RU2791445C2 - New anti-cd19 antibodies - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: present invention relates to an anti-CD19 antibody or its antigen-binding fragment, an isolated polynucleotide sequence encoding them, an expression vector containing the isolated polynucleotide, a host cell for obtaining an antibody or its antigen-binding fragment, a method for expressing an antibody or its antigen-binding fragment, an antibody-drug conjugate, a pharmaceutical composition for the treatment of a CD19-bound disease or condition, a method for treating a CD19-related disease or pathological condition, a chimeric antigenic receptor, a nucleic acid sequence molecule, an expression vector containing a nucleic acid sequence, an isolated T cell, a method for stimulating the T cell immune response, a kit for detecting the presence or amount of CD19 in a sample or for diagnosing a CD19-related disease or condition in an individual.

EFFECT: expansion of the range of solutions for treating CD19.

19 cl, 7 ex, 14 dwg, 14 tbl

Description

The field of technology to which the invention belongs

[0001] The present invention generally relates to new anti-CD19 antibodies and their use.

State of the art

[0002] CD19 (differentiation cluster 19) is a structurally distinct cell surface receptor expressed on the surface of B cells, including, but not limited to, all subtypes of B cell lymphoma, from the indolent to the aggressive form, as well as B cell chronic lymphocytic leukemia and non-T cell acute lymphoblastic leukemia, pre-B cells, early B cells (i.e., immature B cells), mature B cells that have undergone terminal differentiation into plasma cells, and malignant B cells. CD19 is expressed in most pre-B-cell acute lymphoblastic leukemias (ALL), non-Hodgkin's lymphomas, B-cell chronic lymphocytic leukemias (CLL), prolymphocytic leukemias, hairy cell leukemias, advanced acute lymphocytic leukemias, and some phenotype-null acute lymphoblastic leukemias (Nadler 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., 1984. Blood, 63: 1424-1433 (1984), Scheuermann, Leuk Lymphoma, 18: 385-397 (1995)). Expression of CD19 on plasma cells further suggests that it may be expressed on differentiated B cell tumors such as multiple myeloma, plasmacytomas, Waldenström macroglobulinemia tumors (Grossbard et al., Br. J. Haematol., 102: 509-15 (1998 ); Treon et al., Semin. Oncol., 30: 248-52 (2003)). CD19 has also been one of many proposed targets for immunotherapy. Unlike CD20 (another B cell surface receptor), CD19 is thought to be expressed at higher levels and internalized by cells when bound to an anti-CD19 antibody.

[0003] There remains a need for new anti-CD19 antibodies, in particular those with favorable internalization capacity and high binding affinity.

The essence of the invention

[0004] Throughout this disclosure, the articles "a", "an" and "the" are used here to denote one or more than one (i.e., at least one) grammatical object of the article. For example, "antibody" means one antibody or multiple antibodies.

[0005] The present invention relates to novel anti-CD19 monoclonal antibodies, their amino acid and nucleotide sequences, and their uses.

[0006] In one aspect, the present invention provides isolated monoclonal 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) kappa light chain CDR sequences selected from the group consisting of SEQ ID NOs: 4, 5, 6, 10, 11, 12, 16, 17, 18, 40, 41, 42, 137, 138 and 139.

[0007] In some embodiments, the antibodies or antigen-binding fragments thereof comprise 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%, 99%) sequence identity with SEQ ID NOs: 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 or 141. In some embodiments, the antibodies or antigen-binding fragments thereof comprise 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%, 99%) sequence identity with SEQ ID NOs: 4, 5, 6, 10, 11, 12, 16, 17, 18, 40, 41, 42, 137, 138 or 139.

[0008] In some embodiments, the antibodies or antigen-binding fragments thereof comprise a heavy chain variable region selected from the group consisting of:

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;

b) a 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;

c) a 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;

d) a 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;

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;

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;

g) a 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;

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;

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;

j) a 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;

k) a 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;

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;

m) a 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

n) a 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.

[0009] In some embodiments, the antibodies or antigen-binding fragments thereof comprise a kappa light chain variable region selected from the group consisting of:

a) a kappa 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;

b) a kappa 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;

c) a kappa light chain variable region comprising 1, 2 or 3 CDR sequences selected from SEQ ID NO: 16, SEQ ID NO: 17 and/or SEQ ID NO: 18;

d) a kappa 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

e) a kappa light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 137, SEQ ID NO: 138, and SEQ ID NO: 139.

[00010] In some embodiments, the antibodies or antigen-binding fragments thereof comprise:

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 kappa 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;

b) a 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 a kappa 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;

c) a 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 a kappa light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18;

d) a 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 a kappa 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;

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 kappa 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;

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 kappa 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;

g) a 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 a kappa 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;

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 kappa 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;

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 kappa 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;

j) a 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 a kappa 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;

k) a 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 a kappa 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;

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 kappa light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 137, SEQ ID NO: 138, and SEQ ID NO: 139;

m) a 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 a kappa 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; or

n) a 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 a kappa light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18.

[00011] In some embodiments, the antibodies or antigen-binding fragments thereof comprise: a heavy chain CDR3 sequence 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.

[00012] In some embodiments, the antibodies or antigen-binding fragments thereof comprise:

a) a heavy chain CDR1 sequence 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;

b) a heavy chain CDR2 sequence 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: 140 and SEQ ID NO: 141; And

c) a heavy chain CDR3 sequence 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.

[00013] In some embodiments, the antibodies or antigen-binding fragments thereof comprise:

a) a light chain CDR1 sequence selected from SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 16, SEQ ID NO: 40, and SEQ ID NO: 137;

b) a light chain CDR2 sequence selected from SEQ ID NO: 5, SEQ ID NO: 11, SEQ ID NO: 17, SEQ ID NO: 41 and SEQ ID NO: 138; And

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.

[00014] In some embodiments, the antibodies or antigen-binding fragments thereof comprise:

a) a heavy chain CDR1 sequence 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;

b) a heavy chain CDR2 sequence 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: 140 and SEQ ID NO: 141;

c) a heavy chain CDR3 sequence 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;

d) a light chain CDR1 sequence selected from SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 16, SEQ ID NO: 40, and SEQ ID NO: 137;

e) a light chain CDR2 sequence selected from SEQ ID NO: 5, SEQ ID NO: 11, SEQ ID NO: 17, SEQ ID NO: 41, and SEQ ID NO: 138; And

f) 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.

[00015] In some embodiments, the antibodies or antigen-binding fragments thereof further comprise one or more (e.g., 1, 2, 3, or 4) heavy chain framework region (FR) sequences selected from the group consisting of SEQ ID NOs: 54, 55 , 56, 57, 70, 71, 72, 73, 86, 87, 88, and 89, and/or one or more kappa light chain framework region (FR) sequences (e.g., 1, 2, 3, or 4) selected from SEQ ID NOs: 58, 59, 60, 61, 74, 75, 76, 77, 90, 91, 92 and 93.

[00016] In some embodiments, the antibodies or antigen-binding fragments thereof further comprise a heavy chain FR1 sequence selected from SEQ ID NOs: 54, 70, and 86; a heavy chain FR2 sequence selected from SEQ ID NOs: 55, 71 and 87; a heavy chain FR3 sequence selected from SEQ ID NOs: 56, 72 and 88; and/or a heavy chain FR4 sequence selected from SEQ ID NOs: 57, 73 and 89.

[00017] In some embodiments, the antibodies or antigen-binding fragments thereof further comprise a light chain FR1 sequence selected from SEQ ID NOs: 58, 74, and 90; a light chain FR2 sequence selected from SEQ ID NOs: 59, 75 and 91; a light chain FR3 sequence selected from SEQ ID NOs: 60, 76 and 92; and/or a light chain FR4 sequence selected from SEQ ID NOs: 61, 77 and 93.

[00018] In some embodiments, the antibodies or antigen-binding fragments thereof comprise 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 their 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.

[00019] In some embodiments, the antibodies or antigen-binding fragments thereof comprise 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 their 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.

[00020] In some embodiments, the antibodies or antigen-binding fragments thereof comprise all or part of a heavy chain variable region sequence selected from the group consisting of SEQ ID NOs: 94, 98, 102, 106, 108, 110, 112, 114, 116, 118, 122, 124, 128 and 132; and/or all or part of a light chain variable region sequence selected from the group consisting of SEQ ID NOS: 100, 104, 120, 126, 130, and 134. In one embodiment, the antibody, or antigen-binding fragments thereof, is a single domain antibody that consists of all or part of the heavy chain variable region selected from the group consisting of SEQ ID NOS: 94, 98, 102, 106, 108, 110, 112, 114, 116, 118, 122, 124, 128 and 132.

[00021] In certain embodiments, the antibodies or antigen-binding fragments thereof comprise:

a) a heavy chain variable region comprising SEQ ID NO: 94 and a kappa light chain variable region comprising SEQ ID NO: 96;

b) a heavy chain variable region comprising SEQ ID NO: 98 and a kappa light chain variable region comprising SEQ ID NO: 100;

c) a heavy chain variable region comprising SEQ ID NO: 102 and a kappa light chain variable region comprising SEQ ID NO: 104;

d) a heavy chain variable region comprising SEQ ID NO: 106 and a kappa light chain variable region comprising SEQ ID NO: 96;

e) a heavy chain variable region comprising SEQ ID NO: 108 and a kappa light chain variable region comprising SEQ ID NO: 96;

f) a heavy chain variable region comprising SEQ ID NO: 110 and a kappa light chain variable region comprising SEQ ID NO: 96;

g) a heavy chain variable region comprising SEQ ID NO: 112 and a kappa light chain variable region comprising SEQ ID NO: 96;

h) a heavy chain variable region comprising SEQ ID NO: 114 and a kappa light chain variable region comprising SEQ ID NO: 96;

i) a heavy chain variable region comprising SEQ ID NO: 116 and a kappa light chain variable region comprising SEQ ID NO: 96;

j) a heavy chain variable region comprising SEQ ID NO: 118 and a kappa light chain variable region comprising SEQ ID NO: 120;

k) a heavy chain variable region comprising SEQ ID NO: 122 and a kappa light chain variable region comprising SEQ ID NO: 96;

l) a heavy chain variable region comprising SEQ ID NO: 124 and a kappa light chain variable region comprising SEQ ID NO: 126;

m) a heavy chain variable region comprising SEQ ID NO: 128 and a kappa light chain variable region comprising SEQ ID NO: 130; or

n) a heavy chain variable region comprising SEQ ID NO: 132 and a kappa light chain variable region comprising SEQ ID NO: 134.

[00022] In some embodiments, the antibody or antigen-binding fragment thereof further comprises one or more amino acid residue substitutions but retains a specific binding affinity for CD19.

[00023] In some embodiments, the substitution takes place in one or more CDR sequences and/or in one or more FR sequences, in one or both variable region sequences and/or in the Fc region. In some embodiments, at least one (or all) substitution(s) in the CDR sequences, FR sequences, variable region sequences, or Fc region comprises a conservative substitution.

[00024] In some embodiments, the antibody or antigen-binding fragment thereof contains no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue substitutions in one or more CDR sequences selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 136, 137, 138 139, 140 and 141 .

[00025] In some embodiments, the antibody or antigen-binding fragment thereof contains no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue substitutions in one or more FR sequences selected from SEQ ID NO: 54, 55, 56, 57, 58, 59, 60, 61, 70, 71, 72, 73, 74, 75, 76, 77, 86, 87, 88, 89, 90, 91, 92 and 93. In some embodiments, the antibody or antigen-binding fragment thereof contains a total of no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitutions in the heavy chain variable region CDR and/or FR sequences selected from SEQ ID NOs: 94, 98, 102, 106, 108, 110, 112, 114, 116, 118, 122, 124, 128, and 132. In some embodiments, the antibody or antigen-binding fragment thereof contains no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residue substitutions in all light chain variable region FR sequences selected from SEQ ID NOS: 96, 100, 104, 120, 126, 130 and 134.

[00026] In some embodiments, the substitution imparts one or more desirable properties selected from: a) increasing CD19 binding affinity, b) introducing or removing a glycosylation site, c) introducing a free cysteine residue, d) increasing or decreasing ADCC or CDC, e) increasing the half-life of the blood serum; and f) increasing binding to FcRn.

[00027] In some embodiments, the antibody or antigen-binding fragment thereof further comprises an immunoglobulin constant region. In some embodiments, the antibody or antigen-binding fragment thereof comprises an IgG constant region. In a particular embodiment, the antibody, or antigen-binding fragment thereof, comprises a mouse IgG1, mouse IgG2a, mouse IgG2b, or human IgG1 constant region.

[00028] In some embodiments, the antibodies, or antigen-binding fragments thereof, are a "non-human" (eg, mouse or other rodent antibody) antibody or a humanized antibody.

[00029] In some embodiments, the antibodies or antigen-binding fragments thereof are camelid single domain antibody, 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.

[00030] In some embodiments, the antibodies or antigen-binding fragments thereof are bispecific.

[00031] In some embodiments, the antibodies or antigen-binding fragments thereof are linked to one or more conjugates. In some embodiments, the conjugate comprises a chemotherapeutic agent, a toxin, a radioactive isotope, a lanthanide, a fluorescent label, a fluorescent label, or an enzyme-substrate label. In some embodiments, the implementation of the conjugate is a toxin. In some embodiments, the toxin is a cytotoxin, DNA alkylators, a topoisomerase inhibitor, tubulin binding agents, or other antineoplastic agents. In certain embodiments, the anticancer drug is the cytotoxic agent maytansinoid. In certain embodiments, the toxin is DM1.

[00032] In some embodiments, the antibody or antigen-binding fragment thereof is capable of specifically binding to CD19. In some embodiments, the CD19 is from a mouse, rat, monkey, or human.

[00033] In some embodiments, the antibodies or antigen-binding fragments thereof are capable of specifically binding to human CD19 expressed on the cell at a K _D value of not more than 5×10 ^-9 M, not more than 1×10 ^-9 M, not more than 9×10 ^-10 M, no more than 8×10 ^-10 M, no more than 7×10 ^-10 M, no more than 6×10 ^-10 M, no more than 5×10 ^-10 M, no more than 4×10 ^-10 M, no more than 3×10 ^-10 M, not more than 2×10 ^-10 M, not more than 1×10 ^-10 M, as measured by flow cytometry.

[00034] In some embodiments, the antibodies or antigen-binding fragments thereof are capable of specifically binding to human CD19 expressed on a cell with an EC ₅₀ of not more than 0.04 nM, not more than 0.05 nM, not more than 0.1 nM, not more than 0 .2 nM, not more than 0.3 nM, not more than 0.4 nM, not more than 0.5 nM, not more than 0.5 nM, not more than 0.6 nM, not more than 0.7 nM, not more than 0, 8 nM, not more than 0.9 nM or not more than 1 nM according to flow cytometry.

[00035] In some embodiments, the antibodies, or antigen-binding fragments thereof, are capable of specifically binding to Cynomolgus monkey CD19 expressed on the cell, with an EC ₅₀ of not more than 0.2 nM, not more than 0.5 nM, not more than 0.8 nM, not more than 1 nM, not more than 2 nM or not more than 3 nM according to flow cytometry.

[00036] In some embodiments, the antibodies or antigen-binding fragments thereof are capable of being internalized by a CD19-expressing cell with an EC ₅₀ of not more than 1 pM, not more than 2 pM, not more than 3 pM, not more than 4 pM, not more than 5 pM, not more than 6 pM, max 7 pM, max 8 pM, max 9 pM, max 10 pM, max 11 pM, max 12 pM max 13 pM, max 14 pM, max 15 pM, max 16 pM , not more than 17 pM, not more than 18 pM, not more than 19 pM, not more than 20 pM, not more than 21 pM, not more than 22 pM, not more than 23 pM, not more than 24 pM, not more than 25 pM, not more than 30 pM , 35 pM or less, 40 pM or less, 45 pM or less, or 50 μM or less as determined by Fab-Zap analysis.

[00037] In one aspect, the present disclosure relates to antibodies, or antigen-binding fragments thereof, that compete for the same epitope with W7011-4.155.8, W7011-4.202.9, or W7011-4.225.7.

[00038] In one aspect, the present disclosure further provides pharmaceutical compositions comprising the antibody or antigen-binding fragment provided herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition further comprises a second agent that is capable of enhancing the therapeutic effect of the antibody or antigen-binding fragment thereof and/or is capable of reducing the side effect of the antibody or antigen-binding fragment thereof.

[00039] In one aspect, the present disclosure further provides isolated polynucleotides encoding an antibody or antigen-binding fragment provided herein. In some embodiments, the isolated polynucleotide contains a nucleotide sequence selected from the group consisting of SEQ ID NOs: 95, 99, 103, 107, 109, 111, 113, 115, 117, 119, 123, 125, 129, and 133, and/ or a nucleotide sequence selected from the group consisting of SEQ ID NOs: 97, 101, 105, 121, 127, 131, and 135, or a homologous sequence thereof, having at least 80% (e.g., at least 85% , 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity but encoding the same protein sequence.

[00040] In one aspect, the present disclosure further provides vectors containing said isolated polynucleotide.

[00041] In one aspect, the present disclosure further provides host cells containing said vector.

[00042] In one aspect, the present disclosure further provides methods for expressing an antibody, or antigen-binding fragment thereof, provided herein, comprising culturing said host cell under conditions under which said polynucleotide is expressed.

[00043] In one aspect, the present disclosure further relates to antibody-drug conjugates comprising one or more drug molecules covalently linked to the antibodies or antigen-binding moieties provided herein, either directly or through a linker. In some embodiments, the linker is a hydrazine linker, a disulfide linker, a bifunctional linker, a dipeptide linker, a glucuronide linker, a thioether linker. In certain embodiments, the implementation of the linker is SMCC.

[00044] In some embodiments, at least one drug molecule is attached to a specific site of antibodies or antigen-binding fragments thereof. In some embodiments, the specific site is a cysteine residue. In some embodiments, the drug molecules are a toxin or radioactive isotopes. In some embodiments, the drug molecules are a toxin, optionally a cytotoxin, DNA alkylators, a topoisomerase inhibitor, tubulin binding agents or other antineoplastic agents, optionally a maytansinoid cytotoxic agent, optionally the toxin is DM1.

[00045] In one aspect, the present disclosure further provides pharmaceutical compositions comprising the antibodies or antigen-binding fragments provided herein, or the antibody-drug conjugates provided herein, and a pharmaceutically acceptable carrier.

[00046] In one aspect, the present disclosure further provides methods for treating a CD19-associated disease or condition in a subject, comprising administering a therapeutically effective amount of the antibodies or antigen-binding fragments thereof provided herein, the antibody-drug conjugates provided herein, or the pharmaceutical composition provided herein. here, subject. In certain embodiments, the subject is a human. In some embodiments, administration is via oral, intranasal, intravenous, subcutaneous, sublingual, or intramuscular administration. In certain embodiments, said disease or condition is cancer. In some embodiments, said 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. In some embodiments, said disease or condition is B-cell lymphoma, optionally Hodgkin's lymphoma, or non-Hodgkin's lymphoma, wherein the non-Hodgkin's lymphoma includes: diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, marginal zone B-cell lymphoma (MZL), lymphoma gastric mucosal-associated lymphoid tissue (MALT), small lymphocytic lymphoma (chronic lymphocytic leukemia, CLL), mantle cell lymphoma (MCL), acute lymphoblastic leukemia (ALL), or Waldenstrom's macroglobulinemia (WM).

[00047] In one aspect, the present disclosure further provides methods for modulating CD19 activity in a CD19 expressing cell, comprising exposing the CD19 expressing cell to antibodies or antigen-binding fragments thereof provided herein.

[00048] In one aspect, the present disclosure further provides in vivo or in vitro methods for inducing death of a CD19 expressing cell, comprising contacting a CD19 expressing cell with the antibody drug conjugates provided herein.

[00049] In one aspect, the present disclosure further provides a method for determining the presence or amount of CD19 in a sample, comprising contacting the sample with the antibodies or antigen-binding fragments provided herein and determining the presence or amount of CD19 in the sample.

[00050] In one aspect, the present disclosure further provides methods for diagnosing a CD19-associated disease or condition in a subject, comprising: a) obtaining a sample from the subject; b) contacting the sample with the antibodies or antigen-binding fragments provided herein; c) determining the presence or amount of CD19 in the sample; d) correlation of the presence or amount of CD19 with the disease or condition in the subject.

[00051] In one aspect, the present disclosure further provides the use of the antibodies or antigen-binding fragments thereof provided herein in the manufacture of a medicament for the treatment of a disease or condition in a subject, wherein the treatment comprises: administering a therapeutically effective amount of the antibodies or antigen-binding fragments thereof to the subject.

[00052] In one aspect, the present disclosure further provides the use of the antibodies or antigen-binding fragments provided herein in the manufacture of a diagnostic reagent for detecting a disease or condition associated with CD19.

