid sequence of SEQ ID NO. 313 or a conservative modification thereof; and (f) Light chain variable region CDR3 containing the amino acid sequence of SEQ ID NO. 491 or a conservative modification thereof.

[0285] As used herein, the terms “conservative sequence modification” and “conservative modification” refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the CAR of this disclosure (e.g., the extracellular antigen-binding domain), including its amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications may be introduced into the human scFv of the subject of this disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Amino acids may be classified into groups according to their physicochemical properties, such as charge and polarity.

[0286] Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced by an amino acid within the same group. For example, amino acids can be classified by charge: positively charged amino acids include lysine, arginine, and histidine; negatively charged amino acids include aspartic acid and glutamic acid; and neutrally charged amino acids include alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Furthermore, amino acids can be classified by polarity: polar amino acids include arginine (basic polarity), asparagine, aspartic acid (acidic polarity), glutamic acid (acidic polarity), glutamine, histidine (basic polarity), lysine (basic polarity), serine, threonine, and tyrosine; nonpolar amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. Thus, one or more amino acid residues within the CDR region may be replaced with other amino acid residues from the same group, and the modified antibody may be tested for retained function (i.e., the function described in (c) to (l) above) using the functional assays described herein. In certain embodiments, one or fewer, two or fewer, three or fewer, four or five or fewer residues within the identified sequence or CDR region are modified. Exemplary conserved amino acid substitutions are shown in Table 231. [Table 231]

[0287] In certain non-limiting embodiments, the extracellular antigen-binding domain of a CAR may include a linker connecting the heavy-chain variable region and the light-chain variable region of the extracellular antigen-binding domain. As used herein, the term “linker” means a functional group (e.g., a chemical or polypeptide) that covalently links two or more polypeptides or nucleic acids so that they are linked to one another. As used herein, “peptide linker” means a linker that couples two proteins together (e.g., V HDomain and V L This refers to one or more amino acids used to couple a domain. Non-limiting examples of peptide linkers are disclosed in Shen et al., Anal Chem vol. 80(no. 6): pp. 1910-1917 (2008).

[0288] In a non-limiting example, the linker is a G4S linker containing amino acids having the sequence described in SEQ ID NO: 897. In a particular embodiment, the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 897 is described in SEQ ID NO: 898. In a non-limiting example, the linker contains amino acids having the sequence described in SEQ ID NO: 307. In a particular embodiment, the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 307 is described in SEQ ID NO: 305.

[0289] In a particular form, the linker comprises amino acids having the sequence described in Sequence ID No. 901 provided below. GGGGS [Sequence ID 901]

[0290] In a particular form, the linker comprises amino acids having the sequence described in Sequence ID No. 902 provided below. SGGSGGS [Sequence ID 902]

[0291] In a particular form, the linker comprises amino acids having the sequence described in Sequence ID No. 903 provided below. GGGGSGGGS [Sequence ID 903]

[0292] In a particular form, the linker comprises amino acids having the sequence described in Sequence ID No. 904 provided below. GGGGSGGGGS [Sequence No. 904]

[0293] In a particular form, the linker comprises amino acids having the sequence described in Sequence ID No. 905 provided below. GGGGSGGGGSGGGGGGGS [ICS905]

[0294] In a particular form, the linker comprises amino acids having the sequence described in Sequence ID No. 906 provided below. GGGGSGGGGSGGGGSGGGGS [EQ906]

[0295] In a particular form, the linker comprises amino acids having the sequence described in Sequence ID No. 907 provided below. GGGGSGGGGSGGGGSGGGGSGGGGS [ICS907]

[0296] In a particular form, the linker comprises amino acids having the sequence described in Sequence ID No. 908 provided below. GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS [LR908]

[0297] In a particular form, the linker comprises amino acids having the sequence described in Sequence ID No. 909 provided below. GGGGSGGGGSGGGGSGGGGSGGGSGGGGSGGGGGS [LR909]

[0298] In a particular form, the linker comprises amino acids having the sequence described in Sequence ID No. 910 provided below. EPKSCDKTHTCPPCP [Sequence ID 910]

[0299] In a particular form, the linker comprises amino acids having the sequence described in Sequence ID No. 911 provided below. GGGGSGGGSEPKSCDKTHTCPPCP [Sequence ID 911]

[0300] In a particular form, the linker comprises amino acids having the sequence described in Sequence ID No. 912 provided below. ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP [Sequence ID 912]

[0301] In a particular form, the linker comprises amino acids having the sequence described in Sequence ID No. 913 provided below. GSGSGS [Sequence ID 913]

[0302] In a particular form, the linker comprises amino acids having the sequence described in Sequence ID No. 914 provided below. AAA [Sequence ID 914]

[0303] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) includes a heavy chain variable region, a light chain variable region, and a linker peptide between the heavy chain and light chain variable regions. Non-limiting examples of the extracellular antigen-binding domains of this disclosure, e.g., scFv, including the heavy chain variable region, light chain variable region, and linker peptide, are disclosed in Tables 77–152. For example, not as an limitation, the extracellular antigen-binding domain comprising the heavy chain variable region, light chain variable region and linker peptide of this disclosure includes SEQ ID NOs: 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, SEQ ID NOs: 660, 662, 664, 666, SEQ ID NOs: 660 The sequence includes amino acid sequences selected from the group consisting of 668, SEQ ID NO: 670, SEQ ID NO: 672, SEQ ID NO: 674, SEQ ID NO: 676, SEQ ID NO: 678, SEQ ID NO: 680, SEQ ID NO: 682, SEQ ID NO: 684, SEQ ID NO: 686, SEQ ID NO: 688, SEQ ID NO: 690, SEQ ID NO: 692, SEQ ID NO: 694, SEQ ID NO: 696, SEQ ID NO: 698, SEQ ID NO: 700, SEQ ID NO: 702, SEQ ID NO: 704, SEQ ID NO: 706, SEQ ID NO: 708, SEQ ID NO: 710, SEQ ID NO: 712, SEQ ID NO: 714, SEQ ID NO: 716, SEQ ID NO: 718, SEQ ID NO: 720, SEQ ID NO: 722, SEQ ID NO: 724, SEQ ID NO: 726, SEQ ID NO: 728, SEQ ID NO: 730, SEQ ID NO: 732, SEQ ID NO: 734, SEQ ID NO: 736, SEQ ID NO: 738, SEQ ID NO: 740, SEQ ID NO: 742, SEQ ID NO: 744 and their conservative modifications (as shown in Tables 77-152).

[0304] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) having a heavy chain variable region, a light chain variable region, and a linker peptide comprises the amino acid sequence of SEQ ID NO: 650 or a conserved modification thereof.

[0305] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) having a heavy chain variable region, a light chain variable region, and a linker peptide comprises the amino acid sequence of SEQ ID NO: 664 or a conserved modification thereof.

[0306] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) having a heavy chain variable region, a light chain variable region, and a linker peptide comprises the amino acid sequence of SEQ ID NO: 678 or a conserved modification thereof.

[0307] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) having a heavy chain variable region, a light chain variable region, and a linker peptide comprises the amino acid sequence of SEQ ID NO: 700 or a conserved modification thereof.

[0308] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) having a heavy chain variable region, a light chain variable region, and a linker peptide comprises the amino acid sequence of SEQ ID NO: 702 or a conserved modification thereof.

[0309] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) having a heavy chain variable region, a light chain variable region, and a linker peptide comprises the amino acid sequence of SEQ ID NO: 710 or a conserved modification thereof.

[0310] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) having a heavy chain variable region, a light chain variable region, and a linker peptide comprises the amino acid sequence of SEQ ID NO: 726 or a conserved modification thereof.

[0311] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) having a heavy chain variable region, a light chain variable region, and a linker peptide comprises the amino acid sequence of SEQ ID NO: 650 or a conserved modification thereof.

[0312] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) having a heavy chain variable region, a light chain variable region, and a linker peptide comprises the amino acid sequence of SEQ ID NO: 678 or a conserved modification thereof.

[0313] Furthermore, this extracellular antigen-binding domain may contain a leader or signal peptide that directs the nascent protein into the endoplasmic reticulum. The signal peptide or leader may be essential if the CAR is glycosylated and anchored in the cell membrane. This signal sequence or leader may be a peptide sequence (about 5, 10, 15, 20, 25, or 30 amino acids long) located at the N-terminus of the newly synthesized protein that directs them into the secretory pathway. In non-limiting examples, this signal peptide is covalently attached to the 5' end of the extracellular antigen-binding domain. In certain embodiments, this signal peptide comprises a CD8 polypeptide having the amino acids described in Sequence ID No. 26 provided below. MALPVTALLLPLALLLHAAR[SEQ ID NO: 935] The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 935 is described in SEQ ID NO: 936, provided below: ATGGCTCTCCCAGTGACTGCCCTACTGCTTCCCCTAGCGCTTCTCCTGCATGCAGCTCGT[Sequence ID 936]

[0314] In another embodiment, the signal peptide comprises amino acids having the sequence described in SEQ ID NO: 937, provided below. METDTLLLWVLLLWVPGSTG[Sequence ID 937] The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 937 is described in SEQ ID NO: 938, provided below: ATGGAAACCGACACCCTGCTGCTGTGGGTGCTGCTGCTGTGGGTGCCAGGATCCACAGGA[Sequence No. 938]

[0315] In certain embodiments, this extracellular antigen-binding domain, for example, human scFv, includes a heavy chain variable region, a light chain variable region, a linker peptide between the heavy chain variable region and the light chain variable region, and His-tags and HA-tags. In certain embodiments, the amino acid sequences of the His-tags and HA-tags include the amino acid sequence of SEQ ID NO: 308. The nucleotide sequence encoding SEQ ID NO: 308 is SEQ ID NO: 306.

[0316] In certain embodiments, this extracellular antigen-binding domain, e.g., human scFv, binds to a human FcRL5 polypeptide containing the amino acid sequence described in SEQ ID NO: 899. In certain embodiments, this extracellular antigen-binding domain, e.g., human scFv, binds to an epitope in domain 9 (e.g., amino acids 754-835 of SEQ ID NO: 899). In certain embodiments, this extracellular antigen-binding domain, e.g., human scFv, binds to an epitope in domain 8 (e.g., amino acids 658-731 of SEQ ID NO: 899). In certain embodiments, this extracellular antigen-binding domain, e.g., human scFv, binds to an epitope in domain 9 containing amino acids 829-840 of SEQ ID NO: 899. In certain embodiments, this extracellular antigen-binding domain, e.g., human scFv, binds to an epitope in domain 8 containing amino acids 657-667 of SEQ ID NO: 899. For example, but not limited to, this extracellular antigen-binding domain, e.g., human scFv, binds to an epitope containing the amino acid sequence RSETVTLYITGL (SEQ ID NO: 964). In certain embodiments, the antibody or antigen-binding fragment of the Disclosure binds to an epitope comprising the amino acid sequence SRPILTFRAPR (SEQ ID NO: 965). CAR transmembrane domain

[0317] In certain non-limiting embodiments, the transmembrane domain of the CAR comprises a hydrophobic alpha helix extending over at least a portion of the membrane. Different transmembrane domains result in different receptor stabilities. After antigen recognition, the receptors cluster, and signals are transmitted to the cell. According to the subject matter of this disclosure, the transmembrane domain of the CAR may include CD8 polypeptide, CD28 polypeptide, CD3ζ polypeptide, CD4 polypeptide, 4-1BB polypeptide, OX40 polypeptide, ICOS polypeptide, CTLA-4 polypeptide, PD-1 polypeptide, LAG-3 polypeptide, 2B4 polypeptide, BTLA polypeptide, synthetic peptides (not based on proteins associated with the immune response), or combinations thereof.

[0318] In certain embodiments, the transmembrane domain of the CAR of the Disclosure comprises a CD28 polypeptide. This CD28 polypeptide may have an amino acid sequence or fragment thereof that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% homologous to the sequence (Sequence ID 939) having NCBI reference number: P10747 or NP_006130, and / or may optionally contain up to one, up to two, or up to three conserved amino acid substitutions. In non-limiting embodiments, this CD28 polypeptide may have an amino acid sequence that is a contiguous portion of Sequence ID 939 having at least 20, or at least 30, or at least 40, or at least 50, and up to 220 amino acid lengths. Alternatively, in various non-limiting embodiments, the CD28 polypeptide has an amino acid sequence of amino acids 1 to 220, 1 to 50, 50 to 100, 100 to 150, 150 to 200, or 200 to 220 of SEQ ID NO: 939. In certain embodiments, the CAR of the subject of this disclosure comprises a transmembrane domain containing the CD28 polypeptide and an intracellular domain containing a co-stimulatory signaling region containing the CD28 polypeptide. In certain embodiments, the CD28 polypeptide contained in the transmembrane domain and the intracellular domain has an amino acid sequence of amino acids 114 to 220 of SEQ ID NO: 939. Sequence ID 939 is provided below: 1 MLRLLLALNL FPSIQVTGNK ILVKQSPMLV AYDNAVNLSC KYSYNLFSRE FRASLHKGLD 61 SAVEVCVVYG NYSQQLQVYS KTGFNCDGKL GNESVTFYLQ NLYVNQTDIY FCKIEVMYPP 121 PYLDNEKSNG TIIHVKGKHL CPSPLFPGPS KPFWVLVVVG GVLACYSLLV TVAFIIFWVR 181 SKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRS[Sequence ID 939]

[0319] According to the subject matter of this disclosure, “CD28 nucleic acid molecule” refers to a polynucleotide encoding the CD28 polypeptide. In certain embodiments, the CD28 nucleic acid molecule encoding the CD28 polypeptide (amino acids 114 to 220 of SEQ ID NO: 939) contained within the transmembrane domain and intracellular domain (e.g., the co-stimulatory signaling region) of the CAR of this disclosure includes a nucleic acid having the sequence described in SEQ ID NO: 940, provided below. ATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTG CTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC [SEQ ID NO: 940].

[0320] In certain embodiments, the transmembrane domain of the CAR of the Disclosure comprises a CD8 polypeptide. This CD8 polypeptide may have an amino acid sequence or fragment thereof that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% homologous to the sequence having NCBI reference number: AAH25715 (SEQ ID NO: 960), and / or may optionally contain up to one, up to two, or up to three conserved amino acid substitutions. In non-limiting embodiments, this CD8 polypeptide may have an amino acid sequence that is a contiguous portion of SEQ ID NO: 960 having an amino acid length of at least 20, or at least 30, or at least 40, or at least 50, or at least 70, or at least 100, or at least 150, or at least 200 and up to 235 amino acids. Alternatively, in various non-limiting embodiments, the CD8 polypeptide has an amino acid sequence of amino acids 1 to 235, 1 to 50, 50 to 100, 100 to 150, 150 to 200, 130 to 210, or 200 to 235 of SEQ ID NO: 960. In certain embodiments, the CAR of the subject of this disclosure comprises a transmembrane domain containing the CD8 polypeptide. In certain embodiments, the CD8 polypeptide contained within the transmembrane domain has an amino acid sequence of amino acids 137 to 207 of SEQ ID NO: 960. Sequence ID 960 is provided below: 1 malpvtalll plallhaar psqfrvspld rtwnlgetve lkcqvllsnp tsgcswlfqp 61 rgaaasptfl lylsqnkpka aegldtqrfs gkrlgdtfvl tlsdfrrene gcyfcsalsn 121 simyfshfvp vflpakpttt paprpptpap tiasqplslr peacrpaagg avhtrgldfa 181 cdiyiwapla gtcgvlllsl vitlycnhrn rrrvckcprp vvksgdkpsl saryv[Sequence ID 960]

[0321] According to the subject matter of this disclosure, “CD8 nucleic acid molecule” refers to a polynucleotide encoding a CD8 polypeptide. In certain embodiments, a CD8 nucleic acid molecule encoding a CD8 polypeptide (amino acids 137 to 207 of SEQ ID NO: 960) contained within the transmembrane domain and intracellular domain (e.g., the co-stimulatory signaling region) of the CAR of this disclosure includes a nucleic acid having the sequence described in SEQ ID NO: 961, provided below. CCCACCACGACGCCAGCGCCGCGACCACCAACCCCGGCGCCCACGATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAC [SEQ ID NO: 961]

[0322] In certain non-limiting embodiments, the CAR may also include a spacer region that ligates the extracellular antigen-binding domain to the transmembrane domain. This spacer region may be sufficiently flexible to orient the antigen-binding domain to different orientations in order to facilitate antigen recognition. This spacer region may be a hinge region derived from IgG1, or a portion of the CH2CH3 region and CD3 of an immunoglobulin. CAR intracellular domain

