Novel anti-cd19 antibodies

Abstract

The present invention provides antibodies or antigen-binding fragments thereof against CD19, isolated polynucleotides encoding the same, pharmaceutical compositions comprising the same, and uses thereof.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201811100667.3, filed on September 20, 2018, entitled "Novel Anti-CD19 Antibody". The original application claims priority to International Application No. PCT / CN2017 / 102631, filed on September 21, 2017. Invention Field

[0002] This invention relates in general to novel anti-CD19 antibodies and their uses. Background Technology

[0003] CD19 (differentiation cluster 19) is a specially structured cell surface receptor expressed on the surface of B cells, including but not limited to all subtypes of B-cell lymphoma, from indolent to aggressive, as well as B-cell chronic lymphocytic leukemia and non-T-cell acute lymphoblastic leukemia, pre-B cells, B cells in the early developmental stage (i.e., immature B cells), mature B cells undergoing terminal differentiation into plasma cells, and malignant B cells. CD19 is expressed in most pre-B-cell acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma, B-cell chronic lymphocytic leukemia (CLL), lymphoprolymphocytic leukemia, hairy cell leukemia, common acute lymphoblastic leukemia, and some type 0 acute lymphoblastic leukemia (Nadle et al., J. Immunol., 131:244-250 (1983), Loken et al., Blood, 70:1316-1324 (1987), Uckun et al., Blood, 71:13-29 (1988), Anderson et al., Blood, 1984, 63:1424-1433 (1984), Scheuermann, Leuk. Lymphoma, 18:385-397 (1995)). The expression of CD19 on plasma cells further suggests that it can be expressed in differentiated B-cell tumors, such as multiple myeloma, plasmacytoma, and Waldenström tumors (Grossbard et al., Br. J. Haematol., 102:509-15 (1998); Treon et al., Semin. Oncol., 30:248-52 (2003)). CD19 is also one of several recommended targets for immunotherapy. Unlike CD20 (another B-cell surface receptor), CD19 is thought to be expressed at higher levels when bound to anti-CD19 antibodies and is internalized within cells.

[0004] There remains a need for new anti-CD19 antibodies, especially those with superior internalization ability and high binding affinity. Invention Summary

[0005] The articles “a,” “an,” and “described” used throughout this application are hereby used to refer to one or more (i.e., at least one) grammatical objects of the article. For example, “an antibody” refers to one or more antibodies.

[0006] This application provides a novel anti-CD19 monoclonal antibody, its amino acid and nucleotide sequences, and its uses.

[0007] In one aspect, this application provides isolated antibodies or antigen-binding fragments thereof comprising one or more (e.g., 1, 2, or 3) heavy chain complementarity-determining region (CDR) sequences selected from the group consisting of: SEQ ID NO: 1, 2, 3, 7, 8, 9, 13, 14, 15, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 43, 44, 45, 136, 140, and 141, and / or one or more (e.g., 1, 2, or 3) κ light chain CDR sequences selected from the group consisting of: SEQ ID NO: 4, 5, 6, 10, 11, 12, 16, 17, 18, 40, 41, 42, 137, 138, and 139.

[0008] In some embodiments, the antibody or its antigen-binding fragment comprises 1, 2, or 3 heavy chain CDR sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the sequences shown in SEQ ID NO: 1, 2, 3, 7, 8, 9, 13, 37, 38, 39, 43, 44, 45, 136, 140, or 141. In some embodiments, the antibody or its antigen-binding fragment comprises 1, 2 or 3 light chain CDR sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with SEQ ID NO: 4, 5, 6, 10, 11, 12, 16, 17, 18, 40, 41, 42, 137, 138 or 139).

[0009] In some embodiments, the antibody or antigen-binding fragment thereof described in this application includes a heavy chain variable region, wherein the heavy chain variable region is selected from the group consisting of:

[0010] a) Heavy chain variable region, which includes 1, 2 or 3 CDR sequences selected from SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3;

[0011] b) Heavy chain variable region, which includes 1, 2 or 3 CDR sequences selected from SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9;

[0012] c) Heavy chain variable region, which includes 1, 2 or 3 CDR sequences selected from SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15;

[0013] d) Heavy chain variable region, which includes 1, 2 or 3 CDR sequences selected from SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21;

[0014] e) Heavy chain variable region, which includes 1, 2 or 3 CDR sequences selected from SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24;

[0015] f) Heavy chain variable region, which includes 1, 2 or 3 CDR sequences selected from SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:27;

[0016] g) Heavy chain variable region, which includes 1, 2 or 3 CDR sequences selected from SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30;

[0017] h) Heavy chain variable region, which includes 1, 2 or 3 CDR sequences selected from SEQ ID NO:31, SEQ ID NO:32 and SEQ ID NO:33;

[0018] i) Heavy chain variable region, which includes 1, 2 or 3 CDR sequences selected from SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36;

[0019] j) Heavy chain variable region, comprising 1, 2 or 3 CDR sequences selected from SEQ ID NO:37, SEQ ID NO:38 and SEQ ID NO:39;

[0020] k) Heavy chain variable region, which includes 1, 2 or 3 CDR sequences selected from SEQ ID NO:43, SEQ ID NO:44 and SEQ ID NO:45;

[0021] l) Heavy chain variable region, which includes 1, 2 or 3 CDR sequences selected from SEQ ID NO:136, SEQ ID NO:2 and SEQ ID NO:3;

[0022] m) The heavy chain variable region, comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO:7, SEQ ID NO:140, and SEQ ID NO:9; and

[0023] n) Heavy chain variable region, which includes 1, 2 or 3 CDR sequences selected from SEQ ID NO:13, SEQ ID NO:141 and SEQ ID NO:15.

[0024] In some embodiments, the antibody or antigen-binding fragment thereof described in this application includes a κ light chain variable region, wherein the κ light chain variable region is selected from the group consisting of:

[0025] a) The κ light chain variable region, which includes 1, 2 or 3 CDR sequences selected from SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6;

[0026] b) The κ light chain variable region, comprising 1, 2 or 3 CDR sequences selected from SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12;

[0027] c) The κ light chain variable region, which includes 1, 2 or 3 CDR sequences selected from SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18;

[0028] d) The κ light chain variable region, comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO:40, SEQ ID NO:41, and SEQ ID NO:42; and

[0029] e)κ light chain variable region, which includes 1, 2 or 3 CDR sequences selected from SEQ ID NO:137, SEQ ID NO:138 and SEQ ID NO:139.

[0030] In some embodiments, the antibody or its antigen-binding fragment described in this application includes:

[0031] a) a heavy chain variable region comprising 1, 2 or 3 CDR sequences selected from SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3; and a κ light chain variable region comprising 1, 2 or 3 CDR sequences selected from SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6;

[0032] b) The heavy chain variable region, comprising 1, 2 or 3 CDR sequences selected from SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9; and the κ light chain variable region, comprising 1, 2 or 3 CDR sequences selected from SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12;

[0033] c) The heavy chain variable region, comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15; and the κ light chain variable region, comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO:15.

[0034] 16. CDR sequences of SEQ ID NO:17 and SEQ ID NO:18;

[0035] d) The heavy chain variable region, comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21; and the κ light chain variable region, comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO:

[0036] 4. CDR sequences of SEQ ID NO:5 and SEQ ID NO:6;

[0037] e) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24; and a κ light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO:24.

[0038] 4. CDR sequences of SEQ ID NO:5 and SEQ ID NO:6;

[0039] f) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27; and a κ light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO:27.

[0040] 4. CDR sequences of SEQ ID NO:5 and SEQ ID NO:6;

[0041] g) The heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30; and the κ light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30.

[0042] 4. CDR sequences of SEQ ID NO:5 and SEQ ID NO:6;

[0043] h) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33; and a κ light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO:33;

[0044] 4. CDR sequences of SEQ ID NO:5 and SEQ ID NO:6;

[0045] i) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36; and a κ light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO:36.

[0046] 4. CDR sequences of SEQ ID NO:5 and SEQ ID NO:6;

[0047] j) The heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39; and the κ light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO:39.

[0048] 40. CDR sequences of SEQ ID NO:41 and SEQ ID NO:42;

[0049] k) Heavy chain variable region, comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO:43, SEQ ID NO:44, and SEQ ID NO:45; and κ light chain variable region, comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO:45.

[0050] 4. CDR sequences of SEQ ID NO:5 and SEQ ID NO:6;

[0051] l) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO:136, SEQ ID NO:2, and SEQ ID NO:3; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO:136, SEQ ID NO:2, and SEQ ID NO:3.

[0052] 137. CDR sequences of SEQ ID NO:138 and SEQ ID NO:139;

[0053] m) Heavy chain variable region, comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO:7, SEQ ID NO:140, and SEQ ID NO:9; and κ light chain variable region, comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO:7, SEQ ID NO:140, and SEQ ID NO:9.

[0054] 10. The CDR sequences of SEQ ID NO:11 and SEQ ID NO:12; or

[0055] n) Heavy chain variable region, comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO:13, SEQ ID NO:141, and SEQ ID NO:15; and κ light chain variable region, comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO:15.

[0056] 16. CDR sequences of SEQ ID NO:17 and SEQ ID NO:18.

[0057] In some embodiments, the antibody or antigen-binding fragment thereof described in this application includes a heavy chain CDR3 sequence, wherein the heavy chain CDR3 sequence is selected from: SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:15, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:39 and SEQ ID NO:45.

[0058] In some embodiments, the antibody or its antigen-binding fragment described in this application includes:

[0059] a) A heavy chain CDR1 sequence, wherein the heavy chain CDR1 sequence is selected from: SEQ ID NO:1, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:28, SEQ ID NO:31, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:43 and SEQ ID NO:136;

[0060] b) A heavy chain CDR2 sequence, wherein the heavy chain CDR2 sequence is selected from: SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:44, SEQ ID NO:

[0061] 140 and SEQ ID NO:141; and

[0062] c) Heavy chain CDR3 sequence, wherein the heavy chain CDR3 sequence is selected from: SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:15, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:39 and SEQ ID NO:45.

[0063] In some embodiments, the antibody or its antigen-binding fragment described in this application includes:

[0064] a) A light chain CDR1 sequence, wherein the light chain CDR1 sequence is selected from: SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:16, SEQ ID NO:40 and SEQ ID NO:137;

[0065] b) A light chain CDR2 sequence, said light chain CDR2 sequence being selected from: SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:17, SEQ ID NO:41 and SEQ ID NO:138; and

[0066] c) A light chain CDR3 sequence selected from: SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:18, SEQ ID NO:42 and SEQ ID NO:139.

[0067] In some embodiments, the antibody or its antigen-binding fragment described in this application includes:

[0068] a) A heavy chain CDR1 sequence, wherein the heavy chain CDR1 sequence is selected from: SEQ ID NO:1, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:28, SEQ ID NO:31, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:43 and SEQ ID NO:136;

[0069] b) A heavy chain CDR2 sequence, wherein the heavy chain CDR2 sequence is selected from: SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:44, SEQ ID NO:

[0070] 140 and SEQ ID NO:141;

[0071] c) A heavy chain CDR3 sequence, wherein the heavy chain CDR3 sequence is selected from: SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:15, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:39 and SEQ ID NO:45;

[0072] d) A light chain CDR1 sequence, wherein the light chain CDR1 sequence is selected from: SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:16, SEQ ID NO:40 and SEQ ID NO:137;

[0073] e) A light chain CDR2 sequence, said light chain CDR2 sequence being selected from: SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:17, SEQ ID NO:41 and SEQ ID NO:138; and

[0074] f) A light chain CDR3 sequence, wherein the light chain CDR3 sequence is selected from: SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:18, SEQ ID NO:42 and SEQ ID NO:139.

[0075] In some embodiments, the antibody or antigen-binding fragment thereof described in this application further comprises: one or more (e.g., 1, 2, 3 or 4) heavy chain framework region (FR) sequences selected from the group consisting of: SEQ ID NO: 54, 55, 56, 57, 70, 71, 72, 73, 86, 87, 88 and 89, and / or one or more (e.g., 1, 2, 3 or 4) κ light chain framework region (FR) sequences selected from the group consisting of: SEQ ID NO: 58, 59, 60, 61, 74, 75, 76, 77, 90, 91, 92 and 93.

[0076] In some embodiments, the antibody or its antigen-binding fragment described in this application further comprises: a heavy chain FR1 sequence, wherein the heavy chain FR1 is selected from SEQ ID NO: 54, 70 and 86; a heavy chain FR2 sequence, wherein the heavy chain FR2 is selected from SEQ ID NO: 55, 71 and 87; a heavy chain FR3 sequence, wherein the heavy chain FR3 is selected from SEQ ID NO: 56, 72 and 88; and a heavy chain FR4 sequence, wherein the heavy chain FR4 is selected from SEQ ID NO: 57, 73 and 89.

[0077] In some embodiments, the antibody or its antigen-binding fragment described in this application further comprises: a light chain FR1 sequence, wherein the light chain FR1 is selected from SEQ ID NO: 58, 74 and 90; a light chain FR2 sequence, wherein the light chain FR2 is selected from SEQ ID NO: 59, 75 and 91; a light chain FR3 sequence, wherein the light chain FR3 is selected from SEQ ID NO: 60, 76 and 92; and a light chain FR4 sequence, wherein the light chain FR4 is selected from SEQ ID NO: 61, 77 and 93.

[0078] In some embodiments, the antibody or antigen-binding fragment thereof described in this application includes a heavy chain variable region selected from the group consisting of: SEQ ID NO:94, SEQ ID NO:98, SEQ ID NO:102, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:128, SEQ ID NO:132 and homologous sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0079] In some embodiments, the antibody or antigen-binding fragment thereof described in this application includes a light chain variable region selected from the group consisting of: SEQ ID NO:96, SEQ ID NO:100, SEQ ID NO:104, SEQ ID NO:120, SEQ ID NO:126, SEQ ID NO:130, SEQ ID NO:134 and homologous sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with them.

[0080] In some embodiments, the antibody or antigen-binding fragment thereof described in this application comprises all or a portion of the heavy chain variable region sequence selected from the group consisting of: SEQ ID NO: 94, 98, 102, 106, 108, 110, 112, 114, 116, 118, 122, 124, 128, and 132; and / or all or a portion of the light chain variable region sequence selected from the group consisting of: SEQ ID NO: 100, 104, 120, 126, 130, and 134. In one embodiment, the antibody or antigen-binding fragment thereof is a single-domain antibody composed of all or a portion of the heavy chain variable region sequence selected from the group consisting of: SEQ ID NO: 94, 98, 102, 106, 108, 110, 112, 114, 116, 118, 122, 124, 128, and 132.

[0081] In some embodiments, the antibody or its antigen-binding fragment described in this application includes:

[0082] a) Heavy chain variable region, including SEQ ID NO:94 and κ light chain variable region, including SEQ ID NO:96;

[0083] b) Heavy chain variable region, including SEQ ID NO:98 and light chain variable region, including SEQ ID NO:100;

[0084] c) Heavy chain variable region, including SEQ ID NO:102 and κ light chain variable region, including SEQ ID NO:104;

[0085] d) Heavy chain variable region, including SEQ ID NO:106 and κ light chain variable region, including SEQ ID NO:96;

[0086] e) Heavy chain variable region, including SEQ ID NO:108 and κ light chain variable region, including SEQ ID NO:96;

[0087] f) Heavy chain variable region, including SEQ ID NO:110 and light chain variable region, including SEQ ID NO:96;

[0088] g) Heavy chain variable region, including SEQ ID NO:112 and light chain variable region, including SEQ ID NO:96;

[0089] h) Heavy chain variable region, including SEQ ID NO:114 and light chain variable region, including SEQ ID NO:96;

[0090] i) Heavy chain variable region, which includes SEQ ID NO:116 and κ light chain variable region, which includes SEQ ID NO:96;

[0091] j) Heavy chain variable region, including SEQ ID NO:118 and κ light chain variable region, including SEQ ID NO:120;

[0092] k) Heavy chain variable region, which includes SEQ ID NO:122 and κ light chain variable region, which includes SEQ ID NO:96;

[0093] l) Heavy chain variable region, which includes SEQ ID NO:124 and light chain variable region, which includes SEQ ID NO:126;

[0094] m) Heavy chain variable region, including SEQ ID NO:128 and κ light chain variable region, including SEQ ID NO:130; or

[0095] n) Heavy chain variable region, including SEQ ID NO:132 and κ light chain variable region, including SEQ ID NO:134.

[0096] In some embodiments, the antibody or its antigen-binding fragment described in this application further includes one or more amino acid residue substitutions, but still maintains specific binding affinity to CD19.

[0097] In some embodiments, the substitution is in one or more CDR sequences, and / or in one or more FR sequences, in one or two variable region sequences, and / or in the Fc region. In some embodiments, at least one (or all) of the substitutions in the CDR sequence, in the FR sequence, in the variable region sequence, or in the Fc region include conservative substitutions.

[0098] In some embodiments, the antibody or antigen-binding fragment thereof described in this application includes substitutions of no more than 10, 9, 8, 7, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 136, 137, 138, 139, 140, and 141 amino acid residues in one or more CDR sequences.

[0099] In some embodiments, the antibody or antigen-binding fragment thereof described in this application includes no more than 10, 9, 8, 7, 6, 5, 9, 60, 61, 70, 71, 72, 73, 74, 75, 76, 77, 86, 87, 88, 89, 90, 91, 92, and 93 amino acid residue substitutions in one or more FR sequences. In some embodiments, the antibody or antigen-binding fragment thereof described in this application includes no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue substitutions in the CDR and / or FR sequences of the heavy chain variable region sequences selected from SEQ ID NO: 94, 98, 102, 106, 108, 110, 112, 114, 116, 118, 122, 124, 128, and 132. In some embodiments, the antibody or antigen-binding fragment described in this application includes, in all FR sequences of the light chain variable region sequences selected from SEQ ID NO: 96, 100, 104, 120, 126, 130 and 134, substitutions of no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residues.

[0100] In some embodiments, the substitution imparts one or more desired properties selected from: a) increased binding affinity to CD19, b) introduction or removal of glycosylation sites, c) introduction of free cysteine ​​residues, d) increased or decreased ADCC or CDC, e) increased serum half-life; and f) increased FcRn binding.

[0101] In some embodiments, the antibody or antigen-binding fragment thereof described in this application further includes an immunoglobulin constant region. In some embodiments, the antibody or antigen-binding fragment thereof described in this application includes a constant region of IgG. In some embodiments, the antibody or antigen-binding fragment thereof described in this application includes a constant region of mouse IgG1, mouse IgG2a, mouse IgG2b, or human IgG1.

[0102] In some embodiments, the antibody or its antigen-binding fragment described in this application is a non-human (e.g., murine or rodent) antibody or a humanized antibody.

[0103] In some embodiments, the antibody or its antigen-binding fragment described in this application is a camelized single-chain domain antibody, a diabody, scFv, scFv dimer, BsFv, dsFv, (dsFv)2, dsFv-dsFv', Fv fragment, Fab, Fab', F(ab')2, bispecific antibody, ds diabody, nanobody, domain antibody, or bivalent domain antibody.

[0104] In some embodiments, the antibody or its antigen-binding fragment described in this application has bispecificity.

[0105] In some embodiments, the antibody or its antigen-binding fragment described in this application is linked to one or more conjugates. In some embodiments, the conjugate includes a chemotherapeutic agent, a toxin, a radioactive isotope, a lanthanide element, a luminescent label, a fluorescent label, or an enzyme substrate label. In some embodiments, the conjugate is a toxin. In some embodiments, the toxin is a cytotoxic agent, a DNA alkylating agent, a topoisomerase inhibitor, a microtubule binding agent, or other anticancer drug. In some embodiments, the anticancer drug is a maytansine-type cytotoxic agent. In some embodiments, the toxin is DM1.

[0106] In some embodiments, the antibody or its antigen-binding fragment described in this application can specifically bind to CD19. In some embodiments, the CD19 is derived from mice, rats, monkeys, or humans.

[0107] In some embodiments, the antibody or its antigen-binding fragment described in this application can be used at a rate not exceeding 5 × 10⁻⁶. -9 M, not exceeding 1×10 -9 M, not exceeding 9×10 -10 M, not exceeding 8×10 -10 M, not exceeding 7×10 -10 M, not exceeding 6×10 -10 M, not exceeding 5×10 -10 M, not exceeding 4×10 -10 M, not exceeding 3×10 -10 M, not exceeding 2×10 -10 M, not exceeding 1×10 -10 M of K D The K value specifically binds to human CD19 expressed on cells. DThe values ​​were determined by flow cytometry.

[0108] In some embodiments, the antibody or antigen-binding fragment described in this application can be used at EC50 concentrations not exceeding 0.04 nM, 0.05 nM, 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM, 0.5 nM, 0.6 nM, 0.7 nM, 0.8 nM, 0.9 nM, or 1 nM. 50 The value specifically binds to human CD19 expressed on cells, the EC 50 The values ​​were determined by flow cytometry.

[0109] In some embodiments, the antibody or its antigen-binding fragment described in this application can be used at EC50 concentrations not exceeding 0.2 nM, 0.5 nM, 0.8 nM, 1 nM, 2 nM, or 3 nM. 50 The EC specifically binds to CD19 expressed on cells in cynomolgus monkeys. 50 The values ​​were determined by flow cytometry.

[0110] In some embodiments, the antibody or its antigen-binding fragment described in this application can be used at EC50 concentrations not exceeding 1 pM, 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 8 pM, 9 pM, 10 pM, 11 pM, 12 pM, 13 pM, 14 pM, 15 pM, 16 pM, 17 pM, 18 pM, 19 pM, 20 pM, 21 pM, 22 pM, 23 pM, 24 pM, 25 pM, 30 pM, 35 pM, 40 pM, 45 pM, or 50 pM. 50 The value is internalized in cells expressing CD19, the EC 50 The value was determined by the Fab-Zap method.

[0111] In one aspect, this application provides an antibody or an antigen-binding fragment thereof that competes with W7011-4.155.8, W7011-4.202.9 or W7011-4.225.7 for the same epitope.

[0112] In one aspect, this application further provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof described in this application and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition further comprises a second agent capable of enhancing the therapeutic effect of the antibody or antigen-binding fragment thereof and / or reducing the side effects of the antibody or antigen-binding fragment thereof.

[0113] In one aspect, this application further provides isolated polynucleotides encoding the antibodies or antigen-binding fragments thereof described in this application. In some embodiments, the isolated polynucleotides comprise nucleotide sequences selected from the group consisting of SEQ ID NO: 95, 99, 103, 107, 109, 111, 113, 115, 117, 119, 123, 125, 129, and 133, and / or nucleotide sequences selected from the group consisting of SEQ ID NO: 97, 101, 105, 121, 127, 131, and 135, or homologous sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity but encoding the same protein sequence.

[0114] In one aspect, this application further provides a vector comprising the isolated polynucleotide.

[0115] In one aspect, this application further provides a host cell comprising the vector.

[0116] In one aspect, this application further provides a method for expressing the antibody or antigen-binding fragment thereof described in this application, comprising culturing the host cells under conditions of expressing the polynucleotide.

[0117] In one aspect, this application further provides an antibody-drug conjugate comprising one or more pharmaceutical portions, said pharmaceutical portions being directly or covalently linked via a linker to the antibody or its antigen-binding fragment described in this application. In some embodiments, the linker is an hydrazone linker, a disulfide linker, a bifunctional linker, a dipeptide linker, a glucuronide linker, or a thioether linker. In some embodiments, the linker is a SMCC.

[0118] In some embodiments, at least one pharmaceutical portion is linked to a specific site on the antibody or its antigen-binding fragment described in this application. In some embodiments, the specific site is a cysteine ​​residue. In some embodiments, the pharmaceutical portion is a cytotoxin or a radioisotope. In some embodiments, the pharmaceutical portion is a toxin, optionally a cytotoxin, DNA alkylating agent, topoisomerase inhibitor, microtubule binding agent, or other anticancer drug, optionally a maytansine-type cytotoxic agent, optionally DM1.

[0119] In one aspect, this application further provides a pharmaceutical composition comprising the antibody described in this application or an antigen-binding fragment thereof, or an antibody-drug conjugate provided in this application, and a pharmaceutically acceptable carrier.

[0120] In one aspect, this application further provides a method of treating a CD19-related disease or condition in a subject, comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof described in this application, the antibody-drug conjugate described in this application, or the pharmaceutical composition described in this application. In some embodiments, the subject is a human. In some embodiments, the administration is via oral, intranasal, intravenous, subcutaneous, sublingual, or intramuscular administration. In some embodiments, the disease or condition is cancer. In some embodiments, the cancer is lymphoma, lung cancer, liver cancer, cervical cancer, colon cancer, breast cancer, ovarian cancer, pancreatic cancer, melanoma, glioblastoma, prostate cancer, esophageal cancer, or gastric cancer. In some embodiments, the disease or condition is B-cell lymphoma, optionally Hodgkin lymphoma or non-Hodgkin lymphoma, wherein the non-Hodgkin lymphoma includes: diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, marginal zone B-cell lymphoma (MZL), mucosa-associated lymphoid tissue lymphoma (MALT), small lymphocytic lymphoma (chronic lymphocytic leukemia, CLL), mantle cell lymphoma (MCL), acute lymphoblastic leukemia (ALL), or Waldenström macroglobulinemia (WM).

[0121] In one aspect, this application further provides a method for regulating CD19 activity in cells expressing CD19, comprising exposing the CD19-expressing cells to the antibody or antigen-binding fragment thereof described in this application.

[0122] In one aspect, this application further provides a method for killing CD19-expressing cells in vivo or in vitro, comprising contacting the CD19-expressing cells with the antibody-drug conjugate described in this application.

[0123] In one aspect, this application further provides a method for detecting the presence or content of CD19 in a sample, comprising contacting the sample with the antibody or its antigen-binding fragment described in this application, and determining the presence or content of CD19 in the sample.

[0124] In one aspect, this application further provides a method for diagnosing a CD19-related disease or condition in a subject, comprising: a) obtaining a sample from the subject; b) contacting the sample with an antibody or antigen-binding fragment thereof as described in this application; c) determining the presence or amount of CD19 in the sample; and d) associating the presence or amount of CD19 with the disease or condition of the subject.

[0125] In one aspect, this application further provides the use of the antibody or antigen-binding fragment thereof described herein in the preparation of a medicament for treating a disease or condition in a subject, wherein the treatment comprises administering a therapeutically effective amount of the antibody or antigen-binding fragment thereof to the subject.

[0126] In one aspect, this application further provides the use of the antibody or antigen-binding fragment thereof described herein in the preparation of diagnostic reagents for detecting diseases or conditions associated with CD19.

[0127] In one aspect, this application provides a chimeric antigen receptor (CAR) comprising the antigen-binding fragment described in this application and a T-cell activation portion. In some embodiments, the T-cell activation portion comprises the native T-cell activation portion of a T-cell receptor (TCR). In some embodiments, the T-cell activation portion comprises a transmembrane domain of the TCR and an intracellular signal transduction domain of the TCR. In some embodiments, the antigen-binding fragment is scFv.

[0128] In one aspect, this application provides a nucleic acid sequence encoding the CAR described in this application. In some embodiments, the nucleic acid sequence includes a first polynucleotide sequence encoding an antigen-binding fragment of the antibody described in this application, the first polynucleotide sequence being operatively linked to a second polynucleotide sequence encoding a transmembrane domain of the TCR and an intracellular signal transduction domain of the TCR.

[0129] In one aspect, this application provides a vector comprising a nucleic acid sequence encoding the CAR described in this application.

[0130] In one aspect, this application provides isolated T cells expressing the CAR described in this application.

[0131] In one aspect, this application provides a method for activating a T cell-mediated immune response against a CD19-expressing target in a subject, the method comprising administering an effective amount of the T cells described in this application to the subject. Brief description of the attached diagram

[0132] Figure 1 The SDS-PAGE of WBP701-BMK1 and WBP701-BMK2 is shown. M: protein label; lane 1: BMK1, reducing conditions; lane 2: BMK2, reducing conditions; lane 3: BMK1, non-reducing conditions; lane 4: BMK4, non-reducing conditions.