[00053] In one aspect, the present disclosure relates to chimeric antigen receptors (CARs) comprising an antigen-binding fragment provided herein and a T-cell activation fragment. In some embodiments, the T cell activation fragment comprises a native T cell receptor (TCR) activation fragment. In some embodiments, the T cell activation fragment comprises a TCR transmembrane domain and an intracellular TCR signal transduction domain. In some embodiments, the antigen binding fragment is scFv.

[00054] In one aspect, the present disclosure relates to nucleic acids encoding the CAR provided here. In some embodiments, the nucleic acids comprise a first polynucleotide sequence encoding an antigen-binding fragment of the antibodies provided herein, operably linked to a second polynucleotide sequence encoding a TCR transmembrane domain and a TCR intracellular signal transduction domain.

[00055] In one aspect, the present disclosure relates to vectors containing a nucleic acid sequence encoding a CAR provided here.

[00056] In one aspect, the present disclosure provides isolated T cells that express the CAR provided here.

[00057] In one aspect, the present disclosure relates to methods of stimulating a T cell mediated immune response to a CD19 expressing target in a subject, wherein the method comprises administering to the subject an effective amount of T cells provided herein.

Brief description of the figures

[00058] Figure 1 shows the SDS-PAGE of WBP701-BMK1 and WBP701-BMK2. M: protein marker; lane 1: BMK1, restoring conditions; lane 2: BMK2, reducing conditions; lane 3: BMK1, non-reducing conditions; lane 4: BMK4, non-reducing conditions.

[00059] Figure 2 shows the SDS-PAGE of WBP701-BMK3. M: protein marker; lane 1: BMK3, reducing conditions; lane 2: BMK3, non-reducing conditions.

[00060] FIG. 3A shows histograms of flow cytometry for CD19 expression in the 293F cell line transfected with human CD19 (WBP701.293F.hPro1.FL.A2). The peak on the left represents the negative control signal. The right-shifted peak represents CD19 expression in the analyzed cell line.

[00061] FIG. 3B shows histograms of flow cytometry for CD19 expression in the CD19 transfected human CHO-K1 cell line (WBP701.CHO-K1.hPro1.FL.B4). The peak on the left represents the negative control signal. The right-shifted peak represents CD19 expression in the analyzed cell line.

[00062] FIG. 3C shows histograms of flow cytometry for CD19 expression in the 293 cell line transfected with Cynomolgus monkey CD19 (WBP701.293F.cpro1.FL.C1). The peak on the left represents the negative control signal. The right-shifted peak represents CD19 expression in the analyzed cell line.

[00063] FIG. 3D shows histograms of flow cytometry for CD19 expression in the CHO-K1 cell line transfected with Cynomolgus monkey CD19 (WBP701.CHO-K1.cpro1.FL.C9). The peak on the left represents the negative control signal. The right-shifted peak represents CD19 expression in the analyzed cell line.

[00064] FIG. 4A-4F show FACS binding of selected subclones to Ramos cells.

[00065] FIG. 5A-5C show FACS binding of selected subclones to cells expressing Cynomolgus monkey CD19 (WBP701.CHO-K11.cynoPro1).

[00066] FIG. 6A-6E show Fab-Zap analysis of selected subclones.

[00067] FIG. 7A-7C show FACS binning of candidate antibodies against BMK1, BMK2, and BMK3.

[00068] FIG. 8 shows the Scatchard binding affinity analysis of the WBP7011-4.34.11-z1-m5-IgG1k antibody to Ramos cells by FACS.

[00069] FIG. 9 shows the Scatchard binding affinity analysis of the WBP7011-4.87.6-z1-IgG1K (N-S) antibody to Ramos cells by FACS.

[00070] FIG. 10 shows the Scatchard binding affinity analysis of W7011-4.155.8-z1-uIgG1K antibody to Ramos cells by FACS.

[00071] FIG. 11 shows cytotoxicity analysis of W7011-4.155.8-z1-uIgG1K-DM1 and WBP7011-4.87.6-z1-IgG1K (N-S)-DM1 humanized antibody-drug conjugates on Daudi cells.

[00072] FIG. 12 shows cytotoxicity assay of WBP7011-4.87.6-z1-IgG1K(N-S)-DM1 humanized antibody-drug conjugate on Nalm-6 cells.

[00073] FIG. 13 shows cytotoxicity analysis of W7011-4.155.8-z1-uIgG1K-DM1 and WBP7011-4.87.6-z1-IgG1K(N-S)-DM1 humanized antibody-drug conjugates on WSU-DLCL2 cells.

[00074] FIG. 14 shows the antitumor efficacy of reference antibody (W7011-BMK1-DM1) and antibody (W7011-4.87.6-z1-uIgG1k(NS)-DM1), data represent tumor volumes in different treatment groups of CB17-SCID female lymphoma xenograft mice. Nalm-6. Data points are presented as mean ± SEM. The arrow indicates the day on which the introduction was carried out.

Detailed description of the invention

[00075] The following description of the disclosure is only intended to illustrate various embodiments of the invention. As such, the specific modifications discussed should not be construed as limiting the scope of the disclosure. One skilled in the art will appreciate that various equivalents, changes, and modifications may be made without departing from the scope of the invention, and such equivalent embodiments are to be understood to be included herein. All references cited here, including publications, patents and patent applications, are incorporated here by reference in their entirety.

[00076] Definitions

[00077] As used herein, the term "antibody" includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, polyspecific antibody, or bispecific antibody that binds to a specific antigen. A native intact antibody contains two heavy (H) chains and two light (L) chains. Mammalian heavy chains are classified as alpha, delta, epsilon, gamma and mu, each heavy chain is composed of a variable region (VH) and a first, second and third constant region (CH1, CH2, CH3 respectively); mammalian light chains are classified as λ or κ, where each light chain consists of a variable region (V _L for a λ light chain or V _K for a κ light chain, respectively) and a constant region (C _L for a λ light chain or C _K for a κ light chain respectively). The antibody has a "Y" shape, where the Y base consists of the second and third constant regions of two heavy chains linked together via a disulfide bond. Each Y arm includes a variable region and a first constant region of one heavy chain linked to a variable and constant region of one light chain. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions in both chains typically contain three hypervariable loops called complementarity determining regions (CDRs) (light chain CDRs including LCDR1, LCDR2 and LCDR3, heavy chain CDRs including HCDR1, HCDR2, HCDR3). CDR boundaries for antibodies and antigen binding fragments disclosed herein can be defined or identified according to the Kabat, IMGT, Chothia, or Al-Lazikani numbering systems (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., 196901 (1987), Chothia C. et al., Nature 21-28, 342 (6252): 877-83 (1989), Kabat EA et al., National Institutes of Health, Bethesda, Md. (1991)). The three CDRs lie between flanking regions known as framework regions (FRs), which are more conserved than the CDRs and form a scaffold for maintaining hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding, but exhibit different effector functions. Antibodies are categorized based on the amino acid sequence of their heavy chain constant region. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of alpha, delta, epsilon, gamma, and mu heavy chains, respectively. Some of the major classes of antibodies are further subclassed, such as IgG1 (gamma-1 heavy chain), IgG2 (gamma-2 heavy chain), IgG3 (gamma-3 heavy chain), IgG4 (gamma-4 heavy chain), IgA1 (gamma-4 heavy chain). α1 chain) or IgA2 (α2 heavy chain).

[00078] As used here, 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 thereof, is bivalent.

[00079] As used here, the term "bespecifically" antibody refers to an artificial antibody that has fragments derived from two different monoclonal antibodies, and that is able to bind to two different epitopes. The two epitopes may be on the same antigen or may be on two different antigens.

[00080] As used herein, the term "antigen-binding site" refers to an antibody site formed from a portion of an antibody containing 1, 2, or 3 CDRs, or any other antibody site that binds to an antigen but does not contain an intact native antibody structure. Examples of an antigen-binding fragment include, but are not limited to, diabody, Fab, Fab', F(ab') ₂ fragments, Fv fragment, disulfide-stabilized Fv(dsFv), (dsFv) ₂ fragment, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabody (ds diabody), single chain antibody molecule (scFv), scFv dimer (bivalent diabody), bispecific antibody, multispecific antibody, camelid single domain antibody, nanobody, domain antibody, and bivalent domain antibody. The antigen-binding fragment is capable of binding to the same antigen that the parent antibody binds to.

[00081] "Fab" in relation to an antibody refers to that portion of an antibody consisting of a single light chain (both variable and constant region) linked to a variable region and a first constant region of a single heavy chain by a disulfide bond.

[00082] "Fab" refers to a Fab fragment that includes a portion of the hinge region.

[00083] "F(ab') ₂ " refers to a Fab' dimer. "Fv" in relation to an antibody refers to the smallest fragment of an antibody that contains a complete antigen-binding site. An Fv fragment consists of a single light chain variable region linked to a single heavy chain variable region.

[00084] "dsFv" refers to a disulfide-stabilized Fv fragment in which the bond between the variable region of one light chain and the variable region of one heavy chain is a disulfide bond. In some embodiments, "(dsFv) ₂ " or "(dsFv-dsFv')" contains three peptide chains: two V _H groups linked by a peptide linker (e.g., a long flexible linker) and linked to two V _L groups, respectively, through disulfide bridges. In some embodiments, the implementation of dsFv-dsFv' is bispecific, in which each disulfide paired heavy and light chain has a different antigenic specificity.

[00085] "Single chain Fv antibody" or "scFv" refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region connected to each other directly or through a peptide linker sequence (Huston J.S. et al., Proc. Natl Acad. Sci. USA, 85: 5879 (1988)).

[00086] The term "Fc" in relation to an antibody refers to that portion of an antibody that consists 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 a disulfide bond. The Fc region of an antibody is responsible for various effector functions such as antibody dependent cell mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) but does not function in antigen binding.

[00087] "Single chain Fv-Fc antibody" or "scFv-Fc" refers to an engineered antibody consisting of scFv fused to the Fc region of an antibody.

[00088] The term "camel single-domain antibody", "heavy chain antibody" or "HCAb" refers to an antibody that contains two VH domains and does 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. 6005079). Heavy chain antibodies were originally derived from members of the Camelidae family (camels, dromedaries and llamas). Despite the absence of light chains, camelid antibodies have an authentic antigen-binding repertoire (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 V.K. et al. Immunology. May; 109(1):93-101 (2003)). The variable domain of a heavy chain antibody (VHH domain) is the smallest known antigen-binding unit generated by adaptive immune responses (Koch-Nolte F. et al., FASEB J. Nov; 21 (13): 3490-8. Epub 2007 15 Gun . 2007).

[00089] "Nanobody" refers to an antibody fragment that consists of a heavy chain antibody VHH domain and two constant domains, CH2 and CH3.

[00090] "Diabodies" or "dAbs" include small fragments of antibodies with two antigen-binding sites, where the fragments contain a V _H region associated with a V _L region in one polypeptide chain (V _H -V _L or V _L -V _H ) (see eg Holliger P. et al., Proc Natl Acad Sci USA Jul 15;90(14):6444-8 (1993);EP404097;WO93/11161). By using a linker that is too short to allow pairing between two domains on the same strand, the domains are forced to pair with complementary domains on the other strand, thereby creating two antigen-binding sites. Antigen binding sites may target the same or different antigens (or epitopes). In some embodiments, a "bispecific ds diabody" is an antibody directed to two different antigens (or epitopes). In some embodiments, an "scFv dimer" is a bivalent diabody or bivalent scFv (BsFv) containing a V _H -V _L (linked by a peptide linker) dimerized with another V _H -V _L group such that the V _H of one group coordinates with the V _L the other group and form two binding sites that can target the same antigens (or epitopes) or different antigens (or epitopes). In still other embodiments, the "scFv dimer" is a bispecific diabody comprising V _H1 -V _L2 (linked by a peptide linker) linked to V _L1 -V _H2 (also linked by a peptide linker) such that V _H1 and V _L1 and V _H2 and V _L2 coordinate, and each coordinated pair has a different antigenic specificity.

[00091] "Domain antibody" refers to an antibody fragment containing only a heavy chain variable region or a light chain variable region. In some cases, two or more V _H domains are covalently joined by a peptide linker to form a bivalent or multivalent domain antibody. The two V _H domains of a bivalent domain antibody may target the same or different antigens.

[00092] As used herein, the term "chimeric" means an antibody or antigen-binding fragment having a portion of the heavy and/or light chain derived from one species and the remainder of the heavy and/or light chain derived from another species. In an illustrative example, a chimeric antibody may comprise a human-derived constant region and a non-human variable region, such as 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.

[00093] As used herein, the term "humanized" means that the antibody or antigen-binding fragment contains CDRs derived from non-human animals, FR regions derived from a human, and, when applicable, constant regions derived from a human.

[00094] As used here, the term "CD19" refers to the protein cluster of differentiation 19, which is an antigenic determinant defined in leukemic progenitor cells. Human and mouse CD19 amino acid and nucleic acid sequences can be found in a public database such as GenBank, UniProt and Swiss-Prot. For example, the amino acid sequence of human CD19 can be found under UniProt/Swiss-Prot accession number P15391, and the nucleotide sequence encoding human CD19 can be found under accession number NM_001178098. As used herein, 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 tumors of the B cell lineage, including, for example, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin's lymphoma. It is also an early marker for B cell progenitors. See, for example, Nicholson et al. Mol. Immun. 34 (16-17): 1157-1165 (1997). In one aspect, the CD19 protein is expressed on the tumor cell.

[00095] As used here, the term "specific binding" or "specifically binds" refers to a non-random binding reaction between two molecules, such as an antibody and an antigen. In certain embodiments, the antibodies or antigen-binding fragments provided herein specifically bind to human CD19 with a binding affinity (K _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 M, ≤10 ^-8 M, ≤5×10 ^-9 M, ≤4×10 ^-9 M, ≤3×10 ^-9 M , ≤2×10 ^-9 M or ≤10 ^-9 M. As used here, K _D refers to the ratio of dissociation rate to association rate (k _off /k _on ), which can be determined using any conventional method known in the art. techniques including, but not limited to, surface plasmon resonance method, microscale thermophoresis method, HPLC-MS method, and flow cytometry method (such as FACS) In some embodiments, the K _D value can be appropriately determined using a flow cytometry method.

[00096] The ability to "block binding" or "compete for the same epitope" as used herein refers to the ability of an antibody or antigen-binding fragment to inhibit a binding interaction between two molecules (e.g., human CD19 and an anti-CD19 antibody) in any detectable degree. In some embodiments, an antibody or antigen binding fragment that blocks binding between two molecules inhibits the binding interaction between the two molecules by at least 50%, at least 60%, at least 70%, at least , 80%, at least 85%, or at least 90%. In some embodiments, this inhibition may be greater than 60%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, or greater than 90%.

[00097] As used here, the term "epitope" refers to a specific group of atoms or amino acids on an antigen to which an antibody binds. Two antibodies can bind to the same or closely related epitope within an antigen if they exhibit competitive binding to the antigen. For example, if an antibody or antigen-binding fragment blocks 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% %, when it can be considered that at least 80%, at least 85% or at least 90%, then the antibody or its antigen-binding fragment can be considered as binding to the same/closely related epitope, as the reference antibody.

[00098] Those skilled in the art will appreciate that without undue experimentation, it can be determined whether a human monoclonal antibody binds to the same epitope as an antibody of the present invention (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, WBP7011_4.108.3, WBP7011_4.191.6, WBP7011_4.194.10, WBP7011_4.231.5, и гуманизированные антитела W7011- 4.34.11-z1-m5, W7011-4.87.6-z1(N-S), and W7011-4.155.8-z1-P15) by determining how much the first antibody prevents the second antibody from binding to the CD19 antigen polypeptide. If a test antibody competes with an antibody of the present invention, as indicated by reduced binding of the antibody of the present invention to a CD19 antigen polypeptide, then the two antibodies bind to the same or closely related epitope. Or, if the binding of the test antibody to the CD19 antigen polypeptide was inhibited by the antibody of the present invention, then the two antibodies bind to the same or closely related epitope.

[00099] Various symbols used in the names of antibodies provided herein have a different designation: "mIgG2" refers to an antibody with a mouse constant region of the IgG2 isotype; "UIgG1" refers to an antibody with a human constant region of the IgG1 isotype; "K" or "L" refers to an antibody using a kappa or lambda light chain.

[000100] A "conservative substitution" with respect to an amino acid sequence refers to the replacement of an amino acid residue with another amino acid residue having a side chain with similar physicochemical properties. For example, conservative substitutions can be made among amino acid residues with hydrophobic side chains (eg, Met, Ala, Val, Leu, and Ile), among residues with neutral hydrophilic side chains (eg, Cys, Ser, Thr, Asn, and Gln), among residues with acidic side chains (eg Asp, Glu), among amino acids with basic side chains (eg His, Lys and Arg) or among residues with aromatic side chains (eg Trp, Tyr and Phe). As is known in the art, a conservative substitution usually does not cause a significant change in the conformational structure of the protein and, therefore, may preserve the biological activity of the protein.

[000101] As used herein, the terms "homolog" and "homologous" are used interchangeably and refer to nucleic acid sequences (or its complementary strand) or amino acid sequences that share at least 80% sequence identity (e.g., at least , 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) with other sequences with optimal alignment.

[000102] "Percent (%) sequence identity" with respect to an amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleotide) residues in a candidate sequence that are identical to amino acid (or nucleotide) residues in a reference sequence, after alignment sequences and, if necessary, the introduction of gaps, to achieve the maximum number of identical amino acids (or nucleotides). A conservative substitution of amino acid residues may or may not be considered identical residues. Alignment to determine percent amino acid (or nucleotide) sequence identity can be done, for example, using publicly available tools such as BLASTN, BLASTp (available from the US National Center for Biotechnology Information (NCBI) website, see also Altschul S.F. et al, J. Mol. Biol., 215:403-410 (1990); Stephen F. et al, Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (available from the European Bioinformatics Institute website, see also Higgins D. G. et al, Methods in Enzymology, 266:383-402 (1996) Larkin M. A. et al., Bioinformatics (Oxford, England), 23(21): 2947-8 (2007)), and c using ALIGN or Megalign (DNASTAR) software. Those skilled in the art may use the default parameters provided by the tool, or may adjust the parameters in accordance with the alignment, such as selecting an appropriate algorithm.

[000103] As used here, "effector functions" refers to biological activities associated with the binding of the Fc region of an antibody to its effectors, such as the C1 complex and the Fc receptor. Exemplary effector functions include: complement-dependent cytotoxicity (CDC) induced by the interaction of antibodies and C1q on the C1 complex; antibody-dependent cell-mediated cytotoxicity (ADCC) induced by binding of the Fc region of an antibody to an Fc receptor on an effector cell; and phagocytosis.

[000104] As used herein, an antibody that is "internalized" or "capable of internalizing" is an antibody that is taken up by a cell upon binding to its antigen on the cell surface. In some embodiments, the antibody and fragments provided herein can be internalized, at least to some extent, by cells that express CD19 on their surfaces. For example, in some embodiments, an anti-CD19 antibody provided herein can be internalized by a B-cell lymphoma cell upon binding to CD19 expressed on the cell surface. Internalization may occur in vitro or in vivo. For therapeutic use, internalization may occur in vivo. How much an antibody is internalized when bound to a mammalian cell can be determined using various assays, including those described in the examples below (eg, the Fab-Zap method). Methods for determining whether an antibody is internalized by a cell are also described in US Pat. No. 7,619,068, which is incorporated herein by reference in its entirety. In some embodiments, the antibodies and their antigen-binding fragments capable of internalization can be linked or conjugated with antitumor agents, such as cytotoxic molecules, that induce cell death upon internalization. Depending on the activity of the antibody or antibody conjugate, in some cases uptake of a single antibody molecule by a cell is sufficient to induce the death of the target cell to which the antibody binds. For example, some toxins are highly effective in inducing death such that internalization of a single antibody-conjugated toxin molecule is sufficient to kill a tumor cell.

[000105] As used herein, the terms "treating" or "treating" a condition include preventing or alleviating the condition, slowing the onset or rate of progress of the condition, reducing the risk of developing the condition, preventing or delaying the development of symptoms associated with the condition, reducing or stopping symptoms. associated with the condition, generating a complete or partial regression of the condition, treating the condition, or some combination thereof.

[000106] The "isolated" substance has been altered by human hand compared to the natural state. If an "isolated" composition or substance occurs naturally, then it has been altered or removed from its original environment, or both. For example, a polynucleotide or polypeptide naturally present in the body of a living animal is not "isolated", but the same polynucleotide or polypeptide is "isolated" if it has been sufficiently separated from coexisting substances in its natural state to be in an essentially pure state. . The terms isolated "nucleic acid" or "polynucleotide" are used interchangeably and refer to the sequence of an isolated nucleic acid molecule. In some embodiments, an "isolated antibody or antigen-binding fragment thereof" refers to an antibody or antigen-binding fragments having 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%, 99%, as determined by electrophoretic methods ( such as SDS-PAGE, isoelectric focusing, capillary electrophoresis) or chromatographic methods (such as ion exchange chromatography or reverse phase HPLC).