[0323] In certain non-limiting embodiments, the intracellular domain of the CAR may comprise a CD3ζ polypeptide capable of activating or stimulating cells (e.g., lymphoid cells, e.g., T cells). The CD3ζ polypeptide comprises three ITAMs and transmits an activation signal to cells (e.g., lymphoid cells, e.g., T cells) after antigen binding. This CD3ζ polypeptide may have an amino acid sequence or fragment thereof that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous to the sequence described in SEQ ID NO: 941, and / or may optionally comprise up to one, up to two, or up to three conserved amino acid substitutions. In non-limiting embodiments, this CD3ζ polypeptide may have an amino acid sequence that is a contiguous portion of SEQ ID NO: 941, having a length of at least 20, or at least 30, or at least 40, or at least 50, and up to 163 amino acids. Alternatively, in various non-limiting embodiments, the CD3ζ polypeptide has the amino acid sequence of amino acids 1 to 163, 1 to 50, 50 to 100, 100 to 150, or 150 to 163 of SEQ ID NO: 941. In certain embodiments, the CD3ζ polypeptide contained within the intracellular domain of the CAR of this disclosure has the amino acid sequence of amino acids 52 to 163 of SEQ ID NO: 941. Sequence ID 941 is provided below: 1 MKWKALFTAA ILQAQLPITE AQSFGLLDPK LCYLLDGILF IYGVILTALF LRVKFSRSAD 61 APAYQQGQNQ LYNELNLGRR EEYDVLDKRR GRDPEMGGKP RRKNPQEGLY NELQKDKMAE 121 AYSEIGMKGE RRRGKGHDGL YQGLSTATKD TYDALHMQAL PPR[Sequence ID 941]

[0324] According to the subject matter of this disclosure, “CD3ζ nucleic acid molecule” refers to a polynucleotide encoding a CD3ζ polypeptide. In certain embodiments, a CD3ζ nucleic acid molecule encoding a CD3ζ polypeptide (amino acids 52 to 163 of SEQ ID NO: 941) contained within the intracellular domain of the CAR of this disclosure includes a nucleic acid having the sequence described in SEQ ID NO: 942, provided below. AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTG TACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA [SEQ ID NO: 942]

[0325] In certain non-limiting embodiments, the intracellular domain of the CAR further comprises at least one signaling region. This at least one signaling region may include a CD28 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a DAP-10 polypeptide, a PD-1 polypeptide, a CTLA-4 polypeptide, a LAG-3 polypeptide, a 2B4 polypeptide, a BTLA polypeptide, a synthetic peptide (not based on a protein associated with an immune response), or a combination thereof.

[0326] In certain embodiments, this signaling region is a costimulatory signaling region. In certain embodiments, this costimulatory region includes at least one costimulatory molecule that can provide optimal lymphocyte activation. As used herein, “costimulatory molecule” refers to a cell surface molecule other than an antigen receptor or its ligand that is required for an efficient lymphocyte response to an antigen. This at least one costimulatory signaling region may include CD28 polypeptide, 4-1BB polypeptide, OX40 polypeptide, ICOS polypeptide, DAP-10 polypeptide, or a combination thereof. This costimulatory molecule may bind to a costimulatory ligand, which is a cell surface-expressed protein that, upon binding to its receptor, produces a costimulatory response, i.e., an intracellular response that provides the stimulus when an antigen binds to its CAR molecule. Costimulatory ligands include, but are not limited to, CD80, CD86, CD70, OX40L, 4-1BBL, CD48, TNFRSF14, and PD-L1. As an example, the 4-1BB ligand (i.e., 4-1BBL) combines with the CAR signal to form a CAR signal. + To provide intracellular signals that induce effector cell function of T cells, the CAR may bind to 4-1BB (also known as "CD137"). A CAR comprising an intracellular domain containing a costimulatory signaling region comprising 4-1BB, ICOS, or DAP-10 is disclosed in U.S. Patent No. 7,446,190, which is incorporated herein by reference in its entirety (for example, in U.S. Patent No. 7,446,190, the nucleotide sequence encoding 4-1BB is shown in SEQ ID NO: 15, the nucleotide sequence encoding ICOS is shown in SEQ ID NO: 16, and the nucleotide sequence encoding DAP-10 is described in SEQ ID NO: 17). In certain embodiments, the intracellular domain of the CAR comprises a costimulatory signaling region comprising the CD28 polypeptide. In certain embodiments, the intracellular domain of the CAR comprises a costimulatory signaling region comprising two costimulatory molecules: CD28 and 4-1BB or CD28 and OX40.

[0327] 4-1BB may act as a tumor necrosis factor (TNF) ligand and may have stimulating activity. This 4-1BB polypeptide may have an amino acid sequence or fragment thereof that is at least approximately 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homologous to the sequence having NCBI reference number: P41273 or NP_001552 (SEQ ID NO: 943), and / or may optionally contain up to one, two, or three conserved amino acid substitutions. In certain embodiments, the 4-1BB polypeptide contained within the intracellular domain of the CAR of this disclosure has the amino acid sequence of amino acids 214 to 255 of SEQ ID NO: 943. SEQ ID NO: 943 is provided below: 1 MGNSCYNIVA TLLLVLNFER TRSLQDPCSN CPAGTFCDNN RNQICSPCPP NSFSSAGGQR 61 TCDICRQCKG VFRTRKECSS TSNAECDCTP GFHCLGAGCS MCEQDCKQGQ ELTKKGCKDC 121 CFGTFNDQKR GICRPWTNCS LDGKSVLVNG TKERDVVCGP SPADLSPGAS SVTPPAPARE 181 PGHSPQIISF FLALTSTALL FLLFFLTLRF SVVKRGRKKL LYIFKQPFMR PVQTTQEEDG 241 CSCRFPEEEE GGCEL[SEQ ID NO: 943]

[0328] According to the subject matter of this disclosure, “4-1BB nucleic acid molecule” refers to a polynucleotide encoding a 4-1BB polypeptide. In certain embodiments, a 4-1BB nucleic acid molecule encoding a 4-1BB polypeptide (amino acids 214 to 255 of SEQ ID NO: 943) contained within the intracellular domain of the CAR of this disclosure includes a nucleic acid having the sequence described in SEQ ID NO: 962, provided below. AAACGGGGCAGAAAGAAGCTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG [SEQ ID NO: 962]

[0329] OX40 polypeptides may have amino acid sequences or fragments thereof that are at least approximately 85%, approximately 90%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or 100% homologous to the sequence (SEQ ID NO: 944) having NCBI reference number: P43489 or NP_003318, and / or may optionally contain up to one, up to two, or up to three conservative amino acid substitutions. Sequence ID 944 is provided below: 1 MCVGARRLGR GPCAALLLLG LGLSTVTGLH CVGDTYPSND RCCHECRPGN GMVSRCSRSQ 61 NTVCRPCGPG FYNDVVSSKP CKPCTWCNLR SGSERKQLCT ATQDTVCRCR AGTQPLDSYK 121 PGVDCAPCPP GHFSPGDNQA CKPWTNCTLA GKHTLQPASN SSDAICEDRD PPATQPQETQ 181 GPPARPITVQ PTEAWPRTSQ GPSTRPVEVVP GGRAVAAILG LGLVLGLLGP LAILLALYLL 241 RRDQRLPPDA HKPPGGGSFR TPIQEEQADA HSTLAKI[Sequence ID 944]

[0330] According to the subject matter of this disclosure, “OX40 nucleic acid molecule” refers to a polynucleotide that encodes an OX40 polypeptide.

[0331] ICOS polypeptides may have amino acid sequences or fragments thereof that are at least approximately 85%, approximately 90%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or 100% homologous to the sequence (SEQ ID NO: 945) having NCBI reference number: NP_036224, and / or may optionally contain up to one, up to two, or up to three conserved amino acid substitutions. Sequence ID 945 is provided below: 1 MKSGLWYFFL FCLRIKVLTG EINGSANYEM FIFHNGGVQI LCKYPDIVQQ FKMQLLKGGQ 61 ILCDLTKTKG SGNTVSIKSL KFCHSQLSNN SVSFFLYNLD HSHANYYFCN LSIFDPPPFK 121 VTLTGGYLHI YESQLCCQLK FWLPIGCAAF VVVCILGCIL ICWLTKKKYS SSVHDPNGEY181 MFMRAVNTAK KSRLTDVTL[Sequence ID 945]

[0332] According to the subject matter of this disclosure, “ICOS nucleic acid molecule” refers to a polynucleotide that encodes the ICOS polypeptide.

[0333] CTLA-4 is an inhibitory receptor expressed by activated T cells that mediates activated T cell inhibition or anergy when engaged with its corresponding ligands (CD80 and CD86; B7-1 and B7-2, respectively). In both preclinical and clinical studies, systemic antibody blockade of CTLA-4 enhanced the endogenous antitumor response in a clinical setting, although it had significant, unpredictable toxicity.

[0334] CTLA-4 comprises an extracellular V domain, a transmembrane domain, and a cytoplasmic tail. Alternative splice variants encoding different isoforms are characterized. The membrane-bound isoform functions as a homodimer interconnected by disulfide bonds, while the soluble isoform functions as a monomer. This intracellular domain is similar to that of CD28 in that it lacks intrinsic catalytic activity and contains one YVKM motif capable of binding PI3K, PP2A, and SHP-2, as well as one proline-rich motif capable of binding SH3-containing proteins. One role of CTLA-4 in inhibiting T cell responses appears to be direct, via the dephosphorylation of SHP-2 and PP2A of TCR proximal signaling proteins such as CD3 and LAT. CTLA-4 may also indirectly influence signaling by competing with CD28 for CD80 / 86 binding. CTLA-4 has also been shown to bind to PI3K, CD80, AP2M1, and PPP2R5A, and / or interact with PI3K, CD80, AP2M1, and PPP2R5A.

[0335] According to the subject matter of this disclosure, the CTLA-4 polypeptide may have an amino acid sequence or fragment thereof that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous (homonymy as herein can be determined using standard software such as BLAST or FASTA) to UniProtKB / Swiss-Prot reference number: P16410.3 (SEQ ID NO: 946), and / or may optionally contain up to one, up to two, or up to three conserved amino acid substitutions. Sequence ID 946 is provided below: 1 MACLGFQRHK AQLNLATRTW PCTLLFFLLF IPVFCKAMHV AQPAVVLASS RGIASFVCEY 61 ASPGKATEVR VTVLRQADSQ VTEVCAATYM MGNELTFLDD SICTGTSSGN QVNLTIQGLR 121 AMDTGLYICK VELMYPPPYY LGIGNGTQIY VIDPEPCPDS DFLLWILAAV SSGLFFYSFL 181 LTAVSLSKML KKRSPLTTGV YVKMPPTEPE CEKQFQPYFI PIN[Sequence ID 946]

[0336] According to the subject matter of this disclosure, “CTLA-4 nucleic acid molecule” refers to a polynucleotide that encodes a CTLA-4 polypeptide.

[0337] PD-1 is a negative immunomodulator of activated T cells upon engagement with its corresponding ligands, PD-L1 and PD-L2, which are expressed on endogenous macrophages and dendritic cells. PD-1 is a 268-amino acid type I membrane protein. PD-1 has two ligands, PD-L1 and PD-L2, which are members of the B7 family. The structure of this protein includes a transmembrane domain and an extracellular IgV domain followed by an intracellular tail. This intracellular tail contains two phosphorylation sites located within an immunoreceptor tyrosine-based inhibitory motif and an immunoreceptor tyrosine-based switch motif, which are responsible for PD-1's negative regulation of TCR signaling. SHP-I and SHP-2 phosphatases bind to the cytoplasmic tail of PD-1 upon ligand binding. Upregulation of PD-L1 is one mechanism by which tumor cells can evade the host immune system. In preclinical and clinical trials, PD-1 blockade with antagonist antibodies induced an antitumor response mediated through the host endogenous immune system.

[0338] According to the subject matter of this disclosure, the PD-1 polypeptide may have an amino acid sequence or fragment thereof that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous to NCBI reference number: NP_005009.2 (SEQ ID NO: 947), and / or may optionally contain up to one, up to two, or up to three conserved amino acid substitutions. Sequence ID 947 is provided below: 1 MQIPQAPWPV VWAVLQLGWR PGWFLDSPDR PWNPPTFSPA LLVVTEGDNA TFTCSFSNTS 61 ESFVLNWYRM SPSNQTDKLA AFPEDRSQPG QDCRFRVTQL PNGRDFHMSV VRARRNDSGT 121 YLCGAISLAP KAQIKESLRA ELRVTERRAE VPTAHPSPSP RPAGQFQTLV VGVVGGLLGS 181 LVLLVWVLAV ICSRAARGTI GARRTGQPLK EDPSAVPVFS VDYGELDFQW REKTPEPPVP 241 CVPEQTEYAT IVFPSGMGTS SPARRGSADG PRSAQPLRPE DGHCSWPL[Sequence ID 947]

[0339] According to the subject matter of this disclosure, “PD-1 nucleic acid molecule” refers to a polynucleotide that encodes a PD-1 polypeptide.

[0340] Lymphocyte-activating protein 3 (LAG-3) is a negative immunomodulator of immune cells. LAG-3 belongs to the immunoglobulin (Ig) superfamily and contains four extracellular Ig-like domains. The LAG3 gene contains eight exons. Sequence data, exon / intron configuration, and chromosomal localization all indicate a close association of LAG3 with CD4. LAG3 is also known as CD223 (surface antigen classification 223).

[0341] According to the subject matter of this disclosure, LAG-3 polypeptides may have amino acid sequences or fragments thereof that are at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous to UniProtKB / Swiss-Prot reference number: P18627.5 (SEQ ID NO: 948), and / or may optionally contain up to one, up to two, or up to three conserved amino acid substitutions. Sequence ID 948 is provided below: 1 MWEAQFLGLL FLQPLWVAPV KPLQPGAEVP VVWAQEGAPA QLPCSPTIPL QDLSLLRRAG 61 VTWQHQPDSG PPAAAPGHPL APGPHPAAPS SWGPRPRRYT VLSVGPGGLR SGRLPLQPRV 121 QLDERGRQRG DFSLWLRPAR RADAGEYRAA VHLRDRALSC RLRLRLGQAS MTASPPGSLR 181 ASDWVILNCS FSRPDRPASV HWFRNRGQGR VPVRESPHHH LAESFLFLPQ VSPMDSGPWG 241 CILTYRDGFN VSIMYNLTVL GLEPPTPLTV YAGAGSRVGL PCRLPAGVGT RSFLTAKWTP 301 PGGGPDLLVT GDNGDFTLRL EDVSQAQAGT YTCHIHLQEQ QLNATVTLAI ITVTPKSFGS 361 PGSLGKLLCE VTPVSGQERF VWSSLDTPSQ RSFSGPWLEA QEAQLLSQPW QCQLYQGERL 421 LGAAVYFTEL SSPGAQRSGR APGALPAGHL LLFLILGVLS LLLLVTGAFG FHLWRRQWRP 481 RRFSALEQGI HPPQAQSKIE ELEQEPEPEP EPEPEPEPEP EPEQL [Sequence ID 948]

[0342] According to the subject matter of this disclosure, “LAG-3 nucleic acid molecule” refers to a polynucleotide that encodes a LAG-3 polypeptide.

[0343] The natural killer cell receptor 2B4 (2B4) mediates non-MHC-bound cell death on NK cells and a subset of T cells. To date, the function of 2B4 is still under investigation; the 2B4-S isoform is thought to be an activating receptor for immune cells, while the 2B4-L isoform is thought to be a negative immunomodulator for immune cells. 2B4 becomes engaged upon binding to its high-affinity ligand, CD48. 2B4 contains a tyrosine-based switch motif, a molecular switch that associates the protein with various phosphatases. 2B4 is also known as CD244 (surface antigen classification 244).