[0133] Figure 2 The SDS-PAGE of WBP701-BMK3 is shown. M: protein label; lane 1: BMK3, reducing conditions; lane 2: BMK3, non-reducing conditions.

[0134] Figure 3A The flow cytometry histogram shows CD19 expression in the human CD19-transfected 293F cell line (WBP701.293F.hPro1.FL.A2). The peaks on the left represent negative control signals. Peaks shifted to the right represent CD19 expression in the cell lines being tested.

[0135] Figure 3B The flow cytometry histogram shows CD19 expression in the human CD19-transfected CHO-K1 cell line (WBP701.CHO-K1.hPro1.FL.B4). The peak on the left represents the negative control signal. The peak shifted to the right represents CD19 expression in the cell line being tested.

[0136] Figure 3C The flow cytometry histogram shows CD19 expression in the CD19-transfected 293 cell line (WBP701.293F.cpro1.FL.C1) of cynomolgus monkeys. The peaks on the left represent the negative control signal. The peaks shifted to the right represent CD19 expression in the cell lines being tested.

[0137] Figure 3D The flow cytometry histogram shows CD19 expression in the CD19-transfected CHO-K1 cell line (WBP701.CHO-K1.cpro1.FL.C9) from cynomolgus monkeys. The peak on the left represents the negative control signal. The peak shifted to the right represents CD19 expression in the cell line being tested.

[0138] Figures 4A - 4F The binding of selected subclones to Ramos cells was shown by FACS assay.

[0139] Figures 5A - 5C The binding of the selected subclones to cynomolgus CD19-expressing cells (WBP701.CHO-K11.cynoPro1) was shown by FACS assay.

[0140] Figures 6A - 6E The Fab-Zap assay of the selected subclones is shown.

[0141] Figures 7A - 7C The candidate antibody groupings for BMK1, BMK2, and BMK3 antibodies, as determined by FACS, are shown.

[0142] Figure 8 The Scarchard binding affinity analysis of antibody WBP7011-4.34.11-z1-m5-IgG1k to Ramos cells, as determined by FACS, is shown.

[0143] Figure 9 The Scarchard binding affinity analysis of antibody WBP7011-4.87.6-z1-IgG1K (NS) to Ramos cells, as determined by FACS, is shown.

[0144] Figure 10 The results show the affinity analysis of antibody W7011-4.155.8-z1-uIgG1K for Scarchard binding in Ramos cells, as determined by FACS.

[0145] Figure 11 The cytotoxicity assays of the humanized antibody-drug conjugates W7011-4.155.8-z1-uIgG1K-DM1 and WBP7011-4.87.6-z1-IgG1K(NS)-DM1 performed on Daudi cells are shown.

[0146] Figure 12 The cytotoxicity assay of the humanized antibody-drug conjugate WBP7011-4.87.6-z1-IgG1K(NS)-DM1 performed on Nalm-6 cells is shown.

[0147] Figure 13 Cytotoxicity assays of the humanized antibody-drug conjugates W7011-4.155.8-z1-uIgG1K-DM1 and WBP7011-4.87.6-z1-IgG1K(NS)-DM1 on WSU-DLCL2 cells are shown.

[0148] Figure 14 The antitumor potency of the baseline antibody (W7011-BMK1-DM1) and the antibody (W7011-4.87.6-z1-uIgG1k(NS)-DM1) is shown. Data represent tumor volume in different treatment groups of female CB17-SCID mice with Nalm-6 lymphocyte cancer xenografts. Data points are presented as mean + SEM. Arrows indicate the number of days of administration. Invention Details

[0149] The following description of this application is only for illustrating various embodiments of the application. Therefore, the specific modifications discussed herein should not be construed as limiting the scope of the application. Those skilled in the art can readily derive various equivalents, variations, and modifications without departing from the scope of this application, and it should be understood that such equivalent embodiments are included within the scope of this invention. All documents cited in this application, including published materials, patents, and patent applications, are incorporated herein by reference in their entirety.

[0150] Definition

[0151] In this invention, the term "antibody" includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, multispecific antibody, or bispecific antibody capable of binding to a specific antigen. A natural, complete antibody consists of two heavy (H) chains and two light (L) chains. In mammals, the heavy chains can be divided into α, δ, ε, γ, and μ, each heavy chain consisting of a variable region (V... H ) and the first, second and third constant regions (C respectively) H1 C H2 C H3 It consists of ) ; mammalian light chains can be divided into λ or κ, and each light chain consists of a variable region (V of the λ light chain, respectively). L or κ light chain V K ) and a constant region (C for the λ light chain, respectively) L or C of the κ light chain KThe antibody is composed of a "Y"-shaped structure. The neck of the "Y" consists of the second and third constant regions of two heavy chains, which are bound by disulfide bonds. Each arm of the "Y" structure includes a variable region and a first constant region of one heavy chain, which binds to a variable region and a constant region of one light chain. The variable regions of the light and heavy chains determine antigen binding. Each chain's variable region contains three hypervariable regions, called complementarity-determining regions (CDRs) (the CDRs of the light chain include LCDR1, LCDR2, and LCDR3, and the CDRs of the heavy chain include HCDR1, HCDR2, and HCDR3). The CDR boundaries of the antibody and antigen-binding fragments disclosed in this invention can be named or identified using the Kabat, Chothia, or Al-Lazikani nomenclature. (Al-Lazikani, B., Chothia, C., Lesk, AM, J. Mol. Biol., 273(4), 927(1997); Chothia, C. et al., J Mol Biol. Dec 5; 186(3):651-63(1985); Chothia, C. and Lesk, AM, J. Mol. Biol., 196, 901(1987); Chothia, C. et al., Nature. Dec 21-28; 342(6252):877-83(1989); Kabat EA et al., National Institutes of Health, Bethesda, Maryland (1991)). Among these, the three CDRs are separated by lateral continuous portions called the frame region (FR), which is more highly conserved than the CDRs and forms a scaffold supporting the supervariant ring. The constant regions of the heavy and light chains are not involved in antigen binding but possess various effector functions. Antibodies can be classified into several categories based on the amino acid sequence of the heavy chain constant region. According to the presence or absence of α, δ, ε, γ, and μ heavy chains, antibodies can be divided into five main classes or isoforms: IgA, IgD, IgE, IgG, and IgM. Several major antibody classes can also be subdivided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain), etc.

[0152] In this application, the term "bivalent" refers to an antibody or antigen-binding fragment having two antigen-binding sites; the term "monovalent" refers to an antibody or antigen-binding fragment having only one single antigen-binding site; and the term "multivalent" refers to an antibody or antigen-binding fragment having multiple antigen-binding sites. In some embodiments, the antibody or antigen-binding fragment described in this application is bivalent.

[0153] In this application, a "bispecific" antibody refers to an artificial antibody having fragments derived from two different monoclonal antibodies and capable of binding to two different epitopes. The two epitopes may be present on the same antigen or on two different antigens.

[0154] In this application, the term "antigen-binding fragment" refers to an antibody fragment formed by an antibody moiety containing one, two, or three CDRs, or any other antibody fragment that binds to an antigen but does not have a complete antibody structure. Examples of antigen-binding fragments include, but are not limited to, double-chain antibodies (diabody), Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized double-chain antibodies (ds diabody), single-chain antibody molecules (scFv), scFv dimers (bivalent double-chain antibodies), bivalent single-chain antibodies (BsFv), bispecific antibodies, multispecific antibodies, camelized singledomain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies. Antigen-binding fragments can bind the same antigen to the parent antibody.

[0155] The "Fab" fragment of an antibody refers to the portion of the antibody molecule that consists of a light chain (including the variable and constant regions) and a heavy chain whose variable and constant regions are linked together by disulfide bonds.

[0156] A “Fab” segment refers to a Fab segment that contains part of the hinge area.

[0157] "F(ab')2" refers to the Fab' dimer. The "Fv" of an antibody refers to the smallest antibody fragment containing a complete antigen-binding site. An Fv fragment consists of a variable region of one light chain and a variable region of one heavy chain.

[0158] "dsFv" refers to a disulfide-bonded stable Fv fragment, where the variable regions of the single light chain and the single heavy chain are connected by disulfide bonds. In some embodiments, "(dsFv)2" or "(dsFv-dsFv')" contains three peptide chains: two V... H Some are linked by peptide linkers (e.g., long elastic linkers) and are respectively connected to two V via disulfide bonds. L Partial binding. In some embodiments, dsFv-dsFv' has bispecificity, wherein each pair of heavy and light chains paired by disulfide bonds has different antigen specificities.

[0159] "Single-chain Fv antibody" or "scFv" refers to an engineered antibody that is formed by directly linking the variable regions of the light chain and the variable regions of the heavy chain or by linking them through a single peptide chain (Huston JS et al., Proc Natl Acad Sci USA, 85:5879 (1988)).

[0160] The "Fc" of an antibody refers to the portion of the antibody composed of the second and third constant regions of the first heavy chain linked to the second and third constant regions of the second heavy chain via disulfide bonds. The Fc segment of an antibody is responsible for a variety of different effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), but it does not play a role in antigen binding.

[0161] "Single-chain antibody Fv-Fc" or "scFv-Fc" refers to an engineered antibody composed of scFvs linked to the Fc segment of an antibody.

[0162] "Camelized singledomain antibody," "heavy-chain antibody," or "HCAb (Heavy-chain-only antibodies)" all refer to antibodies containing two V's. HAntibodies that do not contain light chains (Riechmann L. and Muyldermans S., J Immunol Methods. Dec 10; 231(1-2):25-38 (1999); Muyldermans S., J Biotechnol. Jun; 74(4):277-302 (2001); WO94 / 04678; WO94 / 25591; US ​​Patent No. 6,005,079). Heavy chain antibodies were originally derived from camels (camels, dromedaries, and llamas). Although lacking the light chain, camelized antibodies have proven full antigen-binding function (Hamers-Casterman C. et al., Nature. Jun 3; 363(6428):446-8(1993); Nguyen VK. et al., “Heavy-chain antibodies in Camelidae; a case of evolutionary innovation” Immunogenetics. Apr; 54(1):39-47(2002); Nguyen VK. et al., Immunology. May; 109(1):93-101(2003)). The variable region (VHH domain) of heavy chain antibodies is the smallest known antigen-binding unit produced by acquired immunity (Koch-Nolte F. et al., FASEB J. Nov; 21(13):3490-8. Epub 2007 Jun 15(2007)).

[0163] "Nanobody" refers to an antibody fragment that consists of a VHH domain derived from a heavy chain antibody and two constant regions CH2 and CH3.

[0164] A "diabody" or "dAb" comprises a small antibody fragment with two antigen-binding sites, wherein the fragment contains V antibodies linked together on the same polypeptide chain. H Domain and V L Domain (V) H -V L or V H -V L(See Holliger P. et al., Proc Natl Acad Sci USA., July 15; 90(14):6444-8(1993); EP404097; WO93 / 11161). The short linker between the two domains prevents them from pairing, forcing them to pair with complementary domains of another chain to form two antibody binding sites. These two antibody binding sites can target the same or different antigens (or epitopes). In some embodiments, a “bispecific ds double-chain antibody” is a double-chain antibody that targets two different antigens (or epitopes). In some embodiments, a “scFv dimer” is a bivalent double-chain antibody or a bivalent single-chain antibody (BsFv) containing two dimerized V… H -V L (Linked by a polypeptide linker) groups, one of which has a V H V with another group L The two binding sites are formed through collaboration, and these two binding sites can target and bind to the same antigen (or epitope) or different antigens (or epitopes). In other embodiments, the "scFv dimer" is a bispecific double-chain antibody containing interconnected V... L1 -V H2 (linked by a polypeptide linker) and V H1 -V L2 (linked by a polypeptide linker), where V H1 and V L1 Collaboration, V H2 and V L2 They cooperate, and each cooperative pair has a different antigen specificity.

[0165] "Domain antibody" refers to an antibody fragment containing only the heavy chain variable region or the light chain variable region. In some cases, two or more V... H Domains are formed by the covalent bonding of peptide linkers, resulting in bivalent or multivalent antibodies. Bivalent antibodies have two V... H The domain can target the same or different antigens.

[0166] As used in this application, the term "chimerism" refers to an antibody or antigen-binding fragment having a portion of a heavy chain and / or light chain derived from one species, and the remainder of the heavy chain and / or light chain derived from a different species. In one exemplary instance, a chimeric antibody may include a constant region derived from a human and a variable region derived from a non-human animal (e.g., a mouse or rat). In some embodiments, the non-human animal is a mammal, such as a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster.

[0167] As used in this application, the term "humanized" refers to antibody or antigen-binding fragments that include CDRs derived from non-human animals, FR regions derived from humans, and constant regions derived from humans (where applicable).

[0168] As used in this application, the term "CD19" refers to the differentiation cluster 19 protein, an antigenic determinant detectable on leukemia progenitor cells. The amino acid and nucleic acid sequences of human and mouse CD19 are available in public databases such as GenBank, UniProt, and SwissProt. For example, the amino acid sequence of human CD19 can be found with UniProt / SwissProt accession number P15391, and the nucleic acid sequence encoding human CD19 can be found with accession number NM_001178098. As used in this application, the term CD19 includes proteins containing mutations, such as point mutations, fragments, insertions, deletions, and splicing variants of full-length wild-type CD19. CD19 is expressed in most B-cell cancers, including, for example, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin's lymphoma. CD19 is also an early marker of progenitor B cells. See, for example, Nicholson et al., Mol. Immun. 34(16-17):1157-1165 (1997). In one respect, CD19 protein is expressed on cancer cells.

[0169] In this application, "specific binding" or "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and an antigen. In some embodiments, the antibody or its antigen-binding fragment of this application specifically binds to human and / or monkey CD19, and its binding affinity (K) is... D ≤10 -6 M (e.g., ≤5×10) -7 M, ≤2×10 -7 M, ≤10 -7 M, ≤5×10 -8 M, ≤2×10 -8 M, ≤10 -8 M, ≤5×10 -9 M, ≤4x10-9M, ≤3x10-9M, ≤2×10 -9 M or ≤10 -9 M). K in this application D It refers to the ratio of dissociation rate to binding rate (k off / k on K can be determined using any conventional method known in the art, including but not limited to surface plasmon resonance, microthermophoresis, HPLC-MS, and flow cytometry (e.g., FACS). In some embodiments, K can be suitably determined using flow cytometry. D value.

[0170] The ability to "block binding" or "competitively identify the same epitope" in this application refers to the ability of an antibody or its antigen-binding fragment to inhibit the interaction of two intermolecular bindings (e.g., human CD19 and anti-CD19 antibody) to any detectable extent. In some embodiments, the antibody or antigen-binding fragment blocking the binding of two molecules can inhibit the interaction of the two intermolecular bindings by at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%. In some embodiments, such inhibition can be greater than 60%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, or greater than 90%.

[0171] As used in this application, "epitope" refers to the portion of an antigen molecule that binds to an antibody. If two antibodies exhibit competitive binding to an antigen, they may bind to the same or closely related epitopes on the antigen. For example, if an antibody or its antigen-binding fragment blocks the binding of a reference antibody to an antigen (e.g., human / monkey CD19) by at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%, then the antibody or its antigen-binding fragment can be considered to bind to the same / closely related epitope as the reference antibody.

[0172] Those skilled in the art will recognize that it is not necessary to conduct excessive experiments to determine whether a human monoclonal antibody is compatible with the antibodies described in this application (e.g., mouse monoclonal antibodies WBP7011-4.34.11, WBP7011-4.87.6, WBP7011_4.155.8, WBP7011_4.56.1, WBP7011-4.15.10, WBP7011-4.100.1, WBP7011-4.106.3, W...). BP7011_4.108.3, WBP7011_4.191.6, WBP7011_4.194.10, WBP7011_4.231.5 and humanized antibodies W7011-4.34.11-z1-m5, W7011-4.87.6-z1 (NS), and W7011-4.155.8-z1-P15) bind to the same epitope. The determination is made by whether the former prevents the latter from binding to the CD19 antigenic peptide. If the test antibody competes with the antibody described in this application, showing a reduced binding of the antibody to the CD19 antigenic peptide, then both antibodies bind to the same or closely related epitope. Alternatively, if the antibody described in this application inhibits the binding of the test antibody to the CD19 antigenic peptide, then both antibodies bind to the same or closely related epitope.

[0173] The symbols used in the antibody names in this application have different meanings: "mIgG2" refers to an antibody with a mouse IgG2 isotype constant region; "uIgG1" refers to an antibody with a human IgG1 isotype constant region; "K" or "L" indicates that the antibody uses a κ light chain or a λ light chain.

[0174] In this application, when "conservative substitution" is used for an amino acid sequence, it refers to replacing one amino acid residue with another amino acid residue from a side chain that has similar physicochemical properties. For example, this can be done between hydrophobic side chain amino acid residues (e.g., Met, Ala, Val, Leu, and Ile) or between neutral hydrophilic side chain residues (e.g., Cys, Ser, Thr, Asn, and...).

[0175] Gln), acidic side chain residues (e.g., Asp, Glu), basic side chain amino acids (e.g., His, Lys, and...)

[0176] Conserved substitutions are made between Arg residues or between directional side chain residues (e.g., Trp, Tyr, and Phe). Conserved substitutions are known in the art to generally not cause significant changes in protein conformation and structure, thus preserving the protein's biological activity.

[0177] The terms “homologous” and “homogeneous” used in this application are used interchangeably to refer to a nucleic acid sequence (or its complementary strand) or amino acid sequence having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identity with another sequence when optimally aligned.

[0178] When "percentage sequence identity" is used for amino acid sequences (or nucleic acid sequences), it refers to the percentage of amino acid (or nucleic acid) residues in the candidate sequence that are identical to those in the reference sequence, after sequence alignment and, where necessary, the introduction of spacers to maximize the number of identical amino acids (or nucleic acids). Conservative substitutions of these amino acid residues may or may not be considered identical residues. Sequences can be aligned to determine the percentage sequence identity of amino acid (or nucleic acid) sequences using tools disclosed in the art, such as BLASTN, BLASTp (National Center for Biotechnology Information (NCBI) website, see also Altschul SF et al., J. Mol. Biol., 215:403–410 (1990); Stephen F. et al., Nucleic Acids Res., 25:3389–3402 (1997)), ClustalW2 (European Institute for Bioinformatics website, see Higgins DG et al., Methods in Enzymology, 266:383-402 (1996); Larkin MA et al., Bioinformatics (Oxford, UK), 23(21):2947-8 (2007)) and ALIGN or Megalign (DNASTAR) software. Those skilled in the art can use the default parameters of these tools or adjust the parameters appropriately according to the needs of the alignment, for example, by selecting a suitable algorithm.

[0179] As used in this application, "effective function" refers to the biological activity of an antibody's Fc region binding to its effectors, such as the C1 complex and Fc receptors. Exemplary effective functions include complement-dependent cytotoxicity (CDC) induced by the interaction of an antibody with C1q on the C1 complex, antibody-dependent cell-mediated cytotoxicity (ADCC) induced by the binding of the antibody's Fc region to Fc receptors on effector cells, and phagocytosis.

[0180] The term "internalized" or "capable of being internalized" as used in this application refers to an antibody that is taken up by a cell after binding to its antigen on the cell surface. In some embodiments, the antibodies and fragments thereof described in this application can be internalized, at least partially, by cells expressing CD19 on their surface. For example, in some embodiments, the anti-CD19 antibody described in this application can be internalized by B lymphocytes after binding to CD19 expressed on the cell surface. Internalization can occur in vitro or in vivo. For therapeutic applications, internalization can occur in vivo. Whether an antigen is internalized after binding to mammalian cells can be determined by various tests, including those described in the following examples (e.g., the Fab-Zap method). A method for detecting whether an antibody has been internalized into a cell is also described in U.S. Patent No. 7,619,068, which is incorporated herein by reference in its entirety. In some embodiments, the internalizable antibody and its antigen-binding fragment can be linked to or conjugated to an anticancer agent (e.g., a cytotoxic portion that kills cells after internalization). Depending on the potency of the antibody or antibody conjugate, in some cases, the uptake of a single antibody molecule into a cell is sufficient to kill the target cell bound to the antibody. For example, some toxins are highly effective at killing tumor cells; internalizing just one molecule of the toxin conjugated with an antibody is sufficient to kill tumor cells.

[0181] "Treatment" or "therapy" for a condition includes preventing or alleviating the condition, slowing the onset or development of the condition, reducing the risk of developing the condition, preventing or delaying the development of symptoms associated with the condition, reducing or stopping the symptoms associated with the condition, producing a complete or partial reversal of the condition, curing the condition, or a combination of the above.

[0182] "Separated" substances have been artificially altered from their natural state. If a substance or component is found in nature that has been "separated," it has either been altered or deviated from its original state, or both. For example, naturally occurring polynucleotides or polypeptides in a living animal may not be separated, but if these polynucleotides or polypeptides are sufficiently separated from substances that coexist in their natural state and exist in a sufficiently pure state, they can be considered "separated." The terms "separated nucleic acid" and "polynucleotide" are used interchangeably, referring to the sequence of the separated nucleic acid molecule. In some embodiments, "isolated antibody or antigen-binding fragment thereof" refers to an antibody or antigen-binding fragment with a purity of at least 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, determined by electrophoretic methods (such as SDS-PAGE, isoelectric focusing, capillary electrophoresis) or chromatographic methods (such as ion exchange chromatography or reversed-phase HPLC).

[0183] In this invention, "vector" refers to a delivery vehicle in which a polynucleotide encoding a protein can be operatively inserted to enable the expression of that protein. Vectors can be used to transform, transduce, or transfect host cells, allowing the genetic material elements they carry to be expressed within the host cells. Examples of vectors include plasmids, phagemids, Cos plasmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC), bacteriophages such as λ phage or M13 phage, and animal viruses. Animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). Vectors may contain various elements controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain a replication initiation site. Vectors may also include components that facilitate their entry into cells, including but not limited to viral particles, liposomes, or protein coats. The vector can be an expression vector or a cloning vector. The vectors provided in this application (e.g., expression vectors) contain a nucleic acid sequence encoding an antigen or its antigen-binding fragment as described in this application, at least one promoter operatively linked to the nucleic acid sequence (e.g., SV40, CMV, EF-1α), and at least one selection marker. Examples of vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, multivacuolar papillomaviruses (e.g., SV40), λ phage and M13 phage, plasmids pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, etc. pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p1 5TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT.RTM., pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, etc.

[0184] In this invention, "host cell" refers to a cell into which exogenous polynucleotides and / or vectors are introduced.

[0185] As used in this application, "CD19-related disease or condition" refers to any disease or condition caused, aggravated, or otherwise associated with increased or decreased expression or activity of CD19. In some embodiments, with

[0186] CD19-related conditions are B-cell lymphomas, optionally Hodgkin lymphomas or non-Hodgkin lymphomas, wherein the non-Hodgkin lymphomas include: diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, marginal zone B-cell lymphoma (MZL), mucosa-associated lymphoid tissue lymphoma (MALT), small lymphocytic lymphoma (chronic lymphocytic leukemia, CLL), mantle cell lymphoma (MCL), acute lymphoblastic leukemia (ALL), or Waldenström macroglobulinemia (WM).

[0187] As used in this application, "cancer" refers to any medical condition characterized by malignant cell growth or tumors, abnormal proliferation, invasion, or metastasis, and includes solid tumors and non-solid cancers (hematologic malignancies) such as leukemia. As used in this application, "solid tumor" refers to a solid mass of tumors and / or malignant cells. Examples of cancer include, but are not limited to, non-small cell lung cancer (squamous / non-squamous), small cell lung cancer, renal cell carcinoma, colorectal cancer, colon cancer, ovarian cancer, breast cancer (including basal breast cancer, ductal carcinoma in situ, and lobular carcinoma), pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, melanoma, myeloma, and mycoses. Fungoids, Merkel cell carcinoma, hepatocellular carcinoma (HCC), fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma and other sarcomas, synovial sarcoma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, malignant lymphoma, basal cell carcinoma, adenocarcinoma, hepatocellular carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchial carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma. Membrane carcinoma, Wilms' tumor, cervical cancer, testicular cancer, seminoma, classical Hodgkin's lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell / histocyte-rich B-cell lymphoma, acute lymphoblastic leukemia, acute myeloid leukemia, acute granulocytic leukemia, chronic myeloid (granulocytic) leukemia, chronic granulocytic leukemia, chronic lymphocytic leukemia, polycythemia vera, mast cell tumor, EBV Positive and negative PTLD, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, marginal zone B-cell lymphoma (MZL), mucosa-associated lymphoid tissue lymphoma (MALT), small lymphocytic lymphoma (chronic lymphocytic leukemia, CLL), mantle cell lymphoma (MCL), acute lymphoblastic leukemia (ALL), plasmablastic lymphoma, extranodal NK / T-cell lymphoma, nasopharyngeal carcinoma, HHV8-related primary exudative lymphoma Tumors, non-Hodgkin's lymphoma, multiple myeloma, Waldenström macroglobulinemia (WM), heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and spinal dysplasia, primary central nervous system lymphoma, spinal tumors, brainstem glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma and retinoblastoma.

[0188] "Pharmaceutical acceptable" means that the carrier, solvent, diluent, excipient and / or salt is generally chemically and / or physically compatible with the other ingredients in the formulation and physiologically compatible with the recipient.

[0189] Anti - CD19 antibody

[0190] This application provides anti-CD19 antibodies comprising one or more (e.g., 1, 2, 3, 4, 5, or 6) CDR sequences selected from the group consisting of: WBP7011-4.34.11, WBP7011-4.87.6, WBP7011_4.155.8, WBP7011_4.56.1, WBP7011-4.15.10, WBP7011- 4.100.1, WBP7011-4.106.3, WBP7011_4.108.3, WBP7011_4.191.6, WBP7011_4.194.10, WBP7011_4.231.5, W7011-4.34.11-z1-m5, W7011-4.87.6-z1(NS) and W7011-4.155.8-z1-P15.

[0191] As used in this application, “WBP7011-4.34.11” refers to a mouse monoclonal antibody having a heavy chain variable region as shown in SEQ ID NO:94 and a κ light chain variable region as shown in SEQ ID NO:96.

[0192] As used in this application, “WBP7011-4.87.6” refers to a mouse monoclonal antibody having a heavy chain variable region as shown in SEQ ID NO:98 and a κ light chain variable region as shown in SEQ ID NO:100.

[0193] As used in this application, “WBP7011_4.155.8” refers to a mouse monoclonal antibody having a heavy chain variable region as shown in SEQ ID NO:102 and a κ light chain variable region as shown in SEQ ID NO:104.

[0194] As used in this application, “WBP7011_4.56.1” refers to a mouse monoclonal antibody having a heavy chain variable region as shown in SEQ ID NO:106 and a κ light chain variable region as shown in SEQ ID NO:96.

[0195] As used in this application, “WBP7011-4.15.10” refers to a mouse monoclonal antibody having a heavy chain variable region as shown in SEQ ID NO:108 and a κ light chain variable region as shown in SEQ ID NO:96.

[0196] As used in this application, “WBP7011-4.100.1” refers to a mouse monoclonal antibody having a heavy chain variable region as shown in SEQ ID NO:110 and a κ light chain variable region as shown in SEQ ID NO:96.

[0197] As used in this application, “WBP7011-4.106.3” refers to a mouse monoclonal antibody having a heavy chain variable region as shown in SEQ ID NO:112 and a κ light chain variable region as shown in SEQ ID NO:96.

[0198] As used in this application, “WBP7011_4.108.3” refers to a mouse monoclonal antibody having a heavy chain variable region as shown in SEQ ID NO:114 and a κ light chain variable region as shown in SEQ ID NO:96.

[0199] As used in this application, “WBP7011_4.191.6” refers to a mouse monoclonal antibody having a heavy chain variable region as shown in SEQ ID NO:116 and a κ light chain variable region as shown in SEQ ID NO:96.

[0200] As used in this application, “WBP7011_4.194.10” refers to a mouse monoclonal antibody having a heavy chain variable region as shown in SEQ ID NO:118 and a κ light chain variable region as shown in SEQ ID NO:120.