[000107] As used here, the term "vector" refers to a carrier into which a polynucleotide encoding a protein can be operably inserted to allow expression of that protein. The vector can be used to transform, transduce, or transfect a host cell to express the genetic element it carries in the host cell. Examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or artificial chromosome with P1 plasmid elements (PAC), bacteriophages such as lambda phage or M13 phage, and viruses. animals. Groups of animal viruses used as vectors include retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (eg, herpes simplex virus), poxvirus, baculovirus, papillomavirus, and papovavirus (eg, SV40). The vector may contain a variety of expression control elements, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. In addition, the vector may contain an origin of replication. The vector may also include substances that facilitate its entry into the cell, including, but not limited to, a viral particle, a liposome, or a protein coat. The vector may be an expression vector or a cloning vector. The present disclosure provides vectors (e.g., expression vectors) comprising a nucleic acid sequence provided herein encoding an antibody or an antigen-binding fragment thereof, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selectable marker. Examples of vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpesvirus (e.g., herpes simplex virus), poxvirus, baculovirus, papillomavirus, papovavirus (e.g., SV40), lambda phage and M13 phage, pcDNA3 plasmids. 3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-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.

[000108] The expression "host cell" in the context of the present description refers to a cell into which an exogenous polynucleotide and/or vector has been introduced.

[000109] "CD19 associated disease or condition" as used herein refers to any disease or condition caused by, exacerbated by, or otherwise associated with increased or decreased CD19 expression or activities. In some embodiments, the CD19-associated condition is B-cell lymphoma, optionally Hodgkin's lymphoma, or non-Hodgkin's lymphoma, wherein the non-Hodgkin's lymphoma includes: diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, marginal zone B-cell lymphoma (MZL ), gastric mucosal-associated lymphoma (MALT), small lymphocytic lymphoma (chronic lymphocytic leukemia, CLL), mantle cell lymphoma (MCL), acute lymphoblastic leukemia (ALL), or Waldenstrom's macroglobulinemia.

[000110] "Cancer" as used herein refers to any medical condition characterized by the growth or neoplasm of malignant cells, abnormal proliferation, infiltration, or metastasis, and includes both solid tumors and non-solid cancers (hematologic malignancies) such as leukemia . As used here, the term "solid tumor" refers to a solid mass of tumor and/or malignant cells. Examples of cancers 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 breast basal cell carcinoma, ductal carcinoma, and lobular carcinoma). breast carcinoma), pancreatic cancer, stomach cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymus cancer, melanoma, myelomas, mycosis fungoides, Merkel cell tumor, hepatocellular carcinoma (HCC), fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, lymphoid neoplasms, basal cell carcinoma, adenocarcinoma, carcinoma sweat gland, medullary thyroid carcinoma, papillary carcinoma thyroid carcinoma, sebaceous pheochromocytomas, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, classic Hodgkin's lymphoma (CHL), primary B- mediastinal large cell lymphoma, T-cell / histiocyte-rich B-cell lymphoma, acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia, chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, polycythemia vera, mast cell tumors, EBV- positive and EBV-negative PTLD, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, marginal zone B-cell lymphoma (MZL), gastric mucosa-associated lymphoid tissue lymphoma (MALT), small lymphocytic lymphoma (chronic lymphocytic leukemia, CLL ), mantle cell lymphoma (MCL), acute lymphoma blast leukemia (ALL), plasmablastic lymphoma, extranodal NK/T cell lymphoma, nasopharyngeal lymphoma, HHV8-associated primary effusion lymphoma, non-Hodgkin's lymphoma, multiple myeloma, Waldenström's macroglobulinemia (WM), heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia , primary CNS lymphoma, spinal tumor, brainstem glioma, astrocytoma, medulloblastoma, craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma.

[000111] The term "pharmaceutically acceptable" indicates that said carrier, solvent, diluent, excipient(s) and/or salt are generally chemically and/or physically compatible with the other ingredients of the formulation and physiologically compatible with its recipient. .

[000112] Anti-CD19 antibodies

[000113] The present disclosure provides anti-CD19 antibodies and antigen-binding fragments thereof, comprising one or more (e.g., 1, 2, 3, 4, 5, or 6) anti-CD19 antibody CDR sequences selected from 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(N-S), and W7011-4.155.8-z1-P15. In the present description, the term "WBP7011" in relation to the names of antibodies is used interchangeably with "W7011". For example, the WBP7011-4.34.11 antibody is also referred to as W7011-4.34.11, and these names refer to the same antibody.

[000114] "WBP7011-4.34.11" as used herein refers to a mouse monoclonal antibody having a heavy chain variable region of SEQ ID NO: 94 and a kappa light chain variable region of SEQ ID NO: 96.

[000115] "WBP7011-4.87.6" as used herein refers to a murine monoclonal antibody having a heavy chain variable region of SEQ ID NO: 98 and a kappa light chain variable region of SEQ ID NO: 100.

[000116] "WBP7011_4.155.8" as used herein refers to a mouse monoclonal antibody having a heavy chain variable region of SEQ ID NO: 102 and a kappa light chain variable region of SEQ ID NO: 104.

[000117] "WBP7011_4.56.1" as used herein refers to a murine monoclonal antibody having a heavy chain variable region of SEQ ID NO: 106 and a kappa light chain variable region of SEQ ID NO: 96.

[000118] "WBP7011-4.15.10" as used herein refers to a mouse monoclonal antibody having a heavy chain variable region of SEQ ID NO: 108 and a kappa light chain variable region of SEQ ID NO: 96.

[000119] "WBP7011-4.100.1" as used herein refers to a murine monoclonal antibody having a heavy chain variable region of SEQ ID NO: 110 and a kappa light chain variable region of SEQ ID NO: 96.

[000120] "WBP7011-4.106.3" as used herein refers to a mouse monoclonal antibody having a heavy chain variable region of SEQ ID NO: 112 and a kappa light chain variable region of SEQ ID NO: 96.

[000121] "WBP7011_4.108.3" as used herein refers to a mouse monoclonal antibody having a heavy chain variable region of SEQ ID NO: 114 and a kappa light chain variable region of SEQ ID NO: 96.

[000122] "WBP7011_4.191.6" as used herein refers to a mouse monoclonal antibody having a heavy chain variable region of SEQ ID NO: 116 and a kappa light chain variable region of SEQ ID NO: 96.

[000123] "WBP7011_4.194.10" as used herein refers to a mouse monoclonal antibody having a heavy chain variable region of SEQ ID NO: 118 and a kappa light chain variable region of SEQ ID NO: 120.

[000124] "WBP7011_4.231.5" as used herein refers to a murine monoclonal antibody having a heavy chain variable region of SEQ ID NO: 122 and a kappa light chain variable region of SEQ ID NO: 96.

[000125] "W7011-4.34.11-z1-m5" as used herein refers to a humanized antibody based on WBP3311_2.166.48 that contains the heavy chain variable region of SEQ ID NO: 124 and the kappa light chain variable region of SEQ ID NO : 126.

[000126] "W7011-4.87.6-z1 (NS)" as used herein refers to a humanized antibody based on WBP3311_2.166.48 that contains the heavy chain variable region of SEQ ID NO 128: and the kappa light chain variable region of SEQ ID NO: 130.

[000127] "W7011-4.155.8-z1-P15" as used herein refers to a humanized antibody based on WBP3311_2.166.48 that contains the heavy chain variable region of SEQ ID NO: 132 and the kappa light chain variable region of SEQ ID NO : 134.

[000128] Table 1 shows the CDR sequences of these 11 mouse anti-CD19 antibodies and three humanized antibodies W7011-4.34.11-z1-m5, W7011-4.87.6-z1 (NS), and W7011-4.155.8-z1-P15. The heavy chain and light chain variable region sequences are also shown below.

[000129]

Table 1 CDR1 CDR2 CDR3 WBP7011-4.34.11 vh SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NO: 3 GYTFTNYVIH YFNPYNDGTEYNEKFKA GPYYYGSSPFDY WBP7011-4.34.11 VK SEQ ID NO: 4 SEQ ID NO: 5 SEQ ID NO: 6 RSSQSLENSNGNTYLN RVSNRFS LQVTHVPYT WBP7011-4.87.6 vh SEQ ID NO: 7 SEQ ID NO: 8 SEQ ID NO: 9 GYAFSTYWMN QIYPGDDDTKYNGKFKG RYFRYDYWYSDV WBP7011-4.87.6 VK SEQ ID NO: 10 SEQ ID NO: 11 SEQ ID NO: 12 RASQDISNYLN YTSRLHS HQGNTLPLT WBP7011_4.155.8 vh SEQ ID NO: 13 SEQ ID NO: 14 SEQ ID NO: 15 GYAFTSYNMY YIDPYNGDTTYNQKFKG TAYAMDY WBP7011_4.155.8 VK SEQ ID NO: 16 SEQ ID NO: 17 SEQ ID NO: 18 SASSTVNYMH STSNLAS HQWSSYPYT WBP7011_4.56.1 vh SEQ ID NO: 19 SEQ ID NO: 20 SEQ ID NO: 21 GYTFTNYVIH YINPYNDGTEYNEKFKG GPYYYGGSPFDY WBP7011_4.56.1 VK SEQ ID NO: 4 SEQ ID NO: 5 SEQ ID NO: 6 RSSQSLENSNGNTYLN RVSNRFS LQVTHVPYT WBP7011-4.15.10 vh SEQ ID NO: 22 SEQ ID NO: 23 SEQ ID NO: 24 GYTFTSYVMH YINPYNDGTEYHEKFKG GPYYYGGSPFDF WBP7011-4.15.10 VK SEQ ID NO: 4 SEQ ID NO: 5 SEQ ID NO: 6 RSSQSLENSNGNTYLN RVSNRFS LQVTHVPYT WBP7011-4.100.1 vh SEQ ID NO: 25 SEQ ID NO: 26 SEQ ID NO: 27 GYTFTSYVIH YINPYNDGAEYTEKFKG GPYYYGGSPFDY WBP7011-4.100.1 VK SEQ ID NO: 4 SEQ ID NO: 5 SEQ ID NO: 6 RSSQSLENSNGNTYLN RVSNRFS LQVTHVPYT WBP7011-4.106.3 vh SEQ ID NO: 28 SEQ ID NO: 29 SEQ ID NO: 30 GYTFSSYVIH YINPYNDGAEYAEKFKG GPYYYGGSPFDY WBP7011-4.106.3 VK SEQ ID NO: 4 SEQ ID NO: 5 SEQ ID NO: 6 RSSQSLENSNGNTYLN RVSNRFS LQVTHVPYT WBP7011_4.108.3 vh SEQ ID NO: 31 SEQ ID NO: 32 SEQ ID NO: 33 GYTFTSYVIH YINPYNDGAEYNEKFKG GPYYYGSSPFDY WBP7011_4.108.3 VK SEQ ID NO: 4 SEQ ID NO: 5 SEQ ID NO: 6 RSSQSLENSNGNTYLN RVSNRFS LQVTHVPYT WBP7011_4.191.6 vh SEQ ID NO: 34 SEQ ID NO: 35 SEQ ID NO: 36 GYTFTDYVIH YINPYNDGSEYSEKFKG GPYYYGGSPFDY WBP7011_4.191.6 VK SEQ ID NO: 4 SEQ ID NO: 5 SEQ ID NO: 6 RSSQSLENSNGNTYLN RVSNRFS LQVTHVPYT WBP7011_4.194.10 vh SEQ ID NO: 37 SEQ ID NO: 38 SEQ ID NO: 39 GYTFTSYVMH YINPYNDGTKYNEKFKG GPYYYGSSPFDY WBP7011_4.194.10 VK SEQ ID NO: 40 SEQ ID NO: 41 SEQ ID NO: 42 RSSQTLENSNGNTYLN RVSNRFS LQVTHVPYT WBP7011_4.231.5 vh SEQ ID NO: 43 SEQ ID NO: 44 SEQ ID NO: 45 GYTFTSYVMH YINPYNDGTQYNEKFKG GPYYYSPSPFDY WBP7011_4.231.5 VK SEQ ID NO: 4 SEQ ID NO: 5 SEQ ID NO: 6 RSSQSLENSNGNTYLN RVSNRFS LQVTHVPYT W7011-4.34.11-z1-m5 vh SEQ ID NO: 136 SEQ ID NO: 2 SEQ ID NO: 3 GYTFTDYVIH YFNPYNDGTEYNEKFKA GPYYYGSSPFDY W7011-4.34.11-z1-m5 VK SEQ ID NO: 137 SEQ ID NO: 138 SEQ ID NO: 139 RSSQSLENSNHNTYIN RVSKRFS HQVTHVPYT W7011-4.87.6-z1(N-S) vh SEQ ID NO: 7 SEQ ID NO: 140 SEQ ID NO: 9 GYAFSTYWMN QIYPGDDDTKYSGKFKG RYFRYDYWYSDV W7011-4.87.6-z1(N-S) VK SEQ ID NO: 10 SEQ ID NO: 11 SEQ ID NO: 12 RASQDISNYLN YTSRLHS HQGNTLPLT W7011-4.155.8-z1-P15 vh SEQ ID NO: 13 SEQ ID NO: 141 SEQ ID NO: 15 GYAFTSYNMY YIDPYNADTTYNQKFKG TAYAMDY W7011-4.155.8-z1-P15 VK SEQ ID NO: 16 SEQ ID NO: 17 SEQ ID NO: 18 SASSTVNYMH STSNLAS HQWSSYPYT

[000130] The kappa heavy or light chain variable region sequences of 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, and humanized W7011-4.34.11-z1-m5, W7011-4.87.6-z1(N-S) and W7011-4.155.8-z1-P15 are below.

[000131] WBP7011-4.34.11-VH

Amino acid sequence (SEQ ID NO: 94):

EVQLQQSGPELVKPGASVKMSCKAS GYTFTNYVIH WVKQKPGQGLEWIG YFNPYNDGTEYNEKFKA KALTSDKSSSTAYMELSSLTSEDSAVYYCAK GPYYYGSSPFDY WGQGTTLTVSS

Nucleic acid sequence (SEQ ID NO: 95):

GAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTAAAGCCTGGGGCTTCAGTGAAGATGTCCTGCAAGGCTTCTGGATACACATTCACTAACTATGTTATTCACTGGGTGAAGCAGAAGCCTGGGCAGGGCCTTGAGTGGATTGGATATTTTAATCCTTACAATGATGGTACTGAATACAATGAGAAGTTCAAAGCCAAGGCCACACTGACTTCAGACAAATCCTCCAGCACAGCCTACATGGAGCTCAGCAGCCTGACCTCTGAGGACTCTGCGGTCTATTACTGTGCAAAAGGTCCCTACTACTACGGTAGTAGCCCCTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA

[000132] WBP7011-4.34.11-VK

Amino acid sequence (SEQ ID NO: 96):

DAVMTQTPLSLPVSLGDQASISC RSSQSLENSNGNTYLN WYLQKPGQSPQLLIY RVSNRFS GVLDRFSGSGSGTDFTLKISRVEAEDLGVYFC LQVTHVPYT FGGGTKLEIK

Nucleic acid sequence (SEQ ID NO: 97):

GATGCTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGGTCTAGTCAGAGCCTTGAAAACAGTAATGGAAACACCTATTTGAACTGGTACCTCCAGAAACCAGGCCAGTCTCCACAGCTCCTGATCTACAGGGTTTCCAACCGATTTTCTGGGGTCCTTGACAGGTTCAGTGGTAGTGGATCAGGGACAGATTTCACACTGAAAATCAGTAGAGTGGAGGCTGAGGATTTGGGAGTTTATTTCTGTCTCCAAGTTACACATGTCCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA

[000133] WBP7011-4.87.6-VH

Amino acid sequence (SEQ ID NO: 98):

QVQLQQSGAELVRPGSSVKISCKAS GYAFSTYWMN WVKQRPGQGLEWIG QIYPGDDDTKYNGKFKG KASLTADKSSSTAYMQLISLTSEDSAVYFCAR RYFRYDYWYSDV WGAGTTVTVTS

Nucleic acid sequence (SEQ ID NO: 99):

CAGGTTCAACTGCAGCAGTCTGGGGCTGAGCTGGTGAGGCCTGGGTCCTCAGTGAAGATTTCCTGCAAGGCTTCTGGCTATGCATTCAGTACCTATTGGATGAACTGGGTGAAGCAGAGGCCTGGACAGGGTCTTGAGTGGATTGGACAGATTTATCCTGGAGATGATGATACTAAGTACAATGGAAAGTTCAAGGGTAAAGCCTCACTGACTGCAGACAAATCCTCCAGCACCGCCTACATGCAGCTCATCAGCCTAACATCTGAGGACTCTGCGGTCTATTTCTGTGCAAGAAGATACTTTAGGTACGACTACTGGTATTCCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCACCTCA

[000134] WBP7011-4.87.6-VK

Amino acid sequence (SEQ ID NO: 100):

DIQMTQTTSSLSASLGDRVTISC RASQDISNYLN WYQQKPDGTVKLLIY YTSRLHS GVPARFSGSGSGTDYSLTISNLEQEDIATYFC HQGNTLPLT FGAGTKLELK

Nucleic acid sequence (SEQ ID NO: 101):

GATATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGCAATTATTTAAACTGGTATCAGCAGAAACCGGATGGAACTGTTAAACTCCTGATCTATTACACATCAAGATTACACTCAGGAGTCCCAGCAAGATTCAGTGGCAGTGGGTCTGGAACAGATTACTCTCTCACCATTAGTAACCTGGAACAAGAAGATATTGCCACTTACTTTTGCCACCAGGGTAATACGCTTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA

[000135] WBP7011-4.155.8-VH

Amino acid sequence (SEQ ID NO: 102):

EIQLQQSGPELVKPGASVKVSCKAS GYAFTSYNMY WVKQSHGKSLEWIG YIDPYNGDTTYNQKFKG KATLTVDKSSSTAYMHLNSLTSEDSAVYYCLT TAYAMDY WGQGTSVTVSS

Nucleic acid sequence (SEQ ID NO: 103):

GAGATCCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGGTATCCTGCAAGGCTTCTGGTTATGCATTCACTAGCTACAACATGTACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGATATATTGATCCTTACAATGGTGATACTACCTACAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTTGACAAGTCCTCCAGCACAGCCTACATGCATCTCAACAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTCTCACTACGGCCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA

[000136] WBP7011-4.155.8-VK

Amino acid sequence (SEQ ID NO: 104):

QIVLTQSPAIMSASLGEEITLTC SASSTVNYMH WYQQKSGTSPKLLIY STSNLAS GVPSRFSGSGSGTFYSLTIRSVEAEDAADYYC HQWSSYPYT FGGGTKLEIK

Nucleic acid sequence (SEQ ID NO: 105):

CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCTAGGGGAGGAGATCACCCTAACCTGCAGTGCCAGCTCGACTGTAAATTACATGCACTGGTACCAGCAGAAGTCAGGCACTTCTCCCAAACTCTTGATTTATAGCACATCCAACCTGGCTTCTGGAGTCCCTTCTCGCTTCAGTGGCAGTGGGTCTGGGACCTTTTATTCTCTCACAATCAGAAGTGTGGAGGCTGAAGATGCTGCCGATTATTACTGCCATCAGTGGAGTAGTTATCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA

[000137] WBP7011_4.56.1-VH

Amino acid sequence (SEQ ID NO: 106):

EVQLQQSGPELVKPGASVKMSCKAS GYTFTNYVIH WVKQKPGQGLEWIG YINPYNDGTEYNEKFKG KATLTSDTSSSTAYMALSSLTSEDSAVYYCTR GPYYYGGSPFDY WGQGTTLTVSS

Nucleic acid sequence (SEQ ID NO: 107):

GAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTAAAGCCTGGGGCTTCAGTGAAGATGTCCTGCAAGGCTTCTGGATACACATTCACTAACTATGTTATACACTGGGTGAAGCAGAAGCCTGGGCAGGGCCTTGAGTGGATTGGATATATTAATCCTTACAATGATGGTACTGAGTACAATGAGAAGTTCAAAGGCAAGGCCACACTGACTTCAGACACATCCTCCAGCACAGCCTACATGGCGCTCAGCAGCCTGACCTCTGAGGACTCTGCGGTCTATTACTGTACAAGAGGACCCTATTACTACGGTGGTAGCCCCTTCGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA

[000138] WBP7011_4.56.1-VK

Amino acid sequence (SEQ ID NO: 96):