[0344] According to the subject matter of this disclosure, the 2B4 polypeptide may have an amino acid sequence or fragment thereof that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous to UniProtKB / Swiss-Prot reference number: Q9BZW8.2 (SEQ ID NO: 949), and / or may optionally contain up to one, up to two, or up to three conserved amino acid substitutions. Sequence ID 949 is provided below: 1 MLGQVVTLIL LLLLKVYQGK GCQGSADHVV SISGVPLQLQ PNSIQTKVDS IAWKKLLPSQ 61 NGFHHILKWE NGSLPSNTSN DRFSFIVKNL SLLIKAAQQQ DSGLYCLEVT SISGKVQTAT 121 FQVFVFESLL PDKVEKPRLQ GQGKILDRGR CQVALSCLVS RDGNVSYAWY RGSKLIQTAG 181 NLTYLDEEVD INGTHTYTCN VSNPVSWESH TLNLTQDCQN AHQEFRFWPF LVIIVILSAL 241 FLGTLACFCV WRRKRKEKQS ETSPKEFLTI YEDVKDLKTR RNHEQEQTFP GGGSTIYSMI 301 QSQSSAPTSQ EPAYTLYSLI QPSRKSGSRK RNHSPSFNST IYEVIGKSQP KAQNPARLSR 361 KELENFDVYS [Sequence ID 949]

[0345] According to the subject matter of this disclosure, “2B4 nucleic acid molecule” refers to a polynucleotide that encodes a 2B4 polypeptide.

[0346] B and T lymphocyte attenuator (BT) BTLA expression is induced during T cell activation, and BTLA remains expressed on Th1 cells but not on Th2 cells. Similar to PD1 and CTLA4, BTLA interacts with the B7 homolog B7H4. However, unlike PD-1 and CTLA-4, BTLA exhibits T cell inhibition not only through interaction with the B7 family cell surface receptor, but also through interaction with tumor necrosis family receptors (TNF-R). BTLA is a ligand for member 14 (TNFRSF14) of the tumor necrosis factor (receptor) superfamily, also known as herpesvirus entry mediator (HVEM). The BTLA-HVEM complex negatively modulates the T cell immune response. BTLA activation is linked to human CD8 + It has been shown to inhibit the function of cancer-specific T cells. BTLA is also known as CD272 (surface antigen classification 272).

[0347] According to the subject matter of this disclosure, BTLA polypeptides may have amino acid sequences or fragments thereof that are at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous to UniProtKB / Swiss-Prot reference number: Q7Z6A9.3 (SEQ ID NO: 950), and / or may optionally contain up to one, up to two, or up to three conserved amino acid substitutions. Sequence ID 950 is provided below: 1 MKTLPAMLGT GKLFWVFFLI PYLDIWNIHG KESCDVQLYI KRQSEHSILA GDPFELECPV 61 KYCANRPHVT WCKLNGTTCV KLEDRQTSWK EEKNISFFIL HFEPVLPNDN GSYRCSANFQ 121 SNLIESHSTT LYVTDVKSAS ERPSKDEMAS RPWLLYRLLP LGGLPLLITT CFCLFCCLRR 181 HQGKQNELSD TAGREINLVD AHLKSEQTEA STRQNSQVLL SETGIYDNDP DLCFRMQEGS 241 EVYSNPCLEE NKPGIVYASL NHSVIGPNSR LARNVKEAPT EYASICVRS[SEQ ID NO: 950]

[0348] According to the subject matter of this disclosure, “BTLA nucleic acid molecule” refers to a polynucleotide that encodes a BTLA polypeptide.

[0349] In certain embodiments, the CAR comprises an extracellular antigen-binding domain that specifically binds to human FcRL5, a transmembrane domain containing a CD28 polypeptide, and an intracellular domain containing a co-stimulatory signaling domain containing a CD3ζ polypeptide and a CD28 polypeptide, as shown in Figure 9. As shown in Figure 9, the CAR also comprises a signal peptide or leader covalently affixed to the 5' end of the extracellular antigen-binding domain. This signal peptide contains a CD8 polypeptide.

[0350] In certain embodiments, the CARs of the subject of this disclosure may further include an inducible promoter for expressing a nucleic acid sequence in human cells. The promoter for use in expressing the CAR gene may be a constitutive promoter, such as a ubiquitin C (UbiC) promoter.

[0351] The subject matter of this disclosure also provides isolated nucleic acid molecules encoding the FcRL5-targeted CAR or a functional portion thereof as described herein. In certain embodiments, this isolated nucleic acid molecule encodes the FcRL5-targeted CAR of this disclosure, comprising an scFv that specifically binds to human FcRL5, a transmembrane domain comprising a CD28 polypeptide, and an intracellular domain comprising a co-stimulatory signaling region comprising a CD3ξ polypeptide and a CD28 polypeptide. In certain embodiments, this scFv is entirely human. In certain embodiments, this scFv is mouse scFv. In certain non-limiting embodiments, this isolated nucleic acid molecule comprises a nucleic acid having the sequence described in Sequence ID No. 951 provided below: The isolated nucleic acid molecule having the sequence encodes a CAR that targets FcRL5, comprising a mouse scFv containing a heavy chain variable region with amino acids having the sequence described in SEQ ID NO: 915, a light chain variable region containing amino acids having the sequence described in SEQ ID NO: 917, and a linker located between the heavy chain variable region and the light chain variable region having the amino acid sequence of SEQ ID NO: 897, a transmembrane domain containing a CD28 polypeptide, and an intracellular domain containing a co-stimulatory signaling region containing a CD3ξ polypeptide and a CD28 polypeptide.

[0352] In another specific, non-limiting example, this isolated nucleic acid molecule includes a nucleic acid having the sequence described in Sequence ID No. 952 provided below:

[0353] The isolated nucleic acid molecule encodes a fully human scFv (encoded by nucleotides 207-998 of SEQ ID NO: 953), which includes a heavy chain variable region containing amino acids having the sequence described in SEQ ID NO: 116, a light chain variable region containing amino acids having the sequence described in SEQ ID NO: 115, and a linker positioned between the heavy chain and light chain variable regions with the amino acid sequence of SEQ ID NO: 307; a transmembrane domain containing a CD8 polypeptide having amino acids 137-207 of SEQ ID NO: 960; and an intracellular domain containing a co-stimulatory signaling region containing a CD3ξ polypeptide having amino acids 52-163 of SEQ ID NO: 941 and a 4-1BB polypeptide having amino acids 214-255 of SEQ ID NO: 943. Nucleotides 270-998 of SEQ ID NO: 953 encode the human scFv. Nucleotides 1008-1220 of SEQ ID NO: 953 encode the CD8 polypeptide contained in the transmembrane domain. Nucleotides 1221–1346 of SEQ ID NO: 953 encode the 4-1BB polypeptide contained within the intracellular domain. Nucleotides 1347–1685 of SEQ ID NO: 953 encode the CD3 zeta polypeptide contained within the intracellular domain. The rest of SEQ ID NO: 953 is shown in Table 232. [Table 232]

[0354] In certain embodiments, this isolated nucleic acid molecule comprises a nucleic acid having the sequence described in Sequence ID No. 954 provided below: The child encodes a fully human scFv (encoded by nucleotides 207-998 of SEQ ID NO: 953) containing a heavy chain variable region containing amino acids having the sequence described in SEQ ID NO: 144, a light chain variable region containing amino acids having the sequence described in sequence number 143, and a linker positioned between the heavy chain variable region and the light chain variable region having the amino acid sequence of SEQ ID NO: 307, a transmembrane domain containing a CD8 polypeptide having amino acids 137-207 of SEQ ID NO: 960, and an intracellular domain containing a co-stimulatory signaling region containing a CD3ξ polypeptide having amino acids 52-163 of SEQ ID NO: 941 and a 4-1BB polypeptide having amino acids 214-255 of SEQ ID NO: 943. Nucleotides 207-1013 of SEQ ID NO: 954 encode a fully human scFv (encoded by nucleotides 207-1013 of SEQ ID NO: 954), and an FcRL5-targeted CAR (named 39 FcRL5-targeted BBz CAR). Nucleotides 207-1013 of SEQ ID NO: 954 encode a human scFv. Nucleotides 1023-1235 of SEQ ID NO: 954 encode the CD8 polypeptide contained in the transmembrane domain. Nucleotides 1236-1361 of SEQ ID NO: 954 encode the 4-1BB polypeptide contained in the intracellular domain. Nucleotides 1362-1700 of SEQ ID NO: 954 encode the CD3 zeta polypeptide contained in the intracellular domain. The rest of SEQ ID NO: 954 is shown in Table 233. [Table 233]

[0355] In certain embodiments, this isolated nucleic acid molecule comprises a nucleic acid having the sequence described in Sequence ID No. 955 provided below: The isolated nucleic acid molecule having the above encodes a CAR (named 69 FcRL5-targeted BBz CAR) that contains a fully human scFv (encoded by nucleotides 207-1037 of SEQ ID NO: 955), comprising an intracellular domain containing a heavy chain variable region with amino acids having the sequence described in SEQ ID NO: 172, a light chain variable region with amino acids having the sequence described in SEQ ID NO: 171, a linker positioned between the heavy chain variable region and the light chain variable region with the amino acid sequence of SEQ ID NO: 307, a transmembrane domain containing a CD8 polypeptide having amino acids 137-207 of SEQ ID NO: 960, and a co-stimulatory signaling region containing a CD3ξ polypeptide having amino acids 52-163 of SEQ ID NO: 941 and a 4-1BB polypeptide having amino acids 214-255 of SEQ ID NO: 943. Nucleotides 207-1037 of SEQ ID NO: 955 encode a human scFv. Nucleotides 1047-1259 of SEQ ID NO: 955 encode a human scFv. Nucleotides 207-1037 of SEQ ID NO: 955 encode a human scFv. Nucleotides 1047-1259 of SEQ ID NO: 955 encode a CD8 polypeptide contained in the transmembrane domain. Nucleotides 1260–1385 of SEQ ID NO: 955 encode the 4-1BB polypeptide contained within the intracellular domain. Nucleotides 1386–1724 of SEQ ID NO: 955 encode the CD3 zeta polypeptide contained within the intracellular domain. The rest of SEQ ID NO: 955 is shown in Table 234. [Table 234]

[0356] In certain embodiments, this isolated nucleic acid molecule comprises a nucleic acid having the sequence described in Sequence ID No. 956 provided below: [Table 235]

[0357] In certain embodiments, this isolated nucleic acid molecule comprises a nucleic acid having the sequence described in Sequence ID No. 957 provided below: The acid molecule encodes a CAR (named 105 FcRL5-targeted BBz CAR) that is entirely human scFv (encoded by nucleotides 207-1019), and includes an intracellular domain containing a heavy chain variable region with amino acids having the sequence described in SEQ ID NO: 220, a light chain variable region with amino acids having the sequence described in SEQ ID NO: 219, a linker positioned between the heavy chain variable region and the light chain variable region with the amino acid sequence of SEQ ID NO: 307, a transmembrane domain containing a CD8 polypeptide having amino acids 137-207 of SEQ ID NO: 960, and a co-stimulatory signaling region containing a CD3ξ polypeptide having amino acids 52-163 of SEQ ID NO: 941 and a 4-1BB polypeptide having amino acids 214-255 of SEQ ID NO: 943. Nucleotides 207-1019 of SEQ ID NO: 957 encode human scFv. Nucleotides 1029-1241 of SEQ ID NO: 957 encode human scFv. Nucleotides 1029-1241 of SEQ ID NO: 957 encode the CD8 polypeptide contained in the transmembrane domain. Nucleotides 1242–1367 of SEQ ID NO: 957 encode the 4-1BB polypeptide contained within the intracellular domain. Nucleotides 1368–1706 of SEQ ID NO: 957 encode the CD3 zeta polypeptide contained within the intracellular domain. The rest of SEQ ID NO: 957 is shown in Table 236. [Table 236]

[0358] In certain embodiments, this isolated nucleic acid molecule comprises a nucleic acid having the sequence described in Sequence ID No. 958 provided below: The isolated nucleic acid molecule encodes a CAR (named 109 FcRL5-targeted BBz CAR) that contains a fully human scFv (encoded by nucleotides 207-1031), comprising a heavy chain variable region containing amino acids having the sequence described in SEQ ID NO: 236, a light chain variable region containing amino acids having the sequence described in SEQ ID NO: 235, a linker positioned between the heavy chain and light chain variable regions having the amino acid sequence of SEQ ID NO: 307, a transmembrane domain containing a CD8 polypeptide having amino acids 137-207 of SEQ ID NO: 960, and an intracellular domain containing a co-stimulatory signaling region containing a CD3ξ polypeptide having amino acids 52-163 of SEQ ID NO: 941 and a 4-1BB polypeptide having amino acids 214-255 of SEQ ID NO: 943. Nucleotides 207-1031 of SEQ ID NO: 957 encode human scFv. Nucleotides 1041-1253 of SEQ ID NO: 958 encode the CD8 polypeptide contained in the transmembrane domain. Nucleotides 1254–1379 of SEQ ID NO: 958 encode the 4-1BB polypeptide contained within the intracellular domain. Nucleotides 1380–1718 of SEQ ID NO: 958 encode the CD3 zeta polypeptide contained within the intracellular domain. The rest of SEQ ID NO: 958 is shown in Table 237. [Table 237]

[0359] In certain embodiments, this isolated nucleic acid molecule comprises a nucleic acid having the sequence described in Sequence ID No. 959 provided below: The resulting nucleic acid molecule encodes a CAR (named 117 FcRL5-targeted BBz CAR) that contains a fully human scFv (encoded by nucleotides 207-1025 of SEQ ID NO: 959), comprising a heavy chain variable region containing amino acids having the sequence described in SEQ ID NO: 268, a light chain variable region containing amino acids having the sequence described in SEQ ID NO: 267, a linker positioned between the heavy chain variable region and the light chain variable region having the amino acid sequence of SEQ ID NO: 307, a transmembrane domain containing a CD8 polypeptide having amino acids 137-207 of SEQ ID NO: 960, and an intracellular domain containing a co-stimulatory signaling region containing a CD3ξ polypeptide having amino acids 52-163 of SEQ ID NO: 941 and a 4-1BB polypeptide having amino acids 214-255 of SEQ ID NO: 943. Nucleotides 207-1025 of SEQ ID NO: 959 encode a human scFv. Nucleotides 1035-1247 of SEQ ID NO: 959 encode a human scFv. Nucleotides 1035-1247 of SEQ ID NO: 959 encode a CD8 polypeptide contained in the transmembrane domain. Nucleotides 1248–1373 of SEQ ID NO: 959 encode the 4-1BB polypeptide contained within the intracellular domain. Nucleotides 1374–1712 of SEQ ID NO: 959 encode the CD3 zeta polypeptide contained within the intracellular domain. The rest of SEQ ID NO: 959 is shown in Table 238. [Table 238]

[0360] In certain embodiments, the isolated nucleic acid molecule encodes a functional moiety of the FcRL5-targeted CAR of the Disclosure. As used herein, the term “functional moiety” means any portion, part, or fragment of the FcRL5-targeted CAR of the Disclosure, which retains the biological activity of the FcRL5-targeted CAR (parent CAR). For example, the functional moiety includes a portion, part, or fragment of the FcRL5-targeted CAR of the Disclosure that retains, to a similar, identical, or even greater degree than, the parent CAR, the ability to recognize target cells, treat diseases, such as multiple myeloma. In certain embodiments, isolated nucleic acid molecules encoding a functional portion of a CAR targeted by FcRL5 of the present disclosure may encode a protein containing, for example, about 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95%, or more, of the parent CAR, e.g., the nucleic acid sequence described in SEQ ID NO: 951, SEQ ID NO: 952, SEQ ID NO: 953, SEQ ID NO: 954, SEQ ID NO: 955, SEQ ID NO: 956, SEQ ID NO: 957, SEQ ID NO: 958, or SEQ ID NO: 959. III. Immune-responding cells

[0361] The subject of this disclosure provides immune response cells expressing a CAR comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular domain, wherein the extracellular antigen-binding domain specifically binds to FcRL5 (e.g., human FcRL5) as described above. In certain embodiments, the extracellular antigen-binding domain specifically binds to domain 7 of FcRL5. In certain embodiments, the extracellular antigen-binding domain specifically binds to domain 8 of FcRL5. In certain embodiments, the extracellular antigen-binding domain specifically binds to domain 9 of FcRL5. Immune response cells can be transduced with the CAR of this disclosure so that the cells express the CAR. The subject of this disclosure also provides methods for using such cells for the treatment of tumors, for example, multiple myeloma (MM). The immune response cells of the subject of this disclosure may be lymphoid cells. Lymphoid cells, including B, T, and natural killer (NK) cells, provide functions such as antibody production, control of the cellular immune system, detection of foreign substances in the blood, and detection of cells that are exogenous to the host. Non-exclusive examples of lymphoid immune response cells include T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, embryonic stem cells, and pluripotent stem cells (e.g., those from which lymphocytes can differentiate). T cells may be lymphocytes that mature in the thymus and are primarily involved in cellular immunity. T cells are involved in the adaptive immune system. The T cells of the subject of this disclosure are not limited to helper T cells, cytotoxic T cells, memory T cells (central memory T cells, stem cell-like memory T cells (or stem-like memory T cells) and two types of effector memory T cells: e.g., T EM Cells and T EMRAThe T cells may be any type of T cell, including regulatory T cells (also known as suppressor T cells), natural killer T cells, mucosa-associated invariant T cells, and γδ T cells. In certain embodiments, CAR-expressing T cells express Foxp3 to achieve and maintain a regulatory T phenotype. Natural killer (NK) cells are lymphocytes that are part of cellular immunity and act in the innate immune response. NK cells do not require prior activation to exert their cytotoxic effects on target cells. Cytotoxic T cells (CTLs or killer T cells) are a subset of T lymphocytes that can induce the death of infected somatic or tumor cells.