[0201] As used in this application, “WBP7011_4.231.5” refers to a mouse monoclonal antibody having a heavy chain variable region as shown in SEQ ID NO:122 and a κ light chain variable region as shown in SEQ ID NO:96.

[0202] The “W7011-4.34.11-z1-m5” used in this application refers to a humanized antibody based on WBP3311_2.166.48, which contains a heavy chain variable region as shown in SEQ ID NO:124 and a κ light chain variable region as shown in SEQ ID NO:126.

[0203] As used in this application, “W7011-4.87.6-z1(NS)” refers to a humanized antibody based on WBP3311_2.166.48, which contains a heavy chain variable region as shown in SEQ ID NO:128 and a κ light chain variable region as shown in SEQ ID NO:130.

[0204] In this application, “W7011-4.155.8-z1-P15” refers to a humanized antibody based on WBP3311_2.166.48, which contains a heavy chain variable region as shown in SEQ ID NO:132 and a κ light chain variable region as shown in SEQ ID NO:134.

[0205] Table 1 shows the CDR sequences of these 11 mouse anti-CD19 antibodies, as well as 3 humanized antibodies W7011-4.34.11-z1-m5, W7011-4.87.6-z1(NS), and W7011-4.155.8-z1-P15. The variable region sequences of the heavy and light chains are also provided below.

[0206] Table 1

[0207]

[0208]

[0209] The following provides the heavy chain or κ light chain variable region sequences of the antibodies WBP7011-4.34.11, WBP7011-4.87.6, WBP7011_4.155.8, WBP7011_4.56.1, WBP7011-4.15.10, WBP7011-4.100.1, WBP7011-4.106.3, WBP7011_4.108.3, WBP7011_4.191.6, WBP7011_4.194.10 or WBP7011_4.231.5, as well as the humanized W7011-4.34.11-z1-m5, W7011-4.87.6-z1(NS) and W7011-4.155.8-z1-P15 antibodies.

[0210] WBP7011-4.34.11-VH

[0211] Amino acid sequence (SEQ ID NO:94):

[0212] EVQLQQSGPELVKPGASVKMSCKAS GYTFTNYVIH WVKQKPGQGLEWIG YFNPYN

[0213] DGTEYNEKFKA KATLTSDKSSSTAYMELSSLTSEDSAVYYCAK GPYYYGSSPFDY W

[0214] GQGTTLTVSS

[0215] Nucleic acid sequence (SEQ ID NO:95):

[0216] GAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTAAAGCCTGGGGCTTCAGTG

[0217] AAGATGTCCTGCAAGGCTTCTGGATACACATTCACTAACTATGTTATTCACTGGG

[0218] TGAAGCAGAAGCCTGGGCAGGGCCTTGAGTGGATTGGATATTTTAATCCTTACAA

[0219] TGATGGTACTGAATACAATGAGAAGTTCAAAGCCAAGGCCACACTGACTTCAGA

[0220] CAAATCCTCCAGCACAGCCTACATGGAGCTCAGCAGCCTGACCTCTGAGGACTCT

[0221] GCGGTCTATTACTGTGCAAAAGGTCCCTACTACTACGGTAGTAGCCCCTTTGACT

[0222] ACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA

[0223] WBP7011-4.34.11-VK

[0224] Amino acid sequence (SEQ ID NO:96):

[0225] DAVMTQTPLSLPVSLGDQASISC RSSQSLENSNGNTYLN WYLQKPGQSPQLLIY RVS

[0226] NRFS GVLDRFSGSGSGTDFTLKISRVEAEDLGVYFC LQVTHVPYT FGGGTKLEIK Nucleic acid sequence (SEQID NO:97):

[0227] GATGCTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCC

[0228] TCCATCTCTTGCAGGTCTAGTCAGAGCCTTGAAAACAGTAATGGAAACACCTATTTG

[0229] AACTGGTACCTCCAGAAACCAGGCCAGTCTCCACAGCTCCTGATCTACAGGGTTTCC

[0230] AACCGATTTTCTGGGGTCCTTGACAGGTTCAGTGGTAGTGGATCAGGGACAGATTTC

[0231] ACACTGAAAATCAGTAGAGTGGAGGCTGAGGATTTGGGAGTTTATTTCTGTCTCCAA

[0232] GTTACACATGTCCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAAWBP7011-4.87.6-VH

[0233] Amino acid sequence (SEQ ID NO:98):

[0234] QVQLQQSGAELVRPGSSVKISCKAS GYAFSTYWMN WVKQRPGQGLEWIG QIYPGD

[0235] DDTKYNGKFKG KASLTADKSSSTAYMQLISLTSEDSAVYFCAR RYFRYDYWYSDV

[0236] WGAGTTVTVTS

[0237] Nucleic acid sequence (SEQ ID NO:99):

[0238] CAGGTTCAACTGCAGCAGTCTGGGGCTGAGCTGGTGAGGCCTGGGTCCTCAGTGAA

[0239] GATTTCCTGCAAGGCTTCTGGCTATGCATTCAGTACCTATTGGATGAACTGGGTGAA

[0240] GCAGAGGCCTGGACAGGGTCTTGAGTGGATTGGACAGATTTATCCTGGAGATGATG

[0241] ATACTAAGTACAATGGAAAGTTCAAGGGTAAAGCCTCACTGACTGCAGACAAATCC

[0242] TCCAGCACCGCCTACATGCAGCTCATCAGCCTAACATCTGAGGACTCTGCGGTCTAT

[0243] TTCTGTGCAAGAAGATACTTTAGGTACGACTACTGGTATTCCGATGTCTGGGGCGCA

[0244] GGGACCACGGTCACCGTCACCTCA

[0245] WBP7011-4.87.6-VK

[0246] Amino acid sequence (SEQ ID NO:100):

[0247] DIQMTQTTSSLSASLGDRVTISC RASQDISNYLN WYQQKPDGTVKLLIY YTSRLHS GV

[0248] PARFSGSGSGTDYSLTISNLEQEDIATYFC HQGNTLPLT FGAGTKLELK

[0249] Nucleic acid sequence (SEQ ID NO:101):

[0250] GATATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTC

[0251] ACCATCAGTTGCAGGGCAAGTCAGGACATTAGCAATTATTTAAACTGGTATCAGCA

[0252] GAAACCGGATGGAACTGTTAAACTCCTGATCTATTACACATCAAGATTACACTCAGG

[0253] AGTCCCAGCAAGATTCAGTGGCAGTGGGTCTGGAACAGATTACTCTCTCACCATTAG

[0254] TAACCTGGAACAAGAAGATATTGCCACTTACTTTTGCCACCAGGGTAATACGCTTCC

[0255] GCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA

[0256] WBP7011-4.155.8-VH

[0257] Amino acid sequence (SEQ ID NO:102):

[0258] EIQLQQSGPELVKPGASVKVSCKAS GYAFTSYNMY WVKQSHGKSLEWIG YIDPYNG

[0259] DTTYNQKFKG KATLTVDKSSSTAYMHLNSLTSEDSAVYYCLT TAYAMDY WGQGTS

[0260] VTVSS

[0261] Nucleic acid sequence (SEQ ID NO:103):

[0262] GAGATCCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAA

[0263] GGTATCCTGCAAGGCTTCTGGTTATGCATTCACTAGCTACAACATGTACTGGGTGAA

[0264] GCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGATATATTGATCCTTACAATGGTG

[0265] ATACTACCTACAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTTGACAAGTCC

[0266] TCCAGCACAGCCTACATGCATCTCAACAGCCTGACATCTGAGGACTCTGCAGTCTAT

[0267] TACTGTCTCACTACGGCCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACC

[0268] GTCTCCTCA

[0269] WBP7011-4.155.8-VK

[0270] Amino acid sequence (SEQ ID NO:104):

[0271] QIVLTQSPAIMSASLGEEITLTCSASSTVNYMH WYQQKSGTSPKLLIY STSNLAS GVPS

[0272] RFSGSGSGTFYSLTIRSVEAEDAADYYC HQWSSYPYT FGGGTKLEIK

[0273] Nucleic acid sequence (SEQ ID NO:105):

[0274] CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCTAGGGGAGGAGATC

[0275] ACCCTAACCTGCAGTGCCAGCTCGACTGTAAATTACATGCACTGGTACCAGCAGAA

[0276] GTCAGGCACTTCTCCCAAACTCTTGATTTATAGCACATCCAACCTGGCTTCTGGAGT

[0277] CCCTTCTCGCTTCAGTGGCAGTGGGTCTGGGACCTTTTATTCTCTCACAATCAGAAGT

[0278] GTGGAGGCTGAAGATGCTGCCGATTATTACTGCCATCAGTGGAGTAGTTATCCGTAC

[0279] ACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA

[0280] WBP7011_4.56.1-VH

[0281] Amino acid sequence (SEQ ID NO:106):

[0282] EVQLQQSGPELVKPGASVKMSCKAS GYTFTNYVIH WVKQKPGQGLEWIG YINPYND

[0283] GTEYNEKFKG KATLTSDTSSSTAYMALSSLTSEDSAVYYCTR GPYYYGGSPFDY WG

[0284] QGTTLTVSS

[0285] Nucleic acid sequence (SEQ ID NO:107):

[0286] GAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTAAAGCCTGGGGCTTCAGTGAA

[0287] GATGTCCTGCAAGGCTTCTGGATACACATTCACTAACTATGTTATACACTGGGTGAA

[0288] GCAGAAGCCTGGGCAGGGCCTTGAGTGGATTGGATATATTAATCCTTACAATGATG

[0289] GTACTGAGTACAATGAGAAGTTCAAAGGCAAGGCCACACTGACTTCAGACACATCC

[0290] TCCAGCACAGCCTACATGGCGCTCAGCAGCCTGACCTCTGAGGACTCTGCGGTCTAT

[0291] TACTGTACAAGAGGACCCTATTACTACGGTGGTAGCCCCTTCGACTACTGGGGCCAA

[0292] GGCACCACTCTCACAGTCTCCTCA

[0293] WBP7011_4.56.1-VK

[0294] Amino acid sequence (SEQ ID NO:96):

[0295] DAVMTQTPLSLPVSLGDQASISC RSSQSLENSNGNTYLN WYLQKPGQSPQLLIY RVS

[0296] NRFS GVLDRFSGSGSGTDFTLKISRVEAEDLGVYFC LQVTHVPYTF GGGTKLEIK Nucleic acid sequence (SEQID NO:97):

[0297] GATGCTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCC

[0298] TCCATCTCTTGCAGGTCTAGTCAGAGCCTTGAAAACAGTAATGGAAACACCTATTTG

[0299] AACTGGTACCTCCAGAAACCAGGCCAGTCTCCACAGCTCCTGATCTACAGGGTTTCC

[0300] AACCGATTTTCTGGGGTCCTTGACAGGTTCAGTGGTAGTGGATCAGGGACAGATTTC

[0301] ACACTGAAAATCAGTAGAGTGGAGGCTGAGGATTTGGGAGTTTATTTCTGTCTCCAA

[0302] GTTACACATGTCCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAAWBP7011_4.15.10-VH

[0303] Amino acid sequence (SEQ ID NO:108):

[0304] EVQLQQSGPELVKPGASVKMSCKAS GYTFTSYVMH WMKQKPGQGLEWIG YINPYN

[0305] DGTEYHEKFKG KATLTSDKSSSTAYMELSSLTSEDSAVFYCAR GPYYYGGSPFDF W

[0306] GQGTTLTVSS

[0307] Nucleic acid sequence (SEQ ID NO:109):

[0308] GAGGTCCAGCTGCAGCAGTCTGGGCCTGAGCTGGTAAAGCCTGGGGCTTCAGTGAA

[0309] GATGTCCTGCAAGGCTTCTGGATACACATTCACTAGCTATGTTATGCACTGGATGAA

[0310] ACAGAAGCCTGGGCAGGGCCTTGAGTGGATTGGATATATTAATCCTTACAATGATG

[0311] GTACTGAGTACCATGAGAAGTTCAAAGGCAAGGCCACACTGACTTCAGACAAATCC

[0312] TCCAGCACAGCCTACATGGAGCTCAGCAGCCTGACCTCTGAGGACTCTGCGGTCTTT

[0313] TACTGTGCAAGAGGACCCTATTACTACGGTGGTAGCCCCTTTGACTTCTGGGGCCAA

[0314] GGCACCACTCTCACGGTCTCCTCA

[0315] WBP7011_4.15.10-VK

[0316] Amino acid sequence (SEQ ID NO:96):

[0317] DAVMTQTPLSLPVSLGDQASISC RSSQSLENSNGNTYLN WYLQKPGQSPQLLIY RVS

[0318] NRFS GVLDRFSGSGSGTDFTLKISRVEAEDLGVYFC LQVTHVPYTF GGGTKLEIK Nucleic acid sequence (SEQID NO:97):

[0319] GATGCTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCC

[0320] TCCATCTCTTGCAGGTCTAGTCAGAGCCTTGAAAACAGTAATGGAAACACCTATTTG

[0321] AACTGGTACCTCCAGAAACCAGGCCAGTCTCCACAGCTCCTGATCTACAGGGTTTCC

[0322] AACCGATTTTCTGGGGTCCTTGACAGGTTCAGTGGTAGTGGATCAGGGACAGATTTC

[0323] ACACTGAAAATCAGTAGAGTGGAGGCTGAGGATTTGGGAGTTTATTTCTGTCTCCAA

[0324] GTTACACATGTCCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAAWBP7011_4.100.1-VH

[0325] Amino acid sequence (SEQ ID NO: 110):

[0326] EVQLQQSGPELVKPGASVKMSCKAS GYTFTSYVIH WVKQKPGQGLEWIG YINPYND

[0327] GAEYTEKFKG KATLTSDKSSSTAYMELSSLTSEDSTVYYCAR GPYYYGGSPFDY WG

[0328] QGTTLTVSS

[0329] Nucleic acid sequence (SEQ ID NO: 111):

[0330] GAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTAAAGCCTGGGGCTTCAGTGAA

[0331] GATGTCCTGCAAGGCTTCTGGATACACATTCACTAGCTATGTTATACACTGGGTGAA

[0332] GCAGAAGCCTGGGCAGGGCCTTGAGTGGATTGGATATATTAATCCTTACAATGATG

[0333] GTGCTGAGTACACTGAGAAGTTCAAGGGCAAGGCCACACTGACTTCAGACAAATCC

[0334] TCCAGTACTGCCTATATGGAGCTCAGCAGCCTGACCTCTGAGGACTCTACGGTCTAT

[0335] TACTGTGCACGAGGACCCTATTACTACGGTGGTAGCCCCTTTGACTACTGGGGCCAA

[0336] GGCACCACTCTCACAGTCTCCTCA

[0337] WBP7011_4.100.1-VK

[0338] Amino acid sequence (SEQ ID NO: 96):

[0339] DAVMTQTPLSLPVSLGDQASISC RSSQSLENSNGNTYLNWYLQKPGQSPQLLIY RVS

[0340] NRFS GVLDRFSGSGSGTDFTLKISRVEAEDLGVYFC LQVTHVPYTF GGGTKLEIK nucleic acid sequence (SEQ ID NO:97):

[0341] GATGCTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCC

[0342] TCCATCTCTTGCAGGTCTAGTCAGAGCCTTGAAAACAGTAATGGAAACACCTATTTG

[0343] AACTGGTACCTCCAGAAACCAGGCCAGTCTCCACAGCTCCTGATCTACAGGGTTTCC

[0344] AACCGATTTTCTGGGGTCCTTGACAGGTTCAGTGGTAGTGGATCAGGGACAGATTTC

[0345] ACACTGAAAATCAGTAGAGTGGAGGCTGAGGATTTGGGAGTTTATTTCTGTCTCCAA

[0346] GTTACACATGTCCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAAWBP7011_4.106.3-VH

[0347] Amino acid sequence (SEQ ID NO:112):

[0348] EVQLQQSGPELVKPGASVKMSCKAS GYTFSSYVIH WVKQKPGQGLEWIG YINPYND

[0349] GAEYAEKFKG KATLTSDKSSSSAYMELGSLTSEDSAVYYCAR GPYYYGGSPFDY WG

[0350] QGTTLTVSS

[0351] Nucleic acid sequence (SEQ ID NO:113):

[0352] GAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTAAAGCCTGGGGCTTCAGTGAA

[0353] GATGTCCTGCAAGGCTTCTGGATACACATTCAGTAGTTATGTTATACACTGGGTGAA

[0354] GCAGAAGCCTGGGCAGGGCCTTGAGTGGATTGGATATATTAATCCTTACAATGATG

[0355] GTGCTGAGTATGCTGAGAAGTTCAAGGGCAAGGCCACACTGACTTCAGACAAATCC

[0356] TCCAGTTCTGCCTATATGGAGCTCGGCAGCCTGACCTCTGAGGACTCTGCGGTCTAT

[0357] TACTGTGCACGAGGACCCTATTACTACGGTGGTAGTCCCTTTGACTACTGGGGCCAA

[0358] GGCACCACTCTCACAGTCTCCTCA

[0359] WBP7011_4.106.3-VK

[0360] Amino acid sequence (SEQ ID NO:96):

[0361] DAVMTQTPLSLPVSLGDQASISC RSSQSLENSNGNTYLN WYLQKPGQSPQLLIY RVS

[0362] NRFS GVLDRFSGSGSGTDFTLKISRVEAEDLGVYFC LQVTHVPYTF GGGTKLEIK Nucleic acid sequence (SEQ ID NO:97):

[0363] GATGCTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCC

[0364] TCCATCTCTTGCAGGTCTAGTCAGAGCCTTGAAAACAGTAATGGAAACACCTATTTG

[0365] AACTGGTACCTCCAGAAACCAGGCCAGTCTCCACAGCTCCTGATCTACAGGGTTTCC

[0366] AACCGATTTTCTGGGGTCCTTGACAGGTTCAGTGGTAGTGGATCAGGGACAGATTTC

[0367] ACACTGAAAATCAGTAGAGTGGAGGCTGAGGATTTGGGAGTTTATTTCTGTCTCCAA

[0368] GTTACACATGTCCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAAWBP7011_4.108.3-VH

[0369] Amino acid sequence (SEQ ID NO:114):

[0370] EVQLQQSGPELVKPGASVEMSCKAS GYTFTSYVIH WLKQKPGQGLEWIG YINPYND

[0371] GAEYNEKFKG KATLTSDKSSSTAYMDLNSLTSEDSAVYYCAR GPYYYGSSPFDY WG

[0372] QGTTLTVSS

[0373] Nucleic acid sequence (SEQ ID NO:115):

[0374] GAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGGA

[0375] GATGTCCTGCAAGGCTTCTGGATACACATTCACTAGCTATGTTATTCACTGGTTGAA

[0376] GCAGAAGCCTGGGCAGGGCCTTGAGTGGATTGGATATATTAATCCTTACAATGATG

[0377] GTGCTGAGTATAATGAGAAGTTCAAGGGCAAGGCCACACTGACTTCAGACAAATCC

[0378] TCCAGTACAGCCTATATGGATCTCAACAGCCTGACCTCTGAGGACTCTGCGGTCTAT

[0379] TACTGTGCAAGAGGACCCTATTACTACGGTAGTAGCCCCTTTGACTACTGGGGCCAA

[0380] GGCACCACTCTCACAGTCTCCTCA

[0381] WBP7011_4.108.3-VK

[0382] Amino acid sequence (SEQ ID NO:96):

[0383] DAVMTQTPLSLPVSLGDQASISC RSSQSLENSNGNTYLN WYLQKPGQSPQLLIY RVS

[0384] NRFS GVLDRFSGSGSGTDFTLKISRVEAEDLGVYFC LQVTHVPYTF GGGTKLEIK Nucleic acid sequence (SEQID NO:97):

[0385] GATGCTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCC

[0386] TCCATCTCTTGCAGGTCTAGTCAGAGCCTTGAAAACAGTAATGGAAACACCTATTTG

[0387] AACTGGTACCTCCAGAAACCAGGCCAGTCTCCACAGCTCCTGATCTACAGGGTTTCC

[0388] AACCGATTTTCTGGGGTCCTTGACAGGTTCAGTGGTAGTGGATCAGGGACAGATTTC

[0389] ACACTGAAAATCAGTAGAGTGGAGGCTGAGGATTTGGGAGTTTATTTCTGTCTCCAA

[0390] GTTACACATGTCCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAAWBP7011_4.191.6-VH

[0391] Amino acid sequence (SEQ ID NO: 116):

[0392] EVQLLQSGPELVKPGASVKMSCKAS GYTFTDYVIH WVKQRPGQGLEWIG YINPYND

[0393] GSEYSEKFKG KATLTSDKSSSTAYMELSSLTSEDSAVYYCAR GPYYYGGSPFDY WG

[0394] QGTTLTVSS

[0395] Nucleic acid sequence (SEQ ID NO: 117):

[0396] GAGGTCCAGCTGCTGCAGTCTGGACCTGAGCTGGTAAAGCCTGGGGCTTCAGTGAA

[0397] GATGTCCTGCAAGGCTTCTGGATACACATTCACTGACTATGTTATACACTGGGTGAA

[0398] GCAGAGGCCTGGGCAGGGCCTTGAGTGGATTGGATATATTAATCCTTACAATGATG

[0399] GTTCTGAGTACAGTGAGAAGTTCAAAGGCAAGGCCACACTGACTTCAGACAAATCC

[0400] TCCAGCACAGCCTACATGGAGCTCAGCAGCCTGACCTCTGAGGACTCTGCGGTCTAT

[0401] TACTGTGCAAGAGGACCCTATTACTACGGTGGTAGTCCCTTTGACTACTGGGGCCAA

[0402] GGCACCACTCTCACAGTCTCCTCA

[0403] WBP7011_4.191.6-VK

[0404] Amino acid sequence (SEQ ID NO: 96):

[0405] DAVMTQTPLSLPVSLGDQASISC RSSQSLENSNGNTYLNWYLQKPGQSPQLLIY RVS

[0406] NRFS GVLDRFSGSGSGTDFTLKISRVEAEDLGVYFC LQVTHVPYTF GGGTKLEIK nucleic acid sequence (SEQ ID NO: 97):

[0407] GATGCTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCC

[0408] TCCATCTCTTGCAGGTCTAGTCAGAGCCTTGAAAACAGTAATGGAAACACCTATTTG

[0409] AACTGGTACCTCCAGAAACCAGGCCAGTCTCCACAGCTCCTGATCTACAGGGTTTCC

[0410] AACCGATTTTCTGGGGTCCTTGACAGGTTCAGTGGTAGTGGATCAGGGACAGATTTC

[0411] ACACTGAAAATCAGTAGAGTGGAGGCTGAGGATTTGGGAGTTTATTTCTGTCTCCAA

[0412] GTTACACATGTCCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAAWBP7011_4.194.10-VH

[0413] Amino acid sequence (SEQ ID NO: 118):

[0414] EVQLQQSGPELVKPGASVKMSCKAS GYTFTSYVMH WVKQKPGQGLEWIG YINPYN

[0415] DGTKYNEKFKG KATLTSDKSSSTAYMELSSLTSEDSAVYYCAR GPYYYGSSPFDY W

[0416] GQGTTLTVSS

[0417] Nucleic acid sequence (SEQ ID NO: 119):

[0418] GAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTAAAGCCTGGGGCTTCAGTGAA

[0419] GATGTCCTGCAAGGCTTCTGGATACACATTCACTAGCTATGTTATGCACTGGGTGAA

[0420] GCAGAAGCCTGGGCAGGGCCTTGAGTGGATTGGATATATTAATCCTTACAATGATG

[0421] GTACTAAGTACAATGAGAAGTTCAAAGGCAAGGCCACACTGACTTCAGACAAATCC

[0422] TCCAGCACAGCCTACATGGAACTCAGCAGCCTGACCTCTGAGGACTCTGCGGTCTAT

[0423] TACTGTGCAAGAGGACCCTATTACTACGGTAGTAGCCCCTTTGACTACTGGGGCCAA

[0424] GGCACCACTCTCACAGTCTCCTCA

[0425] WBP7011_4.194.10-VK

[0426] Amino acid sequence (SEQ ID NO:120):

[0427] DAVMTQTPLSLPVSLGDQASISC RSSQTLENSNGNTYLN WYLQKPGQSPQLLIY RVS

[0428] ,NRFS GVLDRFSGSGSGTDFTLKISRVETEDLGVYFC LQVTHVPYT FGGGTKLEIK Nucleic acid sequence (SEQID NO:121):

[0429] GATGCTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCC

[0430] TCCATCTCTTGCAGGTCTAGTCAGACCCTTGAAAACAGTAATGGAAACACCTATTTG

[0431] AACTGGTACCTCCAGAAACCAGGCCAGTCTCCACAGCTCCTGATCTACAGGGTTTCC

[0432] AACCGATTTTCTGGGGTCCTAGACAGGTTCAGTGGTAGTGGATCAGGGACAGATTTC

[0433] ACACTGAAAATCAGCAGAGTGGAGACTGAGGATTTGGGAGTTTATTTCTGCCTCCAA

[0434] GTTACACATGTCCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAAWBP7011_4.231.5-VH

[0435] Amino acid sequence (SEQ ID NO:122):

[0436] EVQLQQSGPELVKPGASVKMSCKAS GYTFTSYVMH WVKQKPGQGLEWIG YINPYN

[0437] DGTQYNEKFKG KATLTSDKSSSTAYMELSSLTSEDSAVYYCAR GPYYYSPSPFDY W

[0438] GQGTTLTVSS

[0439] Nucleic acid sequence (SEQ ID NO:123):

[0440] GAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTAAAGCCTGGGGCTTCAGTGAA

[0441] GATGTCCTGCAAGGCTTCTGGATACACATTCACTAGCTATGTCATGCACTGGGTGAA

[0442] GCAGAAGCCTGGGCAGGGCCTTGAGTGGATTGGATATATTAATCCTTACAATGATG

[0443] GTACTCAGTACAATGAGAAGTTTAAAGGCAAGGCCACACTGACTTCAGACAAATCC

[0444] TCCAGCACAGCCTACATGGAGCTCAGCAGCCTGACCTCTGAGGACTCTGCGGTCTAT

[0445] TACTGTGCAAGAGGACCCTATTACTACAGTCCTAGCCCCTTTGACTACTGGGGCCAA

[0446] GGCACCACTCTCACAGTCTCCTCA

[0447] WBP7011_4.231.5-VK

[0448] [[ID=1)2]]Amino acid sequence (SEQ ID NO: 96):

[0449] DAVMTQTPLSLPVSLGDQASISC RSSQSLENSNGNTYLN WYLQKPGQSPQLLIY RVS

[0450] NRFS GVLDRFSGSGSGTDFTLKISRVEAEDLGVYFC LQVTHVPYTF GGGTKLEIK Nucleic acid sequence (SEQ ID NO: 97): [[ID=:26]]

[0451] GATGCTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCC

[0452] TCCATCTCTTGCAGGTCTAGTCAGAGCCTTGAAAACAGTAATGGAAACACCTATTTG

[0453] AACTGGTACCTCCAGAAACCAGGCCAGTCTCCACAGCTCCTGATCTACAGGGTTTCC

[0454] AACCGATTTTCTGGGGTCCTTGACAGGTTCAGTGGTAGTGGATCAGGGACAGATTTC

[0455] ACACTGAAAATCAGTAGAGTGGAGGCTGAGGATTTGGGAGTTTATTTCTGTCTCCAA

[0456] GTTACACATGTCCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA [[ID=:44]]

[0457] It is known that CD19 receptor agonists (CDRs) are responsible for antigen binding; however, it has been found that not all six CDRs are indispensable or immutable. In other words, anti-CD19 antibodies WBP7011-4.34.11, WBP7011-4.87.6, WBP7011_4.155.8, WBP7011_4.56.1, WBP7011-4.15.10, WBP7011-4.100.1, WBP7011-4.106.3, and WBP7011_4.108 can be replaced, altered, or modified. .3, WBP7011_4.191.6, WBP7011_4.194.10, WBP7011_4.231.5, W7011-4.34.11-z1-m5, W7011-4.87.6-z1(NS) or W7011-4.155.8-z1-P15, one, two or three of the CDRs, while essentially maintaining an affinity for CD19-specific binding.