DAVMTQTPLSLPVSLGDQASISC RSSQSLENSNGNTYLN WYLQKPGQSPQLLIY RVSNRFS GVLDRFSGSGSGTDFTLKISRVEAEDLGVYFC LQVTHVPYTF GGGTKLEIK

Nucleic acid sequence (SEQ ID NO: 97):

GATGCTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGGTCTAGTCAGAGCCTTGAAAACAGTAATGGAAACACCTATTTGAACTGGTACCTCCAGAAACCAGGCCAGTCTCCACAGCTCCTGATCTACAGGGTTTCCAACCGATTTTCTGGGGTCCTTGACAGGTTCAGTGGTAGTGGATCAGGGACAGATTTCACACTGAAAATCAGTAGAGTGGAGGCTGAGGATTTGGGAGTTTATTTCTGTCTCCAAGTTACACATGTCCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA

[000139] WBP7011_4.15.10-VH

Amino acid sequence (SEQ ID NO: 108):

EVQLQQSGPELVKPGASVKMSCKAS GYTFTSYVMH WMKQKPGQGLEWIG YINPYNDGTEYHEKFKG KALTSDKSSSTAYMELSSLTSEDSAVFYCAR GPYYYGGSPFDF WGQGTTLTVSS

Nucleic acid sequence (SEQ ID NO: 109):

GAGGTCCAGCTGCAGCAGTCTGGGCCTGAGCTGGTAAAGCCTGGGGCTTCAGTGAAGATGTCCTGCAAGGCTTCTGGATACACATTCACTAGCTATGTTATGCACTGGATGAAACAGAAGCCTGGGCAGGGCCTTGAGTGGATTGGATATATTAATCCTTACAATGATGGTACTGAGTACCATGAGAAGTTCAAAGGCAAGGCCACACTGACTTCAGACAAATCCTCCAGCACAGCCTACATGGAGCTCAGCAGCCTGACCTCTGAGGACTCTGCGGTCTTTTACTGTGCAAGAGGACCCTATTACTACGGTGGTAGCCCCTTTGACTTCTGGGGCCAAGGCACCACTCTCACGGTCTCCTCA

[000140] WBP7011_4.15.10-VK

Amino acid sequence (SEQ ID NO: 96):

DAVMTQTPLSLPVSLGDQASISC RSSQSLENSNGNTYLN WYLQKPGQSPQLLIY RVSNRFS GVLDRFSGSGSGTDFTLKISRVEAEDLGVYFC LQVTHVPYTF GGGTKLEIK

Nucleic acid sequence (SEQ ID NO: 97):

GATGCTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGGTCTAGTCAGAGCCTTGAAAACAGTAATGGAAACACCTATTTGAACTGGTACCTCCAGAAACCAGGCCAGTCTCCACAGCTCCTGATCTACAGGGTTTCCAACCGATTTTCTGGGGTCCTTGACAGGTTCAGTGGTAGTGGATCAGGGACAGATTTCACACTGAAAATCAGTAGAGTGGAGGCTGAGGATTTGGGAGTTTATTTCTGTCTCCAAGTTACACATGTCCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA

[000141] WBP7011_4.100.1-VH

Amino acid sequence (SEQ ID NO: 110):

EVQLQQSGPELVKPGASVKMSCKAS GYTFTSYVIH WVKQKPGQGLEWIG YINPYNDGAEYTEKFKG KALTSDKSSSTAYMELSSLTSEDSTVYYCAR GPYYYGGSPFDY WGQGTTLTVSS

Nucleic acid sequence (SEQ ID NO: 111):

GAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTAAAGCCTGGGGCTTCAGTGAAGATGTCCTGCAAGGCTTCTGGATACACATTCACTAGCTATGTTATACACTGGGTGAAGCAGAAGCCTGGGCAGGGCCTTGAGTGGATTGGATATATTAATCCTTACAATGATGGTGCTGAGTACACTGAGAAGTTCAAGGGCAAGGCCACACTGACTTCAGACAAATCCTCCAGTACTGCCTATATGGAGCTCAGCAGCCTGACCTCTGAGGACTCTACGGTCTATTACTGTGCACGAGGACCCTATTACTACGGTGGTAGCCCCTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA

[000142] WBP7011_4.100.1-VK

Amino acid sequence (SEQ ID NO: 96):

DAVMTQTPLSLPVSLGDQASISC RSSQSLENSNGNTYLN WYLQKPGQSPQLLIY RVSNRFS GVLDRFSGSGSGTDFTLKISRVEAEDLGVYFC LQVTHVPYTF GGGTKLEIK

Nucleic acid sequence (SEQ ID NO: 97):

GATGCTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGGTCTAGTCAGAGCCTTGAAAACAGTAATGGAAACACCTATTTGAACTGGTACCTCCAGAAACCAGGCCAGTCTCCACAGCTCCTGATCTACAGGGTTTCCAACCGATTTTCTGGGGTCCTTGACAGGTTCAGTGGTAGTGGATCAGGGACAGATTTCACACTGAAAATCAGTAGAGTGGAGGCTGAGGATTTGGGAGTTTATTTCTGTCTCCAAGTTACACATGTCCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA

[000143] WBP7011_4.106.3-VH

Amino acid sequence (SEQ ID NO: 112):

EVQLQQSGPELVKPGASVKMSCKAS GYTFSSYVIH WVKQKPGQGLEWIG YINPYNDGAEYAEKFKG KATLTSDKSSSSAYMELGSLTSEDSAVYYCAR GPYYYGGSPFDY WGQGTTLTVSS

Nucleic acid sequence (SEQ ID NO: 113):

GAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTAAAGCCTGGGGCTTCAGTGAAGATGTCCTGCAAGGCTTCTGGATACACATTCAGTAGTTATGTTATACACTGGGTGAAGCAGAAGCCTGGGCAGGGCCTTGAGTGGATTGGATATATTAATCCTTACAATGATGGTGCTGAGTATGCTGAGAAGTTCAAGGGCAAGGCCACACTGACTTCAGACAAATCCTCCAGTTCTGCCTATATGGAGCTCGGCAGCCTGACCTCTGAGGACTCTGCGGTCTATTACTGTGCACGAGGACCCTATTACTACGGTGGTAGTCCCTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA

[000144] WBP7011_4.106.3-VK

Amino acid sequence (SEQ ID NO: 96):

DAVMTQTPLSLPVSLGDQASISC RSSQSLENSNGNTYLN WYLQKPGQSPQLLIY RVSNRFS GVLDRFSGSGSGTDFTLKISRVEAEDLGVYFC LQVTHVPYTF GGGTKLEIK

Nucleic acid sequence (SEQ ID NO: 97):

GATGCTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGGTCTAGTCAGAGCCTTGAAAACAGTAATGGAAACACCTATTTGAACTGGTACCTCCAGAAACCAGGCCAGTCTCCACAGCTCCTGATCTACAGGGTTTCCAACCGATTTTCTGGGGTCCTTGACAGGTTCAGTGGTAGTGGATCAGGGACAGATTTCACACTGAAAATCAGTAGAGTGGAGGCTGAGGATTTGGGAGTTTATTTCTGTCTCCAAGTTACACATGTCCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA

[000145] WBP7011_4.108.3-VH

Amino acid sequence (SEQ ID NO: 114):

EVQLQQSGPELVKPGASVEMSCKAS GYTFTSYVIH WLKQKPGQGLEWIG YINPYNDGAEYNEKFKG KATLTSDKSSSTAYMDLNSLTSEDSAVYYCAR GPYYYGSSPFDY WGQGTTLTVSS

Nucleic acid sequence (SEQ ID NO: 115):

GAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGGAGATGTCCTGCAAGGCTTCTGGATACACATTCACTAGCTATGTTATTCACTGGTTGAAGCAGAAGCCTGGGCAGGGCCTTGAGTGGATTGGATATATTAATCCTTACAATGATGGTGCTGAGTATAATGAGAAGTTCAAGGGCAAGGCCACACTGACTTCAGACAAATCCTCCAGTACAGCCTATATGGATCTCAACAGCCTGACCTCTGAGGACTCTGCGGTCTATTACTGTGCAAGAGGACCCTATTACTACGGTAGTAGCCCCTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA

[000146] WBP7011_4.108.3-VK

Amino acid sequence (SEQ ID NO: 96):

DAVMTQTPLSLPVSLGDQASISC RSSQSLENSNGNTYLN WYLQKPGQSPQLLIY RVSNRFS GVLDRFSGSGSGTDFTLKISRVEAEDLGVYFC LQVTHVPYTF GGGTKLEIK

Nucleic acid sequence (SEQ ID NO: 97):

GATGCTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGGTCTAGTCAGAGCCTTGAAAACAGTAATGGAAACACCTATTTGAACTGGTACCTCCAGAAACCAGGCCAGTCTCCACAGCTCCTGATCTACAGGGTTTCCAACCGATTTTCTGGGGTCCTTGACAGGTTCAGTGGTAGTGGATCAGGGACAGATTTCACACTGAAAATCAGTAGAGTGGAGGCTGAGGATTTGGGAGTTTATTTCTGTCTCCAAGTTACACATGTCCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA

[000147] WBP7011_4.191.6-VH

Amino acid sequence (SEQ ID NO: 116):

EVQLLQSGPELVKPGASVKMSCKAS GYTFTDYVIH WVKQRPGQGLEWIG YINPYNDGSEYSEKFKG KALTSDKSSSTAYMELSSLTSEDSAVYYCAR GPYYYGGSPFDY WGQGTTLTVSS

Nucleic acid sequence (SEQ ID NO: 117):

GAGGTCCAGCTGCTGCAGTCTGGACCTGAGCTGGTAAAGCCTGGGGCTTCAGTGAAGATGTCCTGCAAGGCTTCTGGATACACATTCACTGACTATGTTATACACTGGGTGAAGCAGAGGCCTGGGCAGGGCCTTGAGTGGATTGGATATATTAATCCTTACAATGATGGTTCTGAGTACAGTGAGAAGTTCAAAGGCAAGGCCACACTGACTTCAGACAAATCCTCCAGCACAGCCTACATGGAGCTCAGCAGCCTGACCTCTGAGGACTCTGCGGTCTATTACTGTGCAAGAGGACCCTATTACTACGGTGGTAGTCCCTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA

[000148] WBP7011_4.191.6-VK

Amino acid sequence (SEQ ID NO: 96):

DAVMTQTPLSLPVSLGDQASISC RSSQSLENSNGNTYLN WYLQKPGQSPQLLIY RVSNRFS GVLDRFSGSGSGTDFTLKISRVEAEDLGVYFC LQVTHVPYTF GGGTKLEIK

Nucleic acid sequence (SEQ ID NO: 97):

GATGCTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGGTCTAGTCAGAGCCTTGAAAACAGTAATGGAAACACCTATTTGAACTGGTACCTCCAGAAACCAGGCCAGTCTCCACAGCTCCTGATCTACAGGGTTTCCAACCGATTTTCTGGGGTCCTTGACAGGTTCAGTGGTAGTGGATCAGGGACAGATTTCACACTGAAAATCAGTAGAGTGGAGGCTGAGGATTTGGGAGTTTATTTCTGTCTCCAAGTTACACATGTCCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA

[000149] WBP7011_4.194.10-VH

Amino acid sequence (SEQ ID NO: 118):

EVQLQQSGPELVKPGASVKMSCKAS GYTFTSYVMH WVKQKPGQGLEWIG YINPYNDGTKYNEKFKG KATLTSDKSSSTAYMELSSLTSEDSAVYYCAR GPYYYGSSPFDY WGQGTTLTVSS

Nucleic acid sequence (SEQ ID NO: 119):

GAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTAAAGCCTGGGGCTTCAGTGAAGATGTCCTGCAAGGCTTCTGGATACACATTCACTAGCTATGTTATGCACTGGGTGAAGCAGAAGCCTGGGCAGGGCCTTGAGTGGATTGGATATATTAATCCTTACAATGATGGTACTAAGTACAATGAGAAGTTCAAAGGCAAGGCCACACTGACTTCAGACAAATCCTCCAGCACAGCCTACATGGAACTCAGCAGCCTGACCTCTGAGGACTCTGCGGTCTATTACTGTGCAAGAGGACCCTATTACTACGGTAGTAGCCCCTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA

[000150] WBP7011_4.194.10-VK

Amino acid sequence (SEQ ID NO: 120):

DAVMTQTPLSLPVSLGDQASISC RSSQTLENSNGNTYLN WYLQKPGQSPQLLIY RVSNRFS GVLDRFSGSGSGTDFTLKISRVETEDLGVYFC LQVTHVPYT FGGGTKLEIK

Nucleic acid sequence (SEQ ID NO: 121):

GATGCTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGGTCTAGTCAGACCCTTGAAAACAGTAATGGAAACACCTATTTGAACTGGTACCTCCAGAAACCAGGCCAGTCTCCACAGCTCCTGATCTACAGGGTTTCCAACCGATTTTCTGGGGTCCTAGACAGGTTCAGTGGTAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGACTGAGGATTTGGGAGTTTATTTCTGCCTCCAAGTTACACATGTCCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA

[000151] WBP7011_4.231.5-VH

Amino acid sequence (SEQ ID NO: 122):

EVQLQQSGPELVKPGASVKMSCKAS GYTFTSYVMH WVKQKPGQGLEWIG YINPYNDGTQYNEKFKG KATLTSDKSSSTAYMELSSLTSEDSAVYYCAR GPYYYSPSPFDY WGQGTTLTVSS

Nucleic acid sequence (SEQ ID NO: 123):

GAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTAAAGCCTGGGGCTTCAGTGAAGATGTCCTGCAAGGCTTCTGGATACACATTCACTAGCTATGTCATGCACTGGGTGAAGCAGAAGCCTGGGCAGGGCCTTGAGTGGATTGGATATATTAATCCTTACAATGATGGTACTCAGTACAATGAGAAGTTTAAAGGCAAGGCCACACTGACTTCAGACAAATCCTCCAGCACAGCCTACATGGAGCTCAGCAGCCTGACCTCTGAGGACTCTGCGGTCTATTACTGTGCAAGAGGACCCTATTACTACAGTCCTAGCCCCTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA

[000152] WBP7011_4.231.5-VK

Amino acid sequence (SEQ ID NO: 96):

DAVMTQTPLSLPVSLGDQASISC RSSQSLENSNGNTYLN WYLQKPGQSPQLLIY RVSNRFS GVLDRFSGSGSGTDFTLKISRVEAEDLGVYFC LQVTHVPYTF GGGTKLEIK

Nucleic acid sequence (SEQ ID NO: 97):

GATGCTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGGTCTAGTCAGAGCCTTGAAAACAGTAATGGAAACACCTATTTGAACTGGTACCTCCAGAAACCAGGCCAGTCTCCACAGCTCCTGATCTACAGGGTTTCCAACCGATTTTCTGGGGTCCTTGACAGGTTCAGTGGTAGTGGATCAGGGACAGATTTCACACTGAAAATCAGTAGAGTGGAGGCTGAGGATTTGGGAGTTTATTTCTGTCTCCAAGTTACACATGTCCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA

[000153] CDRs are known to be responsible for antigen binding, however, not all of the 6 CDRs have been found to be necessary or unchangeable. In other words, one can replace or change or modify 1, 2 or 3 CDRs in the 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 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-z or W7011-4.155.8-z1-P15, still retaining specific binding affinity for CD19.

[000154] In some embodiments, the anti-CD19 antibodies and antigen-binding fragments provided herein comprise the heavy chain CDR3 sequence of one of the 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 WBP7011_4.108.3 WBP7011_4.191.6 WBP7011_4.194.10 WBP7011_4.231.5 -z1(N-S) or W7011-4.155.8-z1-P15. In some embodiments, the anti-CD19 antibodies and antigen binding fragments provided herein comprise a heavy chain CDR3 sequence selected from the group consisting of SEQ ID NOs: 3, 9, 15, 21, 24, 27, 30, 33, 36, 39 and 45. The CDR3 regions of the heavy chain are located in the center of the antigen-binding site and are therefore considered to have the most contact with the antigen and provide the most free energy to confer the affinity of the antibody for the antigen. It is also believed that the heavy chain CDR3 is the most variable antigen binding site CDR in terms of length, amino acid composition and conformation due to multiple diversification mechanisms (Tonegawa S., Nature. 302: 575-81). Diversity in heavy chain CDR3 is sufficient to obtain most antibody specificities (Xu J.L., Davis M.M. Immunity, 13: 37-45) as well as the desired antigen-binding affinity (Schier R. et al., J. Mol. Biol., 263: 551-67).

[000155] In some embodiments, the antibodies and their antigen-binding fragments provided herein contain suitable framework region (FR) sequences, provided that the antibodies and their antigen-binding fragments can specifically bind to CD19. The CDR sequences shown in Table 1 are derived from mouse antibodies, but they can be grafted onto any suitable FR sequences from any suitable species such as mouse, human, rat, rabbit, among others, using suitable methods known in the art, such as recombinant methods.

[000156] In certain embodiments, the antibodies and antigen-binding fragments provided herein are humanized. A humanized antibody or antigen-binding fragment thereof is desirable due to its low immunogenicity in humans. A humanized antibody is chimeric in its variable regions because "non-human" CDR sequences are grafted onto human or substantially human FR sequences. Humanization of an antibody or antigen-binding fragment can be particularly accomplished by replacing non-human (such as murine) CDR genes with corresponding human CDR genes in the 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).

[000157] Suitable human heavy chain and light chain variable regions can be selected to achieve this goal using methods known in the art. In an illustrative example, a "best fit" approach may be used where the sequence of the variable region of a human (e.g., rodent) antibody is screened or BLASTed against a database of known human variable region sequences and the human sequence closest to the requested non-human sequence is identified. , and is used as a human scaffold for grafting 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 scaffold derived from the consensus sequence of all human antibodies can be used to graft "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).

[000158] In some embodiments, the humanized antibodies or antigen-binding fragments provided herein consist essentially of all human sequences except for CDR sequences that are not human. In some embodiments, the variable region FRs and constant regions, if present, are wholly or essentially human immunoglobulin sequences. Human FR sequences and human constant region sequences can be derived from various human immunoglobulin genes, for example, FR sequences derived from one human antibody and constant regions from another human antibody. In some embodiments, the humanized antibody or antigen-binding fragment thereof comprises a human FR1-4 heavy/light chain.

[000159] In certain embodiments, the humanized antibodies and antigen-binding fragment provided herein comprise one or more FR sequences W7011-4.34.11-z1-m5, W7011-4.87.6-z1(NS) or W7011-4.155.8- z1-P15. Table 2 below lists the FR sequences W7011-4.34.11-z1-m5, W7011-4.87.6-z1(N-S), or W7011-4.155.8-z1-P15. The corresponding native mouse FR sequences are also shown in Table 2. The heavy chain and light chain variable region sequences are also shown below.