[0362] The immune response cells of the subject of this disclosure can express an extracellular antigen-binding domain (e.g., scFV, optionally crosslinked Fab, or F(ab)2) that specifically binds to FcRL5 (e.g., human FcRL5) for the treatment of multiple myeloma. Such immune response cells may be administered to subjects (e.g., human subjects) that require it for the treatment of multiple myeloma. In certain embodiments, the immune response cells are T cells. T cells are CD4 + T cells or CD8 + It may be a T cell. In certain embodiments, the T cell is CD4 + These are T cells. In another embodiment, T cells are CD8 + These are T cells.

[0363] The immune response cells of this disclosure may be further transduced with at least one costimulatory ligand so that the immune response cells co-express or are induced to co-express an FcRL5-specific CAR and at least one costimulatory ligand. The interaction between the FcRL5-specific CAR and at least one costimulatory ligand provides a non-antigen-specific signal that is important for the full activation of the immune response cells (e.g., T cells). Costimulatory ligands include, but are not limited to, members of the tumor necrosis factor (TNF) superfamily and immunoglobulin (Ig) superfamily ligands. TNF is a cytokine involved in systemic inflammation and stimulates the acute phase response. Its primary role is the regulation of immune cells. Members of the TNF superfamily share a number of common properties. The majority of TNF superfamily members are synthesized as type II transmembrane proteins (extracellular C-terminus) containing a short cytoplasmic segment and a relatively long extracellular region. The TNF superfamily members include, non-limitingly, neurotrophic growth factor (NGF), CD40L (CD40L) / CD154, CD137L / 4-1BBL, TNF-α, CD134L / OX40L / CD252, CD27L / CD70, Fas ligand (FasL), CD30L / CD153, tumor necrosis factor beta (TNFβ) / lymphotoxin alpha (LTα), lymphotoxin beta (LTβ), CD257 / B cell activator (BAFF) / Blys / THANK / Tall-1, glucocorticoid-induced TNF receptor ligand (GITRL), and TNF-related apoptosis-inducing ligand (TRAIL), LIGHT (TNFSF14). The immunoglobulin (Ig) superfamily is a large group of cell surface and soluble proteins involved in cell recognition, binding, or adhesion processes. These proteins share structural characteristics with immunoglobulins—they possess an immunoglobulin domain (fold). Immunoglobulin superfamily ligands include, but are not limited to, CD80 and CD86, both ligands for CD28, and PD-L1 / (B7-H1), ligand for PD-1.In certain embodiments, at least one co-stimulatory ligand is selected from the group consisting of 4-1BBL, CD80, CD86, CD70, OX40L, CD48, TNFRSF14, PD-L1, and combinations thereof. In certain embodiments, immune response cells are transduced with one co-stimulatory ligand, which is 4-1BBL. In certain embodiments, immune response cells are transduced with two co-stimulatory ligands, which are 4-1BBL and CD80. A CAR transduced with at least one co-stimulatory ligand is described in U.S. Patent No. 8,389,282, which is incorporated in its entirety by reference.

[0364] Furthermore, the immune response cells of this disclosure may be further transduced with at least one cytokine so that the immune response cells secrete at least one cytokine and express an FcRL5-specific CAR. In certain embodiments, at least one cytokine is selected from the group consisting of IL-2, IL-3, IL-6, IL-7, IL-11, IL-12, IL-15, IL-17, and IL-21. In certain embodiments, the cytokine is IL-12.

[0365] FcRL5-specific or FcRL5-targeted human lymphocytes are peripheral donor lymphocytes, for example, Sadelain, M et al., 2003 Nat Rev Cancer Vol. 3: pp. 35-45 (disclosing peripheral donor lymphocytes genetically modified to express CAR), and Morgan, RA et al., 2006 Science Vol. 314: pp. 126-129 (α- In a report disclosing peripheral donor lymphocytes genetically modified to express a full-length tumor antigen-recognizing T cell receptor complex containing β heterodimers, Panelli, MC et al., 2000 J Immunol 164:495-504 Panelli, MC et al. 2000 J Immunol Volume 164: Pages 4382-4392 (Phase filtration lymphocytes (TILs) derived from tumor biopsy) (Disclosing Pacyl cultures), and Dupont, J et al., 2005 Cancer Res vol. 65: pp. 5417-5427, Papanicolaou, GA et al., 2003 Blood vol. 102: 2 This may be used in the materials disclosed on pages 498-2505 (which disclose antigen-specific peripheral blood leukocytes selectively grown in vitro using artificial antigen-presenting cells (AAPCs) or pulsed dendrites). The immune response cells (e.g., T cells) may be self, non-self (e.g., allogeneic), or may be induced in vitro from engineered progenitor or stem cells.

[0366] In certain embodiments, immune response cells (e.g., T cells) of the Disclosure express vectors with approximately 1 to 4 copies, 2 to 4 copies, 3 to 4 copies, 1 to 2 copies, 1 to 3 copies, or 2 to 3 copies per cell of the FcRL5-specific CAR of the Disclosure.

[0367] The unpurified source of CTLs may be any known in the art, such as bone marrow, fetus, neonatal or adult, or other hematopoietic cell sources, e.g., fetal liver, peripheral blood, or umbilical cord blood. Various techniques may be used to isolate the cells. For example, negative selection methods can remove non-CTLs first. Monoclonal antibodies are particularly useful for both positive and negative selection to identify markers associated with specific cell lineages and / or stages of differentiation.

[0368] Large populations of well-differentiated cells can be initially removed by relatively coarse separation. For example, magnetic bead separation can be used first to remove a large number of irrelevant cells. Preferably, at least about 80%, and usually at least 70%, of all hematopoietic cells are removed prior to cell isolation.

[0369] Procedures for separation include, but are not limited to, density gradient centrifugation, resetting, coupling to particles that alter cell density, magnetic separation with antibody-coated magnetic beads, affinity chromatography, cytotoxic agents including complement and cytotoxic agents used conjugated to or in conjunction with mAbs, as well as panning with antibodies conjugated to a solid matrix such as a plate or chip, elutriation, or any other convenient technique.

[0370] Techniques for separation and analysis are not limited to these, but include flow cytometry, which may have varying degrees of sophistication, such as multiple color channels, small-angle and obtuse-angle light scattering detection channels, and impedance channels.

[0371] Cells can be selected for dead cells by using dead cell-associated dyes such as propidium iodide (PI). Preferably, the cells are collected in a medium containing 2% fetal bovine serum (FCS) or 0.2% bovine serum albumin (BSA) or any other suitable, preferably sterile, isotonic medium. IV. Vectors

[0372] Genetic modification of immune response cells (e.g., T cells, CTL cells, NK cells) can be achieved by transducing recombinant DNA or RNA constructs into a substantially homogeneous cell composition. The vector may be a retroviral vector (e.g., a gamma retrovirus) used for introducing DNA or RNA constructs into the host cell genome. For example, a polynucleotide encoding an FcRL5-specific CAR may be cloned into a retroviral vector, and its expression may be driven from its endogenous promoter, from a retroviral terminal repeat sequence, or from an alternative internal promoter.

[0373] Nonviral vectors or RNA may also be used. Random or targeted integration into chromosomes (e.g., using nucleases, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), and / or clustered, regularly arranged short palindromic sequence repeats (CRISPR)), or transgene expression (e.g., using natural or chemically modified RNA) may be used.

[0374] For the initial genetic modification of cells to provide FcRL5-specific CAR-expressing cells, retroviral vectors are commonly used for transduction; however, any other suitable viral vector or nonviral delivery system may be used. For subsequent genetic modification of cells to provide cells containing an antigen-presenting complex with at least two costimulatory ligands, retroviral gene transfer (transduction) has proven equally effective. A combination of retroviral vector and a suitable packaging system is also preferable, and the capsid protein is functional for infecting human cells. Various bispecific virus-producing viable cell lines are known, but are not limited to PA12 (Miller et al., (1985) Mol Cell). Biol Vol. 5: pp. 431-437), PA317 (Miller et al., (1986) Mol Cell) Biol Vol. 6: pp. 2895-2902) and CRIP (Danos et al., (1988) Proc (Includes Natl Acad Sci USA Vol. 85: pp. 6460-6464). Non-ambiguous directed particles, Pseudotyped particles with VSVG, RD114, or GALV envelopes, and any other known in the art, are also suitable.

[0375] Possible methods of transduction include, for example, the direct co-culture of viable cells with the method described by Bregni et al. (1992) Blood Vol. 80: pp. 1418-1422, or, for example, Xu et al. (1994) Exp Hemat Vol. 22: pp. 223-230 and Hughes et al. (1992) J Suitable growth factors and polycations according to the method in Clin Invest, Vol. 89, p. 1817 This includes culturing viral supernatant alone or with concentrated vector stocks, with or without the virus.

[0376] Transduction viral vectors may be used to express costimulatory ligands (e.g., 4-1BBL and IL-12) in immune-responding cells. Preferably, the selected vector exhibits highly efficient infection and stable integration and expression (e.g., Cayouette et al.). Human Gene Therapy, Vol. 8: pp. 423-430, 1997, Kido et al., Current Eye Research Vol. 15: pp. 833-844, 1996, Bloomer et al.; Journal of Virology Vol. 71: pp. 6641-6649, 1997, Naldini et al.; Science Vol. 272: pp. 263 See also *Proc Natl Acad Sci USA*, Vol. 94: pp. 10319, 1996 (and Miyoshi et al., *Proc Natl Acad Sci USA*, Vol. 94: pp. 10319, 1997). Other viral vectors that may be used include, for example, adenoviruses, lentiviruses and adeno-associated viral vectors, vaccinia viruses, bovine papillomaviruses, or herpesviruses such as Epstein-Barr virus (e.g., Miller, *Human Gene Therapy*, pp. 15-14, 1990; Friedman, *Science*, Vol. 244: pp. 1275-1281, 1989; Eglitis et al., *BioTechniques*, Vol. 6: pp. 608-614, 1988; Tolstoshev et al., *Current Opinion in Biotechnology*, Vol. 1: pp. 55-61, 1990; Sharp, *The Lancet*, Vol. 337: pp. 1277). ~1278 pages, 1991, Cornetta et al., Nucleic Acid Research and Molecular Biology, Vol. 36: pp. 311-322; 1987, Anderson, Science, Vol. 226: 401 ~409 pages, 1984, Moen, Blood Cells 17:407-416, 1991 See also the vectors in Miller et al., Biotechnology Vol. 7: pp. 980-990, 1989; Le Gal La Salle et al., Science Vol. 259: pp. 988-990, 1993; and Johnson, Chest Vol. 107: 77S-83S, 1995. Retrovirus vectors It is particularly well-developed and used in clinical settings (Rosenberg et al., N Engl J Med 323:370, 1990; Anderson et al., U.S. Patent No. 5,399,346).

[0377] In certain non-limiting embodiments, the vector expressing the FcRL5-targeted CAR of this disclosure is a retroviral vector, such as a 293galv9 retroviral vector.

[0378] Nonviral methods can also be used for protein expression in cells. For example, nucleic acid molecules can be expressed by administering nucleic acids in the presence of lipofection (Feigner et al., Proc Nat'l Acad Sci USA vol. 84: 7413, 1987; Ono et al., Neuroscience Letters). (Vol. 17:p. 259, 1990, Brigham et al., Am J Med Sci; Vol. 298:p. 278, 1989, Staubinger et al., Methods in Enzymology; Vol. 101:p. 512, 1983), Asialolosomucoid polylysine conjugation (Wu et al., Journal of Biological Chemistry; Vol. 263:p. 14621, 1988, Wu et al., Journal of Biological Chemistry) Cells can be introduced by microinjection under surgical conditions (Chemistry vol. 264: p. 16985, 1989) or (Wolff et al., Science vol. 247: p. 1465, 1990). Other nonviral means for gene transfer include in vitro transfection using calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes may also be potentially beneficial for DNA delivery to cells. Transplantation of normal genes into the affected tissue of interest may also be achieved by transferring normal nucleic acids to ex vivo cultureable cell types (e.g., autologous or heterologous primary cells or their progeny), after which the cells (or their offspring) are injected into the targeted tissue or systemically. Recombinant receptors may also be induced or obtained using transposases or targeted nucleases (e.g., Zn finger nucleases, meganucleases, or TALE nucleases). Transient expression can be obtained by RNA electroporation.

[0379] cDNA expression for use in polynucleotide therapies may be directed from any suitable promoter (e.g., human cytomegalovirus (CMV), Simianvirus 40 (SV40), or metallothionein promoter) and controlled by any suitable mammalian regulatory element or intron (e.g., elongation factor 1α enhancer / promoter / intron structure). For example, enhancers known to preferentially direct gene expression in a particular cell type may be used to direct nucleic acid expression. The enhancers used may, non-limitingly, include those characterized as tissue or cell-specific enhancers. If a genomic clone is used as an alternative therapeutic construct, control may be mediated by a congeneral regulatory sequence or, optionally, by a regulatory sequence from a heterogeneous source containing any of the promoters or regulatory elements described above.

[0380] The resulting cells may be grown under the same conditions as those for unmodified cells, thereby increasing the number of modified cells which can then be used for various purposes. V. Polypeptides, analogues, and polynucleotides

[0381] Also included in the subject matter of this disclosure are polypeptides or fragments thereof that have been modified in a manner that enhances their antitumor activity when expressed in immune-responding cells, such as FcRL5 (e.g., human FcRL5) (e.g., scFv such as antibodies F56 and F119, Fab or (Fab)2), CD3ζ, CD8, CD28, and extracellular antigen-binding domains that specifically bind to the polynucleotides encoding them. In certain embodiments, the subject matter of this disclosure further provides polypeptides or fragments thereof that have been modified in a manner that enhances their antitumor activity when expressed in immune-responding cells, such as domain 9FcRL5 (e.g., domain 7, domain 8 or domain 9 of human FcRL5) (e.g., scFv, Fab or (Fab)2), CD3ζ, CD8, CD28, and extracellular antigen-binding domains that specifically bind to the polynucleotides encoding them.

[0382] The subject matter of this disclosure provides a method for optimizing an amino acid sequence or nucleic acid sequence by generating changes in the sequence. Such changes may include specific mutations, deletions, insertions, or post-translational modifications. The subject matter of this disclosure further includes analogues of any naturally occurring polypeptide of the subject matter of this disclosure. The analogues may differ from the naturally occurring polypeptide of the subject matter of this disclosure by amino acid sequence differences, by post-translational modifications, or both. Analogues of the subject matter of this disclosure may generally exhibit at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more identity with all or part of the naturally occurring amino acid sequence of the subject matter of this disclosure. The length of the sequence comparison is at least 5, 10, 15, 20, 25, 50, 75, 100, or more amino acid residues. Again, in an exemplary approach to determining the degree of identity, the BLAST program is e-3 from e -100 The probability scores between them can be used to indicate closely related sequences. Modifications include chemical derivatization of polypeptides in vivo and in vitro, e.g., acetylation, carboxylation, phosphorylation, or glycosylation; such modifications may occur during polypeptide synthesis or processing, or after treatment with isolated modified enzymes. Analogues may differ from naturally occurring polypeptides of the subject of this disclosure by changes in the primary sequence. These include both natural and derived genetic variants (e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual (2nd edition), CSH Press, 19 In 1989, or Ausubel et al., irradiation or ethanemethyl sulfate as described above (Resulting from random mutagenesis due to exposure to or site-directed mutagenesis). Similarly included are cyclized peptides, molecules, and analogues containing residues other than L-amino acids, such as D-amino acids or naturally occurring or synthetic amino acids, such as beta (β) or gamma (γ) amino acids.