[0458] In some embodiments, the anti-CD19 antibody and its antigen-binding fragment described in this application comprise anti-CD19 antibodies WBP7011-4.34.11, WBP7011-4.87.6, WBP7011_4.155.8, WBP7011_4.56.1, WBP7011-4.15.10, WBP7011-4.100.1, and WBP7011-4. The heavy chain CDR3 sequence comprises one of the following: 106.3, WBP7011_4.108.3, WBP7011_4.191.6, WBP7011_4.194.10, WBP7011_4.231.5, W7011-4.34.11-z1-m5, W7011-4.87.6-z1(NS), or W7011-4.155.8-z1-P15. In some embodiments, the anti-CD19 antibody and antigen-binding fragment described in this application comprises a heavy chain CDR3 sequence selected from the group consisting of SEQ ID NO: 3, 9, 15, 21, 24, 27, 30, 33, 36, 39, and 45. The heavy chain CDR3 region is located at the center of the antigen-binding site and is therefore considered to have the most contact with the antigen and provide the most free energy for the affinity of the antibody for the antigen. Furthermore, it is believed that heavy chain CDR3 is currently the most diverse CDR in terms of length, amino acid composition, and conformation due to its diverse mechanisms (Tonegawa S., Nature. 302:575-81). The diversity of heavy chain CDR3 is sufficient to produce most antibody specificities (Xu JL, Davis MM. Immunity. 13:37-45) and the required antigen-binding affinity (Schier R et al., J Mol Biol. 263:551-67).

[0459] In some embodiments, the antibody and its antigen-binding fragment described herein contain a frame region (FR) sequence, provided that the antibody and its antigen-binding fragment can specifically bind to CD19. The CDR sequences shown in Table 1 are obtained from mouse antibodies, but they can be transplanted into any suitable FR sequence in any suitable species (such as mice, humans, rats, rabbits, and others) using suitable methods known in the art (such as recombinant techniques).

[0460] In some embodiments, the antibodies and antigen-binding fragments described herein are humanized. Humanized antibodies or antigen-binding fragments ideally possess reduced immunogenicity in the human body. Humanized antibodies or antigen-binding fragments are chimeric in their variable regions because a non-human CDR sequence is transplanted into a human or substantially human FR sequence. Humanization of the antibody or antigen-binding fragment can be substantially accomplished by replacing a non-human (e.g., mouse) CDR gene with a corresponding human CDR gene in a human immunoglobulin gene (see, for example, Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536).

[0461] This can be achieved by selecting appropriate human heavy and light chain variable domains using methods well-known in the art. In an exemplary instance, a best-fit approach can be used, in which non-human (e.g., rodent) antibody variable domain sequences are screened or BLAST-aligned against a database of known human variable domain sequences, and the human sequence closest to the non-human query sequence is identified as the human frame for transplanting non-human CDR sequences (see, for example, Sims et al. (1993) J. Immunol. 151:2296; Chothia et al. (1987) J. Mot. Biol. 196:901). Alternatively, a frame derived from a common sequence of all human antibodies can be used for transplanting non-human CDRs (see, for example, Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; Presta et al. (1993) J. Immunol., 151:2623).

[0462] In some embodiments, the humanized antibody or antigen-binding fragment described herein is substantially composed entirely of human sequences, except for the non-human CDR sequence. In some embodiments, the variable region FR and the constant region (if present) are all or substantially derived from human immunoglobulin sequences. The human FR sequence and the human constant region sequence may originate from different human immunoglobulin genes; for example, the FR sequence may originate from one human antibody, and the constant region from another human antibody. In some embodiments, the humanized antibody or antigen-binding fragment comprises human heavy / light chain FR1-4.

[0463] In some embodiments, the humanized antibody and its antigen-binding fragment described in this application comprise one or more FR sequences of W7011-4.34.11-z1-m5, W7011-4.87.6-z1(NS), or W7011-4.155.8-z1-P15. Table 2 below shows the FR sequences of W7011-4.34.11-z1-m5, W7011-4.87.6-z1(NS), or W7011-4.155.8-z1-P15. The corresponding native mouse FR sequences are also shown in Table 2. Heavy and light chain variable region sequences are also provided below.

[0464] Table 2

[0465]

[0466]

[0467] W7011-4.34.11-z1-m5-VH

[0468] Amino acid sequence (SEQ ID NO:124):

[0469] QVQLVQSGAEVKKPGSSVKVSCKAS GYTFTDYVIH WVRQAPGQGLEWMG YFNPYN

[0470] DGTEYNEKFKA RVTITADKSTSTAYMELSSLRSEDTAVYYCAR GPYYYGSSPFDY W

[0471] GQGTTVTVSS

[0472] Nucleic acid sequence (SEQ ID NO:125):

[0473] CAGGTGCAGCTTGTGCAGTCTGGAGCTGAAGTGAAGAAGCCAGGATCCTCCGTG

[0474] AAGGTCTCCTGTAAGGCTTCTGGCTACACCTTCACCGATTACGTGATCCACTGGG

[0475] TCAGGCAGGCCCCTGGGCAAGGCTTGGAGTGGATGGGGTACTTTAACCCCTACA

[0476] ACGATGGGACTGAGTACAATGAGAAGTTTAAAGCACGGGTGACCATTACCGCCG

[0477] ACAAGAGCACAAGCACAGCCTACATGGAGCTGTCCAGCCTCCGCAGCGAGGATA

[0478] CAGCCGTCTACTACTGCGCCAGAGGCCCTTACTACTATGGGTCCAGCCCCTTCGA

[0479] CTATTGGGGCCAGGGGACTACAGTGACTGTCAGTTCA

[0480] [[ID=第十八条]]W7011-4.34.11-z1-m5-VK

[0481] Amino acid sequence (SEQ ID NO: 126):

[0482] DIVMTQTPLSLPVTPGEPASISC RSSQSLENSNHNTYIN WYLQKPGQSPQLLIY RVSKR

[0483] FS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC HQVTHVPYT FGQGTKLEIK Nucleic acid sequence (SEQ ID NO: 127):

[0484] GATATCGTGATGACCCAGACTCCCCTGTCCCTTCCTGTGACCCCAGGAGAACCAG

[0485] CTTCTATCAGCTGTAGGTCCTCACAGAGCCTGGAGAACTCCAACCACAACACCTA

[0486] CATAAACTGGTACCTCCAGAAGCCTGGGCAGTCTCCCCAGTTGCTGATCTACAGG

[0487] GTCAGCAAACGCTTCTCCGGGGTGCCCGATCGGTTTAGTGGGAGCGGGAGCGGC

[0488] ACAGACTTTACACTCAAGATTTCCAGAGTGGAGGCCGAGGACGTCGGCGTCTATT

[0489] ACTGCCACCAAGTGACACACGTGCCCTACACATTCGGCCAGGGCACTAAACTGG

[0490] AGATTAAG

[0491] W7011-4.87.6-z1(N-S)-VH

[0492] Amino acid sequence (SEQ ID NO:128):

[0493] QVQLVQSGAEVKKPGASVKVSCKAS GYAFSTYWMN WVRQAPGQGLEWMG QIYPG

[0494] DDDTKYSGKFKG RVTITADKSTSTAYMELSSLRSEDTAVYYCAR RYFRYDYWYSDV

[0495] WGQGTTVTVSS

[0496] Nucleic acid sequence (SEQ ID NO:129):

[0497] CAGGTCCAGCTTGTCCAGTCTGGAGCAGAAGTGAAGAAGCCAGGGGCTTCAGTG

[0498] AAGGTGTCTTGCAAGGCTTCCGGATACGCCTTCTCCACTTACTGGATGAACTGGG

[0499] TGCGCCAGGCCCCTGGGCAGGGCTTGGAGTGGATGGGCCAGATCTATCCCGGCG

[0500] ATGACGACACAAAATACAGCGGGAAGTTCAAGGGGCGGGTGACCATTACCGCCG

[0501] ATAAAAGCACCTCCACCGCCTACATGGAGCTCAGTTCCCTGAGAAGCGAGGATA

[0502] CAGCCGTGTACTACTGTGCCAGGAGGTACTTTCGGTACGACTACTGGTATAGCGA

[0503] CGTCTGGGGGCAAGGCACAACTGTCACAGTGAGCAGC

[0504] W7011-4.87.6-z1(N-S)-VK

[0505] Amino acid sequence (SEQ ID NO:130):

[0506] DIQMTQSPSSLSASVGDRVTITC RASQDISNYLN WYQQKPGKVPKLLIY YTSRLHS GV

[0507] PSRFSGSGSGTDFTLTISSLQPEDVATYYC HQGNTLPLT FGQGTKLEIK

[0508] Nucleic acid sequence (SEQ ID NO:131):

[0509] GACATCCAAATGACCCAGAGCCCTTCCTCCTTGTCCGCAAGTGTGGGAGATAGA

[0510] GTGACCATCACCTGCAGGGCTTCTCAGGATATCTCCAACTACCTGAACTGGTATC

[0511] AGCAGAAGCCCGGCAAGGTGCCAAAGCTCCTTATTTACTACACCTCCCGGCTGCA

[0512] CAGCGGAGTCCCATCTCGCTTCAGCGGGTCAGGCAGCGGCACTGACTTTACTCTG

[0513] ACAATTAGCAGCCTCCAGCCTGAAGACGTCGCCACTTACTACTGTCATCAGGGGA

[0514] ATACACTCCCCCTGACATTCGGGCAGGGGACAAAACTGGAGATTAAGW7011-4.155.8-z1-P15-VH

[0515] Amino acid sequence (SEQ ID NO: 132):

[0516] QMQLVQSGPEVKKPGTSVKVSCKAS GYAFTSYNMY WVRQARGQRLEWIG YIDPYN

[0517] ADTTYNQKFKG RVTITRDMSTSTAYMELSSLRSEDTAVYYCLT TAYAMDY WGQGT

[0518] LVTVSS

[0519] Nucleic acid sequence (SEQ ID NO: 133):[[ID=********]] [[ID=********]]

[0520] [[ID=********]]CAAATGCAGCTCGTCCAGTCTGGACCTGAAGTGAAGAAGCCCGGGACATCCGTC[[ID=********]] [[ID=********]]

[0521] [[ID=********]]AAGGTCTCATGTAAGGCTAGCGGGTACGCATTCACTTCCTACAACATGTACTGGG[[ID=********]] [[ID=********]]

[0522] [[ID=********]]TGCGCCAGGCCAGAGGACAGAGGTTGGAGTGGATCGGCTACATCGACCCATACA[[ID=********]] [[ID=********]]

[0523] [[ID=********]]ACGCCGATACTACCTACAATCAGAAGTTTAAAGGGCGGGTGACCATTACCCGGG[[ID=********]] [[ID=********]]

[0524] [[ID=********]]ATATGTCCACCTCCACCGCCTACATGGAGCTGAGCAGCCTGAGGAGCGAGGACA[[ID=********]] [[ID=********]]

[0525] [[ID=********]]CAGCCGTGTACTACTGCCTGACAACAGCCTATGCCATGGACTATTGGGGCCAGG[[ID=********]] [[ID=********]]

[0526] [[ID=********]]GCACACTTGTGACTGTGAGCAGT[[ID=********]] [[ID=********]]

[0527] [[ID=********]]W7011-4.155.8-z1-P15-VK[[ID=********]] [[ID=********]]

[0528] [[ID=********]]Amino acid sequence (SEQ ID NO: 134):

[0529] DIQLTQSPSFLSASVGDRVTITC SASSTVNYMH WYQQKPGKAPKLLIY STSNLAS GVP

[0530] SRFSGSGSGTEFTLTISSLQPEDFATYYC HQWSSYPYT FGQGTKLEIK

[0531] Nucleic acid sequence (SEQ ID NO:135):

[0532] GACATCCAGCTCACCCAATCCCCTTCTTTCCTCTCCGCAAGTGTCGGAGATAGGG

[0533] TGACTATCACCTGCTCAGCTTCTTCAACCGTGAACTACATGCATTGGTACCAGCA

[0534] GAAGCCCGGGAAAGCCCCAAAGCTGCTGATCTACAGCACCTCCAATCTGGCCAG

[0535] TGGAGTGCCAAGCCGGTTTAGCGGGAGCGGCTCCGGCACTGAATTCACTTTGAC

[0536] AATTAGCAGCCTTCAGCCTGAGGACTTTGCCACATATTACTGTCACCAGTGGTCC

[0537] AGCTACCCCTACACATTCGGGCAGGGCACAAAGCTGGAGATTAAG

[0538] Exemplary humanized anti-CD19 antibodies W7011-4.34.11-z1-m5, W7011-4.87.6-z1(NS), or W7011-4.155.8-z1-P15 all maintain affinity for specific binding to CD3-expressing cells (e.g., CD4 T cells), and in this respect are at least comparable to, or even superior to, the parental mouse antibodies.

[0539] In some embodiments, the human-derived FR region may contain the same amino acid sequence as the human immunoglobulin from which it is derived. In some embodiments, one or more amino acid residues of the human FR are replaced by corresponding residues from the parental nonhuman antibody. This is necessary in some embodiments to make the humanized antibody or fragment thereof closely approximate the structure of the nonhuman parent antibody. In some embodiments, the humanized antibody or antigen-binding fragment described herein contains substitutions of no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue in each human FR sequence, or no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue in all FRs in the heavy or light chain variable domains. In some embodiments, such amino acid residue variations may exist only in the heavy chain FR region, only in the light chain FR region, or in both chains.

[0540] In some embodiments, the antibody and its antigen-binding fragment described in this application comprise a heavy chain variable domain sequence selected from the group consisting of: SEQ ID NO:94, SEQ ID NO:98, SEQ ID NO:102, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:128, and SEQ ID NO:132. In some embodiments, the antibody and its antigen-binding fragment described in this application comprise a light chain variable domain sequence selected from the group consisting of: SEQ ID NO:96, SEQ ID NO:100, SEQ ID NO:104, SEQ ID NO:120, SEQ ID NO:126, SEQ ID NO:130, and SEQ ID NO:134.

[0541] In some embodiments, the anti-CD19 antibody and antigen-binding fragment described in this application comprises all or part of the heavy chain variable domain and / or all or part of the light chain variable domain. In one embodiment, the anti-CD19 antibody and antigen-binding fragment described in this application is a single-domain antibody composed of all or part of the heavy chain variable region described in this application. More information on such single-domain antibodies is available in the prior art (see, for example, U.S. Patent No. 6,248,516).

[0542] In some embodiments, the anti-CD19 antibody and its antigen-binding fragment described in this application further comprise an immunoglobulin constant region. In some embodiments, the immunoglobulin constant region comprises a heavy chain and / or a light chain constant region. The heavy chain constant region comprises CH1, hinge, and / or CH2-CH3 regions. In some embodiments, the heavy chain constant region comprises an Fc region. In some embodiments, the light chain constant region comprises Cκ.

[0543] In some embodiments, the anti-CD19 antibody and its antigen-binding fragment have a constant region of IgG1, IgG2a, or IgG2b isotypes, which has reduced or depleted effector functions (such as ADCC or CDC) that can be assessed by various assays known in the art, such as Fc receptor binding assays, C1q binding assays, and cell lysis assays.

[0544] The binding affinity of the antibody and antigen-binding fragment described in this application can be determined by K. D The value represents the ratio of the dissociation rate to the binding rate when the binding between the antigen and antigen-binding molecules reaches equilibrium (k). off / k on Using suitable methods known in the art, including, for example, flow cytometry assays, antigen-binding affinity (e.g., K+) can be appropriately determined. D In some implementations, the binding of antibodies to antigens at different concentrations can be determined by flow cytometry. First, the determined mean fluorescence intensity (MFI) can be plotted against the antibody concentration. Then, using Prism version 5 (GraphPad Software, San Diego, CA), the correlation between specific binding fluorescence intensity (Y) and antibody concentration (X) can be fitted to a single-site saturation equation: Y = B. max *X / (K D +X)(Scarchard analysis), K can be calculated D Value, where B max This refers to testing the maximum specific binding of an antibody to an antigen.

[0545] In some embodiments, the anti-CD19 antibody and its antigen-binding fragment described in this application can specifically bind to human / cynomolgus CD19 naturally or artificially expressed on the cell surface. For example, the human / cynomolgus CD19 DNA sequence can be cloned into an expression vector and subsequently transfected and expressed in 293F cells so that the human / cynomolgus CD19 protein can be expressed on the surface of the transfected 293F cells.

[0546] In some embodiments, the anti-CD19 antibody and its antigen-binding fragment described in this application can be used at a rate not exceeding 5 × 10⁻⁶. -9 M, not exceeding 1×10 -9 M, not exceeding 9×10-10 M, not exceeding 8×10 -10 M, not exceeding 7×10 -10 M, not exceeding 6×10 -10 M, not exceeding 5×10 -10 M, not exceeding 4×10 -10 M, not exceeding 3×10 -10 M, not exceeding 2×10 -10 M or not exceeding 1×10 -10 Binding affinity of M (K) D It specifically binds to human CD19 expressed on the cell surface, the K D The values ​​were determined by flow cytometry.

[0547] In some embodiments, the anti-CD19 antibody and its antigen-binding fragment described in this application cross-react with cynomolgus monkey CD19 expressed on the cell surface.

[0548] The binding of antibodies to CD19 expressed on cells can also be measured using the "half-maximal effector concentration" (EC50). 50 The EC value represents the antibody concentration at which 50% of its maximum effect (e.g., binding or inhibition) is observed. 50 The value can be determined by methods known in the art, such as sandwich assays (e.g., ELISA), Western blotting, flow cytometry, and other binding assays. In some embodiments, the antibodies and fragments described in this application are expressed in EC50 at concentrations not exceeding 0.01 nM, 0.02 nM, 0.03 nM, 0.04 nM, 0.05 nM, 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM, 0.6 nM, 0.7 nM, 0.8 nM, 0.9 nM, or 1 nM. 50 The value specifically binds to human CD19 expressed on cells, the EC 50 The values ​​were determined by flow cytometry.

[0549] In some embodiments, the antibody and its antigen-binding fragment bind to cynomolgus monkey CD19 with a binding affinity similar to human CD19. For example, exemplary antibodies WBP7011-4.34.11, WBP7011-4.87.6, WBP7011_4.155.8, WBP7011_4.56.1, WBP7011-4.15.10, WBP7011-4.100.1, WBP7011-4.106.3, WBP7011_4.108.3, WBP7011_4.191.6, WBP7011_4.194.10, WBP7011_4.225.7, or WBP7011_4.231.5 bind to cynomolgus monkey CD19 with an affinity similar to human CD19 or EC40. 50 The value is associated with CD19 in cynomolgus monkeys.

[0550] In some embodiments, the antibody and its antigen-binding fragment described in this application are expressed at EC50 concentrations not exceeding 0.2 nM, 0.5 nM, 0.8 nM, 1 nM, 2 nM, or 3 nM. 50 The EC specifically binds to CD19 expressed on cells in cynomolgus monkeys. 50 The values ​​were determined by flow cytometry.

[0551] In some embodiments, the antibodies and fragments described in this application are in EC5 concentrations not exceeding 1 pM, 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 8 pM, 9 pM, 10 pM, 11 pM, 12 pM, 13 pM, 14 pM, 15 pM, 16 pM, 17 pM, 18 pM, 19 pM, 20 pM, 21 pM, 22 pM, 23 pM, 24 pM, 25 pM, 30 pM, 35 pM, 40 pM, 45 pM, or 50 pM. 50 The value is internalized in cells expressing CD19, the EC 50 The value was determined by the Fab-Zap method.

[0552] In some embodiments, the antibodies and fragments thereof described in this application have sufficient affinity for specific binding to human CD19 for diagnostic and / or therapeutic purposes. Many B-cell-targeting therapies utilize anti-human CD19 monoclonal antibodies in clinical practice.

[0553] The antibodies or fragments thereof described in this application may be monoclonal antibodies, polyclonal antibodies, humanized antibodies, chimeric antibodies, recombinant antibodies, bispecific antibodies, labeled antibodies, bivalent antibodies, or anti-idiotype antibodies. Recombinant antibodies are antibodies prepared in vitro, rather than in vivo, using recombinant methods.

[0554] Antibody variants

[0555] This application also covers various variants of the antibodies and fragments described in this application. In some embodiments, this application covers variants of the exemplary antibodies described in this application, namely variants of WBP7011-4.34.11, WBP7011-4.87.6, WBP7011_4.155.8, WBP7011_4.56.1, WBP7011-4.15.10, WBP7011-4.100.1, WBP7011-4.106.3, WBP7011_4.108.3, WBP7011_4.191.6, WBP7011_4.194.10, WBP7011_4.231.5, W7011-4.34.11-z1-m5, W7011-4.87.6-z1(NS), or W7011-4.155.8-z1-P15.

[0556] In some embodiments, the antibody variants contain one or more modifications or substitutions in one, two, or three CDR sequences shown in Table 1, one or more FR sequences shown in Table 2, the heavy or light chain variable region sequences described in this application, and / or constant regions (e.g., Fc regions). These antibody variants retain the affinity of their parent for specific binding to CD19, but have the desired properties resulting from one or more of the aforementioned modifications or substitutions. For example, antibody variants may have improved antigen-binding affinity, improved glycosylation patterns, reduced glycosylation risk, reduced deamination, reduced or depleted effector function, improved FcRn receptor binding, increased pharmacokinetic half-life, pH sensitivity, and / or compatibility with conjugations (e.g., one or more introduced cysteine ​​residues).

[0557] Methods known in the art, such as alanine scanning mutagenesis, can be used to screen parental antibody sequences to identify suitable or preferred residues to be modified or substituted (see, for example, Cunningham and Wells, (1989) Science, 244:1081-1085). Briefly, target residues (e.g., positively charged residues such as Arg, Asp, His, Lys, and Glu) can be identified and substituted with uncharged or negatively charged amino acids (e.g., alanine or polyalanine) to generate modified antibodies, which can then be screened for target properties. If substitution at a particular amino acid position exhibits a target functional change, that position can be identified as a potential residue for modification or substitution. These potential residues can be further evaluated by substitution with another residue (e.g., a cysteine ​​residue, a positively charged residue, etc.).

[0558] Affinity variants

[0559] Affinity variants may contain one or more CDR sequences as shown in Table 1, one or more FR sequences as shown in Table 2, or modifications or substitutions in the heavy or light chain variable region sequences described in this application. The affinity variants retain the CD19-specific binding affinity of the parent antibody, or even have improved CD19-specific binding affinity relative to the parent antibody. In some embodiments, at least one (or all) substitutions of the CDR sequence, FR sequence, or variable region sequence comprise conserved substitutions.

[0560] Those skilled in the art will understand that one or more amino acid residues in the CDR and FR sequences shown in Tables 1 and 2 can be substituted, and the resulting antibody or antigen-binding fragment will still maintain its affinity for binding to CD19, or even have improved binding affinity. This can be achieved using various methods known in the art. For example, an antibody variant library (such as Fab or scFv variants) can be generated and expressed using phage display technology, followed by screening for affinity for binding to human CD19. Alternatively, computer software can be used to simulate the binding of an antibody to human CD19 and identify amino acid residues on the antibody that form the binding interface. These residues can be avoided during substitution to prevent a decrease in binding affinity, or they can be used as targets for substitution to obtain a stronger binding.

[0561] In some embodiments, the humanized antibody or antigenic determinant fragment described herein contains one or more amino acid residue substitutions in one or more CDR sequences and / or one or more FR sequences. In some embodiments, the affinity variant contains a total of no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitution in the CDR and / or FR sequences.

[0562] In some embodiments, the anti-CD19 antibody and its antigen-binding fragment comprise one, two, or three CDR sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with the sequences listed in Table 1, while maintaining an affinity for binding to CD19 that is similar to or higher than that of its parent antibody.

[0563] In some embodiments, the anti-CD19 antibody and its antigen-binding fragment comprise one or more FR sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with the sequences listed in Table 2, while maintaining an affinity for binding to CD19 that is similar to or higher than that of its parent antibody.

[0564] In some embodiments, the anti-CD19 antibody and its antigen-binding fragment comprise one or more variable region sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with the following sequences: SEQ ID NO:94, SEQ ID NO:98, SEQ ID NO:102, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:128, SEQ ID NO:132, SEQ ID NO:96, SEQ ID NO:100, SEQ ID NO:104, SEQ ID NO:120, SEQ ID NO:126, SEQ ID NO:130, SEQ ID NO:96, SEQ ID NO:100, SEQ ID NO:104, SEQ ID NO:120, SEQ ID NO:126, SEQ ID NO:130, SEQ ID NO:128, SEQ ID NO:129 ... NO:134, while maintaining an affinity for binding to CD19 similar to or higher than that of its parent antibody. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, or deleted in sequences selected from SEQ ID NO:94, SEQ ID NO:98, SEQ ID NO:102, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:128, SEQ ID NO:132, SEQ ID NO:96, SEQ ID NO:100, SEQ ID NO:104, SEQ ID NO:120, SEQ ID NO:126, SEQ ID NO:130, and SEQ ID NO:134. In some embodiments, the substitution, insertion, or deletion occurs outside the CDR region (i.e., in the FR).

[0565] Glycosylation variants

[0566] The anti-CD19 antibody and antigen-binding fragment described in this application also contain glycosylation variants. These glycosylation variants can be obtained to increase or decrease the degree of glycosylation of the antibody or antigen-binding fragment.

[0567] The anti-CD19 antibody or its antigen-binding fragment may include one or more amino acid residues with side chains to which a carbohydrate moiety (e.g., an oligosaccharide structure) may be attached. Antibody glycosylation is typically N-linked or O-linked. N-linked glycosylation refers to the attachment of a carbohydrate moiety to an aspartic acid residue in the side chain, such as aspartic-X-serine and aspartic-X-threonine in a tripeptide sequence, where X is any amino acid other than proline. O-linked glycosylation refers to the attachment of one of the sugars—N-acetylgalactosamine, galactose, or xylose—to a hydroxyl amino acid, most commonly to serine or threonine. Removal of native glycosylation sites can be readily accomplished, for example, by altering the amino acid sequence so that one of the aforementioned tripeptide sequences (for N-linked glycosylation sites) or serine or threonine residues (for O-linked glycosylation sites) present in that sequence is replaced. Similarly, new glycosylation sites can be created by introducing such tripeptide sequences or serine or threonine residues.

[0568] Cysteine ​​engineered variants

[0569] The anti-CD19 antibody and antigen-binding fragments described in this application also cover cysteine-engineered variants that include one or more introduced free cysteine ​​amino acid residues.

[0570] Free cysteine ​​residues are cysteine ​​residues that are not part of a disulfide bond. Engineered variants of cysteine ​​can be used to conjugate, for example, cytotoxic and / or imaging compounds, tags or radioisotopes, and other substances at the engineered cysteine ​​site via maleimide or haloacetyl groups. Methods for modifying antibody or antigen-binding fragments to introduce free cysteine ​​residues are well known in the art, see, for example, WO2006 / 034488.

[0571] Fc variant

[0572] The anti-CD19 antibody and antigen-binding fragment described in this application also include Fc variants, which include one or more amino acid residue modifications or substitutions in their Fc region and / or hinge region.

[0573] In some embodiments, the anti-CD19 antibody or antigen-binding fragment contains one or more amino acid substitutions that improve pH-dependent binding to the neonatal Fc receptor (FcRn). This variant binds to FcRn at an acidic pH, preventing its degradation in lysosomes and allowing it to be subsequently translocated and released extracellularly; therefore, this variant may have a longer pharmacokinetic half-life. The method of engineering antibodies and their antigen-binding fragments to improve binding affinity to FcRn is well known in the art, see, for example, Vaughn, D. et al., Structure, 6(1):63-73, 1998; Kontermann, R. et al., Antibody Engineering, Vol. 1, Chapter 27: Engineering of the Fc region for improved PK, Springer, 2010; Yeung, Y. et al., Cancer Research, 70:3269-3277 (2010); and Hinton, P. et al., J. Immunology, 176:346-356 (2006).