[000160] W7011-4.34.11-z1-m5-VH

Amino acid sequence (SEQ ID NO: 124):

QVQLVQSGAEVKKPGSSVKVSCKAS GYTFTDYVIH WVRQAPGQGLEWMG YFNPYNDGTEYNEKFKA RVTITADKSTSTAYMELSSLRSEDTAVYYCAR GPYYYGSSPFDY WGQGTTVTVSS

Nucleic acid sequence (SEQ ID NO: 125):

CAGGTGCAGCTTGTGCAGTCTGGAGCTGAAGTGAAGAAGCCAGGATCCTCCGTGAAGGTCTCCTGTAAGGCTTCTGGCTACACCTTCACCGATTACGTGATCCACTGGGTCAGGCAGGCCCCTGGGCAAGGCTTGGAGTGGATGGGGTACTTTAACCCCTACAACGATGGGACTGAGTACAATGAGAAGTTTAAAGCACGGGTGACCATTACCGCCGACAAGAGCACAAGCACAGCCTACATGGAGCTGTCCAGCCTCCGCAGCGAGGATACAGCCGTCTACTACTGCGCCAGAGGCCCTTACTACTATGGGTCCAGCCCCTTCGACTATTGGGGCCAGGGGACTACAGTGACTGTCAGTTCA

[000161] W7011-4.34.11-z1-m5-VK

Amino acid sequence (SEQ ID NO: 126):

DIVMTQTPLSLPVTPGEPASISC RSSQSLENSNHNTYIN WYLQKPGQSPQLLIY RVSKRFS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC HQVTHVPYT FGQGTKLEIK

Nucleic acid sequence (SEQ ID NO: 127):

GATATCGTGATGACCCAGACTCCCCTGTCCCTTCCTGTGACCCCAGGAGAACCAGCTTCTATCAGCTGTAGGTCCTCACAGAGCCTGGAGAACTCCAACCACAACACCTACATAAACTGGTACCTCCAGAAGCCTGGGCAGTCTCCCCAGTTGCTGATCTACAGGGTCAGCAAACGCTTCTCCGGGGTGCCCGATCGGTTTAGTGGGAGCGGGAGCGGCACAGACTTTACACTCAAGATTTCCAGAGTGGAGGCCGAGGACGTCGGCGTCTATTACTGCCACCAAGTGACACACGTGCCCTACACATTCGGCCAGGGCACTAAACTGGAGATTAAG

[000162] W7011-4.87.6-z1(N-S)-VH

Amino acid sequence (SEQ ID NO: 128):

QVQLVQSGAEVKKPGASVKVSCKAS GYAFSTYWMN WVRQAPGQGLEWMG QIYPGDDDTKYSGKFKG RVTITADKSTSTAYMELSSLRSEDTAVYYCAR RYFRYDYWYSDV WGQGTTVTVSS

Nucleic acid sequence (SEQ ID NO: 129):

CAGGTCCAGCTTGTCCAGTCTGGAGCAGAAGTGAAGAAGCCAGGGGCTTCAGTGAAGGTGTCTTGCAAGGCTTCCGGATACGCCTTCTCCACTTACTGGATGAACTGGGTGCGCCAGGCCCCTGGGCAGGGCTTGGAGTGGATGGGCCAGATCTATCCCGGCGATGACGACACAAAATACAGCGGGAAGTTCAAGGGGCGGGTGACCATTACCGCCGATAAAAGCACCTCCACCGCCTACATGGAGCTCAGTTCCCTGAGAAGCGAGGATACAGCCGTGTACTACTGTGCCAGGAGGTACTTTCGGTACGACTACTGGTATAGCGACGTCTGGGGGCAAGGCACAACTGTCACAGTGAGCAGC

[000163] W7011-4.87.6-z1(N-S)-VK

Amino acid sequence (SEQ ID NO: 130):

DIQMTQSPSSLSASVGDRVTITC RASQDISNYLN WYQQKPGKVPKLLIY YTSRLHS GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC HQGNTLPLT FGQGTKLEIK

Nucleic acid sequence (SEQ ID NO: 131):

GACATCCAAATGACCCAGAGCCCTTCCTCCTTGTCCGCAAGTGTGGGAGATAGAGTGACCATCACCTGCAGGGCTTCTCAGGATATCTCCAACTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGTGCCAAAGCTCCTTATTTACTACACCTCCCGGCTGCACAGCGGAGTCCCATCTCGCTTCAGCGGGTCAGGCAGCGGCACTGACTTTACTCTGACAATTAGCAGCCTCCAGCCTGAAGACGTCGCCACTTACTACTGTCATCAGGGGAATACACTCCCCCTGACATTCGGGCAGGGGACAAAACTGGAGATTAAG

[000164] W7011-4.155.8-z1-P15-VH

Amino acid sequence (SEQ ID NO: 132):

QMQLVQSGPEVKKPGTSVKVSCKAS GYAFTSYNMY WVRQARGQRLEWIG YIDPYNADTTYNQKFKG RVTITRDMSTSTAYMELSSLRSEDTAVYYCLT TAYAMDY WGQGTLVTVSS

Nucleic acid sequence (SEQ ID NO: 133):

CAAATGCAGCTCGTCCAGTCTGGACCTGAAGTGAAGAAGCCCGGGACATCCGTCAAGGTCTCATGTAAGGCTAGCGGGTACGCATTCACTTCCTACAACATGTACTGGGTGCGCCAGGCCAGAGGACAGAGGTTGGAGTGGATCGGCTACATCGACCCATACAACGCCGATACTACCTACAATCAGAAGTTTAAAGGGCGGGTGACCATTACCCGGGATATGTCCACCTCCACCGCCTACATGGAGCTGAGCAGCCTGAGGAGCGAGGACACAGCCGTGTACTACTGCCTGACAACAGCCTATGCCATGGACTATTGGGGCCAGGGCACACTTGTGACTGTGAGCAGT

[000165] W7011-4.155.8-z1-P15-VK

Amino acid sequence (SEQ ID NO: 134):

DIQLTQSPSFLSASVGDRVTITC SASSTVNYMH WYQQKPGKAPKLLIY STSNLAS GVPSRFSGSGSGTEFTLTISSLQPEDFATYYC HQWSSYPYT FGQGTKLEIK

Nucleic acid sequence (SEQ ID NO: 135):

GACATCCAGCTCACCCAATCCCCTTCTTTCCTCTCCGCAAGTGTCGGAGATAGGGTGACTATCACCTGCTCAGCTTCTTCAACCGTGAACTACATGCATTGGTACCAGCAGAAGCCCGGGAAAGCCCCAAAGCTGCTGATCTACAGCACCTCCAATCTGGCCAGTGGAGTGCCAAGCCGGTTTAGCGGGAGCGGCTCCGGCACTGAATTCACTTTGACAATTAGCAGCCTTCAGCCTGAGGACTTTGCCACATATTACTGTCACCAGTGGTCCAGCTACCCCTACACATTCGGGCAGGGCACAAAGCTGGAGATTAAG.

[000166] 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 retained specific binding affinity to a CD3-expressing cell (eg, CD4 T cell) and were at least comparable to, or even better than, parental mouse antibodies in that aspect.

[000167] In some embodiments, FR regions derived from a human 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 with the corresponding residues from the parent "non-human" antibody. It may be desirable in certain embodiments for the humanized antibody, or fragment thereof, to closely approximate the structure of the non-human parent antibody. In some embodiments, the humanized antibody or antigen-binding fragment provided herein contains no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue substitutions in each of the human FR sequences, or no more than 10 , 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residue substitutions in all heavy or light chain variable region FRs. In some embodiments, such a change in amino acid residue may occur only in the heavy chain FR regions, only in the light chain FR regions, or in both chains.

[000168] In some embodiments, the antibodies and antigen-binding fragments thereof provided herein comprise a heavy chain variable region 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, SEQ ID NO: 132. In some embodiments, the antibodies and antigen-binding fragments thereof provided herein comprise a light chain variable region 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, SEQ ID NO: 134.

[000169] In some embodiments, the anti-CD19 antibodies and antigen binding fragments provided herein include all or part of the heavy chain variable region and/or all or part of the light chain variable region. In one embodiment, the anti-CD19 antibodies and antigen binding fragments provided herein are a single domain antibody that consists of all or part of the heavy chain variable region provided herein. More information about such a single domain antibody is available in the art (see, for example, US Pat. No. 6,248,516).

[000170] In certain embodiments, the anti-CD19 antibodies and fragments provided herein further comprise an immunoglobulin constant region. In some embodiments, the immunoglobulin constant region comprises a heavy chain and/or light chain constant region. The heavy chain constant region includes CH1 regions, a hinge region, 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.

[000171] In some embodiments, anti-CD19 antibodies and antigen-binding fragments thereof have an immunoglobulin constant region of the IgG1, IgG2a, or IgG2b isotype that has reduced or depleted effector function, such as ADCC or CDC, which can be assessed using various assays known in the art, for example, an Fc receptor binding assay, a C1q binding assay, and a cell lysis assay.

[000172] The binding affinity of the antibody and antigen-binding fragment provided herein can be expressed by the K _D value, which is the ratio of the dissociation rate to the association rate (k _off /k _on ) when the binding between the antigen and the antigen-binding molecule reaches equilibrium. Antigen binding affinity (eg, K _D ) can be appropriately determined using suitable methods known in the art, including, for example, analysis using flow cytometry. In some embodiments, the binding of an antibody to an antigen at various concentrations can be determined using flow cytometry, where the determined mean fluorescence intensity (MFI) can first be plotted against antibody concentration, then the K _D value can be calculated by fitting the dependence of the specific binding fluorescence intensity ( Y) and antibody concentration (X) in a single site saturation equation: Y=Bmax×X/(K _D +X) (Scatchard analysis) using Prism version 5 (GraphPad Software, San Diego, CA) where B _max refers maximum specific binding of the test antibody to the antigen.

[000173] In some embodiments, the anti-CD19 antibodies and antigen-binding fragments provided herein are capable of specifically binding to human/monkey Cynomolgus CD19 naturally or artificially expressed on the cell surface. For example, a Cynomolgus human/monkey CD19 DNA sequence can be cloned into an expression vector and then transfected and expressed in 293F cells such that Cynomolgus human/monkey CD19 protein can be expressed on the surface of the transfected 293F cells.

[000174] In some embodiments, the anti-CD19 antibodies and antigen-binding fragments provided herein are capable of specifically binding to human CD19 expressed on cell surfaces with a binding affinity (K _D ) of not greater than 5×10 ^-9 M, not greater than 1 ×10 ^-9 M, not higher than 9×10 ^-10 M, not higher than 8×10 ^-10 M, not higher than 7×10 ^-10 M, not higher than 6×10 ^-10 M, not higher than 5×10 ^-10 M , not greater than 4×10 ^-10 M, not greater than 3×10 ^-10 M, not greater than 2×10 ^-10 M, or not greater than 1×10 ^-10 M, as measured by flow cytometry.

[000175] In some embodiments, anti-CD19 antibodies and their antigen-binding fragments provided herein cross-react with Cynomolgus monkey CD19 expressed on the cell surface.

[000176] The binding of antibodies to CD19 expressed on a cell can also be represented by the value of "half maximum effective concentration" (EC ₅₀ ), which refers to the concentration of an antibody at which 50% of its maximum effect (for example, binding or inhibition, etc.) .d.). The value of the EU ₅₀ can be determined by methods known in this field, for example, sandwich analysis, such as ELISA, Western blot, analysis using flow cytometry and other analysis of binding. In some embodiments, the antibodies and fragments thereof provided herein specifically bind to human CD19 expressed on a cell with an EC ₅₀ of not more than 0.01 nM, not more than 0.02 nM, not more than 0.03 nM, not more than 0.04 nM, no more than 0.05 nM, no more than 0.1 nM, no more than 0.2 nM, no more than 0.3 nM, no more than 0.4 nM, no more than 0.5 nM, no more than 0.6 nM , not more than 0.7 nM, not more than 0.8 nM, not more than 0.9 nM, or not more than 1 nM according to flow cytometry.

[000177] In some embodiments, the antibodies and antigen-binding fragments thereof bind to Cynomolgus monkey CD19 with a binding affinity similar to human CD19. For example, binding of 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, WBP701.191.6, WBP7011_4.194.10, WBP7011_4.225.7 or WBP7011_4.231.5 with Cynomolgus monkey CD19 has a similar affinity or EC ₅₀ value to that of human CD19.

[000178] In some embodiments, the antibodies and antigen-binding fragments thereof provided herein specifically bind to cell-expressed Cynomolgus monkey CD19 with an EC ₅₀ of not more than 0.2 nM, not more than 0.5 nM, not more than 0.8 nM , not more than 1 nM, not more than 2 nM, or not more than 3 nM according to flow cytometry.

[000179] In some embodiments, the antibodies and fragments thereof provided herein are internalized by a cell expressing CD19 with an EC ₅₀ of no more than 1 pM, no more than 2 pM, no more than 3 pM, no more than 4 pM no more than 5 pM, no more 6 pM, max 7 pM, max 8 pM, max 9 pM, max 10 pM, max 11 pM, max 12 pM, max 13 pM, max 14 pM, max 15 pM, max 16 pM, max 17 pM, max 18 pM, max 19 pM, max 20 pM, max 21 pM, max 22 pM, max 23 pM, max 24 pM, max 25 pM, max 30 pM, 35 pM max, 40 pM max, 45 pM max, or 50 pM max as determined by Fab-Zap analysis.

[000180] In certain embodiments, the antibodies and fragments provided herein have a specific binding affinity for human CD19 that is sufficient to provide diagnostic and/or therapeutic use. In a number of therapeutic strategies based on targeting B cells, monoclonal antibodies against human CD19 are clinically used.

[000181] The antibodies or antigen-binding fragments thereof provided herein can be a monoclonal antibody, a polyclonal antibody, a humanized antibody, a chimeric antibody, a recombinant antibody, a bispecific antibody, a labeled antibody, a bivalent antibody, or an anti-idiotypic antibody. A recombinant antibody is an antibody produced in vitro using recombinant techniques and not in animals.

[000182] Antibody variants

[000183] The present disclosure also includes various types of variant antibodies and antigen-binding fragments provided here. In certain embodiments, the present disclosure includes variants of the exemplary antibody provided herein, i. 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 -P15.

[000184] In some embodiments, antibody variants include one or more modifications or substitutions in 1, 2, or 3 CDR sequences shown in Table 1, one or more FR sequences shown in Table 2, heavy or light chain variable region sequences provided here, and/or a constant region (eg Fc region). Such antibody variants retain the specific CD19 binding affinity of their parent antibodies, but have one or more desirable properties due to the modification(s) or substitution(s). For example, antibody variants may have increased antigen-binding affinity, improved glycosylation pattern, reduced risk of glycosylation, reduced deamination, reduced or depleted effector function(s), improved FcRn receptor binding, increased pharmacokinetic half-life, pH sensitivity, and/or compatibility with conjugation (eg, one or more introduced cysteine residues).

[000185] The sequence of the parent antibody can be screened to identify suitable or preferred residues to be modified or substituted using methods known in the art, such as "scanning alanine mutagenesis" (see, for example, Cunningham and Wells ( 1989) Science, 244: 1081-1085). Briefly, target residues (eg, charged residues such as Arg, Asp, His, Lys, and Glu) can be identified and replaced with a neutral or negatively charged amino acid (eg, alanine or polyalanine), and modified antibodies are produced and screened for the property of interest. If a substitution at a particular amino acid position exhibits a functional change of interest, then the position can be identified as a potential residue for modification or substitution. Potential residues can be further evaluated by replacing a different type of residue (eg, cysteine residue, positively charged residue, etc.).

[000186] Affine variant

[000187] An affinity variant may comprise modifications or substitutions in one or more of the CDR sequences shown in Table 1, one or more FR sequences shown in Table 2, or the heavy or light chain variable region sequences provided here. Affinity variants retain the specific binding affinity for CD19 of the parent antibody, or even have increased binding affinity for CD19 compared to the parent antibody. In certain embodiments, at least one (or all) substitution(s) in CDR sequences, FR sequences, or variable region sequences contains a conservative substitution.

[000188] One skilled in the art will appreciate that in the CDR sequences and FR sequences shown in Table 1 and Table 2, one or more amino acid residues may be substituted, but the resulting antibody or antigen binding fragment still retains CD19 binding affinity, or even have increased binding affinity. To achieve this goal, you can use various methods known in this field. For example, a library of antibody variants (such as Fab or scFv variants) can be generated and expressed using phage display technology and then screened for binding affinity to human CD19. In yet another example, computer software can be used to virtually simulate antibody binding to human CD19 and determine the amino acid residues in the antibodies that form the binding surface. Such residues can either be avoided in the substitution to prevent loss of binding affinity, or targeted for substitution to provide stronger binding.

[000189] In some embodiments, the humanized antibody or antigen binding fragment provided herein comprises 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 no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitutions in the CDR sequences and/or FR sequences.

[000190] In some embodiments, anti-CD19 antibodies and antigen-binding fragments thereof contain 1, 2, or 3 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 one (or those) sequence shown in table 1, and at the same time retain the binding affinity for CD19 at a level similar to or even higher than its parent antibody.

[000191] In some embodiments, anti-CD19 antibodies and antigen-binding fragments thereof 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 one (or those) shown in Table 2, while maintaining CD19 binding affinity for level similar to or even higher than its parent antibody.

[000192] In some embodiments, anti-CD19 antibodies and antigen-binding fragments thereof 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 one (or those) shown in 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: 134, and at the same time retain the binding affinity for CD19 at a level similar to or even higher than that of its parent antibody. In some embodiments, a total of 1-10 amino acids have been substituted, inserted, or deleted in a sequence 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, substitutions, insertions, or deletions occur in regions outside of the CDR (ie, in the FR).

[000193] Glycosylation variant

[000194] The anti-CD19 antibodies and antigen binding fragments provided herein also include a glycosylation variant that can be made to increase or decrease the degree of glycosylation of an antibody or antigen binding fragment.

[000195] An anti-CD19 antibody, or antigen-binding fragment thereof, may contain one or more amino acid residues with a side chain to which a carbohydrate moiety (eg, an oligosaccharide structure) may be attached. Glycosylation of antibodies is usually either N-linked or O-linked. N-linked glycosylation refers to the attachment of a carbohydrate group to the side chain of an asparagine residue, for example an asparagine residue in a tripeptide sequence such as asparagine-X-serine and asparagine-X-threonine, where X is any amino acid other than proline. O-linked glycosylation refers to the addition of one of the sugars: N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine. Removal of a native glycosylation site can conveniently be accomplished, for example, by altering the amino acid sequence such that one of the above tripeptide sequences (for N-linked glycosylation sites) or serine or threonine residues (for O-linked glycosylation sites) present in the sequence are substituted. A new glycosylation site can be similarly created by introducing such a tripeptide sequence or serine or threonine residue.

[000196] Cysteine engineered variant

[000197] The anti-CD19 antibodies and antigen binding fragments provided herein also include a cysteine engineered variant that contains one or more introduced free cysteine amino acid residues.

[000198] The free cysteine residue is a residue that is not part of the disulfide bridge. The cysteine-engineered variant is suitable for conjugation to, for example, a cytotoxic and/or imaging agent, a label, or a radioisotope, among others, at the site of the introduced cysteine, for example, through a group such as maleimide or haloacetyl. Methods for constructing antibodies or antigen-binding fragments by introducing free cysteine residues are known in the art, see eg WO 2006/034488.

[000199] Fc variant

[000200] The anti-CD19 antibodies and antigen binding fragments provided herein also encompass an Fc variant that includes one or more modifications or substitutions of amino acid residues in their Fc region and/or hinge region.

[000201] In some embodiments, the anti-CD19 antibodies or antigen-binding fragments contain one or more amino acid substitutions that increase pH-dependent binding to the neonatal Fc receptor (FcRn). Such a variant may have an increased pharmacokinetic half-life because it binds to FcRn at an acidic pH, allowing it to avoid degradation in the lysosome and then translocate and be released from the cell. Methods for constructing an antibody and its antigen-binding fragment to improve FcRn binding affinity are well known in the art, see, for example, Vaughn, D. et al., Structure, 6(1): 63-73, 1998; Kontermann, R. et al., Antibody Engineering, Volume 1, Chapter 27: Engineering of the Fc region for improved PK, published by Springer, 2010; Yeung, Y. et al., Cancer Research, 70: 3269-3277 (2010); and Hinton, P. et al., J. Immunology, 176:346-356 (2006).

[000202] In some embodiments, the anti-CD19 antibodies or antigen-binding fragments contain one or more amino acid substitutions that alter antibody-dependent cellular cytotoxicity (ADCC). Certain amino acid residues in the CH2 domain of the Fc region can be changed to provide increased ADCC activity. Alternatively or additionally, the carbohydrate structures of the antibody may be altered to enhance ADCC activity. Methods for altering ADCC activity by constructing antibodies have been described in the art, see, for example, Shields RL. et al., J Biol 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 J.O. et al., Mol Cancer Ther. 2008, 7(8): 2517-27; Shields R. L. et al, J. Biol. Chem, 2002, 277: 26733-26740; Shinkawa T. et al, J. Biol. Chem, 2003, 278: 3466-3473.

[000203] In some embodiments, the anti-CD19 antibodies or antigen-binding fragments comprise one or more amino acid substitutions that alter complement-dependent cytotoxicity (CDC), e.g., by increasing or decreasing binding to C1q and/or complement-dependent cytotoxicity (CDC ) (see, for example, WO99/51642; Duncan & Winter Nature 322: 738-40 (1988); US Pat. No. 5,648,260; US Pat. No. 5,624,821; and WO94/29351 for other examples of Fc region variants.

[000204] In some embodiments, anti-CD19 antibodies or antigen-binding fragments contain one or more amino acid substitutions at the border of the Fc region to facilitate and/or promote heterodimerization. Such modifications include the introduction of an overhang into the first Fc polypeptide and a cavity into the second Fc polypeptide, wherein the overhang can be located in the cavity in such a way as to facilitate interaction of the first and second Fc polypeptides to form a heterodimer or complex. Methods for producing antibodies with such modifications are known in the art, for example, as described in US Pat. No. 5,731,168.

[000205] Antigen binding fragments

[000206] Anti-CD19 antigen-binding fragments are also provided. Various types of antigen binding fragments are known in the art and can be designed based on the anti-CD19 antibodies provided herein, including, for example, exemplary antibodies whose CDR and FR sequences are shown in Tables 1 and 2, and various variants thereof (such as affinity variants , glycosylation variants, Fc variants, cysteine engineered variants, and so on).

[000207] In certain embodiments, the anti-CD19 antigen-binding fragment provided herein is a camel single domain antibody, diatelo, single chain Fv fragment (scFv), scFv dimer, BsFv, dsFv, (dsFv) ₂ , dsFv-dsFv', Fv- fragment, Fab, Fab, F(ab') ₂ , bispecific antibody, ds diabody, nanobody, domain antibody, single domain antibody, or bivalent domain antibody.