[0383] In addition to full-length polypeptides, the subject of this disclosure also provides fragments of any one polypeptide or peptide domain of the subject of this disclosure. The fragments may consist of at least 5, 10, 13, or 15 amino acids. In certain embodiments, the fragments may consist of at least 20 neighboring amino acids, at least 30 neighboring amino acids, or at least 50 neighboring amino acids. In certain embodiments, the fragments may consist of at least 60 to 80, 100, 200, 300, or more neighboring amino acids. Fragments of the subject of this disclosure may be generated by methods known to those skilled in the art, or may arise from conventional protein processing (e.g., removal of amino acids not required for biological activity from nascent polypeptides or removal of amino acids by alternative mRNA splicing or alternative protein processing events).

[0384] Non-protein analogs have a chemical structure designed to mimic the functional activity of the protein of the present invention. Such analogs are administered by methods of the subject matter of this disclosure. Such analogs may exceed the physiological activity of the original polypeptide. Methods for designing analogs are well known in the art, and the synthesis of analogs may be carried out by such methods, by modifying the chemical structure such that the resulting analog, when expressed in immune response cells, increases the antineoplastic activity of the original polypeptide. These chemical modifications include, but are not limited to, substituting alternative R groups of the reference polypeptide and altering the degree of saturation at specific carbon atoms. Protein analogs may be relatively resistant to in vivo degradation, resulting in a further extension of the therapeutic effect upon administration. Assays for measuring functional activity include, but are not limited to, those described in the following examples.

[0385] In accordance with the subject matter of this disclosure, polynucleotides encoding extracellular antigen-binding domains that specifically bind to FcRL5 (e.g., scFV (e.g., scFv derived from antibodies F56 and F119), Fab, Fab' or (Fab')2), CD3ζ, CD8, CD28) may be modified by codon optimization. Codon optimization can alter both naturally occurring and recombinant gene sequences to achieve the highest possible level of production in any given expression system. Factors involved in various stages of protein expression include codon compatibility, mRNA structure, and various cis elements of transcription and translation. Any suitable codon optimization methods or techniques known to those skilled in the art, including but not limited to OPTIMUMGENE®, Encor optimization, and Blue Heron, may be used to modify the polynucleotides of the subject matter of this disclosure. VI. Administration

[0386] The FcRL5-specific CARs and immune response cells expressing them of the subject matter of this disclosure may be provided systemically or directly to a subject to treat or prevent neoplasms. In certain embodiments, the FcRL5-specific CARs and immune response cells expressing them are injected directly into the organ of interest (e.g., the organ affected by the neoplasm). Alternatively or additionally, the FcRL5-specific CARs and immune response cells expressing them may be provided indirectly to the organ of interest, for example, by administration into the circulatory system (e.g., tumor vascular structure). Growth and differentiation agents may be provided before, during, or after administration of cells and compositions to increase T cell production in vitro or in vivo.

[0387] The FcRL5-specific CARs and immune response cells expressing them, which are the subject of this disclosure, can be administered normally intravascularly in any physiologically acceptable vehicle, but they may also be introduced into bone or other favorable sites (e.g., the thymus) where cells can find a suitable site for regeneration and differentiation. Typically, at least 1 × 10⁶ cells 5 Individual doses are administered. Ultimately, 1 × 10 10or exceeding that level. The cell population containing immune response cells expressing FcRL5-specific CARs may include a purified population of cells. Those skilled in the art can easily determine the percentage of immune response cells in the cell population using various well-known methods such as fluorescent cell sorting (FACS). The purity range of the cell population containing genetically modified immune response cells expressing FcRL5-specific CARs may be about 50% to about 55%, about 55% to about 60%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100%. The dosage can be easily adjusted by those skilled in the art (for example, a decrease in purity may require an increase in the dosage). The immune response cells may be introduced by injection, catheter, etc. Factors including interleukins, such as IL-2, IL-3, IL-6, IL-11, IL-7, IL-12, IL-15, IL-21 and other interleukins, colony-stimulating factors such as G-, M-, and GM-CSF, and interferons, such as γ-interferon, may also be included, although this is not limited to these.

[0388] The compositions of the subject matter of this disclosure include pharmaceutical compositions comprising immune response cells expressing an FcRL5-specific CAR and a pharmaceutically acceptable carrier. Administration may be autologous or non-autologous. For example, immune response cells expressing an FcRL5-specific CAR and compositions comprising them may be obtained from one subject and administered to the same or different, suitable subjects. Peripheral blood-derived T cells or their progeny (e.g., in vivo, ex vivo, or in vitro-derived) of the subject matter of this disclosure may be administered via catheter administration, systemic injection, local injection, intravenous injection, or local injection, including parenteral administration. When administering the pharmaceutical compositions of the subject matter of this disclosure (e.g., pharmaceutical compositions comprising immune response cells expressing an FcRL5-specific CAR), they may be formulated into injectable unit dose forms (solutions, suspensions, emulsions). VII. Preparations

[0389] In general, the subject matter of this disclosure, immune response cells expressing FcRL5-specific CARs and compositions comprising them, may be conveniently provided as sterile liquid preparations, such as isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may be buffered to a selected pH. Liquid preparations are generally easier to prepare than gels, other viscous compositions, and solid compositions. In addition, liquid compositions are somewhat more convenient for administration by injection. Viscous compositions, on the other hand, may be formulated within a range of viscosity suitable to provide longer contact times with specific tissues. Liquid or viscous compositions may include a carrier, which may be a solvent or dispersion medium containing, for example, water, physiological saline, phosphate-buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.) and suitable mixtures thereof.

[0390] Sterile injectable solutions can be prepared by incorporating a composition containing immune response cells expressing FcRL5-specific CARs throughout the subject matter of this disclosure into a suitable solvent in the required amount, which may optionally contain various amounts of other components. Such compositions may be mixed with suitable carriers, diluents, or excipients such as sterile water, saline, glucose, or dextrose. The compositions may be lyophilized. Depending on the desired route of administration and preparation, the compositions may contain auxiliary substances such as wetting agents, dispersants or emulsifiers (e.g., methylcellulose), pH buffers, gelling agents or viscosity-enhancing additives, preservatives, flavoring agents, and colorants. Standard texts such as "REMINGTON'S PHARMACEUTICAL SCIENCE," 17th edition, 1985, incorporated herein by reference, may be consulted for preparing suitable preparations without excessive experimentation.

[0391] Various additives that enhance the stability and sterility of the composition may be added, including antimicrobial preservatives, antioxidants, chelating agents, and buffers. Prevention of microbial action can be ensured by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, and sorbic acid. The absorption of injectable pharmaceutical forms can be extended by the use of absorption-delaying agents, such as aluminum monostearate and gelatin. This may be the case. However, according to the present invention, any vehicle, diluent or additive used must be compatible with immune response cells expressing FcRL5-specific CARs, which are the subject matter of this disclosure in general.

[0392] The compositions may be isotonic, that is, they may have the same osmotic pressure as blood and tears. The desired isotonicity of the compositions of the subject matter of this disclosure can be achieved using sodium chloride or other pharmaceutically acceptable agents such as glucose, boric acid, sodium tartrate, propylene glycol, or other inorganic or organic solutes. Sodium chloride is particularly preferred as a buffer containing sodium ions.

[0393] The viscosity of the composition may, if desired, be maintained at a selected level using a pharmaceutically acceptable thickener. Methylcellulose may be used because it is readily and economically available and cooperates easily. Other suitable thickeners include, for example, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, and carbomer. The concentration of the thickener may depend on the selected agent. The important point is to use an amount that achieves the desired viscosity. Clearly, the selection of suitable carriers and other additives depends on the precise route of administration and the specific form of dosing, e.g., liquid dosing form (e.g., whether the composition is formulated in solution, suspension, gel, or another liquid form such as sustained-release or liquid-filled form).

[0394] Those skilled in the art will recognize that the components of a composition should be selected to be chemically inert and will not affect the viability or efficacy of the immune response cells described in the subject matter of this disclosure. This does not present any problems to those skilled in the art in terms of chemical and pharmaceutical principles, or any problems can be readily avoided from this disclosure and the documents referenced herein by reference to standard texts or by simple experiments (not excessive experiments).

[0395] One consideration regarding the therapeutic use of immune response cells of the subject of this disclosure is the amount of cells required to achieve optimal efficacy. The amount of cells administered varies depending on the target being treated. In certain embodiments, approximately 10 immune response cells of the subject of this disclosure are used. 4 From one to about 10 10 pieces, about 10 5 From one to about 10 9 pieces or about 10 6 From one to about 10 8 Individuals are administered to the target. More effective cells may be administered in even smaller quantities. In certain embodiments, at least about 1 × 10⁶ immune response cells of the subject of this disclosure 8 pieces, approximately 2×10 8 pieces, about 3×10 8 pieces, about 4×10 8 pieces and approximately 5 × 10 8 The individual dose is administered to human subjects. The precise determination of what is considered an effective dose may be based on individual subject factors, including the size, age, sex, weight, and condition of the specific subject. The dose can be readily determined by those skilled in the art from the present disclosure and knowledge of the art.

[0396] Those skilled in the art can easily determine the amounts of cells administered in the composition and in the methods of the subject matter of this disclosure, as well as the optimal amounts of additives, vehicles and / or carriers. Typically, any additives (in addition to active cells and / or drugs) are present in solution in amounts ranging from about 0.001% to about 50% by weight in phosphate-buffered saline, and the active ingredients are present in amounts ranging from micrograms to milligrams, such as about 0.0001% to about 5% by weight, about 0.0001% to about 1% by weight, about 0.0001% to about 0.05% by weight, about 0.001% to about 20% by weight, about 0.01% to about 10% by weight, or about 0.05% to about 5% by weight. For any composition administered to animals or humans, and for any specific method of administration, toxicity should be determined by determining the lethal dose (LD) and LD50 in a suitable animal model, such as a rodent like a mouse, as well as the dosage of a composition(s) that elicits a suitable response, the concentration of its components, and the timing of administration of the composition(s). Such determinations do not require excessive experimentation by those skilled in the art, based on knowledge from this disclosure and the documents referenced herein. The timing of sequential administration can also be determined without excessive experimentation. VIII. Treatment Methods

[0397] Tumor microenvironment. Tumors have a microenvironment that is hostile to the host immune response, involving a set of mechanisms by malignant cells that protect themselves from immune recognition and elimination. This “hostile tumor microenvironment” is invasive regulatory CD4 +This includes T cells (Treg), myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), immunosuppressive cytokines including IL-10 and TGF-β, and various immunosuppressive factors including the expression of ligands (CTLA-4 and PD-1) targeted to immunosuppressive receptors expressed by activated T cells. While these mechanisms of immunosuppression play a role in maintaining tolerance and suppressing inappropriate immune responses, in the tumor microenvironment, these mechanisms interfere with effective anti-tumor immune responses. Together, these immunosuppressive factors can induce either significant anergy or apoptosis of CAR-modified T cells that have transitioned to adoptive mode upon encountering targeted tumor cells.

[0398] Challenges in tumor immunology. Effective tumor immunity requires recognition of tumor antigens by immune effector cells and unchallenged tumor elimination. Tumor antigens must contain peptide epitopes that are presented by the tumor and can be recognized by specific cytotoxic T lymphocytes (CTLs). Initially stimulated CTLs must be multiplied to a sufficient number and migrate to the tumor site, where they mature into effectors to exert their function, which is enhanced by helper T cells and attenuated by Tregs and inhibitory macrophages.

[0399] Targeted T-cell therapy with manipulated T lymphocytes. T-cell manipulation is a groundbreaking strategy that could address many of the previously observed shortcomings of early immunotherapy approaches. In recent years, researchers have shown that it can address relapsed disease (Brentjens et al., Blood Vol. 118, pp. 4817-4828 (2011) and Brentjens et al., Science Translational Medicine Vol. 5, 177ra138). (2013), chemotherapy-resistant leukemia and metastatic melanoma (Hunder et al., N Engl JMed 358, pp. 2698-2703 (2008), Rosenberg et al., Nat Rev Cancer) In Volume 8, pp. 299-308 (2008) and in Dudley et al., J Clin Oncol Volume 26, pp. 5233-5239 (2008), we reported dramatic complete remission obtained using autologous peripheral blood T cells targeted to specific antigens (CD19 and NY-ESO-1, respectively).

[0400] Rationale for a genetic approach: Cell engineering can be used to redirect T cells toward tumor antigens and enhance T cell function. One driving force behind genetic T cell modification is the potential to enhance T cell survival and proliferation, as well as to counteract T cell death, anergy, and immunosuppression. Genetic targeting of T cells may be refined to prevent undesirable destruction of normal tissues.

[0401] Chimeric antigen receptors (CARs): Tumor-specific T cells can be generated by the transfer of genes encoding CARs (Brentjens et al., ClinCancer Res vol. 13, pp. 5426-5435 (2007); Gade et al., Cancer Res vol. 65, pp. 9080-9088 (2005); Maher et al., NatBiotechnol vol. 20, pp. 70-75 (2002); Kershaw et al., J Immunol vol. 173, pp. 2143-2150 (2004); Sadelain et al., Curr Opin Immunol (2009) and Hollyman et al., J Immunother Vol. 32, pp. 169-180 (200 9 years). Second-generation CARs include a tumor antigen-binding domain fused to an intracellular signaling domain that can activate T cells, as well as a costimulatory domain designed to improve the potency and persistence of T cells (Sadelain et al., Cancer Discovery Vol. 3, 388-39). (Page 8 (2013)). CAR design can therefore harmonize two physiologically mediated functions, antigen recognition and signal transduction, by two separate complexes, the TCR heterodimer and the CD3 complex. The extracellular antigen-binding domain of CAR is typically derived from a mouse monoclonal antibody (mAb), or a receptor or their ligand. Antigen recognition is therefore not limited to MHC (Riviere et al., Curr Hematol Rep vol. 3, pp. 290-297 (2 (2004) and Stephan et al., Nat Med Vol. 13, pp. 1440-1449 (2007) Therefore, the same CAR can be applied to any patient expressing the target antigen. Antigen binding by the CAR triggers phosphorylation of the immune receptor tyrosine-based activation motif (ITAM) in the intracellular domain, initiating a signaling cascade necessary for inducing cell lysis, cytokine secretion, and proliferation. Because MHC limitation of antigen recognition is bypassed, the function of CAR-targeted T cells is not affected by HLA downregulation or defects in the antigen processing mechanism.

[0402] T cell requirements for proliferation and survival: T cell proliferation of tumor-specific T cells is required ex vivo, but undoubtedly preferable in vivo. T cell proliferation must be accompanied by T cell survival to enable complete T cell proliferation and persistence. In order to proliferate in response to an antigen, T cells must receive two types of signals. One is provided by TCR recognition of the antigen peptide / MHC complex displayed on the surface of antigen-presenting cells (APCs) (Sadelain et al., Curr Opin Immunol (2009)). The other is CD28 or 4-1 This is provided by T cell costimulatory receptors such as the BB receptor. On the other hand, while T cell cytolytic activity does not require simultaneous costimulation, as previously demonstrated, the supply of costimulatory signals is essential to maintain the antitumor function of adoptive T cells (Maher et al., NatBiotechnol vol. 20, pp. 70-75 (2002), Sadelain et al., Cancer discovery). Volume 3, pp. 388-398 (2013), Krause et al., J Exp Med Vol. 188, pp. 619. ~626 pages (1998), Gong et al., Neoplasia Vol. 1, pp. 123-127 (1999) ) and Lyddane et al., Jimmunol Vol. 176, pp. 3306-3310 (2006).

[0403] Immune Monitoring: Lymphocytes are multifunctional "drugs" that exhibit dynamically evolving effects after infusion. Upon encountering antigens, tumor-specific T cells activate and / or release various proteins that can trigger tumor lethality, T cell proliferation, and the recruitment or immunomodulation of other immune cells. Therefore, measuring which proteins are secreted from which cells, in what quantities, and at what time provides deep insights into why specific patients respond or do not, and offers crucial feedback for designing more effective clinical trials. These assay systems enable direct and meaningful comparisons of clinical approaches, thereby helping to design rational, next-generation therapeutic strategies.

[0404] The amount administered for treatment is an effective amount to produce the desired effect. The effective amount may be delivered in a single dose or a series of doses. The effective amount may be delivered by a bolus or by continuous perfusion.