[0574] In some embodiments, the anti-CD19 antibody or antigen-binding fragment contains one or more amino acid substitutions that alter antibody-dependent cytotoxicity (ADCC). Certain amino acid residues in the CH2 domain of the Fc region may be substituted to enhance ADCC activity. Alternatively or additionally, the carbohydrate structure on the antibody may be altered to enhance ADCC activity. Methods for altering ADCC activity through antibody engineering have been described in existing technologies, see, for example, Shields RL et al., J Biool Chem. 2001. 276(9):6591-604; Idusogie EE et al., J Immunol. 2000. 164(8):4178-84; Steurer W. et al., J Immunol. 1995. 155(3):1165-74; Idusogie EE et al., J Immunol. 2001. 166(4):2571-5; Lazar GA et al., PNAS, 2006. 103(11):4005-4010; Ryan MC et al., Mol. Cancer Ther. 2007. 6:3009-3018; Richards JO et al., Mol Cancer Ther. 2008. 7(8):2517-27; Shields RL et al., J. Biol. Chem, 2002, 277: 26733-26740; Shinkawa T. et al., J. Biol. Chem, 2003, 278: 3466-3473.

[0575] In some embodiments, the anti-CD19 antibody or antigen-binding fragment contains one or more amino acid substitutions that alter complement-dependent toxicity (CDC), for example by enhancing or weakening C1q binding and / or complement-dependent toxicity (CDC) (for examples of other Fc region variants, see, for example, WO99 / 51642; Duncan & Winter Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO94 / 29351).

[0576] In some embodiments, the anti-CD19 antibody or antigen-binding fragment comprises one or more amino acid substitutions located at the Fc region interface to facilitate and / or promote heterodimerization. These modifications include introducing a protrusion into a first Fc polypeptide and introducing a cavity into a second Fc polypeptide, wherein the protrusion may be located within the cavity to promote the interaction of the first and second Fc polypeptides to form a heterodimer or complex. Methods for generating antibodies with these modifications are well known in the art, for example, as described in U.S. Patent No. 5,731,168.

[0577] antigen-binding fragments

[0578] This application also provides anti-CD19 antigen-binding fragments. Various types of antigen-binding fragments are well known in the art and can be developed based on the anti-CD19 antibodies described in this application, including, for example, exemplary antibodies whose CDR and FR sequences are shown in Tables 1 and 2, and their various variants (such as affinity variants, glycosylation variants, Fc variants, cysteine-engineered antibodies, etc.).

[0579] In some embodiments, the anti-CD19 antigen-binding fragment described in this application is a camelized single-chain domain antibody, a diabody, a single-chain Fv fragment (scFv), an scFv dimer, BsFv, dsFv, (dsFv)2, dsFv-dsFv'Fv fragment, Fab, Fab', F(ab')2, a bispecific antibody, a ds diabody, a nanobody, a domain antibody, a single-domain antibody, or a bivalent domain antibody.

[0580] A variety of techniques can be used to produce such antigen-binding fragments. Exemplary methods include enzymatic digestion of intact antibodies (see, for example, Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992); and Brennan et al., Science, 229:81 (1985)), recombinant expression by host cells (e.g., for Fab, Fv, and ScFv antibody fragments), phage display library screening as discussed above (e.g., for ScFv), and chemical coupling of two Fab'-SH fragments to form the F(ab')2 fragment (Carter et al., Bio / Technology 10:163-167 (1992)). Other techniques for producing antibody fragments will be apparent to those skilled in the art.

[0581] In some embodiments, the antigen-binding fragment is a scFv. The generation of scFv is described, for example, in WO 93 / 16185; U.S. Patent Nos. 5,571,894; and 5,587,458. scFv can be fused to an effector protein at its N-terminus or C-terminus to obtain a fusion protein (see, for example, Antibody Engineering, ed. Borrebaeck).

[0582] The anti-CD19 antibody or its antigen-binding fragment described in this application may be a monoclonal antibody, polyclonal antibody, humanized antibody, chimeric antibody, recombinant antibody, bispecific antibody, labeled antibody, bivalent antibody, or anti-idiotype antibody. Recombinant antibodies are antibodies prepared in vitro using recombinant methods rather than in animals.

[0583] In some embodiments, the antibody and its antigen-binding fragment may be used as the basis for bispecific or multivalent antibodies or antibody-drug conjugates.

[0584] Bispecific antibodies, multivalent antibodies

[0585] In some embodiments, the antibodies and antigen-binding fragments described in this application are bivalent, tetravalent, hexavalent, or multivalent. In some embodiments, the antibodies and antigen-binding fragments described in this application are monospecific or bispecific.

[0586] As used in this application, the term "valence" refers to the presence of a specific number of antigen-binding sites in a given molecule. Thus, the terms "bivalent," "tetravalent," and "hexavalent" indicate that the antigen-binding molecule has two, four, and six binding sites, respectively. A bivalent molecule can be monospecific if both binding sites are used to specifically bind to the same antigen or the same epitope. Similarly, a trivalent molecule can be bispecific, for example, when two binding sites are monospecific against a first antigen (or epitope), and a third binding site is specific against a second antigen (or epitope).

[0587] In some embodiments, the antibodies and antigen-binding fragments described in this application may be monospecific, however, bivalent, trivalent, or valent, having at least two binding sites specific to the same antigen or epitope. In some embodiments, this results in a stronger binding to the antigen or epitope compared to the corresponding monovalent antibody. In some embodiments, in the bivalent antigen-binding portion, the first valent and second valent binding sites are structurally identical (i.e., have the same sequence) or structurally different (i.e., have different sequences but the same specificity).

[0588] In some embodiments, the antibodies and antigen-binding fragments described in this application are bispecific. In some embodiments, the antibodies and antigen-binding fragments described in this application have a first specificity against CD19 and a second specificity. In some embodiments, the second specificity is against CD19 but against a different epitope. In some embodiments, the second specificity is against a second antigen different from CD19 and can promote or facilitate an immune response against CD19-expressing target cells when in close proximity to the second antigen. For example, the bispecific antibody can bring CD19-expressing target cells into close proximity with immune cells such as T cells or NK cells, thereby promoting the recognition or elimination of the target cells by the immune system.

[0589] The bispecific antibodies and antigen-binding fragments described in this application can be prepared using any suitable method known in the art. In conventional methods, two immunoglobulin heavy-light chain pairs with different antigen specificities can be co-expressed in host cells to generate bispecific antibodies in a recombinant manner (see, for example, Milstein and Cuello, Nature, 305:537 (1983)), followed by affinity chromatography purification.

[0590] Alternatively, a recombinant approach can be used, in which sequences encoding the variable domains of two specific antibody heavy chains are fused to immunoglobulin constant domain sequences, respectively, and then inserted into an expression vector, which is co-transfected with an expression vector of the light chain sequence into suitable host cells to recombinantly express the bispecific antibody (see, for example, WO 94 / 04690; Suresh et al., Methods in Enzymology, 121:210 (1986)). Similarly, scFv dimers can also be recombinantly constructed and expressed from host cells (see, for example, Gruber et al., J. Immunol., 152:5368 (1994)).

[0591] In another approach, leucine zipper peptides from Fos and Jun proteins can be fused to the Fab' portion of two different antibodies via gene fusion. The fused antibodies are reduced to four half-antibodies (i.e., monomers) in the hinge region and then oxidized to form heterodimers (Kostelny et al., J. Immunol., 148(5):1547-1553(1992)).

[0592] The two antigen-binding domains can also be conjugated or cross-linked to form bispecific antibodies or antigen-binding fragments. For example, one antibody can be coupled to biotin and the other to avidin, and the strong link between biotin and avidin can cause the two antibodies to complex together to form a bispecific antibody (see, for example, U.S. Patent Nos. 4,676,980; WO 91 / 00360, WO 92 / 00373, and EP 03089). As another example, the two antibodies or antigen-binding fragments can be cross-linked using conventional methods known in the art, for example, as described in U.S. Patent No. 4,676,980.

[0593] Bispecific antigen-binding fragments can be generated from bispecific antibodies, for example, through proteolytic cleavage or chemical linkage. For example, an antigen-binding fragment of an antibody (e.g., Fab') can be prepared and converted into a Fab'-thiol derivative, which is then mixed with and reacted with another converted Fab' derivative having different antigen specificity to form a bispecific antigen-binding fragment (see, for example, Brennan et al., Science, 229:81 (1985)).

[0594] In some embodiments, the bispecific antibody or antigen-binding fragment can be modified at the interface to form a knot-into-hole link, thereby promoting heterodimerization of two distinct antigen-binding sites. As used in this application, "knob-into-hole" refers to an interaction between two polypeptides (such as Fc), one of which has a protrusion (i.e., a "knob") due to the presence of amino acid residues with large side chains (e.g., tyrosine or tryptophan), and the other polypeptide has a cavity (i.e., a "hole") containing small side-chain amino acid residues (e.g., alanine or threonine), and the protrusion can be positioned within the cavity to promote the interaction of the two polypeptides to form a heterodimer or complex. Methods for generating polypeptides using knots-into-holes are well known in the art, for example, as described in U.S. Patent No. 5,731,168.

[0595] Conjugate

[0596] In some embodiments, the anti-CD19 antibody and its antigen-binding fragment are linked to conjugates. A conjugate is a non-protein portion that can attach to the antibody or its antigen-binding fragment. It is conceivable that the antibody or antigen-binding fragment described in this application can be linked to a variety of conjugates (see, for example, "Conjugate Vaccines," Contributions to Microbiology and Immunology, J.M. Curuse and RE. Lewis, Jr. (eds.), Carger Press, New York (1989)). These conjugates can be linked to the antibody or antigen-binding fragment by covalent binding, affinity binding, intercalation, coordinate binding, complexation, binding, mixing, or addition, among other methods. In some embodiments, the antibody or its antigen-binding fragment is linked to one or more conjugates via a linker. In some embodiments, the linker is a hydrazone linker, a disulfide linker, a bifunctional linker, a dipeptide linker, a glucuronide linker, or a thioether linker.

[0597] In some embodiments, the anti-CD19 antibody and antigen-binding fragment disclosed in this application can be engineered to contain a specific site in addition to the epitope-binding portion, said specific site being available for binding to one or more conjugates. For example, the site may include one or more reactive amino acid residues (e.g., cysteine ​​or histidine residues) to facilitate covalent linkage with the conjugate.

[0598] The conjugate may be a therapeutic agent (e.g., a chemotherapeutic agent), a radioisotope, a detectable label (e.g., a lanthanide element, a luminescent label, a fluorescent label, or an enzyme-substrate label), a pharmacokinetic modified portion, or a purified portion (e.g., magnetic beads or nanoparticles).

[0599] Examples of detectable markers may include fluorescent markers (e.g., fluorescein, rhodamine, dansyl, phycoerythrin, or Texas red), enzyme-substrate markers (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, glucosyl amylase, lysozyme, sugar oxidase, or β-D-galactosidase), radioisotopes, other lanthanides, luminescent markers, chromophores, digoxigenin, biotin / avidin, DNA molecules, or gold for detection.

[0600] Examples of radioactive isotopes may include 123 I, 124 I, 125 I, 131 I, 35 S, 3 H, 111 In、 112 In、 14 C 64 Cu、 67 Cu、 86 Y、 88 Y、 90 Y、 177 Lu、 211 At、 186 Re、 188 Re、 153 Sm、 212 Bihe 32 P. Radioisotope-labeled antibodies can be used in receptor-targeted imaging experiments.

[0601] In some embodiments, the conjugate may be a pharmacokinetic modification, such as PEG, which helps prolong the antibody's half-life. Other suitable polymers include, for example, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, ethylene glycol / propylene glycol copolymers, etc.

[0602] In some embodiments, the conjugate may be a purified fraction, such as magnetic beads or nanoparticles.

[0603] Antibody-drug conjugates

[0604] In some embodiments, this disclosure provides antibody-drug conjugates (ADCs) comprising any of the anti-CD19 antibodies or antigen-binding fragments conjugated to a cytotoxic agent (such as a chemotherapeutic agent, drug, growth inhibitor, toxin, or radioisotope (i.e., a radioconjugate)) as described above.

[0605] Antibody-drug conjugates can be used for the local delivery of cytotoxic agents, such as in cancer treatment. This allows for the targeted delivery of cytotoxic agents to tumors, where they can accumulate intracellularly. This effect is particularly significant because systemic administration of these unconjugated cytotoxic agents to normal cells and to eliminate tumor cells could result in unacceptable levels of toxicity (Baldwin et al. (1986) Lancet pp. (March 15, 1986): 603-05; Thorpe, (1985) “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review” in Monoclonal Antibodies' 84: Biological and Clinical Applications, A. Pinchera et al. (eds.), pp. 475-506; Syrigos and Ebenetos (1999) Anticancer Research 19: 605-614; Niculescu-Duvaz and Springer (1997) Adv. Drg Del. Rev. 26: 151-172; US Patent No. 4,975,278).

[0606] In some embodiments, the cytotoxic agent can be any agent that is harmful to cells or may damage or kill cells. In some embodiments, the cytotoxic agent is optionally a cytotoxin, a DNA alkylating agent, a topoisomerase inhibitor, a microtubule binding agent, or other anticancer drug.

[0607] Examples of enzymatically active cytotoxins include bacterial and plant toxins such as diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin, absinthecin, saccharitoxin, spatholobus suberectus toxin, tung oil protein, caryophyllin protein, pokeweed protein (PARI, PAPII, and PAP-S), bitter melon inhibitor, jatropha toxin, croton toxin, soapwort inhibitor, white tree toxin, localized aspergillin, phenolmycin, enoxacin, and trichothecene toxins (see, for example, WO 93 / 21232). When acted upon by methods known in the art (e.g., as described by Vitetta et al. (1987) Science, 238:1098), this macromolecular toxin may be conjugated to the antibody or antigen-binding fragment described in this application.

[0608] The cytotoxic agents can also be small molecule toxins and drugs, such as gerdemycin (Mandler et al. (2000) Jour. of the Nat. Cancer Inst. 92(19):1573-1581; Mandler et al. (2002) Bioconjugate Chem. 13:786-791), maytansine compounds (EP 1391213; Liu et al. (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623), cazithromycin (Lode et al. (1998) Cancer Inst. 92(19):1573-1581; Mandler et al. (2002) Bioconjugate Chem. 13:786-791), maytansine compounds (EP 1391213; Liu et al. (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623), and cazithromycin (Lode et al. (1998) Cancer Inst. 92(19):1573-1581; Mandler et al. (2002) Bioconjugate Chem. 13:786-791), maytansine compounds (EP 1391213; Liu et al. (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623), maytansine (Lode et al. (1998) Cancer Inst. 92(19):1573-1581; Mandler et al. (2002) Bioconjugate Chem. 13:786-791), maytansine compounds (EP 1391213; Liu et al. (1996) Proc. Natl. Acad. Sci. USA Res. 58:2928; Hinman et al. (1993) Cancer Res. 53:3336-3342), paclitaxel, cytochalasin B, bacitracin D, ethidium bromide, ipecacine, mitomycin, etoposide, teniposide, vincristine, vinblastine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthraxetine dione, mitoxantrone, photomycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and its analogues, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine), alkylating agents (e.g., nitrogen mustard, thiotepa). Thioepachlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and dichlorodiamineplatin(II)(DDP)cisplatin), anthracycline antibiotics (e.g. daunorubicin (formerly donomycin) and doxorubicin), antibiotics (e.g. dermatomycin (formerly actinomycin), bleomycin, photomycin and amycin (AMC)), and antimitotic agents (e.g. vincristine and vinblastine), kazimidoxine, maytansine compounds, sea haretoxin, orlistatine, trichothecene toxins and CC1065, and their derivatives with cytotoxic activity.

[0609] The cytotoxic agent can also be a highly radioactive isotope. Examples include At. 211 I 131 I 125 Y 90 Re 186 、Sm 153 Bi 212 P 32 Pb 212And radioactive isotopes of Lu. Methods for conjugating radioactive isotopes to antibodies are well known in the art, for example via suitable ligand reagents (see, for example, WO94 / 11026; Current Protocols in Immunology, Volumes 1 and 2, edited by Coligen et al., Wiley-Interscience, New York, NY, Pubs. (1991)). The ligand reagent has a chelating ligand capable of binding, chelating, or otherwise complexing with the radioactive isotope metal, and also has a functional group that readily reacts with the thiol group of cysteine ​​in the antibody or antigen-binding fragment. Exemplary chelating ligands include DOTA, DOTP, DOTMA, DTPA, and TETA (Macrocyclics, Dallas, Texas).

[0610] The antibody (or antigen-binding fragment) conjugate with the cytotoxic agent described in this application can be generated using various bifunctional protein linkers. Exemplary bifunctional linkers include N-succinimide-3-(2-pyridinedithio)propionic acid (SPDP), succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid (SMCC), iminothioalkyl (IT), bifunctional derivatives of imine esters (such as dimethyl hexamethylenediimide hydrochloride), active esters (such as disuccinimide octanoate), aldehydes (such as glutaraldehyde), diazid compounds (such as bis(p-azidobenzoyl)hexamethylenediamine), dinitrogen salt derivatives (such as bis(p-diazidobenzoyl)ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and biactive fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).

[0611] In some embodiments, the ADC described in this application is prepared using a ligation reagent selected from the group consisting of: BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPRH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfonated EMCS, sulfonated GMBS, sulfonated KMUS, sulfonated MBS, sulfonated SIAB, sulfonated SMCC, and sulfonated SMPB, as well as SVSG (succinimide-(4-vinyl sulfone)benzoic acid). These ligation reagents are commercially available (e.g., from Pierce Biotechnology, Rockford, Illinois, USA; see 2003-2004 Applications Handbook and Catalog, pp. 467-498).

[0612] In some embodiments, the linker is cleavable under specific physiological conditions, thereby facilitating the release of intracellular cytotoxic drugs. For example, the linker may be acid-labile, peptidase-sensitive, light-labile, dimethyl, or disulfide-containing (Chari et al., Cancer Research 52:127-131 (1992); US Patent No. 5,208,020). In some embodiments, the linker may comprise amino acid residues such as dipeptides, tripeptides, tetrapeptides, or pentapeptides. The amino acid residues in the linker may be native or non-native amino acid residues. Examples of such linkers include: valine-citrulline (ve or val-cit), alanine-phenylalanine (af or ala-phe), glycine-valine-citrulline (gly-yal-cit), glycine-glycine-gly (gly-gly-gly), and valine-citrulline-p-aminobenzyloxycarbonyl (“vc-PAB”). In terms of the selectivity of amino acid linker components for enzyme cleavage, specific enzymes can be designed and optimized, such as tumor-associated proteases, cathepsins B, C and D, or plasminogen lysins.

[0613] In some embodiments, in the ADC described in this application, an antibody (or antigen-binding fragment) is conjugated to one or more cytotoxic agents in an antibody:agent ratio of about 1 to about 20, about 1 to about 6, about 2 to about 6, about 3 to about 6, about 2 to about 5, about 2 to about 4, or about 3 to about 4.

[0614] The ADC described in this application can be prepared by any suitable method known in the art. In some embodiments, the nucleophilic group of the antibody (or antigen-binding fragment) first reacts with a bifunctional adapter reagent and then is linked to the cytotoxic agent, or vice versa, i.e., the nucleophilic group of the cytotoxic agent first reacts with the bifunctional adapter and then is linked to the antibody.

[0615] In some embodiments, the cytotoxic agent may contain (or be modified to contain) a thiol-reactive functional group that can react with the cysteine ​​thiol group of the free cysteine ​​of the antibody or antigen-binding fragment described in this application. Exemplary thiol-reactive functional groups include, for example, maleimide, iodoacetamide, pyridine disulfide, haloacetyl, succinimide esters (e.g., NHS, N-hydroxysuccinimide), isothiocyanates, sulfonyl chlorides, 2,6-dichlorotriazine, pentafluorophenyl esters, or phosphoramides (Haugland, 2003, Molecular Probes Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Inc.; Brinkley, 1992, Bioconjugate Chem. 3:2; Garman, 1997, Non-Radioactive Labelling: A Practical Approach, Academic Press, London (1990) Bioconjugate Chem. 1:2; Hermanson, G. in Bioconjugate Techniques (1996) Academic Press, San Diego, pp. 40-55, 643-671).

[0616] The cytotoxic agent or the antibody may react with a linker before being conjugated to form the ADC. For example, N-hydroxysuccinimide ester (NHS) of the cytotoxic agent may be pre-formed, isolated, purified, and / or characterized, or the NHS may be formed in situ and react with the nucleophilic group of the antibody. Typically, the carboxyl form of the conjugate is activated by certain combinations of reactions with the following reagents: carbodiimide reagents (e.g., dicyclohexylcarbodiimide; diisopropylcarbodiimide) or urea reagents (e.g., TsTu (O-(N-succinimide)-N,N,N',N'-tetramethylurea tetrafluoroborate), HBTU (O-benzotriazol-1-yl)-N,N,N'N'-tetramethylurea hexafluorophosphate) or HATU (O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate)), activators (such as 1-hydroxybenzotriazole (HOBt), and N-hydroxysuccinimide giving an NHS ester). In some cases, the cytotoxic agent and the antibody can be linked by in situ activation and reaction to form the ADC in one step. Other activations and linkages... Reagents include TBTU (2-(1H-benzotriazol-1-yl)-1-1,3,3-tetramethylurea hexafluorophosphate), TFFH (N,N',N”,N”'-tetramethylurea-2-fluoro-hexafluorophosphate), PyBOP (benzotriazol-1-yl-oxytripyrrolylphosphine hexafluorophosphate), EEDQ (2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline), DCC (dicyclohexylcarbodiimide); DIPCDI (diisopropylcarbodiimide), MSNT (1-(trimethylmethyl-2-sulfonyl)-3-nitro-1H-1,2,4-triazole), and arylsulfonyl halides (e.g., triisopropylbenzenesulfonyl chloride). In another example, the antibody or antigen-binding fragment may be conjugated to biotin, followed by indirect conjugation to a second conjugate conjugated to avidin.

[0617] Maytansin and Maytansin-like compounds

[0618] In one embodiment, any anti-CD19 antibody or antigen-binding fragment described in this application is conjugated to one or more maytansine molecules. Maytansine compounds are mitotic inhibitors that inhibit the polymerization of tubulin.

[0619] Maytansine compounds suitable for use as fractions in maytansine drugs (such as maytanol and C-3 maytanol ester) are well known in the art, and can be isolated from natural sources and produced using genetic engineering techniques according to known methods (see Yu et al. (2002) PNAS). Maytanol and its analogues can be synthesized by known methods (see, for example, U.S. Patent Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533).

[0620] Suitable maytansine compounds are disclosed, for example, in U.S. Patent No. 5,208,020, and in other patent or non-patent publications mentioned in this application. Exemplary maytansine compounds are maytanol, and maytanol analogues modified at the aromatic ring or other positions of the maytanol molecule, see, for example, C-49-dechloro (U.S. Patent No. 4,256,746); C-20-hydroxy (or C-20-demethyl) + / - C-19-dechloro (U.S. Patent Nos. 4,361,650 and 4,307,016); C-20-demethoxy, C-20-acyloxy (--OCOR) + / -dechloro (U.S. Patent No. 4,294,757), C-9-SH (U.S. Patent No. 4,424,2190); C-14 -Alkoxymethyl (demethoxy / CH2OR) (US Patent No. 4,331,598); C-14-hydroxymethyl or acylmethyl (CH2OH or CH2OAc) (US Patent No. 4,450,254); C-15-hydroxy / acyloxy (US Patent No. 4,364,866); C-15-methoxy (US Patent Nos. 4,313,946 and 4,315,929); C-18-N-demethyl (US Patent Nos. 4,362,663 and 4,322,348); and 4,5-deoxy (US Patent No. 4,371,533). In some embodiments, the maytansine compound conjugated with the antibody described in this application is DM1 (N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)-matansine) or DM4 (N2'-deacetyl-N2'-(4-mercapto-4-methyl-1-oxopentyl)-6-methylmatansine).

[0621] Anti-CD19 antibody-matansine conjugates can be prepared by chemically linking an antibody to a maytansine molecule without significantly reducing the biological activity of the antibody or the maytansine molecule. See, for example, U.S. Patent No. 5,208,020 (the disclosure of which is expressly incorporated herein by reference). In some embodiments, each antibody molecule is conjugated to an average of 1 to 4, 2 to 4, or 3 to 4 maytansine molecules without negatively affecting the function or solubility of the antibody.

[0622] The maytansine derivative can be linked to an antibody or antigen-binding fragment via any suitable linker known in the art, see, for example, U.S. Patent Nos. 5,208,020, 6,441,163 or European Patent Nos. 0,425,235B1, Chari et al., Cancer Research 52:127-131 (1992), and US2005 / 0169933 A1, the disclosures of which are expressly incorporated herein by reference. The linkers in the ACD described in this application include disulfide groups, thioether groups, acid-labile groups, light-labile groups, peptidase-labile groups, or esterase-labile groups. In some embodiments, the linker in the ACD described in this application is a bifunctional protein coupling agent, such as N-succinimide-3-(2-pyridinedithio)propionic acid (SPDP), succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid (SMCC), N-succinimide-4-(2-pyridinethio)valerate (SPP), iminothia (IT), bifunctional derivatives of imine esters (such as dimethyl hexamethylenediimide hydrochloride), active esters (such as disuccinimide octanoate), aldehydes (such as glutaraldehyde), diazid compounds (such as bis(p-azidobenzoyl)hexamethylenediamine), dinitrogen salt derivatives (such as bis(p-diazidobenzoyl)ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and histamine-reactive fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).

[0623] Depending on the type of connector, the connector can attach to the maytansine molecule at various positions. For example, using conventional coupling techniques, an ester connector can be formed by reacting with a hydroxyl group. This reaction can occur at the C-3 position with a hydroxyl group, the C-14 position modified with a hydroxymethyl group, the C-15 position modified with a hydroxyl group, and the C-20 position with a hydroxyl group. In a preferred embodiment, the connector is formed at the C-3 position of maytansine or a maytansine analogue.

[0624] Orlistatine and sea hare toxin

[0625] In one embodiment, any anti-CD19 antibody or antigen-binding fragment described in this application is conjugated with one or more saurus toxins, saurus toxin peptide analogs and derivatives, or orlistatine (US Patent Nos. 5,635,483; 5,780,588). Saurus toxins and orlistatine are presumed to have anticancer and antifungal activities by interfering with microtubule dynamics, GTP hydrolysis, and nuclear and cell division (see, for example, US Patent Nos. 5,663,149; Pettit et al. (1998) Antimicrob. Agents Chemother. 42:2961-2965; Woyke et al. (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584).

[0626] Exemplary orlistatine includes MMAE and MMAF. The orlistatine / saurus toxin pharmaceutical portion can be prepared according to the following methods: US2005 / 0238649; US Patent No. 5,635,483; US Patent No. 5,780,588; Pettit et al. (1989) J. Am. Chem. Soc. 111:5463-5465; Pettit et al. (1998) Anti-Cancer Drug Design 13:243-277; Pettit, GR et al., Synthesis, 1996, 719-725; Pettit et al. (1996) J. Chem. Soc. Perkin Trans. 1 5:859-863; and Doronina (2003) Nat Biotechnol 21(7):778-784.

[0627] The saurus toxin or orlistatine drug moiety can be attached to the antibody (WO 02 / 088172) via the N (amino) or C (carboxyl) terminus of the peptide drug moiety.

[0628] Chimeric antigen receptor (CAR) compositions

[0629] This disclosure also provides a chimeric antigen receptor (CAR) comprising an antigen-binding fragment of an antibody that specifically binds to CD19 and the T-cell activation moiety as described in this application. In some embodiments, the T-cell activation moiety comprises the native T-cell activation moiety of a TCR. In some embodiments, the T-cell activation moiety comprises a transmembrane domain and an intracellular signaling domain of a TCR.

[0630] antigen-binding fragments

[0631] In some embodiments, the antigen-binding fragment may be any fragment derived from any one or more antibodies described in this application that binds to CD19, including but not limited to an antigen recognition domain. In some embodiments, the antigen-binding fragment is preferably derived from the same species in which the CAR will ultimately be used. For example, when used in humans, the antigen-binding fragment for the CAR is preferably derived from a human antibody or a humanized antibody. In some embodiments, the antigen-binding fragment is a single-chain variable fragment (scFv). In some embodiments, the antigen-binding fragment may be present in a variety of other forms, including, for example, Fv, Fab, and (Fab')2, as well as bifunctional (i.e., bispecific) hybrid antibody fragments (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)).