[000208] Various techniques can be used to obtain such antigen-binding fragments. Illustrative methods include enzymatic cleavage 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 of host cells such as E. coli (eg, for Fab, Fv, and scFv antibody fragments), screening a phage display library as discussed above (eg, for scFv), and chemically linking the two Fab'-SH fragments to form fragments F(ab') ₂ (Carter et al., Bio/Technology, 10: 163-167 (1992)). Other methods for obtaining antibody fragments are known to those skilled in the art.

[000209] In certain embodiments, the antigen-binding fragment is a scFv. The production of scFv is described, for example, in WO 93/16185; US Pat. Nos. 5,571,894 and 5,587,458. scFv can be fused to an effector protein at the amino or carboxy terminus to provide a fusion protein (see, for example, Antibody Engineering, ed. Borrebaeck).

[000210] The anti-CD19 antibodies or antigen-binding fragments thereof provided herein can be a monoclonal antibody, a polyclonal antibody, a humanized antibody, a chimeric antibody, a recombinant antibody, a bispecific antibody, a labeled antibody, a bivalent antibody, or an anti-idiotypic antibody. A recombinant antibody is an antibody produced in vitro using recombinant techniques and not in animals.

[000211] In some embodiments, antibodies and antigen-binding fragments thereof can be used as the basis for bispecific or multivalent antibodies or antibody-drug conjugates.

[000212] Bispecific antibodies, multivalent antibodies

[000213] In some embodiments, the antibodies and antigen-binding fragments provided herein are bivalent, tetravalent, hexavalent, or multivalent. In some embodiments, the antibodies and antigen-binding fragments provided herein are monospecific or bispecific.

[000214] As used here, the term "valence" refers to the presence of a certain number of antigen binding sites in a given molecule. As such, the terms "bivalent", "tetravalent" and "hexavalent" molecule refer to the presence of two binding sites, four binding sites and six binding sites, respectively, in one antigen-binding molecule. A bivalent molecule may be monospecific if both binding sites are designed to specifically bind the same antigen or the same epitope. Similarly, a trivalent molecule can be bispecific, for example, when two binding sites are monospecific for the first antigen (or epitope) and the third binding site is specific for the second antigen (or epitope).

[000215] In some embodiments, the antibodies and antigen-binding fragments thereof provided herein may be monospecific, but bivalent, trivalent, or tetravalent, with at least two binding sites specific for the same antigen or epitope. In some embodiments, this provides stronger binding to the antigen or epitope than the monovalent counterpart. In some embodiments, in a bivalent antigen-binding fragment, the first binding site valency and the second binding site valency are structurally identical (ie, have the same sequences) or structurally different (ie, have different sequences, although they have the same specificity).

[000216] In some embodiments, the antibodies and antigen-binding fragments provided herein are bispecific. In some embodiments, the bispecific antibodies and antigen-binding fragments provided herein have a first specificity for CD19 and a second specificity. In some embodiments, the second specificity is for CD19, but for different epitopes. In some embodiments, the second specificity is for a second antigen other than CD19 and is capable of stimulating or facilitating an immune response to target cells expressing CD19 when they are in close proximity. For example, a bispecific antibody can provide target cells expressing CD19 in close proximity to an immune cell, such as a T cell or NK cell, thereby facilitating recognition or elimination of such target cell by the immune system.

[000217] The bispecific antibodies and antigen-binding fragments provided herein can be made by any suitable methods known in the art. In a traditional approach, two immunoglobulin heavy chain-light chain pairs having different antigenic specificities can be co-expressed in a host cell to produce bispecific antibodies in a recombinant manner (see, e.g., Milstein and Cuello, Nature, 305: 537 (1983)) with subsequent purification by affinity chromatography.

[000218] A recombinant approach can also be used where sequences encoding antibody heavy chain variable regions for two specificities are respectively fused to immunoglobulin constant region sequences, followed by insertion into an expression vector that is co-transfected with the expression vector for the light chain sequence in an appropriate host cell for recombinant expression of a bispecific antibody (see, for example, WO 94/04690; Suresh et al., Methods in Enzymology, 121:210 (1986)). Similarly, scFv dimers can also be recombinantly engineered and expressed from a host cell (see, for example, Gruber et al., J. Immunol., 152:5368 (1994).)

[000219] In yet another way, the leucine zipper peptides from the Fos and Jun proteins can be linked to the Fab' fragments of two different antibodies via gene fusion. Bound antibodies are reduced in the hinge region to four half antibodies (ie, monomers) and then re-oxidized to form heterodimers (Kostelny et al., J. Immunol., 148 (5): 1547-1553 (1992)).

[000220] Two antigen-binding domains can also be conjugated or “cross-linked” to form a bispecific antibody or antigen-binding fragment. For example, one antibody can bind to biotin while another antibody to avidin, and a strong association between biotin and avidin can bind two antibodies together to form a bispecific antibody (see, for example, US Pat. No. 4,676,980; WO 91/00360 , WO 92/00373 and EP 03089). In yet another example, two antibodies or antigen-binding fragments can be "crosslinked" by conventional methods known in the art, for example, as described in US patent No. 4676980.

[000221] Bispecific antigen-binding fragments can be obtained from a bispecific antibody, for example, by proteolytic cleavage or chemical coupling. For example, an antigen-binding fragment (eg, Fab') of an antibody can be prepared and converted to a Fab'-thiol derivative, and then mixed and reacted with another converted Fab' derivative having a different antigen specificity to form a bispecific antigen-binding fragment (see, e.g., , Brennan et al., Science, 229: 81 (1985)).

[000222] In some embodiments, the bispecific antibody or antigen-binding fragments thereof can be designed at the interface so that a ridge-in-trough association can be formed to promote heterodimerization of two different antigen-binding sites. As used herein, the term "lug-in-cavity" refers to an interaction between two polypeptides (such as Fc), where one polypeptide has a bulge (i.e., "bulge") due to the presence of an amino acid residue having a bulky side chain (e.g. , tyrosine, or tryptophan), and the other polypeptide has a cavity (i.e., a "trough") in which a small side chain amino acid residue (e.g., alanine or threonine) resides, and the protrusion is located in the cavity in such a way as to facilitate the interaction of the two polypeptides to form a heterodimer or complex. Methods for producing ridge-in-gap polypeptides are known in the art, for example, as described in US Pat. No. 5,731,168.

[000223] Conjugates

[000224] In some embodiments, anti-CD19 antibodies and antigen-binding fragments thereof are linked to a conjugate. A conjugate is a non-protein fragment that can be attached to an antibody or antigen-binding fragment thereof. It is believed that various conjugates may be linked to the antibodies or antigen binding fragments provided herein (see, for example, "Conjugate Vaccines", Contributions to Microbiology and Immunology, J. M. Cruse and R. E. Lewis, Jr. (eds.), Carger Press, New York, (1989)). Such conjugates can be linked to antibodies or antigen-binding moieties via covalent linkage, affinity linkage, intercalation, coordination linkage, complexation, association, mixing, or addition, among other methods. In some embodiments, the antibodies or antigen-binding fragments thereof are linked to one or more conjugates via a linker. In some embodiments, the linker is a hydrazine linker, a disulfide linker, a bifunctional linker, a dipeptide linker, a glucuronide linker, a thioether linker.

[000225] In some embodiments, the anti-CD19 antibodies and antigen binding fragments disclosed herein can be designed to contain specific sites outside the epitope binding site that can be used to bind to one or more conjugates. For example, such a site may include one or more reactive amino acid residues, such as, for example, cysteine or histidine residues, to facilitate the formation of a covalent bond with the conjugate.

[000226] The conjugate can be a therapeutic agent (e.g., a chemotherapeutic agent), a radioactive isotope, a detectable label (e.g., a lanthanide, a luminescent label, a fluorescent label, or an enzyme-substrate label), a pharmacokinetic-modifying molecule, or a purifying agent (such as a magnetic bead or nanoparticle). ).

[000227] Examples of a detectable label may include fluorescent labels (eg, fluorescein, rhodamine, dansyl, phycoerythrin, or Texas red), enzyme-substrate labels (eg, horseradish peroxidase, alkaline phosphatase, luceriferase, glucoamylase, lysozyme, saccharide oxidase, or β-D- galactosidase), radioisotopes, other lanthanides, luminescent labels, chromophore fragment, digoxigenin, biotin/avidin, DNA molecule or gold for detection.

[000228] Examples of radioisotopes 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 Bi, and ³² P. Radioisotopically labeled antibodies are useful in receptor-targeted imaging experiments.

[000229] In some embodiments, the implementation of the conjugate may be a pharmacokinetic-modifying molecule, such as PEG, which helps to increase the half-life of the antibody. Other suitable polymers include, for example, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, ethylene glycol/propylene glycol copolymers, and the like.

[000230] In some embodiments, the implementation of the conjugate may be a cleaning agent, such as a magnetic bead or a nanoparticle.

[000231] Antibody drug conjugates

[000232] In some embodiments, the present disclosure relates to antibody-drug conjugates (ADC) containing any of the above anti-CD19 antibodies or antigen-binding fragments conjugated to a cytotoxic agent, such as a chemotherapeutic agent, a drug, an agent that inhibits growth, toxin, or radioactive isotope (i.e., radioconjugate).

[000233] Antibody-drug conjugates may be useful for local delivery of cytotoxic agents, for example, in the treatment of cancer. This provides targeted delivery of cytotoxic agents to tumors and intracellular accumulation in them, which is especially beneficial when the systemic administration of these unconjugated cytotoxic agents can lead to unacceptable levels of toxicity to normal cells, as well as to those tumor cells whose death must be caused (Baldwin et al. ., (1986) Lancet pp. (Mar. 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 (ed.s), pp. 475-506, Syrigos and Epenetos (1999) Anticancer Research 19:605-614, Niculescu-Duvaz and Springer (1997) Adv. Drg Del. Rev. 26:151- 172; US patent No. 4975278).

[000234] In some embodiments, the implementation of the cytotoxic agent can be any agent that is toxic to cells or that can cause damage or cell death. In certain embodiments, the cytotoxic agent is optionally a cytotoxin, DNA alkylators, a topoisomerase inhibitor, tubulin binders, or other anticancer drugs.

[000235] Examples of an enzymatically active cytotoxin include bacterial toxins and plant toxins such as, for example, diphtheria toxin, exotoxin A-chain (Pseudomonas aeruginosa), ricin, abrin, modecin, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca proteins americana (PARI, PAPII and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, restrictocin, fenomycin, enomycin and trichothecenes (see, for example, WO 93/21232). Such a large molecule toxin can be conjugated to the antibodies or antigen-binding fragments provided herein using methods known in the art, for example, as described in Vitetta et al. (1987) Science, 238: 1098.

[000236] The cytotoxic agent can also be small molecule toxins and drugs such as geldanamycin (Mandler et al. (2000) Jour. Of Nat. Cancer Inst., 92 (19): 1573-1581; Mandler et al. (2002) Bioconjugate Chem., 13: 786-791), maytansinoids (EP 1391213; Liu et al. (1996) Proc. Natl. Acad. Sci. USA, 93: 8618-8623), calicheamicin (Lode et al. (1998) Cancer Res., 58: 2928; Hinman et al. (1993) Cancer Res., 53: 3336-3342), taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, vindesine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and its analogues, antimetabolites (eg, methotrexate, 6-mercaptopurine, 6-mercaptoguanine, cytarabine, 5-fluorouracil, decarbazine), alkylating agents (eg, mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU) and scrap stin (CCNU), cyclophosphamide, busulfan, dibromannitol, streptozotocin, mitomycin C and cis-dichlorodiaminoplatinum (II) (DDP), cisplatin), anthracyclines (eg, daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (eg, dactinomycin (formerly actinomycin ), bleomycin, mithramycin and anthramycin (AMC)) and antimitotic agents (eg vincristine and vinblastine), calicheamicin, maytansinoids, dolastatins, auristatins, trichothecene and CC1065 and their derivatives having cytotoxic activity.

[000237] The cytotoxic agent may 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 ²¹² and Lu radioactive isotopes. Methods for conjugating a radioisotope to an antibody are known in the art, for example, using a suitable ligand reagent (see, for example, WO94/11026; Current Protocols in Immunology, Volumes 1 and 2, Coligen et al, Ed. Wiley-Interscience, New York, NY, Pubs (1991)). The ligand reagent has a chelating ligand that can bind, chelate, or otherwise complex with a radioactive metal isotope, and also has a functional group that reacts with the cysteine thiol of the antibody or antigen-binding fragment. Exemplary chelating ligands include DOTA, DOTP, DOTMA, DTPA and TETA (Macrocyclics, Dallas, Tex.).

[000238] Conjugates provided here, antibodies (or antigennegative fragments) and cytotoxic agents can be obtained using various bifunctional protein-binding agents. Typical bifunctional coupling reagents include, for example, N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional imidoester derivatives (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido derivatives (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p- diazonium benzoyl)ethylenediamine), diisocyanates (such as toluene-2,6-diisocyanate) and active bis-fluorine derivatives (such as 1,5-difluoro-2,4-dinitrobenzene).

[000239] In some embodiments, the ADC provided herein is prepared with linker reagents selected from the group consisting of: BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPRH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC and sulfo-SMPB and SVSG (succinimidyl-(4-vinylsulfone)benzoate). These linker reagents are commercially available (eg, from Pierce Biotechnology, Inc., Rockford, Ill., U.S.A., see pages 467-498, 2003-2004 Applications Handbook and Catalog).

[000240] In some embodiments, the implementation of the linker is cleaved in a specific physiological environment, thereby promoting the release of cytotoxic drugs in the cell. For example, the linker may be an acid-sensitive linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker (Chari et al., Cancer Research, 52: 127-131 (1992); US Pat. No. 5,208,020). In some embodiments, the linker may contain amino acid residues such as a dipeptide, tripeptide, tetrapeptide, or pentapeptide. Amino acid residues in the linker may be naturally occurring or non-naturally occurring 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-glycine (gly-gly-gly ), valine-citrulline-p-aminobenzyloxycarbonyl ("vc-PAB"). Amino acid linker components can be designed and optimized for their selectivity for enzymatic cleavage by specific enzymes, for example tumor associated protease, cathepsin B, C and D, or plasmin protease.

[000241] In some embodiments, in the ADC provided herein, the antibody (or antigen-binding fragment thereof) is conjugated to one or more cytotoxic agents at an antibody:agent ratio of about 1 to about 20, about 1 to about 6, about 2 to 6, about 3 to about 6, about 2 to about 5, about 2 to about 4, or about 3 to about 4.

[000242] The ADC provided here can be prepared by any suitable methods known in the art. In some embodiments, the nucleophilic group of the antibody (or antigen-binding fragment) is first reacted with a bifunctional linker reagent and then coupled to a cytotoxic agent, or vice versa, i. first, the nucleophilic group of the cytotoxic agent reacts with a bifunctional linker and then binding to the antibody occurs.

[000243] In some embodiments, the implementation of the cytotoxic agent may contain (or modified to contain) a reactive thiol functional group that can react with cysteine thiol free cysteine antibodies or antigen-binding fragments provided here. Representative reactive thiol functional groups include, for example, maleimide, iodoacetamide, pyridyl disulfide, haloacetyl, succinimidyl ether (e.g. NHS, N-hydroxysuccinimide), isothiocyanate, sulfonyl chloride, 2,6-dichlorotriazinyl, pentafluorophenyl ether, or phosphoramidite (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; Means (1990) Bioconjugate Chem. 1:2; Hermanson, G. in Bioconjugate Techniques (1996) Academic Press, San Diego, pp. 40-55, 643-671).

[000244] The cytotoxic agent or antibody may be reacted with the coupling agent before it is conjugated to form the ADC. For example, the N-hydroxysuccinimide ester (NHS) of the cytotoxic agent may be preformed, isolated, purified and/or characterized, or it may be generated in situ and reacted with the nucleophilic group of the antibody. Typically, the carboxylic form of the conjugate is activated by reaction with some combination of a carbodiimide reagent, such as dicyclohexylcarbodiimide; diisopropylcarbodiimide or uronic reagent, e.g. -tetramethyluronium hexafluorophosphate) or HATU (O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate), an activator such as 1-hydroxybenzotriazole (HOBt) and N-hydroxysuccinimide, to obtain broadcast NHS. In some cases, the cytotoxic agent and the antibody may be linked by in situ activation and interaction to form ADC in one step. Other activating and binding reagents include TBTU (2-(1H-benzotriazo-1-yl)-1-1,3,3-tetramethyluronium hexafluorophosphate), TFFH (N, N',N'',N'''-tetramethyluronium 2 -fluoro-hexafluorophosphate), PyBOP (benzotriazol-1-yloxy-tris-pyrrolidino-phosphonium hexafluorophosphate), EEDQ (2-ethoxy-1-ethoxycarbonyl-1,2-dihydro-quinoline), DCC (dicyclohexylcarbodiimide); DIPCDI (diisopropylcarbodiimide), MSNT (1-(mesitylene-2-sulfonyl)-3-nitro-1H-1,2,4-triazole, and arylsulfonyl halides such as triisopropylbenzenesulfonyl chloride. In yet another example, the antibody or antigen-binding fragments thereof may be conjugated to biotin , then indirectly conjugated to a second conjugate that is conjugated to avidin.

[000245] Maytansine and maytansinoids

[000246] In one embodiment, any of the anti-CD19 antibodies or antigen-binding fragments provided herein is conjugated to one or more maytansinoid molecules. Maytansinoids are mitotic spindle inhibitors that act by inhibiting tubulin polymerization.

[000247] Maytansine compounds (such as maytansinol and maytansinol C-3 esters) suitable for use as maytansinoid drug molecules are well known in the art and can be isolated from natural sources according to known methods prepared with using genetic engineering methods (see Yu et al. (2002) PNAS 99: 7968-7973) maytansinol or maytansinol analogs obtained synthetically in accordance with known methods (see, for example, US patents No. 4137230; 4248870; 4256746; 4260608 ; 4265814; 4294757; 4307016; 4308268; 4308269; 4309428; 4313946; 4315929; 4317821; 4322348; 4331598; 4361650; 4364866; 4424219; 4450254; 4362663 and 437153).

[000248] Suitable maytansinoids are disclosed, for example, in US patent No. 5208020 and other patents and non-patent publications mentioned here. Examples of maytansinoids are maytansinol and maytansinol analogs modified at the aromatic ring or at other positions on the maytansinol molecule, see, for example, C-49-dechlor (US Pat. No. 4,256,746); C-20-hydroxy (or C-20-demethyl) +/- C-19-dechlor (US Pat. Nos. 4,361,650 and 4,307,016); C-20-demethoxy, C-20-acyloxy (-OCOR), +/- dechlor (US Pat. No. 4,294,757), C-9-SH (US Pat. No. 4,424,219); C-14-alkoxymethyl(demethoxy/CH ₂ OR) (US patent No. 4331598); C-14-hydroxymethyl or acyloxymethyl (CH ₂ OH or CH ₂ OAc) (US Pat. No. 4,450,254); C-15-hydroxy/acyloxy (US Pat. No. 4,364,866); C-15-methoxy (US Pat. Nos. 4,313,946 and 4,315,929); C-18-N-demethyl (US Pat. Nos. 4,362,663 and 4,322,348); and 4,5-deoxy (US Pat. No. 4,371,533). In some embodiments, the maytansinoid conjugated to the antibodies provided herein is DM1 (N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)-mytansine) or DM4 (N2'-deacetyl-N2'-(4 -mercapto-4-methyl-1-oxopentyl)-6-methylmytansine).

[000249] Anti-CD19 antibody-maytansinoid conjugates can be prepared by chemically linking an antibody to a maytansinoid molecule without significantly reducing the biological activity of the antibody or maytansinoid molecule. See, for example, US patent No. 5208020 (the disclosure of which is incorporated here by reference). In some embodiments, an average of 1-4, 2-4, or 3-4 maytansinoid molecules are conjugated per antibody molecule without adversely affecting antibody function or solubility.

[000250] The maytansinoid molecule can be linked to the antibody or antigen-binding moiety via any suitable linkers known in the art, see, for example, US Pat. No. 5,208,020, US Pat. 52: 127-131 (1992) and US Patent Application 2005/0169933 A1, the disclosure of which is hereby incorporated by reference. Linkers in the ADCs provided herein include disulfide linkers, thioether linkers, acid sensitive linkers, photolabile linkers, peptidase sensitive linkers, or esterase sensitive linkers. In certain embodiments, the linker in the ADC provided herein is a bifunctional protein-binding agent such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP), iminothiolane (IT), difunctional imidoester derivatives (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde) , bis-azido derivatives (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazonium benzoyl)ethylenediamine), diisocyanates (such as toluene-2,6-diisocyanate) and its active fluorine compounds bis derivatives (such as 1,5-difluoro-2,4-dinitrobenzene).