[0405] An effective dose (or therapeutic effective dose) is the amount sufficient to act on a beneficial or desirable clinical outcome in a treatment. An effective dose may be administered to a subject in one or more doses. In a treatment, an effective dose is the amount sufficient to mitigate, reverse, stabilize, reverse or slow the progression of a disease, or otherwise reduce the pathological consequences of the disease. The effective dose is generally determined on a case-by-case basis by a physician and is within the scope of the skill of a person skilled in the art. Several factors are typically considered when determining a suitable dose to achieve an effective dose. These factors include the age, sex, and weight of the subject, the condition being treated, the severity of the condition, and the morphology and effective concentration of immune response cells being administered.

[0406] Approximately 10 adoptive immunotherapy using antigen-specific T cells 6 From one to about 10 10 A range of (for example, about 10) 9 A typical dose of (1) cells is injected. Administration of immune response cells to a subject and subsequent differentiation induces immune response cells that are specifically directed to one particular antigen (e.g., FcRL5). T cell "induction" may include inactivation of antigen-specific T cells, such as by deletion or anergy. Inactivation is particularly useful for establishing or re-establishing tolerance in autoimmune disorders, etc. The immune response cells of the subject of this disclosure may be administered by any method known in the art, including, but not limited to, pleural administration, intravenous administration, subcutaneous administration, intranodal administration, intratumoral administration, subarachnoid administration, intrapleural administration, intraperitoneal administration, and direct administration to the thymus. In certain embodiments, immune response cells and compositions comprising them are administered intravenously to the subject requiring them.

[0407] The subject matter of this disclosure provides various methods of using immune response cells (e.g., T cells) expressing FcRL5-specific CARs. For example, the subject matter of this disclosure provides a method for reducing tumor burden in a subject. In one non-limiting example, a method for reducing tumor burden includes administering an effective amount of the immune response cells of this disclosure to a subject, thereby inducing tumor cell death in the subject. The immune response cells of this disclosure can reduce the number of tumor cells, reduce tumor size, and / or eradicate the tumor in the subject. Non-limiting examples of suitable tumors include multiple myeloma, non-Hodgkin lymphoma (particularly mantle cell), Hodgkin lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, Burket lymphoma, and Waldenström macroglobulinemia.

[0408] The subject matter of this disclosure also provides methods for increasing or extending the survival of subjects having neoplasms. In one non-limiting example, a method for increasing or extending the survival of a subject having neoplasms includes administering an effective amount of the immune response cells of this disclosure to the subject, thereby increasing or extending the survival of the subject. The method can reduce or eradicate the tumor burden in the subject. The subject matter of this disclosure further provides methods for treating or preventing neoplasms in a subject, including administering the immune response cells of this disclosure to the subject.

[0409] As used herein, the term “neoplasm” refers to a disease characterized by the pathological proliferation of cells or tissues and their subsequent migration or invasion into other tissues or organs. Neoplasmic proliferation is typically unregulated, progressive, and occurs under conditions that do not induce or cause quiescence of normal cell proliferation. Neoplasms may affect a variety of cell types, tissues, or organs, including, but are not limited to, organs selected from the group consisting of the bladder, colon, bone, brain, breast, cartilage, glia, esophagus, fallopian tubes, gallbladder, heart, intestines, kidneys, liver, lungs, lymph nodes, nerve tissue, ovaries, pleura, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid, trachea, urogenital tract, ureters, urethra, uterus, and vagina or their tissues or cell types. Neoplasms include cancers such as sarcomas, carcinomas, or plasmacytomas (malignant tumors of plasma cells).

[0410] Cancers whose growth can be inhibited using immune response cells of the subject matter of this disclosure typically include cancers that respond to immunotherapy. Non-limiting examples of cancers to be treated include multiple myeloma, non-Hodgkin lymphoma (particularly mantle cell), Hodgkin lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, Burket lymphoma, and Waldenström macroglobulinemia. In certain embodiments, the cancer is multiple myeloma.

[0411] In addition, the subject matter of this disclosure provides a method for increasing the production of immune-activating cytokines in response to cancer cells in a subject. In one non-limiting example, the method includes administering immune-responsive cells of this disclosure to a subject. The immune-activating cytokines may be granulocyte-macrophage colony-stimulating factor (GM-CSF), IFN-α, IFN-β, IFN-γ, TNF-α, IL-2, IL-3, IL-6, IL-11, IL-7, IL-12, IL-15, IL-21, interferon regulator 7 (IRF7), and combinations thereof. In certain embodiments, immune-responsive cells containing FcRL5-specific CARs of the subject matter of this disclosure increase the production of GM-CSF, IFN-γ, and / or TNF-α.

[0412] Human subjects suitable for treatment typically include two treatment groups that can be identified by clinical criteria. Subjects with “progressive disease” or “high tumor burden” are those who have a clinically measurable tumor (e.g., multiple myeloma). A clinically measurable tumor is one that is detectable based on tumor volume (e.g., by palpation, CAT scan, ultrasound imaging, mammography, or radiography; positivity of biochemical or histopathological markers of the tumor itself is insufficient to identify this population). The pharmaceutical compositions exemplified in the subject matter of this disclosure are administered to these subjects to induce an antitumor response for the purpose of alleviating their condition. Ideally, a reduction in tumor volume results, but any clinical improvement constitutes a benefit. Clinical improvement includes a reduction in the risk or rate of progression of the tumor (e.g., multiple myeloma) or a reduction in pathological outcomes.

[0413] A second group of suitable subjects is known in the art as the “adjuvant group.” These are individuals with a history of neoplasms (e.g., multiple myeloma) who have responded to other forms of treatment. Prior treatments include, but are not limited to, surgical resection, radiotherapy, and conventional chemotherapy. As a result, these individuals do not have clinically measurable tumors. However, they are suspected to be at risk of disease progression, either near the original tumor site or through metastasis. This group can be further subdivided into high-risk and low-risk individuals. Subdivision is based on characteristics observed before and after the initial treatment. These characteristics are known in the clinical field and are appropriately defined for each different neoplasm. Typical characteristics of the high-risk subgroup are that the tumor (e.g., multiple myeloma) has invaded neighboring tissues or shows lymph node involvement. Another group consists of individuals with a genetic predisposition to neoplasms (e.g., multiple myeloma) but who have not yet shown clinical signs of neoplasms (e.g., multiple myeloma). For example, a woman who has tested positive for a gene mutation associated with breast cancer but is still of childbearing age and who wishes to receive one or more antigen-binding fragments described herein in a prophylactic treatment to prevent the development of a neoplasm until she is suitable to undergo prophylactic surgery.

[0414] Patients may have a progressive form of disease (e.g., multiple myeloma), in which case treatment goals may include reducing or reversing disease progression and / or mitigating side effects. Patients may have a history of previously treated conditions, in which case treatment goals typically include reducing or delaying the risk of relapse.

[0415] Further modifications may be introduced into FcRL5-specific CAR-expressing immune response cells (e.g., T cells) to prevent or minimize the risk of immunological complications (known as "malignant T cell transformation"), such as graft-versus-host disease (GvHD), or when healthy tissue expresses the same target antigens as tumor cells, leading to similar consequences as GvHD. A potential solution to this problem is to manipulate CAR-expressing T cells with a suicide gene. Suitable suicide genes include, but are not limited to, herpes simplex virus thymidine kinase (hsv-tk), the inducible caspase 9 suicide gene (iCasp-9), and the truncated human epidermal growth factor receptor (EGFRt) polypeptide. In certain embodiments, the suicide gene is the EGFRt polypeptide. The EGFRt polypeptide can eliminate T cells by administering an anti-EGFR monoclonal antibody (e.g., cetuximab). EGFRt can be covalently bound to the 3' end of the intracellular domain of the FcRL5-specific CAR. The suicide gene may be contained within a vector containing the nucleic acid encoding the FcRL5-specific CAR of this disclosure. Thus, administration of a prodrug designed to activate the suicide gene during malignant T cell transformation (e.g., GVHD) (e.g., a prodrug (e.g., AP1903 can activate iCasp-9)) induces apoptosis in suicide gene-activated CAR-expressing T cells. IX. Kit

[0416] The subject matter of this disclosure provides kits for the treatment or prevention of neoplasms (e.g., multiple myeloma). In certain embodiments, the kit comprises a therapeutic or prophylactic composition containing an effective amount of immune-responding cells containing an FcRL5-specific CAR in a unit dose form. In specific embodiments, the cells further express at least one co-stimulatory ligand. In certain embodiments, the kit comprises a sterile container containing the therapeutic or prophylactic vaccine; such a container may be a box, ampoule, bottle, vial, tube, bag, pouch, blister container or other suitable container form known in the art. Such a container may be made of plastic, glass, laminated paper, metal foil or other material suitable for holding pharmaceuticals.

[0417] If desired, the immune response cells are provided with instructions for administering the cells to subjects with or at risk of developing a neoplasm (e.g., multiple myeloma). The instructions include general information about the use of the composition for the treatment or prevention of neoplasms (e.g., multiple myeloma). In other embodiments, the instructions include at least one of the following: description of the therapeutic agent; dosing schedule and administration for the treatment or prevention of neoplasms (e.g., multiple myeloma) or its symptoms; precautions for use; warnings; indications; contraindications; overdose information; adverse reactions; animal pharmacological effects; clinical studies; and / or references. The instructions may be printed directly on the container (if any), as a label attached to the container, or on a separate sheet, pamphlet, card, or folder inside or supplied together with the container. X. Exemplary extracellular antigen-binding domain (e.g., scFv) [Table 1]

[0418] [Table 2]

[0419] [Table 3]

[0420] [Table 4]

[0421] [Table 5]

[0422] [Table 6]

[0423] [Table 7]

[0424] Table 8

[0425] Table 9

[0426] Table 10

[0427] Table 11

[0428] Table 12

[0429] Table 13

[0430] Table 14

[0431] Table 15

[0432] Table 16

[0433] Table 17

[0434] Table 18

[0435] Table 19

[0436] Table 20

[0437] Table 21

[0438] Table 22

[0439] Table 23

[0440] Table 24

[0441] Table 25

[0442] Table 26

[0443] Table 27

[0444] Table 28

[0445] Table 29

[0446] Table 30

[0447] Table 31

[0448] Table 32

[0449] Table 33

[0450] Table 34

[0451] Table 35

[0452] Table 36

[0453] Table 37

[0454] Table 38

[0455] Table 39

[0456] Table 40

[0457] Table 41

[0458] Table 42

[0459] Table 43

[0460] Table 44

[0461] Table 45

[0462] Table 46

[0463] Table 47

[0464] Table 48

[0465] Table 49

[0466] Table 50

[0467] Table 51

[0468] Table 52

[0469] Table 53

[0470] Table 54

[0471] Table 55

[0472] Table 56

[0473] Table 57

[0474] Table 58

[0475] Table 59

[0476] Table 60

[0477] Table 61

[0478] Table 62

[0479] Table 63

[0480] Table 64

[0481] Table 65

[0482] Table 66

[0483] Table 67

[0484] Table 68

[0485] Table 69

[0486] [Table 70]

[0487] [Table 71]

[0488] [Table 72]

[0489] [Table 73]

[0490] [Table 74]

[0491] [Table 75]

[0492] [Table 76]

[0493] Exemplary extracellular antigen-binding domains (e.g., scFv) including the XI heavy chain variable region, light chain variable region, and linker peptide. [Table 77]

[0494] [Table 78]

[0495] [Table 79]

[0496] Table 80

[0497] Table 81

[0498] Table 82

[0499] Table 83

[0500] Table 84

[0501] Table 85

[0502] Table 86

[0503] Table 87

[0504] Table 88

[0505] Table 89

[0506] Table 90

[0507] Table 91

[0508] Table 92

[0509] Table 93

[0510] Table 94

[0511] Table 95

[0512] Table 96

[0513] Table 97

[0514] Table 98

[0515] Table 99

[0516] Table 100

[0517] Table 101

[0518] Table 102

[0519] Table 103

[0520] Table 104

[0521] Table 105

[0522] Table 106

[0523] Table 107

[0524] Table 108

[0525] Table 109

[0526] Table 110

[0527] Table 111

[0528] Table 112

[0529] Table 113

[0530] Table 114

[0531] Table 115

[0532] Table 116

[0533] Table 117

[0534] Table 118

[0535] Table 119

[0536] Table 120

[0537] Table 121

[0538] Table 122

[0539]

Table 123

[0540] Table 124

[0541] Table 125

[0542] Table 126

[0543] Table 127

[0544] Table 128

[0545] Table 129

[0546] Table 130

[0547] Table 131

[0548] Table 132

[0549] Table 133

[0550] Table 134

[0551] Table 135

[0552] Table 136

[0553] Table 137

[0554] Table 138

[0555] Table 139

[0556] Table 140

[0557] Table 141

[0558] Table 142

[0559] Table 143

[0560] Table 144

[0561] Table 145

[0562] Table 146

[0563] Table 147

[0564] Table 148

[0565] Table 149

[0566] Table 150

[0567] Table 151

[0568] [Table 152]

[0569] XII heavy chain variable region, light chain variable region, linker peptide, and His-tag and HA - Exemplary extracellular antigen-binding domains including tags (e.g., scFv) [Table 153]

[0570] [Table 154]

[0571] [Table 155]

[0572] [Table 156]

[0573] [Table 157]

[0574] [Table 158]

[0575] [Table 159]

[0576] [Table 160]

[0577] Table 161

[0578] Table 162

[0579] Table 163

[0580] Table 164

[0581] Table 165

[0582] Table 166

[0583] Table 167

[0584] Table 168

[0585] Table 169

[0586] Table 170

[0587] Table 171

[0588] Table 172

[0589] Table 173

[0590] Table 174

[0591] Table 175

[0592] Table 176

[0593] Table 177

[0594] Table 178

[0595] Table 179

[0596] Table 180

[0597] Table 181

[0598] Table 182

[0599] Table 183

[0600] Table 184

[0601] Table 185

[0602] Table 186

[0603] Table 187

[0604] Table 188

[0605] Table 189

[0606] Table 190

[0607] Table 191

[0608] Table 192

[0609] Table 193

[0610] Table 194

[0611] Table 195

[0612] Table 196

[0613] Table 197

[0614] Table 198

[0615] Table 199

[0616] Table 200

[0617] Table 201

[0618] Table 202

[0619] Table 203

[0620] Table 204

[0621] Table 205

[0622] Table 206

[0623] Table 207

[0624] Table 208

[0625] Table 209

[0626] Table 210

[0627] Table 211

[0628] Table 212

[0629] Table 213

[0630] Table 214

[0631] Table 215

[0632] Table 216

[0633] Table 217

[0634] Table 218

[0635] Table 219

[0636] Table 220

[0637] Table 221

[0638] Table 222

[0639] Table 223

[0640] Table 224

[0641] Table 225

[0642] Table 226

[0643] Table 227

[0644] Table 228

[0645] Table XIII-1

[0646] Table XIII-2

[0647] Table XIII-3

[0648] Table XIII-4

[0649] [Table XIII-5]

[0650] [Table 230] [Examples]

[0651] The implementation of this invention will utilize, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology that are well within the scope of the art. Such techniques include "Molecular Cloning: A Laboratory Manual," 2nd edition (Sambrook, 1989), "Oligonucleotide Synthesis" (Gait, 1984), and "Animal Cell These techniques are fully described in literature such as "Culture" (Freshney, 1987); "Methods in Enzymology" and "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction" (Mullis, 1994); and "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of polynucleotides and polypeptides of the present invention and can therefore be considered in the preparation and implementation of the present invention. Techniques particularly useful for specific embodiments are discussed in the following sections.