[0632] Transmembrane domain

[0633] Regarding the transmembrane domain, in various embodiments, the CAR is designed to include a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, a transmembrane domain naturally linked to a domain of the CAR is used. In some cases, the transmembrane domain may be selectively modified, or modified by amino acid substitution, to prevent the transmembrane domain from binding to the transmembrane domains of the same or different surface membrane proteins, thereby minimizing interactions with other components of the receptor complex.

[0634] The transmembrane domain can be derived from natural or synthetic sources. When the source is natural, the domain can be derived from any membrane-binding or transmembrane protein. The transmembrane domain for a particular purpose in this invention can be derived from the following group (i.e., transmembrane regions comprising at least the following group): the α, β, or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. In some cases, various human hinges, including human Ig (immunoglobulin) hinges, may also be used.

[0635] In one embodiment, the transmembrane domain may be synthetic, in which case it comprises predominantly hydrophobic residues such as leucine and valine. In one aspect, a triplet of phenylalanine, tryptophan, and valine is present at each end of the synthetic transmembrane domain. Optionally, short oligopeptides or polypeptide linkers of 2 to 10 amino acids in length can form a link between the transmembrane domain and the cytoplasmic signaling domain of the CAR. Glycine-serine duplexes provide particularly suitable linkers.

[0636] Signal transmission domain

[0637] The signal transduction domain of the CAR described in this disclosure is responsible for activating at least one normal TCR effector function of the T cell in which the CAR resides. The TCR effector function of the T cell can be, for example, cytotoxic activity or helper activity, including cytokine secretion. Therefore, the term "signal transduction domain" refers to a portion of a protein that transduces TCR effector function signals and guides the T cell to perform a specified function. While the entire intracellular signal transduction domain can generally be used, in many cases it is not necessary to use the entire strand. Where a truncated portion of the intracellular signal transduction domain is used, that truncated portion can be used in place of the complete strand as long as it transduces the effector function signal. The term "intracellular signal domain" is therefore intended to include any truncated portion of the intracellular signal transduction domain sufficient to transduce the TCR effector function signal.

[0638] Examples of intracellular signaling domains used in the CARs described in this disclosure include cytoplasmic sequences of T-cell receptors (TCRs), co-receptors that work synergistically to initiate signal transduction upon the involvement of antigen receptors, and any derivatives or variants of these sequences, and any synthetic sequences having the same functional properties.

[0639] It is known that the signals generated by the TCR alone are insufficient to fully activate T cells, and secondary or co-stimulatory signals are also required. Therefore, T cell activation can be said to be mediated by two different types of cytoplasmic signaling sequences: one type initiates antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences), and the other type acts in an antigen-independent manner to provide secondary or co-stimulatory signals (secondary cytoplasmic signaling sequences).

[0640] Primary intracellular signal transduction sequences regulate primary activation of the TCR complex in a stimulatory or inhibitory manner. Primary intracellular signaling sequences that act in a stimulatory manner may contain signaling motifs called immune receptor-based tyrosine activation motifs or ITAMs.

[0641] Examples of ITAMs containing primary cytoplasmic signaling sequences that are particularly useful in this invention include ITAMs derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. Particularly preferred is that the cytoplasmic signaling molecule in the CAR of this invention comprises a cytoplasmic signaling sequence derived from CD3-ζ.

[0642] In a preferred embodiment, the intracellular signaling domain of the CAR is designed to contain only the CD3-ζ signaling domain itself, or a combination thereof with any other desirable cytoplasmic domain that can be used in the context of the CAR described herein. For example, the intracellular signaling domain of the CAR may contain a CD3ζ chain portion and a co-stimulatory signaling region. The co-stimulatory signaling region refers to a portion of the intracellular domain of the CAR containing a co-stimulatory molecule. The co-stimulatory molecule is a cell surface molecule, rather than an antigen receptor or its ligand, that is required for an effective lymphocyte response to an antigen. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83. The CD3ζ chain portion and the co-stimulatory signaling region may be linked to each other in a random or specified order. Optionally, short oligopeptides or polypeptide linkers of 2 to 10 amino acids in length may form the link. The glycine-serine dinucleotide provides an exceptionally suitable linker.

[0643] In one aspect, this disclosure further provides a nucleic acid sequence encoding the CAR described in this application, comprising a first polynucleotide sequence encoding an antigen-binding fragment of the antibody described in this application, and optionally, a second polynucleotide sequence encoding a transmembrane domain and an intracellular signaling domain of a TCR. In some embodiments, the sequence encoding the antigen-binding fragment is operatively linked to the sequences encoding the transmembrane domain and the signaling domain of the TCR. In some embodiments, the signaling domain includes a co-stimulatory signaling region and / or a portion of the CD3ζ chain. The nucleic acid sequence encoding the desired molecule can be obtained using recombination methods known in the art, such as by screening a library from cells expressing the gene, obtaining the gene from a vector known to include the gene, or by direct isolation from cells and tissues containing the gene. Alternatively, the target gene can be generated synthetically rather than by cloning.

[0644] In one aspect, this disclosure provides a vector comprising a nucleic acid sequence encoding the CAR described in this application. In some embodiments, the vector is constructed from a retroviral or lentiviral vector expressing the CAR described in this disclosure that can be directly transduced into cells, or from an RNA vector that can be directly transduced into cells.

[0645] In one aspect, this disclosure provides isolated host cells expressing the CAR described in this application.

[0646] In one aspect, this disclosure further provides a method for activating a T cell-mediated immune response against a CD19-expressing target in an individual, the method comprising administering to the individual an effective amount of T cells expressing the CAR described in this application.

[0647] Polynucleotides and methods

[0648] This disclosure provides isolated polynucleotides encoding the anti-CD19 antibody and its antigen-binding fragment. In some embodiments, the isolated polynucleotides comprise one or more nucleotide sequences encoding the variable region of an exemplary antibody, as shown below: SEQ ID NO: 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133 and / or 135. The DNA encoding the monoclonal antibody can be readily isolated and sequenced using conventional steps (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody). The encoded DNA can also be obtained by synthetic methods.

[0649] Using recombination techniques known in the art, isolated polynucleotides encoding the anti-CD19 antibody and its antigen-binding fragment (e.g., sequences including those shown below: SEQ ID NO: 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133 and / or 135) can be inserted into a vector for further cloning (DNA amplification) or for expression. A variety of vectors are available. Vector components typically include, but are not limited to, one or more of the following: a signal sequence, a replication origin, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1α), and a transcription termination sequence.

[0650] In some embodiments, the vector system includes mammalian, bacterial, yeast, and other systems, and contains plasmids, such as, but not limited to, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pCMV, pEGFP, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMD18-T, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS420, pLexA, pACT2.2, etc., as well as other vectors available from the laboratory or commercially available. Suitable vectors may include plasmids or viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses).

[0651] Vectors containing a multinucleotide sequence encoding the antibody or antigen-binding fragment can be introduced into host cells for cloning or gene expression. The host cells suitable for cloning or expressing the DNA in the vectors described in this application are the aforementioned prokaryotic, yeast, or higher eukaryotic cells. Prokaryotic cells suitable for the purposes of this application include eubacteria, such as Gram-negative or Gram-positive bacteria, for example, Enterobacteriaceae, such as *Escherichia coli*, *Enterobacter* spp., *Erwinia* spp., *Klebsiella* spp., *Proteus* spp., *Salmonella* spp., such as *Salmonella typhimurium*, *Serratia* spp., such as *Serratia marcescens*, and *Shigella* spp., and *Bacillus* spp., such as *Bacillus subtilis* and *Bacillus licheniformis*, and *Pseudomonas* spp., such as *Pseudomonas aeruginosa* and *Streptomyces*.

[0652] Besides prokaryotic cells, eukaryotic microorganisms such as filamentous fungi or yeast can also serve as host cell clones or vectors for expressing anti-CD19 antibodies. Saccharomyces cerevisiae and baker's yeast are the most commonly used lower eukaryotic host microorganisms. However, many other genera, species, and strains are commonly used and applicable in this invention, such as *Schizosaccharomyces cerevisiae*; hosts of the *Kluyveromyces* genus such as *Kluyveromyces lactis*, *Kluyveromyces brittle* (ATCC 12,424), *Kluyveromyces bulgaricus* (ATCC 16,045), *Kluyveromyces wilfordii* (ATCC 24,178), *Kluyveromyces spp.* (ATCC 56,500), *Kluyveromyces drosophila* (ATCC 36,906), *Kluyveromyces thermostrophicus*, and *Kluyveromyces marx*; *Yersinia lipolytica* (EP 402,226); *Pichia pastoris* (EP183,070); *Candida*; *Trichoderma reesei* (EP 244,234); *Neurospora*; *Schwannium spp.*, such as *Schwannium spp.*; and filamentous fungi such as *Neurospora*, *Penicillium*, *Cyclophorus*, and *Aspergillus*, such as *Aspergillus niger* and *Aspergillus niger*.

[0653] The host cells provided in this invention suitable for expressing glycosylated antibodies or their antigen-binding fragments are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Various baculoviral strains and their variants, along with corresponding permissive insect host cells, have been discovered from hosts such as the grass armyworm (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and silkworm. Several viral strains for transfection are publicly available, such as the alfalfa silver-striped armyworm nucleopolyhedrovirus and the Bm-5 variant of the silkworm nucleopolyhedrovirus, which can be used in this invention, particularly for transfecting grass armyworm cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be used as hosts.

[0654] However, the most interesting cells are spinal cells, and the culture of spinal cells (tissue culture) has become a routine procedure. Examples of available mammalian host cells include: SV40-transformed monkey kidney cell line CV1 (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 or 293 cell subclones in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); young hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse testicular supporting cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 1651). 2); canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (HepG2, HB 8065); mouse mammary tumors (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and human hepatocellular carcinoma cell line (Hep G2). In some preferred embodiments, the host cell is 293F cell.

[0655] The host cells are transformed using the aforementioned expression or cloning vector capable of producing anti-CD19 antibodies, and then cultured in a conventional nutrient medium modified to induce promoters, select transformed cells, or amplify genes encoding the target sequence. In another embodiment, the antibody can be prepared using homologous recombination methods known in the art.

[0656] The host cells used to produce the antibodies or their antigen-binding fragments in this invention can be cultured in a variety of culture media. Commercially available culture media such as Ham's F10 (Sigma), Minimal Basic Medium (MEM (Sigma)), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM, Sigma) can be used to culture the host cells. Additionally, any culture medium described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Patent Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90 / 03430; WO 87 / 00195; or U.S. Patent Application Re. 30,985 can be used as the culture medium for the host cells. These culture media may be fortified with necessary hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium chloride, magnesium chloride, and phosphates), buffers (such as HEPES), nucleotides (such as adenosine and thymine), antibiotics (such as gentamicin), trace elements (defined as inorganic compounds typically in the micromolar range), and glucose or an equivalent energy source. The culture media may also contain any other necessary additives at appropriate concentrations known in the art. The conditions of the culture media, such as temperature, pH, and similar conditions, are those previously used for selecting the host cells for expression and are well known to those skilled in the art.

[0657] Anti-CD19 antibodies or their antigen-binding fragments obtained from the cells can be purified using methods such as hydroxyapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being the preferred purification technique.

[0658] In some embodiments, protein A immobilized on a solid phase is used for immunoaffinity purification of the antibody and its antigen-binding fragments. The type of antibody and the presence of any Fc domains of immunoglobulins in the antibody determine the suitability of protein A as an affinity ligand. Protein A can be used to purify antibodies based on human γ1, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is suitable for all murine isoforms and human γ3 (Guss et al., EMBO J. 5:1567 1575 (1986)). Agarose is the most commonly used affinity ligand attachment matrix, but other matrices can also be used. Mechanically stable matrices such as controlled-porosity glass or poly(styrene)benzene allow for faster flow rates and shorter processing times compared to agarose. If the antibody contains a CH3 domain, purification can be performed using Bakerbond ABX.™ resin (JTBaker, Phillipsburg, New Jersey). Other protein purification techniques may be determined based on the antibodies obtained, such as fractionation in ion exchange columns, ethanol precipitation, reversed-phase HPLC, silica gel chromatography, heparin agarose gel chromatography based on anion or cation exchange resins (e.g., polyaspartic acid columns), chromatography focusing, SDS-PAGE, and ammonium sulfate precipitation.

[0659] Following any preliminary purification step, the mixture containing the antibody of interest and impurities can be treated with low-pH hydrophobic interaction chromatography using a wash buffer of about pH 2.5–4.5, preferably at low salt concentrations (e.g., from about 0 to 0.25 M).

[0660] Pharmaceutical Composition

[0661] This application further provides a pharmaceutical composition comprising the anti-CD19 antibody described herein or its antigen-binding fragment or antibody-drug conjugate, and one or more pharmaceutically acceptable carriers.

[0662] Pharmaceutically acceptable carriers used in the pharmaceutical compositions disclosed in this application may include, for example, pharmaceutically acceptable liquid, gel or solid carriers, aqueous solvents, non-aqueous solvents, antimicrobial substances, isotonic substances, buffers, antioxidants, anesthetics, suspending / dispersing agents, chelating agents, diluents, adjuvants, excipients or non-toxic excipients, and other components known in the art, or combinations thereof.

[0663] Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickeners, colorants, emulsifiers, or stabilizers such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercaptoglycerol, mercaptoacetic acid, mercaptosorbitol, butylated methyl anisole, butylated hydroxytoluene, and / or propyl gallate. As disclosed in this invention, including one or more antioxidants such as methionine in a composition containing an antibody or antigen-binding fragment disclosed in this invention can reduce the oxidation of said antibody or antigen-binding fragment. Reduction of oxidation can prevent or reduce the decrease in binding affinity, thereby improving antibody stability and extending shelf life. Therefore, in some embodiments, the compositions provided by this invention contain one or more of the antibodies or antigen-binding fragments described herein and one or more antioxidants such as methionine. The present invention further provides various methods for preventing oxidation of the antibody or its antigen-binding fragment, extending its shelf life, and / or increasing its activity by mixing the antibody or its antigen-binding fragment provided in the present invention with one or more antioxidants, such as methionine.

[0664] Furthermore, pharmaceutically acceptable carriers may include, for example, aqueous media such as sodium chloride injection, Ringer's solution injection, isotonic glucose injection, sterile water injection, or glucose and lactated Ringer's solution injection; non-aqueous media such as non-volatile plant-derived oils, cottonseed oil, corn oil, sesame oil, or peanut oil; antimicrobial substances at bacterial or fungal inhibitory concentrations; isotonic agents such as sodium chloride or glucose; buffers such as phosphate or citrate buffers; antioxidants such as sodium bisulfate; local anesthetics such as procaine hydrochloride; suspending and dispersing agents such as sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone; emulsifiers such as polysorbate 80 (Tween-80); chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol bis(2-aminoethyl ether)tetraacetic acid); ethanol; polyethylene glycol; propylene glycol; sodium hydroxide; hydrochloric acid; citric acid; or lactic acid. Antimicrobial agents acting as carriers can be added to pharmaceutical compositions in multiple-dose containers and include phenols or cresols, mercury preparations, benzyl alcohol, chlorobutanol, methyl and propylparabens, thiamethoxam, chlorobenzamide, and chlorophenoxyacetamide. Suitable excipients may include, for example, water, salts, glucose, glycerol, or ethanol. Suitable non-toxic excipients may include, for example, emulsifiers, pH buffers, stabilizers, solubilizers, or substances such as sodium acetate, dehydrosorbitan laurate, triethanolamine oleate, or cyclodextrin.

[0665] The pharmaceutical composition may be a liquid solution, suspension, emulsion, pill, capsule, tablet, sustained-release formulation, or powder. Oral formulations may include standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharin, cellulose, magnesium carbonate, etc.

[0666] In some embodiments, the pharmaceutical composition is formulated as an injectable composition. Injectable pharmaceutical compositions can be prepared in any conventional form, such as liquid solvents, suspensions, emulsifiers, or solid forms suitable for producing liquid solvents, suspensions, or emulsifiers. Injectable formulations may include pre-existing sterile and / or pyrogen-free solutions, sterile dried soluble products bound to a solvent before use, such as lyophilized powders, including subcutaneous tablets, sterile suspensions ready for injection, sterile dried insoluble products bound to a medium before use, and sterile and / or pyrogen-free emulsions. The solvent may be aqueous or non-aqueous.

[0667] In some embodiments, a unit dose of the injectable formulation is packaged in an ampoule, a tube, or a syringe with a needle. It is known in the art that all injectable formulations should be sterile and pyrogen-free.

[0668] In some embodiments, a sterile lyophilized powder can be prepared by dissolving the antibody or its antigen-binding fragment disclosed in this application in a suitable solvent. The solvent may contain other pharmacological components that improve the stability of the powder or recombinant solution derived from the powder, or enhance the powder or recombinant solution. Suitable excipients include, but are not limited to, water, glucose, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose, or other suitable substances. The solvent may contain a buffer solution, such as citrate buffer, sodium phosphate or potassium phosphate buffer, or other buffer solutions known to those skilled in the art; in one embodiment, the buffer solution is neutral at pH. The dissolution is subsequently sterilized by filtration under standard conditions known in the art, and then lyophilized to obtain the desired formulation. In one embodiment, the resulting solvent is aliquoted into tubes and lyophilized. Each tube may contain a single or multiple dose of the anti-CD19 antibody or its antigen-binding fragment, or a combination thereof. The amount loaded into each tube may be slightly higher than required for each or multiple doses (e.g., 10% excess) to ensure accurate sampling and administration. The freeze-dried powder can be stored under appropriate conditions, such as in the range of about 4°C to room temperature.

[0669] The lyophilized powder is reconstituted with water for injection to obtain a formulation for injection. In one embodiment, the lyophilized powder can be reconstituted in sterile, pyrogen-free water or other suitable liquid carrier. The precise amount depends on the chosen therapy and can be determined empirically.

[0670] How to use

[0671] This disclosure also provides a treatment method comprising: administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof as described herein to an individual in need, thereby treating or preventing a CD19-related condition or disease. In some embodiments, the CD19-related condition or disease is cancer. In some embodiments, the cancer is selected from the group consisting of B-cell lymphoma, optionally Hodgkin lymphoma, or non-Hodgkin lymphoma, wherein the non-Hodgkin lymphoma includes: diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, marginal zone B-cell lymphoma (MZL), mucosa-associated lymphoid tissue lymphoma (MALT), small lymphocytic lymphoma (chronic lymphocytic leukemia, CLL) or mantle cell lymphoma (MCL), acute lymphoblastic leukemia (ALL), or Waldenström macroglobulinemia (WM). In some embodiments, the individual is a person.

[0672] In another aspect, a method is provided for treating a condition in individuals who would benefit from regulation of an immune response by CD19, the method comprising administering to the individual in need a therapeutically effective amount of an antibody or antigen-binding fragment as described in this application.

[0673] The therapeutically effective dose of the antibody or its antigen-binding fragment described in this application depends on a variety of factors known in the art, such as weight, age, medical history, current treatment, the subject's health status and potential for cross-infection, allergies, hypersensitivity and side effects, as well as the route of administration and the extent of tumor development. Those skilled in the art (e.g., physicians or veterinarians) may proportionally reduce or increase the dose based on these or other conditions or requirements.

[0674] In some embodiments, the anti-CD19 antibody or antigen-binding fragment as described in this application may be administered at a therapeutically effective dose between about 0.01 mg / kg and about 100 mg / kg (e.g., about 0.01 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 65 mg / kg, about 70 mg / kg, about 75 mg / kg, about 80 mg / kg, about 85 mg / kg, about 90 mg / kg, about 95 mg / kg, or about 100 mg / kg). In some embodiments, the antibody or antigen-binding fragment is administered at a dose of about 50 mg / kg or less, and in some such embodiments, the dose is 10 mg / kg or less, 5 mg / kg or less, 3 mg / kg or less, 1 mg / kg or less, 0.5 mg / kg or less, or 0.1 mg / kg or less. In some embodiments, the dosage may vary with the course of treatment. For example, in some embodiments, the initial dosage may be higher than subsequent dosages. In some embodiments, the dosage is adjusted during the course of treatment based on the subject's response.

[0675] Dosing regimens can be adjusted to achieve optimal response (e.g., therapeutic response). For example, a single dose can be administered or multiple doses can be administered over a period of time.

[0676] The anti-CD19 antibody and antigen-binding fragment disclosed in this application can be administered via methods known in the art, such as parenteral administration (e.g., subcutaneous injection, intraperitoneal injection, intravenous injection, including intravenous infusion, intramuscular injection or intradermal injection) or non-parenteral administration routes (e.g., oral administration, nasal administration, sublingual administration, rectal administration or topical administration).

[0677] In some embodiments, the anti-CD19 antibody or antigen-binding fragment disclosed in this application can be administered alone or in combination with one or more other therapeutic agents or substances. For example, the antibody and antigen-binding fragment disclosed in this invention can be administered in combination with another therapeutic agent, such as a chemotherapeutic agent or an anticancer drug.

[0678] In some such embodiments, when the anti-CD19 antibody or antigen-binding fragment disclosed in this application is used in combination with one or more other therapeutic substances, it may be administered simultaneously with said one or more other therapeutic substances. In some such embodiments, the antibody and antigen-binding fragment may be administered simultaneously as part of the same pharmaceutical composition. However, the anti-CD19 antibody or antigen-binding fragment "used in combination" with other therapeutic substances does not need to be administered simultaneously or in the same composition as the therapeutic substance. The meaning of "used in combination" in this application also includes that an anti-CD19 antibody or antigen-binding fragment administered before or after another therapeutic substance is also considered to be "used in combination" with that therapeutic substance, even if the antibody or its antigen-binding fragment is administered with the second substance via a different route of administration. Where possible, other therapeutic substances used in combination with the antibodies or antigen-binding fragments disclosed in this application may be administered according to the instructions for use of the other therapeutic substance, or according to Physicians' Desk Reference 2003 (57th edition; Medical Economics Company; ISBN: 1563634457; 57th edition (November 2002)), or according to other methods known in the art.

[0679] This disclosure further provides methods for using the anti-CD19 antibody or its antigen-binding fragment. In some embodiments, this disclosure provides a method for inhibiting the growth of CD19-expressing cells in vivo or in vitro, comprising: contacting the CD19-expressing cells with the antibody or its antigen-binding fragment described in this application. In some embodiments, this disclosure provides a method for regulating CD19 activity in CD19-expressing cells, comprising exposing the CD19-expressing cells to the antibody or its antigen-binding fragment described in this application.

[0680] In some embodiments, this disclosure provides a method for detecting the presence or content of CD19 in a sample, comprising contacting the sample with the antibody or its antigen-binding fragment, and determining the presence or content of CD19 in the sample.

[0681] In some embodiments, this disclosure provides a method for diagnosing a CD19-related disease or condition in an individual, comprising: a) obtaining a sample from the individual; b) contacting the sample with an antibody or antigen-binding fragment thereof as described in this application; c) determining the presence or amount of CD19 in the sample; and d) determining the presence of the CD19-related disease or condition in the individual.

[0682] In some embodiments, this disclosure provides a kit comprising an antibody or antigen-binding fragment thereof optionally conjugated to a detectable portion. The kit can be used to detect CD19 or diagnose related conditions.

[0683] CD19-related diseases.

[0684] In some embodiments, this disclosure also provides the use of the antibody or antigen-binding fragment thereof described in this application in the preparation of medicaments for treating CD19-related diseases or conditions in an individual, and in the preparation of diagnostic reagents for diagnosing CD3-related diseases or conditions.

[0685] This document also includes the following implementation methods:

[0686] Implementation Method 1. An isolated antibody or its antigen-binding fragment, comprising one or more heavy chain complementarity-determining region (CDR) sequences selected from the group consisting of: SEQ ID NO: 1, 2, 3, 7, 8, 9, 13, 14, 15, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 43, 44,

[0687] 45, 136, 140 and 141, and / or one or more κ light chain CDR sequences selected from the following group: SEQ ID NO: 4, 5, 6, 10, 11, 12, 16, 17, 18, 40, 41, 42, 137, 138 and 139.

[0688] Implementation Method 2. The antibody or antigen-binding fragment thereof according to Implementation Method 1 includes a heavy chain variable region selected from the group consisting of:

[0689] a) Heavy chain variable region, which includes 1, 2 or 3 CDR sequences selected from SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3;

[0690] b) Heavy chain variable region, which includes 1, 2 or 3 CDR sequences selected from SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9;

[0691] c) Heavy chain variable region, which includes 1, 2 or 3 CDR sequences selected from SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15;

[0692] d) Heavy chain variable region, which includes 1, 2 or 3 CDR sequences selected from SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21;

[0693] e) Heavy chain variable region, which includes 1, 2 or 3 CDR sequences selected from SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24;

[0694] f) Heavy chain variable region, which includes 1, 2 or 3 CDR sequences selected from SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:27;

[0695] g) Heavy chain variable region, which includes 1, 2 or 3 CDR sequences selected from SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30;

[0696] h) Heavy chain variable region, which includes 1, 2 or 3 CDR sequences selected from SEQ ID NO:31, SEQ ID NO:32 and SEQ ID NO:33;

[0697] i) Heavy chain variable region, which includes 1, 2 or 3 CDR sequences selected from SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36;

[0698] j) Heavy chain variable region, comprising 1, 2 or 3 CDR sequences selected from SEQ ID NO:37, SEQ ID NO:38 and SEQ ID NO:39;

[0699] k) Heavy chain variable region, which includes 1, 2 or 3 CDR sequences selected from SEQ ID NO:43, SEQ ID NO:44 and SEQ ID NO:45;

[0700] l) Heavy chain variable region, which includes 1, 2 or 3 CDR sequences selected from SEQ ID NO:136, SEQ ID NO:2 and SEQ ID NO:3;

[0701] m) The heavy chain variable region, comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO:7, SEQ ID NO:140, and SEQ ID NO:9; and

[0702] n) Heavy chain variable region, which includes 1, 2 or 3 CDR sequences selected from SEQ ID NO:13, SEQ ID NO:141 and SEQ ID NO:15.

[0703] Implementation Method 3. The antibody or antigen-binding fragment thereof according to any of the foregoing embodiments includes a κ light chain variable region, wherein the κ light chain variable region is selected from the group consisting of:

[0704] a) The κ light chain variable region, which includes 1, 2 or 3 CDR sequences selected from SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6;

[0705] b) The κ light chain variable region, comprising 1, 2 or 3 CDR sequences selected from SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12;

[0706] c) The κ light chain variable region, which includes 1, 2 or 3 CDR sequences selected from SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18;

[0707] d) The κ light chain variable region, comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO:40, SEQ ID NO:41, and SEQ ID NO:42; and

[0708] e)κ light chain variable region, which includes 1, 2 or 3 CDR sequences selected from SEQ ID NO:137, SEQ ID NO:138 and SEQ ID NO:139.

[0709] Implementation Method 4. The antibody or antigen-binding fragment thereof according to any of the foregoing embodiments comprises:

[0710] a) a heavy chain variable region comprising 1, 2 or 3 CDR sequences selected from SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3; and a κ light chain variable region comprising 1, 2 or 3 CDR sequences selected from SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6;

[0711] b) The heavy chain variable region, comprising 1, 2 or 3 CDR sequences selected from SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9; and the κ light chain variable region, comprising 1, 2 or 3 CDR sequences selected from SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12;

[0712] c) The heavy chain variable region, comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15; and the κ light chain variable region, comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO:15.

[0713] 16. CDR sequences of SEQ ID NO:17 and SEQ ID NO:18;

[0714] d) The heavy chain variable region, comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21; and the κ light chain variable region, comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO:

[0715] 4. CDR sequences of SEQ ID NO:5 and SEQ ID NO:6;

[0716] e) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24; and a κ light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO:24.