[000251] The linker can be attached to the maytansinoid molecule at various positions, depending on the type of bond. For example, an ester bond can be formed by reaction with a hydroxyl group using conventional coupling techniques. The reaction can take place at the C-3 position having a hydroxyl group, the C-14 position modified with hydroxymethyl, the C-15 position modified with a hydroxyl group, and the C-20 position having a hydroxyl group. In a preferred embodiment, the bond is formed at the C-3 position of maytansinol or a maytansinol analog.

[000252] Auristatins and dolostatins

[000253] In one embodiment, any of the anti-CD19 antibodies or antigen-binding fragments provided herein is conjugated to one or more dolastatins, peptide analogs and dolastatin derivatives, or auristatins (US Pat. Nos. 5,635,483; 5,780,588). Dolastatins and auristatins have antitumor and antifungal activity, presumably by disrupting microtubule dynamics, GTP hydrolysis, and nuclear and cell division (see, for example, US Pat. No. 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).

[000254] Exemplary auristatins include MMAE and MMAF. Auristatin/dolastatin drug molecules can be prepared according to the methods described in US Patent Application 2005/0238649; US Pat. No. 5,635,483; US Pat. No. 5,780,588; publications Pettit et al (1989) J. Am. Chem. Soc. 111:5463-5465; Pettit et al (1998) Anti-Cancer Drug Design., 13:243-277; Pettit, G.R., et al. Synthesis, 1996, 719-725; Pettit et al. (1996) J. Chem. soc. Perkin Trans. 15:859-863; and Doronina (2003) Nat. Biotechnol., 21(7):778-784.

[000255] A dolastatin or auristatin drug moiety can be attached to an antibody through the N(amino)-terminus or C(carboxyl)-terminus of the peptide drug moiety (WO 02/088172).

[000256] Chimeric antigen receptor (CAR) composition

[000257] The present disclosure also relates to chimeric antigen receptors (CARs) comprising an antigen-binding antibody fragment provided herein that specifically binds to CD19 and a T cell activation fragment. In some embodiments, the T cell activation fragment comprises a native TCR T cell activation fragment. In some embodiments, the T cell activation fragment comprises a transmembrane domain and an intracellular TCR signaling domain.

[000258] Antigen binding fragment

[000259] In some embodiments, an antigen-binding fragment may be any fragment that binds to CD19, including, but not limited to, antigen recognition domains derived from any one or more of the antibodies provided herein. In some embodiments, it is advantageous for the antigen binding fragment to be from the same species in which the CAR will ultimately be used. For example, for use in humans, it may be useful to use an antigen-binding fragment in a CAR 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 exist in a variety of other forms, including, for example, Fv, Fab, and (Fab') ₂ , as well as bifunctional (ie, bi-specific) fusion antibody fragments (eg, Lanzavecchia et al., Eur J Immunol 17, 105 (1987)).

[000260] Transmembrane domain

[000261] With respect to the transmembrane domain, in various embodiments, the CAR is designed to include a transmembrane domain that is fused to the extracellular domain of the CAR. In one embodiment, a transmembrane domain is used that is naturally associated with one of the domains in the CAR. In some instances, the transmembrane domain may be selected or modified by amino acid substitution to avoid binding such domains to transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

[000262] The transmembrane domain can be obtained from a natural or synthetic source. If the source is natural, then the domain can be derived from any membrane-bound or transmembrane protein. The transmembrane domains that can be used in this invention can be derived from (i.e., include at least the transmembrane region(s)) from the alpha, beta or zeta chain of the T cell receptor, CD28, CD3- epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. In some cases, various human hinge regions can be used, including the human Ig (immunoglobulin) hinge region.

[000263] In one embodiment, the transmembrane domain may be synthetic, in which case it will contain predominantly hydrophobic residues such as leucine and valine. In one aspect, a triplet of phenylalanine, tryptophan and valine will be at each end of the synthetic transmembrane domain. Optionally, a short oligo- or polypeptide linker of 2 to 10 amino acids in length can form a link between the transmembrane domain and the cytoplasmic signaling domain of CAR. The glycine-serine doublet provides a particularly suitable linker.

[000264] Signal transduction domain

[000265] The CAR signal transduction domain of the present invention is responsible for activating at least one of the normal TCR effector functions of the T cell into which the CAR has been placed. The TCR effector function of a T cell, for example, may be cytolytic activity or helper activity, including cytokine secretion. Thus, the term "signal transduction domain" refers to the portion of the protein that signals the effector function of the TCR and directs the T cell to perform a specialized function. Although it is usually possible to use the entire intracellular signal transduction domain, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signal transduction domain is used, such a truncated portion can be used in place of the intact strand as long as it signals the effector function. The term "intracellular signaling domain" thus implies the inclusion of any truncated portion of the intracellular signal transduction domain sufficient to signal the effector function of the TCR.

[000266] Examples of intracellular signaling domains for use in the CARs of the present invention include cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act together to initiate signal transduction upon antigen receptor activation, as well as any derivative or variant of these sequences, and any synthetic a sequence that has similar functional activities.

[000267] It is known that signals generated only through the TCR are not sufficient for full activation of the T cell and that a secondary or costimulatory signal is also needed. Thus, T cell activation can be said to be mediated by two different groups of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via TCRs (primary cytoplasmic signaling sequences) and those that function in an antigen-independent manner, providing a secondary or costimulatory signal (secondary cytoplasmic signal sequences).

[000268] Primary intracellular signal transduction sequences regulate the primary activation of the TCR complex in either a stimulatory or inhibitory manner. Primary intracellular signaling sequences that function in a stimulatory manner may contain signaling motifs, which are known as immunoreceptor tyrosine-based activation motifs or ITAMs.

[000269] Examples of ITAMs containing primary cytoplasmic signal sequences that are particularly useful in the invention include sequences derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d . It is particularly preferred that the cytoplasmic signaling molecule in the CAR of the invention contains a cytoplasmic signaling sequence derived from CD3 zeta.

[000270] In a preferred embodiment, the CAR intracellular signaling domain is designed to include the CD3 zeta signaling domain alone or in combination with any other desired cytoplasmic domain(s) useful in the context of the CAR of the present invention. For example, a CAR intracellular signaling domain may contain a region of the CD3 zeta chain and a co-stimulatory signaling region. The costimulatory signaling region refers to the portion of the CAR containing the intracellular domain of a costimulatory molecule. A co-stimulatory molecule is a cell surface molecule, other than the antigen receptor or its ligands, 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 a ligand that specifically binds to CD83 and the like. The CD3 zeta chain portion and the costimulatory signaling region may be linked to each other in a random or specific order. Optionally, a short oligo- or polypeptide linker, for example 2 to 10 amino acids in length, may form a bond. The glycine-serine doublet provides a particularly suitable linker.

[000271] In one aspect, the present disclosure further provides nucleic acid sequences encoding CARs provided herein, comprising a first polynucleotide sequence encoding an antigen-binding fragment of the antibodies provided herein, and optionally a second polynucleotide sequence encoding a transmembrane domain and an intracellular signal transduction domain of a TCR . In some embodiments, a sequence encoding an antigen-binding fragment is operably linked to a sequence encoding a transmembrane domain and a TCR signal transduction domain. In some embodiments, the signal transduction system comprises a costimulatory signaling region and/or a portion of the CD3 zeta chain. Nucleic acid sequences encoding the desired molecules can be obtained using recombinant techniques known in the art, such as screening libraries from cells expressing the gene, obtaining the gene from a vector known to contain it, or isolating directly from cells and tissues containing it using standard techniques. Alternatively, the gene of interest can be obtained synthetically rather than cloned.

[000272] In one aspect, the present disclosure relates to vectors containing a nucleic acid sequence encoding a CAR provided here. In some embodiments, the vector is a retroviral and lentiviral vector construct expressing a CAR of the present invention that can be directly transfected into a cell, or an RNA construct that can be directly transfected into a cell.

[000273] In one aspect, the present disclosure provides isolated host cells that express the CAR provided here.

[000274] In one aspect, the present disclosure further provides methods for stimulating a T cell immune response to a CD19 expressing target in a subject, wherein the method comprises administering to the subject an effective amount of a T cell expressing the CAR provided herein.

[000275] Polynucleotides and recombinant methods

[000276] The present invention provides isolated polynucleotides that encode anti-CD19 antibodies and antigen-binding fragments thereof. In some embodiments, isolated polynucleotides comprise one or more of the nucleotide sequences shown in SEQ IN NOs: 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125 , 127, 129, 131, 133 and/or 135, which encode the variable region of typical antibodies. DNA encoding a monoclonal antibody can be easily isolated and sequenced using conventional procedures (eg, using oligonucleotide probes that are capable of specifically binding to the genes encoding the heavy and light chains of the antibody). Coding DNA can also be obtained synthetically.

[000277] An isolated polynucleotide that encodes anti-CD9 antibodies and antigen-binding fragments thereof (for example, including the sequences shown in SEQ IN 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 using recombinant techniques known in the art. Many vectors are available. Vector components typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (eg, SV40, CMV, EF-1α), and a transcription termination sequence.

[000278] In some embodiments, the vector system includes mammalian, bacterial, yeast systems, and so on. 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, pex2, pTD, pRS, etc., and other laboratory and commercially available vectors. Suitable vectors may include plasmid or viral vectors (eg, replication-defective retroviruses, adenoviruses, and adeno-associated viruses).

[000279] Vectors containing a polynucleotide sequence encoding an antibody or antigen-binding fragment can be introduced into a host cell for gene cloning or expression. Suitable host cells for cloning or expression of DNA in the vectors of the present invention are prokaryotic, yeast or higher eukaryotic cells as described above. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive microorganisms, for example Enterobacteriaceae, such as Escherichia, for example, E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, for example, Salmonella typhimurium, Serratia, for example, Serratia marcescans and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis, Pseudomonas such as P. aeruginosa, and Streptomyces.

[000280] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are suitable hosts for cloning or expression of vectors encoding anti-CD19 antibodies. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, a number of other genera, species and strains are generally available and applicable here, such as Schizosaccharomyces pombe; Kluyveromyces hosts such as K. lactis, K. fragilis (ATCC 12424), K. bulgaricus (ATCC 16045), K. wickeramii (ATCC 24178), K. waltii (ATCC 56500), K. drosophilarum (ATCC 36906) ), K. thermotolerans and K. marxianus; Yarrowia (EP 402226); Pichia pastoris (EP 183070); Candida Trichoderma reesia (EP 244234); Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi such as Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.

[000281] Suitable host cells for expression of the glycosylated antibodies or antigen-binding fragment provided herein are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains and variants and corresponding permissive insect host cells have been identified from hosts such as Spodoptera frugiperda (corn fallworm), Aedes aegypti (yellow fever mosquito), Aedes albopictus (bloodsucking mosquito), Drosophila melanogaster (drosophila), and Bombyx mori . A variety of virus strains for transfection are commonly available, such as Autographa californica NPV variant L-1 and Bombyx mori NPV strain Bm-5, and such viruses can be used as a virus in accordance with the present invention, in particular for transfection of Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be used as hosts.

[000282] However, vertebrate cells are of most interest, and propagation of vertebrate cells in culture (tissue culture) has become a common procedure. Examples of suitable mammalian host cell lines are monkey kidney line CV1 transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby 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 Sertoli 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 carcinoma cells (HELA, ATCC CCL 2); dog kidney cells (MDCK, ATCC CCL 34); Buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor cells (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N. Y. Acad. Sci. 383: 44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma lineage (Hep G2). In some preferred embodiments, the host cell is a 293F cell.

[000283] Host cells are transformed with the expression or cloning vectors described above to produce anti-CD19 antibodies and cultured in conventional nutrient media suitably modified to induce promoters, select transformants, or amplify genes encoding the desired sequences. In yet another embodiment, the antibody can be obtained by homologous recombination, known in this field.

[000284] The host cells used to produce the antibodies or antigen-binding fragments provided herein can be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium (MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM, Sigma) are suitable for culturing host cells . In addition, any medium described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102: 255 (1980) US Pat. No. 4,767,704; 4657866; 4927762; 4560655 or 5122469; W090/03430; WO 87/00195; or US Patent Re 30985 can be used as a culture medium for host cells. Any of these media can optionally be supplemented with hormones and/or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium and phosphate), buffers (such as HEPES), nucleotides ( such as adenosine and thymidine), antibiotics (such as GENTAMYCIN™), trace elements (defined as inorganic compounds typically present in final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary additives can also be included at appropriate concentrations that are known to those skilled in the art. Culture conditions such as temperature, pH, and the like are those previously used for the host cell selected for expression, and will be apparent to a person skilled in the art.

[000285] Anti-CD19 antibodies or antigen-binding fragments thereof derived from cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, where affinity chromatography is the preferred purification method.

[000286] In some embodiments, protein A immobilized on a solid phase is used for immunoaffinity purification of an antibody and its antigen-binding fragment. The suitability of Protein A as an affinity ligand depends on the species and isotype of the Fc region of any immunoglobulin that is present in the antibody. Protein A can be used to purify antibodies based on human gamma-1, gamma-2 or gamma-4 heavy chains (Lindmark et al., J. Immunol. Meth., 62: 1-13 (1983)). Protein G is recommended for all mouse isotypes and for human gamma-3 (Guss et al., EMBO J., 5: 1567 1575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or polystyrenedivinylbenzene provide higher flow rates and shorter processing times than can be achieved with agarose. When the antibody contains a CH3 domain, then the Bakerbond ABX.TM. (J. T. Baker, Phillipsburg, N.J.) is cleanable. Other protein purification methods such as ion exchange column fractionation, ethanol precipitation, reverse phase HPLC, silica gel chromatography, heparin-SEPHAROSE™ chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), focus chromatography, SDS-PAGE, and ammonium sulfate precipitation is also available depending on the antibody to be extracted.

[000287] After any pre-purification step(s), the mixture containing the antibody of interest and contaminants can be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH of about 2.5-4.5, preferably carried out at low salt concentrations (eg, about 0-0.25 M salt).

[000288] Pharmaceutical compositions

[000289] The present disclosure further provides pharmaceutical compositions comprising anti-CD19 antibodies or antigen-binding fragments thereof or an antibody-drug conjugate provided herein and one or more pharmaceutically acceptable carriers.

[000290] Pharmaceutically acceptable carriers for use in the pharmaceutical compositions disclosed herein may include, for example, pharmaceutically acceptable liquid, gel or solid carriers, aqueous carriers, non-aqueous carriers, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending/distributing agents, isolating or chelating agents, diluents, adjuvants, excipients or non-toxic excipients, other components known in the art, or various combinations thereof.

[000291] Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavors, 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, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxanisole, butylated hydroxytoluene, and/or propyl gallate. As disclosed herein, the inclusion of one or more antioxidants, such as methionine, in a composition containing an antibody or antigennegative fragment and the conjugates provided herein reduces oxidation of the antibody or antigennegative fragment. This reduction in oxidation prevents or reduces the loss of binding affinity, thereby improving antibody stability and maximizing shelf life. Therefore, in certain embodiments, compositions are provided that contain one or more antibodies or antigen-binding fragments, as described herein, and one or more antioxidants, such as methionine. In addition, methods are provided to prevent oxidation, extend shelf life, and/or increase the potency of an antibody or antigen-binding fragment provided herein by mixing the antibody or antigen-binding fragment with one or more antioxidants, such as methionine.

[000292] For further illustration, pharmaceutically acceptable carriers may include, for example, aqueous vehicles such as sodium chloride injection, Ringer's solution for injection, isotonic dextrose injection, sterile water injection, or Ringer's dextrose and lactate injection, non-aqueous vehicles such as fixed vegetable oils, cottonseed oil, corn oil, sesame oil or peanut oil, antimicrobial agents at bacteriostatic or fungistatic concentrations, isotonic agents such as sodium chloride or dextrose, 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 carboxymethylcellulose, hydroxypropylmethylcellulose or polyvinylpyrrolidone, emulsifying agents such as polysorbate 80 (tween-80), sequestering or chelating agents such as ED TA (ethylenediaminetetraacetic acid), EGTA (ethylene glycoltetraacetic acid), ethyl alcohol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid or lactic acid. Antimicrobial agents used as carriers may be added to pharmaceutical compositions in multi-dose containers which include phenols or cresols, mercury-containing agents, benzyl alcohol, chlorobutanol, esters of methyl and propyl p-hydroxybenzoic acid, thimerosal, benzalkonium chloride and benzethonium chloride. Suitable excipients may include, for example, water, saline, dextrose, glycerol or ethanol. Suitable non-toxic excipients may include, for example, wetting or emulsifying agents, pH buffering agents, stabilizers, solubilizers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrin.

[000293] Pharmaceutical compositions can be a liquid solution, suspension, emulsion, pill, capsule, tablet, sustained release formulation, or powder. Formulations for oral administration may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharin, cellulose, magnesium carbonate, and the like.

[000294] In some embodiments, the pharmaceutical compositions are formulated into an injectable composition. Injectable pharmaceutical compositions can be prepared in any conventional form, such as liquid solution, suspension, emulsion, or solid forms suitable for preparing a liquid solution, suspension or emulsion. Preparations for injection may include sterile and/or non-pyrogenic solutions ready for injection, sterile dry soluble products such as lyophilized powders ready to be combined with a solvent immediately before use, including water-soluble tablets for subcutaneous injection, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with the carrier immediately before use, and sterile and/or non-pyrogenic emulsions. Solutions may be aqueous or non-aqueous.

[000295] In some embodiments, parenteral unit dose preparations are packaged in an ampoule, vial, or syringe with a needle. All preparations for parenteral administration must be sterile, and non-pyrogenic, as is known and practiced in the art.

[000296] In certain embodiments, a sterile lyophilized powder is prepared by dissolving the antibody or antigen-binding fragment disclosed herein in a suitable solvent. The solvent may include an excipient that enhances stability or other pharmacological components of the powder or reconstituted solution prepared from the powder. Excipients that may be used include, but are not limited to, water, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable agent. The solvent may include a buffer such as citrate, sodium or potassium phosphate, or other such buffer known to those skilled in the art, in one embodiment at approximately neutral pH. Subsequent sterilization by filtering the solution, followed by lyophilization under standard conditions known to those skilled in the art, yields the desired composition. In one embodiment, the resulting solution is filled into lyophilization vials. Each vial may contain a single dose or multiple doses of the anti-CD19 antibody or antigen-binding fragment thereof, or a composition thereof. Overfilling the vials with a small amount in excess of what is needed for a dose or series of doses (eg, about 10%) is acceptable to ensure accurate sample collection and accurate dosing. The lyophilized powder can be stored under suitable conditions, for example at 4° C. to room temperature.

[000297] Reconstitution of the lyophilized powder with water for injection provides a formulation for parenteral use. In one embodiment, sterile and/or non-pyrogenic water or other suitable liquid carrier is added to the lyophilized powder for reconstitution. The exact amount depends on the chosen therapy and can be determined empirically.

[000298] Methods of use

[000299] The present disclosure also provides therapeutic methods comprising: administering a therapeutically effective amount of an antibody or antigen-binding fragment provided herein to a subject in need thereof, thereby providing treatment or prevention of a CD19-associated condition or disorder. In some embodiments, the CD19-associated condition or disorder is cancer. In some embodiments, the cancer is selected from the group consisting of B-cell lymphoma, optionally Hodgkin's lymphoma, or non-Hodgkin's lymphoma, wherein the non-Hodgkin's lymphoma includes: diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, marginal zone B-cell lymphoma (MZL), lymphoid tissue lymphoma associated with the gastric mucosa (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 subject is a human.

[000300] In another aspect, methods are provided for treating a condition in a subject that would benefit from modulating CD19 in an immune response, comprising administering a therapeutically effective amount of an antibody or antigen binding fragment provided herein to a subject in need thereof.

[000301] The therapeutically effective amount of the antibody or antigen-binding fragment provided herein will depend on various factors known in the art, such as, for example, body weight, age, past medical history, currently used medications, the subject's health status and the potential for cross-reactivity, allergies, sensitivities, and side effects, as well as the route of administration and stage of disease progression. The dose may be proportionally reduced or increased by a person of ordinary skill in the art (eg, physician or veterinarian) as dictated by these and other circumstances or requirements.

[000302] In some embodiments, the anti-CD19 antibody or antigen binding fragment provided herein can be administered at a therapeutically effective dosage of about 0.01 to 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 of these embodiments, the antibody or antigen-binding fragment thereof is administered at a dosage of about 50 mg/kg or less, and in some of these embodiments, the dosage is 10 mg/kg or less, 5 mg/kg or less, 3 mg/kg, or below, 1 mg/kg or below, 0.5 mg/kg or below, or 0.1 mg/kg or below. In some embodiments, the implementation of the administered dose may be adjusted during the course of treatment. For example, in certain embodiments, the initial dose administered may be higher than the subsequent dose administered. In some embodiments, the implementation of the administered dose may vary during the course of treatment depending on the response of the subject.