[0652] The following examples are provided to give a complete disclosure and description of how the assays, screenings, and therapeutic methods of the present invention are prepared and used, and are not intended to limit the scope that the inventors consider to be their invention. (Example 1) FcRL5 expression in various tissues

[0653] Human FcRL5 expression was evaluated and assessed in various tissues. As shown in Figure 1, human FcRL5 was highly expressed in lymphoma and multiple myeloma, but not in other tissues. The upper panel of Figure 1 shows differential expression of human FcRL5 in tumor cell lines derived from the Cancer Cell Line Encyclopedia (CCLE). The lower panel of Figure 1 shows differential expression of human FcRL5 in normal tissues derived from BioGPS. As shown in Figure 1, human FcRL5 expression is limited to MM and lymphoma compared to other malignant cells. Normal expression appeared to be limited to B cells and plasma cells. Based on our patient experience with CD19-targeted CAR T cells, eradication of CAR T cells with latent FcRL5 in these normal cell types may not have significant adverse effects. Any deficiency of physiological antibody products can be addressed with intravenous immunoglobulin treatment. (Example 2) Selection of ScFv specific to FcRL5 using a complete human phage display library

[0654] Phage display selection for FcRL5 was performed using a cell panning strategy with 31 human scFv-untreated and semi-synthetic phage sublibraries. FcRL5-overexpressing 3T3 cells were used for positive panning, and FcRL1, 2, 3, 4, and 6-overexpressing 3T3 cells (a total of 5 cell lines) were used for negative panning (Figure 2). Binding clones were then eluted and used to infect with E. coli XL1-Blue. The scFv phage clones expressed in bacteria were purified as previously described (Yasmina et al., Probing the binding mechanism and affinity of tanezumab, a recombinant humanized anti-NGF monoclonal antibody, using a repertoire of biosensors Protein Science). 2008, Vol. 17 (No. 8): pp. 1326-1335, Roberts et al., Vaccination with CD20 peptides induces a biologically active, specific immune response in mice Blood, 2002, Vol. 99 (No. 10): pp. 3748-3755. Panning was performed for approximately 3 to 4 cycles to enrich scFv phage clones that specifically bind to FcRL5. Positive clones were identified by ELISA for His-tagged FcRL5.

[0655] Positive clones were further examined by flow cytometry using FcRL5-overexpressing cell lines, 3T3 and Raji, for their binding to FcRL5 on the surface of viable cells. The cells were then washed and staining was performed using the following steps: Cells could be stained first with purified scFv phage clones, followed by staining with mouse anti-M13 mAb, and finally with equine anti-mouse Ig conjugated to PE. Each staining step was completed on ice for 30–60 minutes, and cells were washed twice between each staining step.

[0656] Seventy-six unique clones specific to FcRL5 were identified and confirmed by screening (see Tables 1-229 and Figure 2). (Example 3) Selection of ScFv specific to domain 9 of FcRL5

[0657] FcRL5 contains nine extracellular immunoglobulin-like domains (domains 1-9) and can exist intracellularly as a soluble isoform, a glycosylphosphatidyl (phosphotidyl) inositol (GPI)-anchored isoform, and a transmembrane isoform (Figure 3A). As shown in Figure 2A, the transmembrane isoform of FcRL5 has domain 9; on the other hand, the soluble isoform and the GPI-anchored isoform do not.

[0658] To examine whether scFv is specific to domain 9 of FcRL5, 76 clones were further screened in 3T3 cells overexpressing a vector encoding FcRL5 lacking domain 9 (FcRL5Δdom9), and then further screened in Raji cells overexpressing full-length FcRL5 (Figures 4B-D). Several clones showed reduced or decreased binding to FcRL5 domain 9-deleted overexpressing 3T3 cells compared to binding to FcRL5-overexpressing 3T3 cells. Figures 4, 5, 6, 7, and 8 show the specificity of ET200-39, ET200-104, ET200-105, ET200-109, and ET200-117 to domain 9 of FcRL5, respectively. (Example 4) Construction of mouse FcRL5-specific CAR

[0659] To generate scFvs targeting human FcRL5, two commercially available mouse hybridomas (Franco (2013), Ise et al. (2005), Ise et al. (2006)) that bind to different extracellular epitopes on human FcRL5 were obtained. Two scFvs targeting human FcRL5 were obtained from these hybridomas. One scFv containing a G4S linker with the amino acid sequence of SEQ ID NO: 897 was described in Ise et al. (2005). As described above, it was generated by synthesizing the heavy and light chain variable regions of the mouse anti-human FcRL5 antibody F56. A second scFv containing a G4S linker having the amino acid sequence of SEQ ID NO: 897 was generated from the mouse anti-human FcRL5 antibody F119 as described by Ise et al. (2005). It was generated by synthesizing the heavy chain and light chain variable regions.

[0660] Two FcRL5 CARs were generated: F56 FcRL5-28z CAR and F119 FcRL5-28z CAR. F56 FcRL5-28z CAR and F119 FcRL5-28z CAR have similar structures, as shown in Figure 9A, for example, each containing a transmembrane domain with a CD28 polypeptide, an intracellular domain with a CD3ξ polypeptide, and a co-stimulatory signaling region with a CD28 polypeptide. The FcRL5-28z CAR contains scFv derived from the antibody F56, and the F119 FcRL5-28z CAR contains scFv derived from the antibody F119. The F56 FcRL5-28z CAR and the F119 FcRL5-28z CAR were cloned into the retroviral vector 293galv9, respectively. Human T cells (unselected (CD4 and CD8) human T cells from healthy donors) were transduced to express either the F56 FcRL5-28z CAR or the F119 FcRL5-28z CAR, respectively. (Example 5) Construction of human FcRL5-specific CARs

[0661] This embodiment discloses the generation of human FcRL5-targeted CARs using the fully human scFv described herein. ET200-31 scFv, ET200-39 scFv, ET200-69 scFv, ET200-104 scFv, ET200-105 scFv, ET200-109 scFv, and ET200-117 scFv were used to generate second-generation FcRL5-targeted CARs, referred to as 31FcRL5 BBz CAR, 39FcRL5 BBz CAR, 69FcRL5 BBz CAR, 104FcRL5 BBz CAR, 105FcRL5 BBz CAR, 109FcRL5 BBz CAR, and 117FcRL5 BBz CAR, each having either a 4-1BB co-stimulatory domain or a CD3ξ polypeptide (see Figures 9B-916). 4-1BB-containing CARs that target FcRL5 have similar structures, for example, as shown in Figure 9B, each containing a CD8a polypeptide transmembrane domain and an intracellular domain containing a co-stimulatory signaling region composed of CD3ξ polypeptide and 4-1BB polypeptide. Each of these FcRL5-targeted CARs was cloned into SFG retroviral vectors, as an example of a 4-1BB-containing CAR vector shown in Figures 10-16. These viral vectors were then transduced into HEK 293galv9 virus-packaging cells to generate stable packaging strains for CAR+ T cell generation. (Example 6) Human FcRL5-specific CAR expression in T cells

[0662] Human primary T cells were transduced using supernatant derived from retrovirus galv9 HEK 293 packaging cells to express the FcRL5-targeted 4-1BBz CAR (generated using anti-FcRL5 ET200-104 scFv). As shown in Figure 17, cell surface expression of the FcRL5-targeted CAR was determined by binding of a PE-conjugate anti-HIS-tagged secondary antibody to recombinant human FcRL5 modified to contain a His tag. Cell surface detection was confirmed by flow cytometry. As shown in Figure 17, the FcRL5-targeted CAR was expressed on transduced T cells. (Example 7) Cytotoxicity of human FcRL5-specific CARs

[0663] The cytotoxic effects of 4-1BB CAR T cells targeted by FcRL5 were analyzed using Raji (Burkett lymphoma cell line) cells transduced to express luciferase and FcRL5 antigen or control GPRC5D (an unrelated antigen). T cells expressing 4-1BB CAR, generated using anti-FcRL5 ET200-69 scFv, were used in these experiments (referred to herein as "αFcRL5-69 BBz"). The number of viable cells was determined by bioluminescence imaging (BLI) after 36 hours of co-culture. As shown in Figure 18, FcRL5-targeted CAR T cells specifically lysed transduced Raji cells expressing luciferase and FcRL5 antigen (Figure 18A), but did not lyse control GPRC5D-expressing Raji cells, which were lysed by GPRC5D-targeted CAR T cells (Figure 18B). (Example 8) Human FcRL5-specific CAR cytokine secretion

[0664] This example describes cytokine secretion by FcRL5-targeted CAR T cells. T cells expressing the 4-1BB CAR, generated using anti-FcRL5 ET200-69 scFv, were used in these experiments. IL-2, INFγ, and TNFα secretion from FcRL5-targeted CAR T cells after 24-hour co-culture on a monolayer of transduced 3T3 cells with either FcRL5 or CD19 was evaluated by Luminex multiplex analysis. As shown in Figure 19, CAR-mediated signaling by FcRL5-targeted CAR T cells induced cytokine secretion consistent with T cell activation. (Example 9) Human FcRL5-specific CAR proliferation

[0665] This example describes the proliferation of FcRL5-targeted CAR T cells upon antigen stimulation. T cells expressing 4-1BB CAR, generated using anti-FcRL5 ET200-69 scFv, were used in these experiments. 500,000 FcRL5 or CD19-targeted CAR+ T cells / ml were placed on a monolayer of 3T3 cells transduced with either FcRL5 or CD19 (3T3-FcRL; 3T3-CD19). After 4 days, CAR+ T cells were stained and counted by flow cytometry with known concentrations of counting microbeads. As shown in Figure 20, antigen stimulation of FcRL5-targeted CAR T cells induced proliferation. FcRL5-targeted CAR T cells co-cultured with 3T3-FcRL5 cells increased 2.9 times more than FcRL5-targeted CAR T cells co-cultured with 3T3-CD19 cells, while control CD19-targeted CAR T cells co-cultured with 3T3-CD19 cells similarly increased 2.5 times more than CD19-targeted CAR T cells co-cultured with 3T3-FcRL5 cells (Figure 20). (Example 10) Epitope mapping of human anti-FcRL5 antibodies

[0666] Two anti-FcRL5 bispecific antibodies, ET200-104 and ET200-117, were analyzed by Pepscan to determine epitope specificity. See Table 239. The target protein is human FcRL5 containing amino acids 1-851 of SEQ ID NO: 899. [Table 239] method

[0667] The principle of CLIPS technology. CLIPS technology structurally fixes peptides into a defined three-dimensional structure. This produces functional mimics of most complex binding sites. CLIPS technology is currently routinely used to shape peptide libraries into single, double, or triple-loop structures, as well as sheets and helix-like folds (Figure 21).

[0668] Detailed combinatorial CLIPS library screening. CLIPS library screening begins with the conversion of up to 10,000 duplicate peptide constructs of the target protein into a library using combinatorial matrix design. On a solid carrier, a matrix of linear peptides is synthesized, which is then shaped into spatially defined CLIPS constructs (Figure 22). Constructs representing both parts of a discontinuous epitope with precise conformation bind to the antibody with high affinity and are detected and quantified. Constructs showing an incomplete epitope bind to the antibody with low affinity, while constructs that do not contain the epitope do not bind at all. Affinity information is used in repeated screening to define the sequence and conformation of the epitope in detail.

[0669] Heatmap analysis. A heatmap is a graphical representation of data where values ​​taken by variables in a two-dimensional map are shown in color. For double-loop CLIPS peptides, such a two-dimensional map can be derived from the independent sequences of the first and second loops. For example, the sequences of 16 CLIPS peptides shown in Figure 24 are a valid rearrangement of four unique subsequences in loop 1 (colored blue in Figure 23) and four unique subsequences in loop 2 (colored green in Figure 23). Thus, the observed ELISA data (colored red in Figure 24A) may be plotted in a 4x4 matrix, where each X coordinate corresponds to the sequence of the first loop and each Y coordinate corresponds to the sequence of the second loop. For example, the observed ELISA values ​​for the CLIPS peptide CLSSERERVEDLFEYECELLTSEPIFHCRQEDC (indicated by the arrow in Figure 23A) may be found in the third column, the third column in Figure 24B (indicated by the arrow and red square). To facilitate further visualization, ELISA values ​​may be replaced with a continuous gradient of colors. In this case, extremely low values ​​are colored green, extremely high values ​​are colored red, and average values ​​are colored black (see Figure 24C). For the example above, the average value is 0.71. Applying this color map to the data matrix shown in Figure 24B yields a color heatmap (see Figure 24D, the original data is also shown for further clarity).

[0670] Peptide synthesis. A library of peptides was synthesized to reconstruct the epitopes of target molecules. This was obtained by bonding an amino-functionalized polypropylene support to a dedicated hydrophilic polymer formulation, followed by the reaction of t-butyloxycarbonyl-hexamethylenediamine (BocHMDA) with dicyclohexylcarbodiimide (DCC) together with N-hydroxybenzotriazole (HOBt), and subsequent cleavage of the Boc group using trifluoroacetic acid (TFA). Standard Fmoc peptide synthesis was used to synthesize peptides on amino-functionalized solid supports using custom-modified JANUS liquid handling stations (Perkin Elmer). Structural mimetic synthesis was performed using Pepscan's dedicated Chemically Linked Peptides on Scaffold (CLIPS) technology. CLIPS technology allows peptides to be structured into single-loop, double-loop, triple-loop, sheet-like folds, helix-like folds, and combinations thereof. CLIPS templates are coupled to cysteine ​​residues. Multiple cysteine ​​side chains in the peptide were coupled to one or two CLIPS templates. For example, a 0.5 mM solution of P2 CLIPS (2,6-bis(bromomethyl)pyridine) was dissolved in ammonium bicarbonate (20 mM, pH 7.8) / acetonitrile (1:3 (v / v)). This solution was added to the peptide array. The CLIPS templates were coupled to two cysteine ​​side chains present in the solid-bound peptides of the peptide array (a 455-well plate with 3 μl wells). The peptide array was completely covered with the solution and gently shaken in the solution for 30 to 60 minutes. Finally, the peptide array was thoroughly w...

Claims

1. A chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular domain that binds to Fc receptor-like 5 (FcRL5), wherein the extracellular antigen-binding domain is (a) A light chain variable region comprising CDR1 containing the amino acid sequence described in SEQ ID NO: 318, CDR2 containing the amino acid sequence described in SEQ ID NO: 319, and CDR3 containing the amino acid sequence described in SEQ ID NO: 419; and a heavy chain variable region comprising CDR1 containing the amino acid sequence described in SEQ ID NO: 411, CDR2 containing the amino acid sequence described in SEQ ID NO: 412, and CDR3 containing the amino acid sequence described in SEQ ID NO: 463; (b) A light chain variable region comprising CDR1 containing the amino acid sequence described in SEQ ID NO: 318, CDR2 containing the amino acid sequence described in SEQ ID NO: 319, and CDR3 containing the amino acid sequence described in SEQ ID NO: 531; and a heavy chain variable region comprising CDR1 containing the amino acid sequence described in SEQ ID NO: 515, CDR2 containing the amino acid sequence described in SEQ ID NO: 516, and CDR3 containing the amino acid sequence described in SEQ ID NO: 517; (c) A light chain variable region comprising CDR1 containing the amino acid sequence described in SEQ ID NO: 533, CDR2 containing the amino acid sequence described in SEQ ID NO: 534, and CDR3 containing the amino acid sequence described in SEQ ID NO: 535; and a heavy chain variable region comprising CDR1 containing the amino acid sequence described in SEQ ID NO: 403, CDR2 containing the amino acid sequence described in SEQ ID NO: 404, and CDR3 containing the amino acid sequence described in SEQ ID NO: 532; (d) A light chain variable region comprising CDR1 containing the amino acid sequence described in SEQ ID NO: 544, CDR2 containing the amino acid sequence described in SEQ ID NO: 448, and CDR3 containing the amino acid sequence described in SEQ ID NO: 545, and a heavy chain variable region comprising CDR1 containing the amino acid sequence described in SEQ ID NO: 411, CDR2 containing the amino acid sequence described in SEQ ID NO: 412, and CDR3 containing the amino acid sequence described in SEQ ID NO: 543; or (e) A light chain variable region comprising CDR1 containing the amino acid sequence described in SEQ ID NO: 571, CDR2 containing the amino acid sequence described in SEQ ID NO: 572, and CDR3 containing the amino acid sequence described in SEQ ID NO: 573, and a heavy chain variable region comprising CDR1 containing the amino acid sequence described in SEQ ID NO: 372, CDR2 containing the amino acid sequence described in SEQ ID NO: 475, and CDR3 containing the amino acid sequence described in SEQ ID NO:

570. CAR, including.

2. A chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular domain that binds to Fc receptor-like 5 (FcRL5), wherein the extracellular antigen-binding domain is (a) A light chain variable region comprising CDR1, CDR2, and CDR3 of the light chain variable region sequence described in Sequence ID No. 143, and a heavy chain variable region comprising CDR1, CDR2, and CDR3 of the heavy chain variable region sequence described in Sequence ID No. 144; (b) A light chain variable region comprising CDR1, CDR2, and CDR3 of the light chain variable region sequence described in Sequence ID No. 215, and a heavy chain variable region comprising CDR1, CDR2, and CDR3 of the heavy chain variable region sequence described in Sequence ID No. 216; (c) A light chain variable region comprising CDR1, CDR2, and CDR3 of the light chain variable region sequence described in Sequence ID No. 219, and a heavy chain variable region comprising CDR1, CDR2, and CDR3 of the heavy chain variable region sequence described in Sequence ID No. 220; (d) A light chain variable region comprising CDR1, CDR2, and CDR3 of the light chain variable region sequence described in SEQ ID NO: 235, and a heavy chain variable region comprising CDR1, CDR2, and CDR3 of the heavy chain variable region sequence described in SEQ ID NO: 236; or (e) A light chain variable region comprising CDR1, CDR2, and CDR3 of the light chain variable region sequence described in Sequence ID No. 267, and a heavy chain variable region comprising CDR1, CDR2, and CDR3 of the heavy chain variable region sequence described in Sequence ID No. 268 CAR, including.