[0717] 4. CDR sequences of SEQ ID NO:5 and SEQ ID NO:6;

[0718] f) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27; and a κ light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO:27.

[0719] 4. CDR sequences of SEQ ID NO:5 and SEQ ID NO:6;

[0720] g) The heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30; and the κ light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30.

[0721] 4. CDR sequences of SEQ ID NO:5 and SEQ ID NO:6;

[0722] h) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33; and a κ light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO:33;

[0723] 4. CDR sequences of SEQ ID NO:5 and SEQ ID NO:6;

[0724] i) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36; and a κ light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO:36.

[0725] 4. CDR sequences of SEQ ID NO:5 and SEQ ID NO:6;

[0726] j) The heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39; and the κ light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO:39.

[0727] 40. CDR sequences of SEQ ID NO:41 and SEQ ID NO:42;

[0728] k) Heavy chain variable region, comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO:43, SEQ ID NO:44, and SEQ ID NO:45; and κ light chain variable region, comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO:45.

[0729] 4. CDR sequences of SEQ ID NO:5 and SEQ ID NO:6;

[0730] l) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO:136, SEQ ID NO:2, and SEQ ID NO:3; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO:136, SEQ ID NO:2, and SEQ ID NO:3.

[0731] 137. CDR sequences of SEQ ID NO:138 and SEQ ID NO:139;

[0732] m) Heavy chain variable region, comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO:7, SEQ ID NO:140, and SEQ ID NO:9; and κ light chain variable region, comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO:9;

[0733] 10. CDR sequences of SEQ ID NO:11 and SEQ ID NO:12;

[0734] n) Heavy chain variable region, comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO:13, SEQ ID NO:141, and SEQ ID NO:15; and κ light chain variable region, comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO:15.

[0735] 16. CDR sequences of SEQ ID NO:17 and SEQ ID NO:18.

[0736] Implementation Method 5. The antibody or antigen-binding fragment thereof according to any of the foregoing embodiments, comprising:

[0737] a) A heavy chain CDR1 sequence, wherein the heavy chain CDR1 sequence is selected from: SEQ ID NO:1, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:28, SEQ ID NO:31, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:43 and SEQ ID NO:136;

[0738] b) A heavy chain CDR2 sequence, wherein the heavy chain CDR2 sequence is selected from: SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:44, SEQ ID NO:

[0739] 140 and SEQ ID NO:141; and

[0740] c) Heavy chain CDR3 sequence, wherein the heavy chain CDR3 sequence is selected from: SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:15, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:39 and SEQ ID NO:45.

[0741] Implementation Method 6. The antibody or antigen-binding fragment thereof according to any of the foregoing embodiments, comprising:

[0742] a) A light chain CDR1 sequence, wherein the light chain CDR1 sequence is selected from: SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:16, SEQ ID NO:40 and SEQ ID NO:137;

[0743] b) A light chain CDR2 sequence, said light chain CDR2 sequence being selected from: SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:17, SEQ ID NO:41 and SEQ ID NO:138; and

[0744] c) A light chain CDR3 sequence selected from: SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:18, SEQ ID NO:42 and SEQ ID NO:139.

[0745] Implementation Method 7. The antibody or antigen-binding fragment thereof according to any of the foregoing embodiments comprises:

[0746] a) A heavy chain CDR1 sequence, wherein the heavy chain CDR1 sequence is selected from: SEQ ID NO:1, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:28, SEQ ID NO:31, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:43 and SEQ ID NO:136;

[0747] b) A heavy chain CDR2 sequence, wherein the heavy chain CDR2 sequence is selected from: SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:44, SEQ ID NO:

[0748] 140 and SEQ ID NO:141;

[0749] c) A heavy chain CDR3 sequence, wherein the heavy chain CDR3 sequence is selected from: SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:15, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:39 and SEQ ID NO:45;

[0750] d) A light chain CDR1 sequence, wherein the light chain CDR1 sequence is selected from: SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:16, SEQ ID NO:40 and SEQ ID NO:137;

[0751] e) A light chain CDR2 sequence, said light chain CDR2 sequence being selected from: SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:17, SEQ ID NO:41 and SEQ ID NO:138; and

[0752] f) A light chain CDR3 sequence, wherein the light chain CDR3 sequence is selected from: SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:18, SEQ ID NO:42 and SEQ ID NO:139.

[0753] Implementation Method 8. The antibody or antigen-binding fragment thereof according to any of the foregoing embodiments further comprises: one or more heavy chain framework region (FR) sequences selected from the group consisting of: SEQ ID NO: 54, 55, 56, 57, 70, 71, 72, 73, 86, 87, 88 and 89, and / or one or more κ light chain framework region (FR) sequences selected from the group consisting of: SEQ ID NO: 58, 59, 60, 61, 74, 75, 76, 77, 90, 91, 92 and 93.

[0754] Embodiment 9. The antibody or antigen-binding fragment thereof according to any of the foregoing embodiments further comprises: a heavy chain FR1 sequence, wherein the heavy chain FR1 is selected from SEQ ID NO: 54, 70 and 86; a heavy chain FR2 sequence, wherein the heavy chain FR2 is selected from SEQ ID NO: 55, 71 and 87; a heavy chain FR3 sequence, wherein the heavy chain FR3 is selected from SEQ ID NO: 56, 72 and 88; and a heavy chain FR4 sequence, wherein the heavy chain FR4 is selected from SEQ ID NO: 57, 73 and 89.

[0755] Embodiment 10. The antibody or antigen-binding fragment thereof according to any of the foregoing embodiments further comprises: a light chain FR1 sequence, wherein the light chain FR1 is selected from SEQ ID NO: 58, 74 and 90; a light chain FR2 sequence, wherein the light chain FR2 is selected from SEQ ID NO: 59, 75 and 91; a light chain FR3 sequence, wherein the light chain FR3 is selected from SEQ ID NO: 60, 76 and 92; and a light chain FR4 sequence, wherein the light chain FR4 is selected from SEQ ID NO: 61, 77 and 93.

[0756] Implementation Method 11. An antibody or antigen-binding fragment thereof according to any of the foregoing embodiments, comprising: a heavy chain variable region selected from the group consisting of: SEQ ID NO:94, SEQ ID NO:98, SEQ ID NO:102, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:128, SEQ ID NO:132 and homologous sequences having at least 80% sequence identity with them.

[0757] Implementation Method 12. An antibody or antigen-binding fragment thereof according to any of the foregoing embodiments, comprising: a κ light chain variable region selected from the group consisting of: SEQ ID NO:96, SEQ ID NO:100, SEQ ID NO:104, SEQ ID NO:120, SEQ ID NO:126, SEQ ID NO:130, SEQ ID NO:134 and homologous sequences having at least 80% sequence identity with them.

[0758] Implementation Method 13. The antibody or antigen-binding fragment thereof according to any of the foregoing embodiments, comprising:

[0759] a) Heavy chain variable region, including SEQ ID NO:94; and κ light chain variable region, including SEQ ID NO:96;

[0760] b) Heavy chain variable region, including SEQ ID NO:98; and light chain variable region, including SEQ ID NO:100;

[0761] c) Heavy chain variable region, including SEQ ID NO:102; and κ light chain variable region, including SEQ ID NO:104;

[0762] d) Heavy chain variable region, including SEQ ID NO:106; and κ light chain variable region, including SEQ ID NO:96;

[0763] e) Heavy chain variable region, including SEQ ID NO:108; and κ light chain variable region, including SEQ ID NO:96;

[0764] f) Heavy chain variable region, including SEQ ID NO:110; and κ light chain variable region, including SEQ ID NO:96;

[0765] g) Heavy chain variable region, including SEQ ID NO:112; and κ light chain variable region, including SEQ ID NO:96;

[0766] h) Heavy chain variable region, including SEQ ID NO:114; and κ light chain variable region, including SEQ ID NO:96;

[0767] i) Heavy chain variable region, which includes SEQ ID NO:116; and κ light chain variable region, which includes SEQ ID NO:96;

[0768] j) Heavy chain variable region, including SEQ ID NO:118; and κ light chain variable region, including SEQ ID NO:120;

[0769] k) Heavy chain variable region, which includes SEQ ID NO:122; and κ light chain variable region, which includes SEQ ID NO:96;

[0770] l) Heavy chain variable region, which includes SEQ ID NO:124; and κ light chain variable region, which includes SEQ ID NO:126;

[0771] m) Heavy chain variable region, including SEQ ID NO:128; and κ light chain variable region, including SEQ ID NO:130; or

[0772] n) Heavy chain variable region, including SEQ ID NO:132; and κ light chain variable region, including SEQ ID NO:134.

[0773] Implementation 14. The antibody or antigen-binding fragment thereof according to any of the foregoing embodiments further comprises one or more amino acid residue substitutions, but still maintains specific binding affinity to CD19.

[0774] Implementation 15. The antibody or antigen-binding fragment thereof according to Implementation 14, wherein the substitution is in one or more CDR sequences, and / or in one or more FR sequences, in one or more variable region sequences, and / or in the Fc region.

[0775] Implementation Method 16. An antibody or antigen-binding fragment thereof according to Implementation Method 14 or 15, wherein the substitution imparts one or more desired properties, the desired properties being selected from:

[0776] a) Increase binding affinity with CD19,

[0777] b) Introduce or remove glycosylation sites.

[0778] c) Introduce free cysteine ​​residues.

[0779] d) Increase or decrease ADCC or CDC,

[0780] e) Increase serum half-life, and

[0781] f) Increase FcRn binding.

[0782] Implementation 17. The antibody or antigen-binding fragment thereof according to any of the foregoing embodiments further includes an immunoglobulin constant region, optionally including a constant region of IgG, optionally including a constant region of human IgG1.

[0783] Implementation Method 18. The antibody or antigen-binding fragment thereof described in any of the foregoing embodiments is a humanized antibody.

[0784] Implementation Method 19. The antibody or its antigen-binding fragment according to any of the foregoing embodiments is a camelized single-chain domain antibody, a diabody, scFv, scFv dimer, BsFv, dsFv, (dsFv)2, dsFv-dsFv', Fv fragment, Fab, Fab', F(ab')2, ds diabody, nanobody, domain antibody, or bivalent domain antibody.

[0785] Implementation Method 20. The antibody or its antigen-binding fragment according to any of the foregoing embodiments has bispecificity.

[0786] Implementation Method 21. An antibody or antigen-binding fragment thereof according to any of the foregoing embodiments, which is linked to one or more conjugates.

[0787] Implementation Method 22. The antibody or antigen-binding fragment thereof according to Implementation Method 21, wherein the conjugate includes a chemotherapeutic agent, a toxin, a radioactive isotope, a lanthanide element, a luminescent label, a fluorescent label, or an enzyme substrate label.

[0788] Implementation Method 23. The antibody or antigen-binding fragment thereof according to any of the foregoing embodiments is specifically capable of binding to CD19, and optionally, is specifically capable of binding to CD19 derived from humans or monkeys.

[0789] Implementation Method 24. The antibody or antigen-binding fragment thereof according to any of the foregoing embodiments is capable of producing at a concentration not exceeding 5 × 10⁻⁶. -9 M of K D The K value specifically binds to human CD19 expressed on cells. DThe values ​​were determined by flow cytometry.

[0790] Implementation Method 25. The antibody or antigen-binding fragment thereof according to any of the foregoing embodiments is capable of being distributed at an EC50 concentration not exceeding 0.9 nM or not exceeding 1 nM. 50 The value specifically binds to human CD19 expressed on cells, the EC 50 The values ​​were determined by flow cytometry.

[0791] Implementation Method 26. The antibody or antigen-binding fragment thereof according to any of the foregoing embodiments is capable of EC50 at a concentration not exceeding 3 nM. 50 The EC specifically binds to CD19 expressed on cells in cynomolgus monkeys. 50 The values ​​were determined by flow cytometry.

[0792] Implementation Method 27. The antibody or antigen-binding fragment thereof according to any of the foregoing embodiments is capable of being generated at an EC50 concentration not exceeding 50 pM. 50 The value is internalized in cells expressing CD19, the EC 50 The value was determined by the Fab-Zap method.

[0793] Implementation 28. An antibody or an antigen-binding fragment thereof, which competes with W7011-4.155.8 for the same epitope.

[0794] Implementation 29. An isolated polynucleotide sequence encoding an antibody or antigen-binding fragment thereof according to any of the foregoing embodiments.

[0795] Implementation 30. The isolated polynucleotide sequence according to Implementation 29, comprising nucleotide sequences selected from the group consisting of SEQ ID NO: 95, 99, 103, 107, 109, 111, 113, 115, 117, 119, 123, 125, 129 and 133, and / or nucleotide sequences selected from the group consisting of SEQ ID NO: 97, 101, 105, 121, 127, 131 and 135, or homologous sequences having at least 80% sequence identity but encoding the same protein sequence.

[0796] Embodiment 31. A carrier comprising isolated polynucleotides according to Embodiment 29 or 30.

[0797] Embodiment 32. A host cell comprising the vector according to Embodiment 31.

[0798] Embodiment 33. A method for expressing an antibody or antigen-binding fragment thereof according to any one of Embodiments 1-28, comprising culturing host cells according to Embodiment 32 under conditions of expressing the vector according to Embodiment 31.

[0799] Embodiment 34. An antibody-drug conjugate comprising one or more drug portions, said drug portions being directly or covalently linked via a linker to an antibody or an antigen-binding fragment thereof according to any one of Embodiments 1-28.

[0800] Example 35. The antibody-drug conjugate according to Example 34, wherein the linker is a hydrazone linker, a disulfide linker, a bifunctional linker, a dipeptide linker, a glucuronide linker, or a thioether linker; optionally, the linker is an SMCC. Example 36. The antibody-drug conjugate according to Example 34 or 35, wherein at least one drug portion is linked to a specific site on the antibody or its antigen-binding fragment; optionally, the specific site is a cysteine ​​residue. Example 37. The antibody-drug conjugate according to Example 36, wherein the drug portion is a cytotoxin or a radioisotope.

[0801] Implementation Method 38. The antibody or antigen-binding fragment thereof according to Implementation Method 21, wherein the conjugate is a toxin, optionally a cytotoxic agent, DNA alkylating agent, topoisomerase inhibitor, microtubule binding agent, or other anticancer drug, optionally a maytansine-type cytotoxic agent, optionally the toxin is DM1.

[0802] Embodiment 39. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of Embodiments 1-28, or an antibody-drug conjugate according to any one of Embodiments 34-37, and a pharmaceutically acceptable carrier.

[0803] Implementation 40. A method of treating a CD19-related disease or condition in a subject, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to any one of Implementations 1-28, an antibody-drug conjugate according to any one of Implementations 34-37, or a pharmaceutical composition according to Implementation 39.

[0804] Implementation 41. The method according to implementation 40, wherein the disease or condition is cancer.

[0805] Implementation Method 42. The method according to Implementation Method 41, wherein the cancer is lymphoma, lung cancer, liver cancer, cervical cancer, colon cancer, breast cancer, ovarian cancer, pancreatic cancer, melanoma, glioblastoma, prostate cancer, esophageal cancer, or stomach cancer.

[0806] Implementation Method 43. The method according to Implementation Method 40, wherein the disease or condition is B-cell lymphoma, optionally Hodgkin lymphoma or non-Hodgkin lymphoma, wherein the non-Hodgkin lymphoma includes: diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, marginal zone B-cell lymphoma (MZL), mucosa-associated lymphoid tissue lymphoma (MALT), small lymphocytic lymphoma (chronic lymphocytic leukemia, CLL), mantle cell lymphoma (MCL), acute lymphoblastic leukemia (ALL), or Waldenström macroglobulinemia (WM).

[0807] Implementation 44. The method according to implementation 40, wherein the administration is via oral, intranasal, intravenous, subcutaneous, sublingual, or intramuscular administration.

[0808] Implementation 45. The method according to implementation 40, wherein the subject is a human.

[0809] Implementation 46. A method for regulating CD19 activity in CD19-expressing cells, comprising exposing the CD19-expressing cells to an antibody or antigen-binding fragment thereof according to any one of Implementation 1-28.

[0810] Embodiment 47. A method for killing CD19-expressing cells in vivo or in vitro, comprising contacting the CD19-expressing cells with an antibody-drug conjugate according to any one of Embodiments 34-37.

[0811] Implementation 48. A method for detecting the presence or content of CD19 in a sample, comprising contacting the sample with an antibody or antigen-binding fragment thereof according to any one of Implementations 1-28, and determining the presence or content of CD19 in the sample.

[0812] Implementation 49. A method for diagnosing a CD19-related disease or condition in a subject, comprising: a) obtaining a sample from the subject; b) contacting the sample with an antibody or antigen-binding fragment thereof according to any one of Implementations 1-28; c) determining the presence or amount of CD19 in the sample; and d) associating the presence or amount of CD19 with the disease or condition of the subject.

[0813] Example 50. Use of the antibody or antigen-binding fragment thereof according to any one of Examples 1-28, the antibody-drug conjugate according to any one of Examples 34-37, or the pharmaceutical composition according to Example 39 in the preparation of a medicament for treating a disease or condition in a subject who requires it, wherein the treatment comprises administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof.

[0814] Implementation 51. Use of the antibody or antigen-binding fragment thereof according to any one of Implementations 1-28 in the preparation of a diagnostic reagent for diagnosing diseases or conditions related to CD19.

[0815] Embodiment 52. A chimeric antigen receptor (CAR) comprising an antigen-binding fragment according to any one of Embodiments 1-28 and a T-cell activation portion, wherein the T-cell activation portion comprises a transmembrane domain of a T-cell receptor and an intracellular signal transduction domain of a T-cell receptor.

[0816] Implementation 53. The chimeric antigen receptor (CAR) according to Implementation 52, wherein the antigen-binding fragment is scFv.

[0817] Implementation 54. A nucleic acid sequence encoding a CAR as described in Implementation 52.

[0818] Implementation 55. The nucleic acid sequence according to Implementation 54, comprising the polynucleotide sequence according to Implementation 29 or 30, the polynucleotide sequence being operatively linked to a second polynucleotide sequence encoding a transmembrane domain of a T cell receptor and a signal transduction domain of a T cell receptor.

[0819] Embodiment 56. A vector comprising a nucleic acid sequence according to Embodiment 54 or 55.

[0820] Embodiment 57. An isolated T cell expressing CAR according to Embodiment 52.

[0821] Embodiment 58. A method for activating a T cell-mediated immune response against a CD19-expressing target in a subject, the method comprising administering to the subject an effective amount of the T cells according to Embodiment 57.

[0822] The following examples are intended to better illustrate the invention and should not be construed as limiting the scope of the invention. All specific compositions, materials, and methods described below, in whole or in part, are within the scope of the invention. These specific compositions, materials, and methods are not intended to limit the invention, but merely to illustrate specific embodiments within the scope of the invention. Those skilled in the art can develop equivalent compositions, materials, and methods without adding inventive step or departing from the scope of the invention. It should be understood that various modifications made to the methods of the invention may still be included within the scope of the invention. The inventors intend to include such modifications within the scope of the invention.

[0823] Example 1: Material Production

[0824] 1.1 Reference antibody production

[0825] The genes of each region of the anti-CD19 reference antibody (WBP701-BMK1 corresponds to huB4 in patent US20140072587A1; WBP701-BMK2 corresponds to hBU12 in patent US8242252B2; WBP701-BMK3 corresponds to 21D4 in patent US8097703B2) were cloned into an expression vector containing human Fc region genes.

[0826] The expression plasmid was transfected into Expi293 cells using the ExpiFectamine293 transfection kit (Invitrogen-A14524). The cells were cultured in Expi293 expression medium (Invitrogen-A1435101) at 37°C on a fixed-orbit oscillation platform rotating at 135 rpm. The collected supernatant was purified using a Protein A column (GE Healthcare 17543802).

[0827] The reference antibodies WBP701-BMK1, WBP701-BMK2, and WBP701-BMK3, generated according to the above method, were analyzed by SDS-PAGE. Figure 1 and 2 The results showed that all three generated reference antibodies migrated significantly at molecular weights of 25 kDa and 55 kDa on SDS-PAGE under reducing conditions, corresponding to the light and heavy chains of the antibodies, respectively. The main band on non-reducing SDS-PAGE corresponded to intact IgG molecules with a molecular weight of approximately 150 kDa. The purity of the reference antibodies was greater than 95% (see [link to relevant documentation]). Figure 1 and 2 ).

[0828] 1.2 Generation of human or cynomolgus monkey CD19-expressing cell lines

[0829] The full-length human or cynomolgus monkey CD19 gene was cloned into the pcDNA3.3 vector. Briefly, using PlasfectReagent (Bioline-46025), 30 μg of DNA was transfected into a 30 mL volume at a density of 1 × 10⁻⁶. 6 / mL of FreeStyle293F cells (ThermoFisher-R79007) were transfected. The transfected cells were cultured in an incubator set at 37°C, 8% CO2, and 100 rpm. Twenty-four hours after transfection, a stable library was generated using blastcinon (Invitrogen-A1113902) at a final concentration of 4–10 μg / mL. Selected clones were examined by FACS using an anti-CD19 antibody. After 2–3 rounds of selection, the cells were enriched using PE-conjugated anti-CD19 antibody and anti-PE microbeads (Miltenyi-013-048-801). Stable single-cell clones were isolated by limiting dilution and by FACS selection using an anti-CD19 antibody.

[0830] The full-length human or cynomolgus monkey CD19 gene was cloned into the pcDNA3.3 vector. Subsequently, each expression vector was transfected into CHO-K1 cells using Lipofectamine 2000. The cells were cultured in F12-K containing 10% FBS. Phenythiocyanate was added 24-48 hours after transfection. After 2-3 rounds of selection, the selected clones were examined by FACS. Following 2-3 rounds of selection, the cells were enriched using PE-conjugated anti-CD19 antibody and anti-PE microbeads (Miltenyi-013-048-801). Stable single-cell clones were isolated by limiting dilution and selection using anti-CD19 antibody via FACS.

[0831] Using an anti-CD19 antibody, flow cytometry was used to detect the expression of human CD19 and cynomolgus monkey CD19 in transfected cell lines. The transfected cell lines WBP701.293F.hPro1.FL.A2, WBP701.CHO-K1.hPro1.FL.B4, WBP701.cPro1.293F.FL.C1, and WBP701.CHO-K1.cpro1.FL.C9 all showed high expression of either human or monkey CD19. Figures 3A-3D ).

[0832] Example 2: Antibody Production

[0833] 2.1 Immunity

[0834] Balb / c mice were immunized with CD19-transfected 293F cells. Cell membrane lysis products were mixed with adjuvants (including CpG-ODN and Adju-Phos or Titer-Max). The mice were immunized twice via paw, subcutaneous, or intraperitoneal routes, with a two-week interval between the immunizations. The immunization was performed with 1×10⁻⁶ cells dissolved in PBS. 6 Cell membrane lysis products from cells / animals and 10 μg of ECD protein / animal were administered to mice with high serum titers for booster immunization.

[0835] 2.2 Serum titer detection

[0836] Serum titers were detected by flow cytometry. CD19-transfected CHO-K1 cells were cultured at a rate of 1×10⁻⁶. 5 Cells were seeded at a density of 1:3 in 96-well U-shaped plates (BD). Mouse serum was diluted 1:3 from a 100-fold dilution using staining buffer (1×PBS / 1% BSA). Serum samples were incubated with cells at 4°C for 1 hour. After washing the cells twice with staining buffer, PE-conjugated goat anti-mouse IgG Fc antibody (Jackson) was added, and the cells were incubated at 4°C in the dark for 30 minutes. The cells were then washed twice and resuspended in 100 μL of staining buffer. Fluorescence intensity was measured by flow cytometry (BD Canto II) and analyzed using FlowJo.

[0837] All mice showed CD19-specific titers. Mice with high serum titers were selected for hybridoma cell fusion (Table 3).

[0838] Table 3. Serum titers

[0839] Mice# 1 2 3 4 5 Pre-bloodletting of CHO-K1.CD19 cells <100 <100 <100 <100 <100 The titer of CHO-K1CD19 cells 1968300 656100 218700 656100 72900 The titer of parental CHO-K1 cells 2700 2700 900 300 2700

[0840] 2.3 Production of hybridoma cells

[0841] Following a standard electrofusion procedure, lymph node cells were fused with Sp2 / 0 myeloma cells via electrofusion. After cell fusion, the cells were then... 4 Lymphocytes / well were seeded into 96-well plates with DMEM medium containing 20% ​​FBS and 1% HAT. The 96-well plates were incubated at 37°C for 10–12 days.

[0842] 2.4 Antibody Screening

[0843] 2.4.1 Binding with human CD19

[0844] CHO-K1 cells transfected with human CD19 were used at a rate of 1×10⁻⁶. 5Cells were plated at a density per well in 96-well U-shaped plates (BD). Hybridoma cell supernatant was transferred to the 96-well plates and incubated with the cells at 4°C for 1 hour. The cells were then washed twice with staining buffer (BSA / 1×PBS). PE-conjugated goat anti-mouse IgG Fc antibody (Jackson 115-115-164) was added, and the plates were incubated at 4°C in the dark for 30 minutes. The cells were then washed twice and resuspended in 100 μL of staining buffer. Fluorescence intensity was measured by flow cytometry (BD Canto II) and analyzed using FlowJo.

[0845] 2.4.2 Binding with CD19 of cynomolgus monkeys

[0846] CHO-K1 cells transfected with CD19 from cynomolgus monkeys were used at a rate of 1×10⁻⁶. 5 Cells were plated at a density per well in 96-well U-shaped plates (BD). Hybridoma supernatant was transferred to the 96-well plates and incubated with the cells at 4°C for 1 hour. The cells were then washed twice with staining buffer (BSA / 1×PBS). PE-conjugated goat anti-mouse IgG Fc antibody (Jackson115-115-164) was added, and the plates were incubated at 4°C in the dark for 30 minutes. The cells were then washed twice and resuspended in 100 μL of staining buffer. Fluorescence intensity was measured by flow cytometry (BD Canto II) and analyzed using FlowJo.

[0847] 2.4.3 Internalization Test

[0848] Fab-ZAP is a chemical conjugate of a goat anti-human monovalent antibody and the ribosome-inactivating protein saponin. The internalization ability of the antibody was determined using Fab-ZAP. The concentration of IgG in the hybridoma cell supernatant was determined by ELISA. Normalized hybridoma cell supernatant was mixed with Fab-ZAP at a molar ratio of 1:3. Ramos cells (5000 / well) were cultured with different concentrations of the conjugate in a 37°C, 5% CO2 incubator for 96 hours. Cytotoxicity was determined by CellTiter Glo (Promega). Cell viability (%) was calculated as follows: Cell viability (%) = RLU of sample / RLU of control × 100%, where RLU represents relative light units.

[0849] result

[0850] The first round of screening was performed using hybridoma cell supernatant. The initial binding screening identified 116 hybridomas capable of producing antibodies that specifically bind to the antigen. Binding of these antigen-specific hybridomas was subsequently confirmed on CD19-transfected CHO-K1 cells, and reverse screening was performed on parental CHO-K1 cells. Binding of the selected 40 hybridoma cell lines was confirmed on Ramos cells. Positive binders were then screened in the Fab-Zap assay. Thirteen hybridoma cell lines were selected for subcloning based on binding and internalization aptitude.

[0851] 2.5 Subcloning of hybridoma cells

[0852] Hybridoma cells from each selected cell line were seeded at a density of 1 cell / well in 96-well plates. The 96-well plates were stored in a humidified incubator at 37°C and 6% CO2 for 10–12 days. Single clones were selected and examined using FACS.

[0853] 2.6 Homotype

[0854] Antibody isotypes were identified using ELISA. Plates (Nunc) were coated overnight at 4°C with goat anti-mouse IgG1, anti-mouse IgG2a, anti-mouse IgG2b, anti-mouse IgG3, and anti-mouse IgM antibodies at a concentration of 2 μg / ml. After blocking and washing, hybridoma cell supernatant was transferred to the coated plates and incubated at room temperature for 1 hour. The plates were then incubated with goat anti-mouse kappa HRP or goat anti-mouse lambda HRP secondary antibody (Southern Biotech) for 45 minutes. After washing, TMB substrate chromogenic buffer was added, and chromogenic development was terminated with 2M HCl. The absorbance at 450 nm was read using a molecular device.