3. A CAR according to claim 1 or 2, (a) The light chain variable region includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence described in SEQ ID NO: 143, and the heavy chain variable region includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence described in SEQ ID NO: 144; (b) The light chain variable region includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence described in SEQ ID NO: 215, and the heavy chain variable region includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence described in SEQ ID NO: 216; (c) The light chain variable region includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence described in SEQ ID NO: 219, and the heavy chain variable region includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence described in SEQ ID NO: 220; (d) The light chain variable region includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence described in SEQ ID NO: 235, and the heavy chain variable region includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence described in SEQ ID NO: 236; or (e) The light chain variable region includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence described in SEQ ID NO: 267, and the heavy chain variable region includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence described in SEQ ID NO:

268. CAR.

4. A CAR according to any one of claims 1 to 3, (a) The light chain variable region comprises the amino acid sequence described in SEQ ID NO: 143, and the heavy chain variable region comprises the amino acid sequence described in SEQ ID NO: 144; (b) The light chain variable region comprises the amino acid sequence described in SEQ ID NO: 215, and the heavy chain variable region comprises the amino acid sequence described in SEQ ID NO: 216; (c) The light chain variable region comprises the amino acid sequence described in SEQ ID NO: 219, and the heavy chain variable region comprises the amino acid sequence described in SEQ ID NO: 220; (d) The light chain variable region comprises the amino acid sequence described in SEQ ID NO: 235, and the heavy chain variable region comprises the amino acid sequence described in SEQ ID NO: 236; or (e) The light chain variable region comprises the amino acid sequence described in SEQ ID NO: 267, and the heavy chain variable region comprises the amino acid sequence described in SEQ ID NO:

268. CAR.

5. A CAR according to any one of claims 1 to 3, (1) The light chain variable region comprises CDR1 containing the amino acid sequence described in SEQ ID NO: 318, CDR2 containing the amino acid sequence described in SEQ ID NO: 319, and CDR3 containing the amino acid sequence described in SEQ ID NO: 419, and the heavy chain variable region comprises CDR1 containing the amino acid sequence described in SEQ ID NO: 411, CDR2 containing the amino acid sequence described in SEQ ID NO: 412, and CDR3 containing the amino acid sequence described in SEQ ID NO: 463; (2) The light chain variable region comprises CDR1 containing the amino acid sequence described in SEQ ID NO: 318, CDR2 containing the amino acid sequence described in SEQ ID NO: 319, and CDR3 containing the amino acid sequence described in SEQ ID NO: 531, and the heavy chain variable region comprises CDR1 containing the amino acid sequence described in SEQ ID NO: 515, CDR2 containing the amino acid sequence described in SEQ ID NO: 516, and CDR3 containing the amino acid sequence described in SEQ ID NO: 517; (3) The light chain variable region comprises CDR1 containing the amino acid sequence described in SEQ ID NO: 533, CDR2 containing the amino acid sequence described in SEQ ID NO: 534, and CDR3 containing the amino acid sequence described in SEQ ID NO: 535, and the heavy chain variable region comprises CDR1 containing the amino acid sequence described in SEQ ID NO: 403, CDR2 containing the amino acid sequence described in SEQ ID NO: 404, and CDR3 containing the amino acid sequence described in SEQ ID NO: 532; (4) The light chain variable region comprises CDR1 containing the amino acid sequence described in SEQ ID NO: 544, CDR2 containing the amino acid sequence described in SEQ ID NO: 448, and CDR3 containing the amino acid sequence described in SEQ ID NO: 545, and the heavy chain variable region comprises CDR1 containing the amino acid sequence described in SEQ ID NO: 411, CDR2 containing the amino acid sequence described in SEQ ID NO: 412, and CDR3 containing the amino acid sequence described in SEQ ID NO: 543; or (5) The light chain variable region comprises CDR1 containing the amino acid sequence described in SEQ ID NO: 571, CDR2 containing the amino acid sequence described in SEQ ID NO: 572, and CDR3 containing the amino acid sequence described in SEQ ID NO: 573, and the heavy chain variable region comprises CDR1 containing the amino acid sequence described in SEQ ID NO: 372, CDR2 containing the amino acid sequence described in SEQ ID NO: 475, and CDR3 containing the amino acid sequence described in SEQ ID NO:

570. CAR.

6. A CAR according to any one of claims 1 to 5, (1) The light chain variable region includes the amino acid sequence described in SEQ ID NO: 143, and the heavy chain variable region includes the amino acid sequence described in SEQ ID NO: 144; (2) The light chain variable region includes the amino acid sequence described in SEQ ID NO: 215, and the heavy chain variable region includes the amino acid sequence described in SEQ ID NO: 216; (3) The light chain variable region includes the amino acid sequence described in SEQ ID NO: 219, and the heavy chain variable region includes the amino acid sequence described in SEQ ID NO: 220; (4) The light chain variable region comprises the amino acid sequence described in SEQ ID NO: 235, and the heavy chain variable region comprises the amino acid sequence described in SEQ ID NO: 236; or (5) The light chain variable region includes the amino acid sequence described in SEQ ID NO: 267, and the heavy chain variable region includes the amino acid sequence described in SEQ ID NO:

268. CAR.

7. The CAR according to any one of claims 1 to 6, further comprising a linker located between the heavy chain variable region and the light chain variable region.

8. The CAR according to claim 7, wherein the linker comprises the amino acid sequence described in SEQ ID NO: 307 or SEQ ID NO:

897.

9. The CAR according to any one of claims 1 to 8, wherein the extracellular antigen-binding domain comprises a single-chain variable fragment (scFv).

10. The CAR according to any one of claims 1 to 9, wherein the extracellular antigen-binding domain comprises human scFv.

11. A CAR according to any one of claims 1 to 10, wherein the extracellular antigen-binding domain comprises the amino acid sequence described in SEQ ID NO: 664, SEQ ID NO: 700, SEQ ID NO: 702, SEQ ID NO: 710, or SEQ ID NO:

726.

12. A CAR according to any one of claims 1 to 11, (1) The extracellular antigen-binding domain comprises the amino acid sequence described in Sequence ID No. 664; (2) The extracellular antigen-binding domain comprises the amino acid sequence described in Sequence ID No. 700; (3) The extracellular antigen-binding domain comprises the amino acid sequence described in SEQ ID NO: 702; (4) The extracellular antigen-binding domain comprises the amino acid sequence described in SEQ ID NO: 710; or (5) The extracellular antigen-binding domain comprises the amino acid sequence described in SEQ ID NO: 726, CAR.

13. The CAR according to any one of claims 9 to 12, wherein the scFv is contained within the fusion protein so as to form the extracellular antigen-binding domain together with a heterologous sequence.

14. The CAR according to any one of claims 1 to 13, wherein the extracellular antigen-binding domain is entirely a human antibody or an antigen-binding fragment thereof.

15. The CAR according to any one of claims 1 to 14, wherein the FcRL5 comprises the amino acid sequence described in Sequence ID No.

899.

16. The CAR according to any one of claims 1 to 15, wherein the extracellular antigen-binding domain binds to an epitope comprising the amino acid sequence described in SEQ ID NO: 964 or SEQ ID NO:

965.

17. The CAR according to any one of claims 1 to 16, wherein the transmembrane domain comprises a CD8 polypeptide, a CD28 polypeptide, a CD3ζ polypeptide, a CD4 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a CTLA-4 polypeptide, a PD-1 polypeptide, a LAG-3 polypeptide, a 2B4 polypeptide, a BTLA polypeptide, a synthetic peptide, or a combination thereof.

18. The CAR according to any one of claims 1 to 17, wherein the transmembrane domain comprises a CD8 polypeptide.

19. The CAR according to any one of claims 1 to 17, wherein the transmembrane domain comprises a CD28 polypeptide.

20. The CAR according to any one of claims 1 to 19, wherein the intracellular domain comprises a CD3ζ polypeptide.

21. The CAR according to any one of claims 1 to 20, wherein the intracellular domain further comprises at least one signal transduction region.

22. The CAR according to claim 21, wherein the at least one signaling region comprises a CD28 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a PD-1 polypeptide, a CTLA-4 polypeptide, a LAG-3 polypeptide, a 2B4 polypeptide, a BTLA polypeptide, a synthetic peptide, or a combination thereof.

23. The CAR according to claim 21 or 22, wherein the at least one signaling region includes at least one co-stimulatory signaling region.

24. The CAR according to claim 23, wherein the at least one co-stimulatory signaling region comprises a CD28 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a DAP-10 polypeptide, or a combination thereof.

25. The CAR according to claim 23 or 24, wherein the at least one co-stimulatory signaling region comprises a CD28 polypeptide.

26. The CAR according to claim 23 or 24, wherein the at least one co-stimulatory signaling region comprises a 4-1BB polypeptide.

27. ​​A CAR according to any one of claims 24 to 26, (a) The transmembrane domain comprises a CD28 polypeptide, the intracellular domain comprises a CD3ζ polypeptide, and the at least one co-stimulatory signaling region comprises a CD28 polypeptide; (b) The transmembrane domain comprises a CD8 polypeptide, the intracellular domain comprises a CD3ζ polypeptide, and the at least one co-stimulatory signaling region comprises a 4-1BB polypeptide; or (c) The transmembrane domain comprises a CD28 polypeptide, the intracellular domain comprises a CD3ζ polypeptide, and the at least one co-stimulatory signaling region comprises a 4-1BB polypeptide. CAR.

28. A nucleic acid molecule encoding a CAR according to any one of claims 1 to 27.

29. The nucleic acid molecule according to claim 28, comprising the nucleotide sequence described in SEQ ID NO: 954, SEQ ID NO: 956, SEQ ID NO: 957, SEQ ID NO: 958, or SEQ ID NO:

959.

30. A vector comprising the nucleic acid molecule according to claim 28 or 29.

31. The vector according to claim 30, which is a γ-retrovirus vector.

32. The vector according to claim 30, which is a lentiviral vector.

33. An immune response cell comprising a CAR according to any one of claims 1 to 27, a nucleic acid molecule according to claim 28 or 29, or a vector according to any one of claims 30 to 32.

34. The immune response cell according to claim 33, transduced with the nucleic acid molecule encoding the CAR.

35. The immune response cell according to claim 33 or 34, wherein the CAR is constitutively expressed on the surface of the immune response cell.

36. The immune response cell according to any one of claims 33 to 35, wherein the immune response cell is further transduced with a nucleic acid molecule encoding the at least one costimulatory ligand so that the immune response cell expresses the at least one costimulatory ligand.

37. The immune response cell according to claim 36, wherein the at least one co-stimulatory ligand is selected from the group consisting of 4-1BBL, CD80, CD86, CD70, OX40L, CD48, TNFRSF14, or a combination thereof.

38. The immune response cell according to any one of claims 33 to 37, wherein the immune response cell is further transduced with a nucleic acid molecule encoding the at least one cytokine so that the immune response cell secretes the at least one cytokine.

39. The immune response cell according to claim 38, wherein the at least one cytokine is selected from the group consisting of IL-2, IL-3, IL-6, IL-7, IL-11, IL-12, IL-15, IL-17, IL-21, and combinations thereof.

40. The immune response cell according to any one of claims 33 to 39, wherein the immune response cell is selected from the group consisting of T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, human embryonic stem cells, lymphocyte precursor cells, T cell-progenitor cells, and pluripotent stem cells that can differentiate into lymphoid cells.

41. The immune response cell according to any one of claims 33 to 40, wherein the immune response cell is a T cell.

42. A T cell comprising a CAR according to any one of claims 1 to 27, a nucleic acid molecule according to claim 28 or 29, or a vector according to any one of claims 30 to 32.

43. A composition for use in reducing tumor burden in a subject, increasing or extending the survival of a subject having a tumor, or in treating a tumor, comprising immune response cells according to any one of claims 33 to 41 or T cells according to claim 42, characterized in that an effective amount of the immune response cells or T cells is administered to the subject.

44. The composition for use according to claim 43, wherein the use reduces the number of tumor cells, reduces the size of the tumor, or eradicates the tumor in the subject.

45. The composition for use according to claim 43 or 44, wherein the tumor is selected from the group consisting of multiple myeloma, non-Hodgkin lymphoma, mantle cell lymphoma, Hodgkin lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, Burkitt lymphoma, and Waldenström macroglobulinemia.

46. The composition for use according to any one of claims 43 to 45, wherein the tumor is multiple myeloma.

47. The composition for use according to any one of claims 43 to 46, wherein the subject is a human.

48. A composition for producing immune response cells that bind to Fc receptor-like 5 (FcRL5), comprising a nucleic acid molecule encoding a CAR according to any one of claims 1 to 27, a nucleic acid molecule according to claim 28 or 29, or a vector according to any one of claims 30 to 32, wherein the composition is introduced into the immune response cells.

49. A pharmaceutical composition comprising an effective amount of immune response cells according to any one of claims 33 to 41 or T cells according to claim 42, and a pharmaceutically acceptable excipient.

50. The pharmaceutical composition according to claim 49, for treating a tumor.

51. The pharmaceutical composition according to claim 50, wherein the tumor is selected from the group consisting of multiple myeloma, non-Hodgkin lymphoma, mantle cell lymphoma, Hodgkin lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, Burkitt lymphoma, and Waldenström macroglobulinemia.

52. The pharmaceutical composition according to any one of claims 49 to 51, wherein the tumor is multiple myeloma.

53. A kit for treating a tumor, comprising an immune response cell according to any one of claims 33 to 41 or a T cell according to claim 42.

54. The kit according to claim 53, further comprising instructions for the use of the immune response cells or T cells for treating a subject having a tumor.

55. The kit according to claim 54, wherein the tumor is selected from the group consisting of multiple myeloma, non-Hodgkin lymphoma, mantle cell lymphoma, Hodgkin lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, Burkitt lymphoma, and Waldenström macroglobulinemia.

56. The kit according to claim 54 or 55, wherein the tumor is multiple myeloma.

57. Use of an effective amount of immune response cells according to any one of claims 33 to 41 or T cells according to claim 42 in the manufacture of a pharmaceutical product for treating a tumor in a subject.

58. Use of a composition comprising an effective amount of immune response cells according to any one of claims 33 to 41 or T cells according to claim 42 in the manufacture of a pharmaceutical for treating a tumor in a subject, wherein the pharmaceutical increases or prolongs the survival of the subject.

59. The use according to claim 57 or 58, wherein the pharmaceutical agent reduces the number of tumor cells, reduces the size of the tumor, or eradicates the tumor in the subject.

60. The use according to any one of claims 57 to 59, wherein the tumor is selected from the group consisting of multiple myeloma, non-Hodgkin lymphoma, mantle cell lymphoma, Hodgkin lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, Burkitt lymphoma, and Waldenström macroglobulinemia.

61. The use according to any one of claims 57 to 60, wherein the tumor is multiple myeloma.

62. The use according to any one of claims 57 to 61, wherein the subject is a human.

63. An antitumor composition comprising an effective amount of immune response cells according to any one of claims 33 to 41 or T cells according to claim 42.

64. The antitumor composition according to claim 63, wherein the tumor is selected from the group consisting of multiple myeloma, non-Hodgkin lymphoma, mantle cell lymphoma, Hodgkin lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, Burkitt lymphoma, and Waldenström macroglobulinemia.

65. The antitumor composition according to claim 63 or 64, wherein the tumor is multiple myeloma.

66. An antitumor composition according to any one of claims 63 to 65, for administration to a human subject.

67. The antitumor composition according to claim 66, wherein administration of the composition to a human subject reduces or eradicates the tumor in the subject.

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