[0855] result:

[0856] Subclones of hybridoma cells were validated by binding to the CD19 cell line, and their isotypes were also detected (see Table 4). The selected subclones were purified and further evaluated in binding tests, internalization tests, cross-family binding tests, and binning tests.

[0857] Table 4. Antibody isotypes

[0858]

[0859]

[0860] Example 3: Characterization of candidate antibodies

[0861] 3.1 Antibody purification

[0862] After adjusting the pH to 7.0, the collected hybridoma supernatant was loaded onto a protein A column (MabSelect SuRe, GE). Antibodies were eluted with Glycine and immediately neutralized with 1M Tris. Antibody concentration was determined using Nano Drop (Thermal-Fisher). Protein purity was assessed by SDS-PAGE (Invitrogen, NuPAGE 4%–12% Bis-Tris gel) and HPLC-SEC (Agilent).

[0863] 3.2 Affinity detection by FACS

[0864] The CD19-transfected CHO-K1 cells or Ramos cells were used at 5 × 10⁻⁶. 4 Cells were plated at a density of 96-well plates (BD). The test antibody was serially diluted in staining buffer (1×PBS / 1% BSA) and cultured with cells at 4°C for 1 hour. After discarding the supernatant, PE-conjugated goat anti-mouse IgG Fc antibody (Jackson115-1154-164) was added, and the cells were incubated at 4°C in the dark for 30 minutes. The cells were washed once and resuspended in 100 μL of staining buffer. Fluorescence intensity was measured by flow cytometry (BD Canto II) and analyzed using FlowJo. The concentrations of bound and free IgG were calculated based on the fluorescence intensity from the quantitative beads (PE PCR kit, BD340495). K was calculated using Scatchard analysis. D .

[0865] result

[0866] The affinity of the selected candidate antibodies was detected by flow cytometry on CD19-transfected CHO-K1 cells. D The values ​​are summarized in Table 5. All candidate antibodies exhibited sub-nanomolar binding affinity to human CD19.

[0867] Table 5. Affinity of candidate antibodies

[0868]

[0869] 3.3 Binding with human CD19

[0870] Ramos cells at 1×10 5Cells were plated at a density of 96-well U-shaped plates (BD). Purified antibody was serially diluted in staining buffer (1×PBS / 1% BSA) and incubated with cells at 4°C for 1 hour. The cells were then washed twice with staining buffer (BSA / 1×PBS). PE-conjugated goat anti-mouse IgG Fc antibody (Jackson 115-115-164) was added, and the cells were incubated at 4°C in the dark for 30 minutes. The cells were then washed twice and resuspended in 100 μL of staining buffer. Fluorescence intensity was measured by flow cytometry (BD Canto II) and analyzed using FlowJo.

[0871] The binding affinity of the selected subclones was examined on Ramos cells by flow cytometry (Figure 4). (EC) 50 The values ​​are summarized in Table 6. All candidate antibodies exhibited sub-nanomolar affinity for human CD19.

[0872] Table 6. Binding activities of the selected subclones

[0873]

[0874]

[0875] 3.4 Binding with CD19 of cynomolgus monkeys

[0876] CHO-K1 cells transfected with CD19 from cynomolgus monkeys were used at a rate of 1×10⁻⁶. 5 Cells were plated at a density of 96-well U-shaped plates (BD). Purified antibody was serially diluted in staining buffer (1×PBS / 1% BSA) and cultured with cells at 4°C for 1 hour. Cells were washed twice with staining buffer (1×PBS / 1% BSA). PE-conjugated goat anti-mouse IgG Fc antibody (Jackson 115-115-164) was added, and the cells were incubated at 4°C in the dark for 30 minutes. The cells were then washed twice and resuspended in 100 μL of staining buffer. Fluorescence intensity was measured by flow cytometry (BD Canto II) and analyzed using FlowJo.

[0877] The binding activity of the candidate antibody to cynomolgus CD19 was assessed using the CHO-K1 cell line transfected with cynomolgus macaque CD19 (Figure 5). The antibody was then used to bind EC... 50 The values ​​are summarized in Table 7. All selected clones showed strong binding to cynomolgus monkey CD19 cells.

[0878] Table 7. Binding activity with CD19 in cynomolgus monkeys

[0879]

[0880] 3.5 Internalization Test

[0881] The internalization ability of the antibody was determined using Fab-ZAP. Serially diluted antibodies were mixed with Fab-ZAP at a molar ratio of 1:3. Ramos cells (5000 / well) were incubated with different concentrations of conjugates in a 37°C, 5% CO2 incubator for 96 hours. Cytotoxicity was determined by CellTiter Glo (Promega). Cell viability (%) was calculated as follows: Cell viability (%) = RLU of sample / RLU of control × 100%.

[0882] The internalization activity of selected subclones was examined on Ramos cells using the Fab-Zap assay (Figure 6). EC50 of cell viability was also measured. 50 A summary is provided in Table 8. All candidate antibodies were internalized on Ramos cells and showed picomolar-level EC50 in the Fab-Zap assay. 50 value.

[0883] Table 8. Fab-Zap Testing

[0884]

[0885]

[0886] 3.6 Epitope Binning

[0887] CD19-transfected cells WBP701.CHO-K1.hPro1.B4 were used at a dose of 1×10⁻⁶. 5 Cells were plated at a density per well in 96-well U-shaped plates (BD). The assay antibody was serially diluted and mixed with a reference antibody. The mixture was added to the 96-well plates and incubated at 4°C for 30 min. After washing, PE-conjugated goat anti-human IgG Fc antibody (Jackson) was added, and the plates were incubated at 4°C in the dark for 30 min. The cells were then washed twice and resuspended in 100 μL of staining buffer (1×PBS / 1% BSA). Fluorescence intensity was measured by flow cytometry (BD Canto II) and analyzed using FlowJo.

[0888] The selected candidate clones were tested for competitive binding to the BMK1, BMK2, and BMK3 reference antibodies. Some candidate antibodies could block the binding of the reference antibodies to CD19. W7011-4.155.8, W7011-4.202.9, and...

[0889] W7011-4.225.7 does not compete with the reference antibody (Figure 7). Based on the results of competitive binding, the antibody was assigned to two epitope groups (bins) (Table 9).

[0890] Table 9. Epitope grouping of candidate antibodies

[0891]

[0892]

[0893] After sequencing the antibody clones, we found that the amino acid sequences of antibody clones W7011-4.155.8, W7011-4.202.9, and W7011-4.225.7 were identical. The amino acid and nucleic acid sequences of the antibody clones are listed in the detailed description of the invention.

[0894] Example 4: Antibody Humanization and Affinity Maturation

[0895] 4.1 Hybridoma cell sequencing

[0896] RNA was isolated from hybridoma cells using a Trizol kit (Invitrogen-15596018), and cDNA was amplified using a 5'-RACE kit (Takara-28001488). The cDNA was then amplified by PCR using 3'-degenerate primers and 3'-adaptor primers (ExTaq: Takara-RR001B). The PCR fragment was inserted into the pMD18-T vector (Takara-D101C) and sequenced (Shanghai Platinum Technology Co., Ltd.).

[0897] The antibody sequence (mouse) from hybridoma cells is shown in SEQ ID NO:94-123.

[0898] 4.2 Humanization

[0899] The antibody light and heavy chains were humanized using a best-fit method. The amino acid sequence of the corresponding V gene light chain was compared with the company's internal human V gene database. Using the Kabat CDR definition, the mouse CDR sequence was replaced with the highest-hitting human CDR sequence to obtain the humanized VL gene sequence. For the heavy chain, four humanized sequences were obtained. For the light chain, one humanized sequence was first obtained using the above method, and then three more sequences were generated by comparing the mouse framework with the human V gene database. The framework was defined using the extended CDR definition, where Kabat CDR1 was extended by 5 amino acids at the N-terminus. The humanized V gene was obtained using the three highest-hitting sequences. The humanized gene was back-translated, codon-optimized for mammalian expression, and synthesized via GeneArt Costum Gene Synthesis (LifeTechnologies). The synthesized gene was recloned into an IgG expression vector, expressed, and purified.

[0900] 4.3 Affinity Maturity

[0901] Using a hybridization mutagenesis method (Kunkel, 1985), each amino acid in the six complementarity-determining regions (CDRs) was independently mutated to 20 different amino acids. Mutations were introduced into each target CDR location using DNA primers containing NNS codons encoding the 20 amino acids. Individual degenerate primers were used in the hybridization mutagenesis reactions. The results were collected separately.

[0902] The synthetic products of VH and VL CDRs were obtained. 200 ng of the collected library DNA was transfected into BL21 to produce the scFv fragment.

[0903] First, bacterial periplasmic extracts were used to screen for mutations via capture ELISA. 96-well Maxisorp immunoassay plates (Nunc) were prepared with anti-c-myc antibody in coating buffer (200 mM Na₂CO₃ / NaHCO₃, pH 9.2).

[0904] The sample was coated overnight at 4°C. After blocking with casein at room temperature for 1 hour, the periplasmic extract sample was added to a 96-well plate and incubated at room temperature for 1 hour. After washing, biotinylated CD19 ECD protein was added and incubated at room temperature for 1 hour, followed by incubation with streptavidin-HRP for 1 hour. After washing, TMB substrate was added, and the reaction was stopped with 2M HCl. The absorbance was read at 450 nm using a molecular device.

[0905] Clones exhibiting an optical density (OD) signal greater than that of the parental clone at a wavelength of 450 nm were selected for sequencing. These unique clones were confirmed by FACS at normalized scFv concentrations to determine the relative binding affinity between the mutant scFv and the parental antibody.

[0906] Point mutations in VH and VL that were identified as beneficial for antigen binding were further combined to obtain additional binding synergies. These combined mutations were expressed as scFv and screened using a capture ELISA. Clones that showed an optical density (OD) signal greater than that of the parental clones at 450 nm were sequenced and further confirmed by binding FACS.

[0907] 4.4 Binding affinity of engineered antibodies

[0908] 4.4.1WBP7011-4.34.11-z1-m5-IgG1k

[0909] The antibody WBP7011-4.34.11 is humanized and affinity-matured. The affinity of the engineered antibody WBP7011-4.34.11-z1-m5 was measured on Ramos cells using FACS. Figure 8 ).use

[0910] Scatchard analysis and calculation of K D The affinity of WBP7011-4.34.11-z1-m5-IgG1k is 0.23 nM.

[0911] 4.4.2WBP7011-4.87.6-z1-IgG1k(NS)

[0912] The antibody WBP7011-4.87.6 is humanized and engineered with PTM risk residues. The affinity of the final antibody WBP7011-4.87.6-z1-IgG1k(NS) was measured by FACS on Ramos cells. Figure 9 K was calculated using Scatchard analysis. D The affinity of WBP7011-4.87.6-z1-IgG1k(NS) is 0.25 nM.

[0913] 4.4.3W7011-4.155.8-z1-uIgG1K

[0914] The antibody W7011-4.155.8 is humanized. The affinity of the humanized antibody W7011-4.155.8-z1-uIgG1K was measured by FACS on CD19-transfected CHO-K1 cells. Figure 10 K was calculated using Scatchard analysis. D The affinity of W7011-4.155.8-z1-uIgG1K is 0.82 nM.

[0915] 4.5 Engineered antibody sequences

[0916] The modified antibody sequence is shown in SEQ ID NO:124-135.

[0917] Example 5: Generation of Antibody-Drug Conjugates (ADCs)

[0918] The antibody was exchanged into PBS (pH 7.4) buffer via buffer exchange and mixed with DMA (Alfa Aesar). Then DM1-SMCC (BrightGene) was added, and the mixture was incubated at 22°C with gentle rotation for conjugation.

[0919] To remove free drug, the ADC product buffer was exchanged using a 30 kDa ultrafiltration tube (Millipore).

[0920] The ADC product was stored in ADC storage buffer. After eight buffer exchanges, the ADC product was filtered through a 0.22 μm membrane for final characterization.

[0921] The concentration of the ADC was characterized using UV-vis (NanoDrop). The DAR value was determined using UV-vis and SEC-HPLC. Aggregation level and purity were determined using SEC-HPLC. Free drug was determined using RP-HPLC. Endotoxin levels were determined using dynamic turbidity assays.

[0922] Candidate antibodies were conjugated to DM1. Concentration, purity, DAR, aggregation level, and percentage of free drug were assessed after conjugation (Table 10).

[0923] Table 10. Characterization of DM1 conjugated antibodies

[0924]

[0925] Example 6: Cytotoxicity analysis of ADC

[0926] B lymphocytes (5000 / well) were cultured with different concentrations of DM1-conjugated antibodies at 37°C for 72 hours. Cytotoxicity was determined using CellTiter Glo (Promega). Cell viability (%) was calculated as follows: Cell viability (%) = RLU of sample / RLU of control × 100%.

[0927] DM1-conjugated antibodies were tested in a cytotoxicity assay on Daudi, Nalm-6, and WSU-DLCL2 cells. Figure 11 , 12 13). EC 50 The values ​​are summarized in Tables 11, 12, and 13. ADC WBP7011-4.87.6-z1-IgG1K(NS)-DM1 showed superior cytotoxicity to WBP701-BMK1-DM1 on all tested tumor cells. ADC WBP7011-4.34.11-z1-m5-uIgG1K-DM1 showed comparable cytotoxicity to WBP701-BMK1-DM1.

[0928] Table 11. Cytotoxicity tests on Daudi

[0929]

[0930] Table 12. Cytotoxicity assays on Nalm-6 cells

[0931]

[0932] Table 13. Cytotoxicity assays on WSU-DLCL2 cells

[0933]

[0934] Example 7: Antitumor Analysis of ADCs

[0935] 7.1 Cell Culture

[0936] Nalm-6 tumor cells were cultured in suspension in RPMI-1640 with 10% fetal bovine serum under humid conditions (95% air and 5% CO2). The tumor cells were passaged twice weekly using standard methods. Cells in the exponential growth phase were collected and counted for tumor inoculation.

[0937] 7.2 Tumor inoculation and grouping

[0938] Each mouse underwent subcutaneous implantation of Nalm-6 tumor cells (10 million+ Matrigel) on the right side for tumorigenesis. The tumor volume reached an average of 113 mm². 3 Treatment began at that time. The administration of experimental materials and the number of mice in each group are shown in the table below.

[0939] Table 14. Administration of experimental ADCs and number of mice in each group

[0940]

[0941] 7.3 Observation

[0942] The animal care and use protocols involved in this study, as well as any modifications or procedures, will be reviewed and approved by WuXi AppTec's Institutional Animal Care and Use Committee (IACUC) before implementation. During the study, animal care and use will be conducted in accordance with the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Morbidity and mortality rates in mice will be checked daily post-vaccination. During routine monitoring, mice will be checked for any effects on tumor growth, and management of normal behaviors such as mobility, food and water consumption, weight gain / loss (measured daily), dull eyes / fur, and any other abnormal effects. Mortality and observed clinical symptoms will be recorded based on the number of mice in each subset.

[0943] 7.4 Tumor Measurement and Endpoints

[0944] The primary endpoint was to see if tumor growth could be slowed or if the mice could be cured. Tumor size was measured twice weekly in two dimensions using calipers, and volume was calculated using the following formula and expressed in mm. 3 The unit representation is: V = 0.5a × b2 Where a and b are the long and short diameters of the tumor, respectively. The size of the tumor is used for calculation.

[0945] T / C value and TGI. The T / C value (in percentage) is an indicator of antitumor effect; T and C are the mean volumes in the treatment group and control group on day 21 and day 28, respectively. The TGI for each group was calculated using the following formula: TGI (%) = [1 - (T / C) / (T / C)] i -T0) / (V i -V0)]×100;T i V is the mean tumor volume of the treatment group on days 21 and 28, T0 is the mean tumor volume of the treatment group on the day treatment began, and V is the mean tumor volume of the treatment group on day 21. i V0 is the mean tumor volume of the load-controlled group on days 21 and 28, while V0 is the mean tumor volume of the load-controlled group on the day treatment began.

[0946] Record all groups according to the plan on day 28.

[0947] All mice maintained their body weight well during the experimental phase.

[0948] 7.5 Efficacy study in the Nalm-6 lymphoma xenograft model

[0949] In this study, the efficacy of the reference antibody-drug conjugates W7011-BMK1-DM1 and W7011-4.87.6-z1-uIgG1k(NS)-DM1 was evaluated in Nalm-6 lymphoma xenografts in female CB17-SCID mice. Tumor volumes at different time points for all groups are shown in [data missing]. Figure 14 middle.

[0950] On PG-D21, the mean tumor volume in the allotype control group reached 840 mm. 3 Use 1 mg / kg (TV = 364 mm) 3 (TGI = 66%, p < 0.01) and 10 mg / kg (TV = 327 mmHg). 3 Treatment with W7011-BMK1-DM1 (TGI = 71%, p < 0.001) showed significant antitumor activity. 1 mg / kg (TV = 398 mmHg) 3 , TGI=61%, p<0.01), 3mg / kg (TV=387mm 3 The ADC W7011-4.87.6-z1-uIgG1k(NS)-DM1 at 10 mg / kg (TV=332 mm3, TGI=70%, p<0.001) showed significant antitumor activity.

[0951] One week after suspension administration, the average tumor volume in the isotype control group reached 1266 mm. 3 Use 1 mg / kg (TV = 593 mm) 3 (TGI = 58%, p < 0.01) and 10 mg / kg (TV = 499 mmHg). 3 Treatment with W7011-BMK1-DM1 (TGI = 67%, p < 0.001) showed significant antitumor activity. (1 mg / kg (TV = 562 mmHg) 3 , TGI=61%, p<0.01), 3mg / kg (TV=556mm 3 (TGI = 62%, p < 0.01) and 10 mg / kg (TV = 502 mmHg). 3 The ADCs W7011-4.87.6-z1-uIgG1k(NS)-DM1 (TGI=66%, p<0.001) showed significant antitumor activity.

[0952] All mice maintained their body weight well during the experimental phase.

Claims

1. An isolated anti-CD19 antibody or its antigen-binding fragment, comprising: a) Heavy chain variable region, including HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:2 and HCDR3 as shown in SEQ ID NO:3; and light chain variable region, including LCDR1 as shown in SEQ ID NO:4, LCDR2 as shown in SEQ ID NO:5 and LCDR3 as shown in SEQ ID NO:6; b) Heavy chain variable region, including HCDR1 as shown in SEQ ID NO:7, HCDR2 as shown in SEQ ID NO:8 and HCDR3 as shown in SEQ ID NO:9; and light chain variable region, including LCDR1 as shown in SEQ ID NO:10, LCDR2 as shown in SEQ ID NO:11 and LCDR3 as shown in SEQ ID NO:12; c) Heavy chain variable regions, including HCDR1 as shown in SEQ ID NO:136, HCDR2 as shown in SEQ ID NO:2, and HCDR3 as shown in SEQ ID NO:3; and κ light chain variable regions, including LCDR1 as shown in SEQ ID NO:137, LCDR2 as shown in SEQ ID NO:138, and LCDR3 as shown in SEQ ID NO:139; or d) Heavy chain variable regions, including HCDR1 as shown in SEQ ID NO:7, HCDR2 as shown in SEQ ID NO:140 and HCDR3 as shown in SEQ ID NO:9; and light chain variable regions, including LCDR1 as shown in SEQ ID NO:10, LCDR2 as shown in SEQ ID NO:11 and LCDR3 as shown in SEQ ID NO:

12.

2. The antibody or antigen-binding fragment thereof according to claim 1, comprising: The heavy chain variable region is selected from the group consisting of: SEQ ID NO:94, SEQ ID NO:98, SEQ ID NO:124 and SEQ ID NO:

128.

3. The antibody or antigen-binding fragment thereof according to claim 1, comprising: The κ light chain variable region is selected from the group consisting of: SEQ ID NO:96, SEQ ID NO:100, SEQ ID NO:126 and SEQ ID NO:

130.

4. The antibody or antigen-binding fragment thereof according to claim 1, comprising: The heavy chain variable region, wherein the amino acid sequence of the heavy chain variable region has at least 97% sequence identity with SEQ ID NO:124; and the κ light chain variable region, wherein the amino acid sequence of the κ light chain variable region has at least 97% sequence identity with SEQ ID NO:

126.

5. The antibody or antigen-binding fragment thereof according to claim 1, comprising: The heavy chain variable region, wherein the amino acid sequence of the heavy chain variable region has substitutions and / or deletions of no more than 3 amino acid residues in the frame region relative to SEQ ID NO:124; and the κ light chain variable region, wherein the amino acid sequence of the κ light chain variable region has substitutions and / or deletions of no more than 3 amino acid residues in the frame region relative to SEQ ID NO:

126.

6. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, comprising: a) Heavy chain variable region, which includes SEQ ID NO:94; And the κ light chain variable region, which includes SEQ ID NO:96; b) Heavy chain variable region, which includes SEQ ID NO:98; And the κ light chain variable region, which includes SEQ ID NO:100; c) Heavy chain variable region, which includes SEQ ID NO:124; And the κ light chain variable region, which includes SEQ ID NO:126; or d) Heavy chain variable region, which includes SEQ ID NO:128; and the κ light chain variable region, which includes SEQ ID NO:

130.

7. The antibody or antigen-binding fragment thereof according to claim 1, further comprising an immunoglobulin constant region.

8. The antibody or antigen-binding fragment thereof according to claim 7, wherein the immunoglobulin constant region is the constant region of IgG.

9. The antibody or antigen-binding fragment thereof according to claim 8, wherein the constant region of IgG is the constant region of human IgG1.

10. The antibody or antigen-binding fragment thereof according to claim 1, wherein it is a humanized antibody.

11. The antibody or antigen-binding fragment thereof according to claim 1, wherein it is a double-chain antibody (diabody), scFv, scFv dimer, BsFv, dsFv, (dsFv)2, dsFv-dsFv', Fv fragment, Fab, Fab', F(ab')2 or ds double-chain antibody (dsdiabody).

12. The antibody or its antigen-binding fragment according to claim 1, which has bispecificity.

13. The antibody or antigen-binding fragment thereof according to claim 1, wherein it is linked to one or more conjugates.

14. The antibody or antigen-binding fragment thereof according to claim 13, wherein the conjugate is a toxin, a radioactive isotope, a lanthanide element, a luminescent label, a fluorescent label, or an enzyme substrate label.

15. The antibody or antigen-binding fragment thereof according to claim 13, wherein the conjugate is a chemotherapeutic agent.

16. The antibody or antigen-binding fragment thereof according to claim 1, which is capable of specifically binding to CD19 derived from humans or monkeys.

17. The antibody or antigen-binding fragment thereof according to claim 1, which can bind at a rate not exceeding 5 × 10⁻⁶. -9 M of K D The K value specifically binds to human CD19 expressed on cells. D The values ​​were determined by flow cytometry.

18. The antibody or antigen-binding fragment thereof according to claim 1, which is capable of EC50 at a concentration not exceeding 0.9 nM or not exceeding 1 nM. 50 The value specifically binds to human CD19 expressed on cells, the EC 50 The values ​​were determined by flow cytometry.

19. The antibody or antigen-binding fragment thereof according to claim 1, which is capable of EC50 up to 3 nM. 50 The EC specifically binds to CD19 expressed on cells in cynomolgus monkeys. 50 The values ​​were determined by flow cytometry.

20. The antibody or antigen-binding fragment thereof according to claim 1, which is capable of EC50 at a concentration not exceeding 50 pM. 50 The value is internalized in cells expressing CD19, the EC 50 The value was determined by the Fab-Zap method.

21. An isolated polynucleotide encoding an antibody or an antigen-binding fragment thereof according to any one of the preceding claims.

22. The isolated polynucleotide according to claim 21, comprising nucleotide sequences selected from the group consisting of SEQ ID NO: 95, 99, 125 and 129, and / or nucleotide sequences selected from the group consisting of SEQ ID NO: 97, 101, 127 and 131.

23. A carrier comprising the isolated polynucleotide according to claim 21 or 22.

24. A host cell comprising the vector according to claim 23.

25. A method for expressing an antibody or an antigen-binding fragment thereof according to any one of claims 1-20, comprising culturing a host cell according to claim 24 under conditions that allow expression of the vector according to claim 23.

26. An antibody-drug conjugate comprising one or more drug portions, said drug portions being directly or covalently linked via a linker to an antibody or an antigen-binding fragment thereof according to any one of claims 1-20.

27. The antibody-drug conjugate of claim 26, wherein the linker is a bifunctional linker.

28. The antibody-drug conjugate according to claim 26, wherein the linker is an hydrazone linker, a disulfide linker, a dipeptide linker, a glucuronide linker, or a thioether linker.

29. The antibody-drug conjugate of claim 27, wherein the linker is SMCC.

30. The antibody-drug conjugate according to any one of claims 26-29, wherein at least one drug portion is linked to a specific site of the antibody or its antigen-binding fragment.

31. The antibody-drug conjugate according to claim 30, wherein the specific site is a cysteine ​​residue.

32. The antibody-drug conjugate of claim 30, wherein the drug portion is a cytotoxin or a radioisotope.

33. The antibody or antigen-binding fragment thereof according to claim 13, wherein the conjugate is a toxin.

34. The antibody or antigen-binding fragment thereof according to claim 13, wherein the conjugate is a cytotoxin or other anticancer drug.

35. The antibody or antigen-binding fragment thereof according to claim 13, wherein the conjugate is a DNA alkylating agent, a topoisomerase inhibitor, or a microtubule binding agent.

36. The antibody or antigen-binding fragment thereof according to claim 13, wherein the conjugate is a maytansin-type cytotoxic agent.

37. The antibody or antigen-binding fragment thereof according to claim 33, wherein the toxin is DM1.

38. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1-20, or an antibody-drug conjugate according to any one of claims 26-32, and a pharmaceutically acceptable carrier.

39. Use of the antibody according to any one of claims 1-20, the antibody-drug conjugate according to any one of claims 26-32, or the pharmaceutical composition according to claim 38 in the preparation of a medicament for treating CD19-positive B-cell lymphoma in a subject, comprising administering a therapeutically effective amount of the medicament to the subject.

40. The use according to claim 39, wherein the B-cell lymphoma is Hodgkin lymphoma or non-Hodgkin lymphoma, wherein the non-Hodgkin lymphoma comprises: Diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, marginal zone B-cell lymphoma (MZL), small lymphocytic lymphoma (chronic lymphocytic leukemia, CLL), mantle cell lymphoma (MCL), acute lymphoblastic leukemia (ALL), or Waldenström macroglobulinemia (WM).

41. The use according to claim 39, wherein the B-cell lymphoma is mucosa-associated lymphoid tissue lymphoma (MALT).

42. The use according to claim 39, wherein the administration is via oral, intranasal, intravenous, subcutaneous, sublingual, or intramuscular administration.

43. The use according to claim 39, wherein the subject is a human.

44. Use of the antibody-drug conjugate according to any one of claims 26-32 in the preparation of a medicament for killing CD19-expressing lymphoma cells in vivo or in vitro, wherein the lymphoma cells are B-cell lymphoma cells.

45. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-20 in the preparation of a kit for detecting the presence or content of CD19 in a sample.

46. ​​A chimeric antigen receptor (CAR) comprising an antigen-binding fragment according to any one of claims 1-20 and a T-cell activation portion, wherein the T-cell activation portion comprises a transmembrane domain of a T-cell receptor and an intracellular signal transduction domain of a T-cell receptor.

47. The chimeric antigen receptor (CAR) of claim 46, wherein the antigen-binding fragment is scFv.

48. A nucleic acid encoding the CAR according to claim 46.

49. The nucleic acid of claim 48, comprising the polynucleotide of claim 21 or 22, said polynucleotide being operatively linked to a second polynucleotide encoding a transmembrane domain of a T cell receptor and a signal transduction domain of the T cell receptor.

50. A vector comprising the nucleic acid according to claim 48 or 49.

51. An isolated T cell expressing the CAR according to claim 46.

52. Use of the T cell according to claim 51 in the preparation of a medicament for treating CD19-positive B-cell lymphoma in a subject.

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