[000303] Dosage regimens may be adjusted to provide the optimal desired response (eg, therapeutic response). For example, a single dose or multiple divided doses over time may be administered.

[000304] Anti-CD19 antibodies and antigen-binding fragments disclosed herein can be administered by any method known in the art, such as parenteral (eg, subcutaneous, intraperitoneal, intravenous, including intravenous infusion, intramuscular or intradermal injection) or non-parenteral (eg, oral, intranasal, intraocular, sublingual, rectal, or topical) routes.

[000305] In some embodiments, anti-CD19 antibodies or antigen-binding fragments disclosed herein can be administered alone or in combination with one or more additional therapeutic agents or agents. For example, the antibodies or antigen-binding fragments disclosed herein may be administered in combination with another therapeutic agent, such as a chemotherapeutic agent or anticancer drug.

[000306] In some of these embodiments, the anti-CD19 antibody or antigen binding fragment disclosed herein, which is administered in combination with one or more additional therapeutic agents, can be administered simultaneously with one or more additional therapeutic agents, and in some of these embodiments embodiment, the antibody or antigen-binding fragment thereof and the additional therapeutic agent(s) may be administered as part of the same pharmaceutical composition. However, an anti-CD19 antibody or antigen-binding fragment thereof administered "in combination" with another therapeutic agent should not be administered at the same time or in the same composition as the agent. It is believed that the anti-CD19 antibody or antigen-binding fragment administered before or after another agent, the expression "in combination" with that agent is used even if the antibody or antigen-binding fragment and the second agent are administered by different routes. Where possible, additional therapeutic agents administered in combination with the antibodies or antigen-binding fragments disclosed herein are administered according to the schedule provided in the product information leaflet for the additional therapeutic agent, or according to the Physicians' Desk Reference 2003 (Physicians ' Desk Reference, ^57th Ed; Medical Economics Company; ISBN: 1563634457; ^57th edition (November 2002)) or protocols well known in the art.

[000307] The present disclosure further provides methods for using anti-CD19 antibodies or antigen-binding fragments thereof. In some embodiments, the present disclosure relates to methods for inhibiting the growth of cells expressing CD19, in vivo or in vitro, including: contacting cells expressing CD19 with an antibody or antigen-binding fragment provided here. In some embodiments, the present disclosure relates to methods for modulating CD19 activity in a CD19-expressing cell, comprising exposing the CD19-expressing cell to an antibody or antigen-binding fragment provided herein.

[000308] In some embodiments, the present disclosure provides methods for detecting the presence or amount of CD19 in a sample, comprising contacting the sample with an antibody or antigen-binding fragment thereof and determining the presence or amount of CD19 in the sample.

[000309] In some embodiments, the present disclosure provides methods for diagnosing a disease or condition associated with CD19 in a subject, comprising: a) obtaining a sample from the subject; b) contacting the sample with an antibody or antigen-binding fragment provided herein; c) determining the presence or amount of CD19 in the sample; and d) determining the presence of a disease or condition associated with CD19 in the subject.

[000310] In some embodiments, the present disclosure provides kits containing the antibody or antigen-binding fragment provided herein, optionally conjugated to a detectable molecule. Kits may be useful for detecting CD19 or diagnosing diseases associated with CD19.

[000311] In some embodiments, the present disclosure also provides the use of an antibody or antigen-binding fragment provided herein in the manufacture of a medicament for the treatment of a disease or condition associated with CD19 in a subject, in the manufacture of a diagnostic reagent for diagnosing a disease or condition associated with CD3.

[000312] The following examples are provided to better illustrate the claimed invention and should not be interpreted as limiting the scope of the invention. All specific compositions, materials and methods described below, in whole or in part, are included in the scope of the present invention. These specific compositions, materials, and methods are not intended to limit the invention, but only to illustrate specific embodiments that fall within the scope of the invention. A person skilled in the art can develop equivalent compositions, materials and methods without inventive efforts and without deviating from the scope of the invention. It is understood that many changes can be made to the procedures described herein while still remaining within the scope of the present invention. The applicants of the invention assume that such options are included in the scope of the invention.

Example 1: Receiving Materials

[000313] 1.1 Preparation of reference antibody

[000314] The reference anti-CD19 antibody variable region gene (WBP701-BMK1 corresponds to huB4 in US Patent Application US20140072587A1; WBP701-BMK2 corresponds to hBU12 in US Patent Application US8242252B2; WBP701-BMK3 corresponds to 21D4 in US Patent Application US8097703B2) was cloned into an expression vector containing the human Fc region gene. Expression plasmids were transfected into Expi293 cells (Invitrogen-A14527) using the ExpiFectamine293 transfection kit (Invitrogen-A14524). Cells were cultured in Expi293 expression medium (Invitrogen-A1435101) on an orbital shaker with a platform rotating at 135 rpm in a thermostat at 37°C. The collected supernatant was purified using a protein A column (GE Healthcare 17543802).

[000315] The reference antibodies WBP701-BMK1, WBP701-BMK2 and WBP701-BMK3, obtained in accordance with the above method, were analyzed using SDS-PAGE. In FIG. 1 and 2 show that all three reference antibodies produced migrated, respectively, to an apparent molecular weight of 25 kDa and 55 kDa in SDS-PAGE under reducing conditions that correspond to the light chain and heavy chain. The main band under non-reducing conditions corresponds to total IgG with a molecular weight of 150 kDa. The purity of the reference antibodies is above 95% (see FIGS. 1 and 2).

[000316] 1.2 Generation of human or cynomolgus monkey cell lines expressing CD19

[000317] The full-length human or monkey Cynomolgus CD19 gene was cloned into the pcDNA3.3 vector. Briefly, FreeStyle 293F cells in a volume of 30 ml (ThermoFisher-R79007) at a density of 1×10 ⁶ /ml were transfected with 30 μg of DNA using the Plasfect reagent (Bioline-46025). The transfected cells were cultured in an incubator at 37°C, 8% CO ₂ and a shaking speed of 100 rpm. 24 h after transfection, blastidine (Invitrogen-A1113902) was used at a final concentration of 4-10 μg/ml to obtain a stable pool. Selected clones were tested by FACS using an anti-CD19 antibody. After two to three selection passages, cells were enriched in PE-conjugated anti-CD19 antibody and anti-PE antibody microbeads (Miltenyi-013-048-801). Stable monocellular clones were isolated by limiting dilution and screened by FACS using an anti-CD19 antibody.

[000318] The full-length human or monkey Cynomolgus CD19 gene was cloned into the pcDNA3.3 vector. Each expression vector was then transfected into CHO-K1 cells, respectively, using Lipofectamine 2000. Cells were cultured in F12-K medium with 10% FBS. Blastidin was added 24-48 hours after transfection. After two to three selection passages, cells were enriched in PE-conjugated anti-CD19 antibody and anti-PE antibody microbeads (Miltenyi-013-048-801). Stable monocellular clones were isolated by limiting dilution and screened by FACS using an anti-CD19 antibody.

[000319] Expression of human CD19 and Cynomolgus CD19 in transfected cell lines was determined using an anti-CD19 antibody by flow cytometry. All 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 showed a high level human or monkey CD19 expression (FIGS. 3A-3D).

Example 2: Obtaining an Antibody

[000320] 2.1 Immunization

[000321] Balb/c mice were immunized with transfected CD19 293F cells. The cell membrane lysate was mixed with an adjuvant including CpG-ODN and Adju-Phos or Titer-Max. Mice were immunized twice by injection into the paw pad, subcutaneously or intraperitoneally with an interval of two weeks. Mice with high serum titer were final boosted with cell membrane lysate at 1×10 ⁶ cells/animal and 10 μg ECD protein/animal in PBS.

[000322] 2.2 Serum titer determination

[000323] Serum titer was determined by flow cytometry. CHO-K1 cells transfected with CD19 were distributed in 96-well U-bottom (BD) plates at 1×10 ⁵ cells/well. Mouse serum was diluted 1:3 starting at 100-fold using staining buffer (1X PBS/1% BSA). Serum samples were incubated with cells for 1 h at 4°C. After washing the cells with staining buffer, a PE-conjugated goat anti-mouse anti-Fc IgG antibody (Jackson) was added and incubated at 4°C in the dark for 30 min. The cells were then washed twice and resuspended in 100 μl of staining buffer. Fluorescence intensity was measured using flow cytometry (BD Canto II) and analyzed using FlowJo.

[000324] All mice were found to have a CD19-specific titer. Mice with high serum titer were selected to obtain hybridoma fusion (table 3).

Table 3
Serum titer Mouse# 1 2 3 4 5 Before blood sampling on CHO-K1.CD19 cells <100 <100 <100 <100 <100 Titer on CHO-K1.CD19 cells 1968300 656100 218700 656100 72900 Titer on parental CHO-K1 cells 2700 2700 900 300 2700

[000325] 2.3 Preparation of hybridoma

[000326] Lymph node cells were fused with Sp2/0 myeloma cells by electrofusion according to general electrofusion procedures. After confluence, cells were seeded in 96-well plates at the rate of 1×10 ⁴ lymphocytes/well with DMEM medium with 20% FBS and 1% HAT. The plates were incubated at 37°C for 10-12 days.

[000327] 2.4 Antibody screening

[000328] 2.4.1 Binding to human CD19

[000329] CHO-K1 cells transfected with human CD19 were seeded in 96-well U-bottom (BD) plates at a density of 1×10⁵ cells/well. Hybridoma supernatants were transferred onto dishes and incubated with cells for 1 h at 4°C. The cells were then washed twice with staining buffer (BSA/1X PBS). PE-conjugated goat anti-mouse anti-Fc IgG (Jackson 115-115-164) was added and incubated at 4° C. in the dark for 30 min. The cells were then washed twice and resuspended in 100 μl of staining buffer. Fluorescence intensity was measured using flow cytometry (BD Canto II) and analyzed using FlowJo.

[000330] 2.4.2 Cynomolgus monkey CD19 binding

[000331] Cynomolgus CD19 transfected CHO-K1 cells were plated in 96-well U-bottom (BD) plates at a density of 1×10 ⁵ cells/well. Hybridoma supernatants were transferred onto dishes and incubated with cells for 1 h at 4°C. The cells were then washed twice with staining buffer (BSA/ 1X PBS). PE-conjugated goat anti-mouse anti-Fc IgG (Jackson 115-115-164) was added and incubated at 4° C. in the dark for 30 min. The cells were then washed twice and resuspended in 100 μl of staining buffer. Fluorescence intensity was measured using flow cytometry (BD Canto II) and analyzed using FlowJo.

[000332] 2.4.3 Internalization Analysis

[000333] Fab-ZAP is a chemical conjugate of a goat monovalent anti-human antibody and a saporin ribosome inactivating protein. Fab-ZAP is used to determine the ability of antibodies to internalize. The IgG concentration in hybridoma supernatants was determined by ELISA. Normalized hybridoma supernatants and Fab-ZAP were mixed in a 1:3 molar ratio. Ramos cells (5000/well) were incubated with various concentrations of the conjugate in an incubator at 37° C., 5% CO ₂ for 96 hours. Cell cytotoxicity was determined using CellTiter Glo (Promega). Cell viability (%) was calculated as follows: cell viability (%) = RLU sample/RLU control × 100%, where RLU denotes relative light units.

[000334] Results

[000335] The hybridoma supernatant was used for primary screening. In the primary binding screen, 116 hybridomas were identified that can produce antigen-specific binding antibodies. The binding of antigen-specific hybridomas to transfected CD19 CHO-K1 cells was then confirmed and a control screen was performed on parental CHO-K1 cells. Binding of 40 selected hybridoma lines on Ramos cells was confirmed. Positive binders were then screened in the Fab-Zap assay. For subcloning were selected 13 hybridoma lines based on the ability to bind and internalization.

[000336] 2.5 Hybridoma subcloning

[000337] Hybridoma cells of each selected line were seeded in 96-well plates at the rate of 1 cell/well. The tablets were kept in a humid thermostat at 37°C, 6% CO ₂ for 10-12 days. Single clones were selected and analyzed by FACS.

[000338] 2.6 Isotype

[000339] The antibody isotype was identified by ELISA. Plates (Nunc) were coated with goat anti-mouse IgG1, anti-mouse IgG2a, anti-mouse IgG2b, anti-mouse IgG3, anti-mouse IgM antibodies at a concentration of 2 μg/ml overnight at 4°C. After blocking and washing, hybridoma supernatants were transferred to coated plates and incubated at room temperature for 1 hour. 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 was added and the reaction was stopped with 2 M HCl. Optical density at 450 nm was determined using a microplate reader (Molecular Device).

[000340] Results

[000341] The hybridoma subclones were confirmed to bind to the CD19 cell line, and their isotypes were also determined (see Table 4). Selected subclones were purified and further evaluated in binding assay, internalization assay, cross-family assay and binning assay.

Table 4
Antibody isotype Antibody Isotype WBP7011_4.34.11 mouse IgG2a, kappa WBP7011_4.87.6 mouse IgG2a, kappa WBP7011_4.100.1 mouse IgG2a, kappa WBP7011_4.106.3 mouse IgG2a, kappa WBP7011_4.155.8 mouse IgG2a, kappa WBP7011_4.15.10 mouse IgG1, kappa WBP7011_4.56.1 mouse IgG2a, kappa WBP7011_4.202.9 mouse IgG2a, kappa WBP7011_4.231.5 mouse IgG2b, kappa WBP7011_4.108.3 mouse IgG2a, kappa WBP7011_4.191.3 mouse IgG1, kappa WBP7011_4.194.10 mouse IgG2b, kappa WBP7011_4.225.7 mouse IgG2a, kappa

Example 3: characterization of candidate antibodies

[000342] 3.1 Purification of antibodies

[000343] The harvested hybridoma supernatants were loaded onto a Protein A column (MabSelect SuRe, GE) after adjusting the pH to 7.0. Antibodies were eluted with glycine followed by immediate neutralization using 1 M Tris buffer. Antibody concentration was determined by Nano Drop (Thermal-Fisher). Protein purity was assessed by SDS-PAGE (Invitrogen, NuPAGE 4%-12% bis-Tris gel) and HPLC-SEC (Agilent).

[000344] 3.2 Affinity using FACS

[000345] Transfected CD19 CHO-K1 cells or Ramos cells were seeded in 96-well plates (BD) at a density of 5×10 ⁴ cells/well. Serial dilutions of antibodies to be tested were prepared in staining buffer (1X PBS/1% BSA) and incubated with cells at 4°C for 1 h. 1154-164) and incubated at 4°C in the dark for 30 min. Cells were washed once and resuspended in 100 µl of staining buffer. Fluorescence intensity was measured using flow cytometry (BD Canto II) and analyzed using FlowJo. The concentration of bound IgG and free IgG was calculated from the fluorescence intensities of the quantitative beads (PE Fluorescence Quantification Kit, BD 340495). K _D was calculated using Scatchard analysis.

[000346] Results

[000347] The affinity of the selected candidate antibodies was tested on CD19 transfected CHO-K1 cells by flow cytometry. The K _D values are summarized in Table 5. All candidate antibodies exhibited subnanomolar binding affinity for human CD19.

Table 5
Candidate antibody affinity Antibody K _D (M) W7011-4.34.11 1.81E-10 W7011-4.87.6 9.55E-11 W7011-4.100.1 2.13E-10 W7011-4.106.3 2.31E-10 W7011-4.155.8 9.49E-10 W7011-4.15.10 1.70E-10 W7011-4.56.1 1.18E-10 W7011-4.202.9 8.47E-10 W7011-4.231.5 1.70E-10 W7011-4.108.3 2.52E-10 W7011-4.191.3 1.14E-10 W7011-4.194.10 4.20E-10 W7011-4.225.7 7.35E-10

[000348] 3.3 Binding to human CD19

[000349] Ramos cells were seeded in 96-well U-bottom (BD) plates at a density of 1×10 ⁵ cells/well. Serial dilutions of purified antibody were prepared in staining buffer (1X PBS/1% BSA) and incubated with cells for 1 h at 4°C. The cells were then washed twice with staining buffer (BSA/1XPBS). PE-conjugated goat anti-mouse anti-Fc IgG (Jackson 115-115-164) was added and incubated at 4° C. in the dark for 30 min. The cells were then washed twice and resuspended in 100 μl of staining buffer. Fluorescence intensity was measured using flow cytometry (BD Canto II) and analyzed using FlowJo.

[000350] The binding activity of the selected subclones was analyzed on Ramos cells by flow cytometry (FIG. 4). Binding EC ₅₀ values are summarized in Table 6. All candidate antibodies showed sub-nanomolar EC ₅₀ binding assays.

Table 6
Binding activity of selected subclones Clone# EC ₅₀ (nM) W7011-4.34.11 0.23 W7011-4.34.17 0.21 W7011-4.34.18 0.24 W7011-4.87.6 0.10 W7011-4.87.8 0.09 W7011-4.87.18 0.09 W7011-4.100.1 0.17 W7011-4.100.14 0.17 W7011-4.100.18 0.18 W7011-4.106.3 0.23 W7011-4.106.9 0.18 W7011-4.106.20 0.19 W7011-4.155.8 0.85 W7011-4.155.14 0.82 W7011-4.155.17 0.69 W7011-4.15.10 0.31 W7011-4.15.13 0.32 W7011-4.56.1 0.10 W7011-4.56.2 0.08 W7011-4.202.9 0.64 W7011-4.231.5 0.12 W7011-4.231.6 0.05 W7011-4.231.15 0.09 W7011-4.108.3 0.19 W7011-4.108.6 0.09 W7011-4.108.11 0.05 W7011-4.202.3 0.58 W7011-4.202.8 0.34 W7011-4.191.3 0.04 W7011-4.191.6 0.07 W7011-4.191.16 0.13 W7011-4.194.10 0.15 W7011-4.194.11 0.26 W7011-4.194.13 0.34 W7011-4.225.7 0.45 W7011-4.225.9 0.39 WBP701.BMK3 0.11

[000351] 3.4 Cynomogus monkey CD19 binding

[000352] Cynomolgus CD19 transfected CHO-K1 cells were plated in 96-well U-bottom (BD) plates at a density of 1 x 10 ⁵ cells/well. Serial dilutions of purified antibody were prepared in staining buffer (1X PBS/1% BSA) and incubated with cells for 1 h at 4°C. The cells were then washed twice with staining buffer (BSA/1XPBS). PE-conjugated goat anti-mouse anti-Fc IgG (Jackson 115-115-164) was added and incubated at 4° C. in the dark for 30 min. The cells were then washed twice and resuspended in 100 μl of staining buffer. Fluorescence intensity was measured using flow cytometry (BD Canto II) and analyzed using FlowJo.

[000353] Candidate antibody binding activity to Cynomolgus monkey CD19 was assessed using the CHO-K1 cell line transfected with Cynomolgus monkey CD19 (FIG. 5). Binding EC ₅₀ values are summarized in Table 7. All clones selected showed strong binding to Cynomolgus monkey CD19 expressing cells.

Table 7
Cynomolgus monkey CD19 binding activity Antibody EC ₅₀ (nM) W7011-4.34.11 0.4427 W7011-4.87.6 0.285 W7011-4.100.1 0.4384 W7011-4.106.3 0.4959 W7011-4.155.8 1.542 W7011-4.15.10 0.4598 W7011-4.56.1 0.4381 W7011-4.202.9 2.299 W7011-4.231.5 0.628 W7011-4.108.3 0.8634 W7011-4.191.3 0.5984 W7011-4.194.10 0.9959 W7011-4.225.7 1.77 WBP701-BMK1 0.4473 WBP701-BMK2 0.7407 WBP701-BMK3 0.1936

[000354] 3.5 Internalization analysis

[000355] Fab-ZAP is used to determine the ability of antibodies to internalize. Serial dilutions of antibodies were mixed with Fab-Zap in a 1:3 molar ratio. Ramos cells (5000/well) were incubated with various concentrations of conjugate in an incubator with 37°C, 5% CO₂ within 96 hours Cytotoxicity for cells was determined using CellTiter Glo (Promega). Cell viability (%) was calculated as follows: cell viability (%) = sample RLU/control RLU × 100%.

[000356] The internalization activity of the selected subclones was tested on Ramos cells using the Fab-Zap assay (FIG. 6). Cell viability EC ₅₀ values are summarized in Table 8. All candidate antibodies can be internalized into Ramos cells and showed EC ₅₀ values at the picomolar level in the Fab-Zap assay.

Table 8
Fab-Zap Analysis Antibody EC ₅₀ (pM) W7011-4.34.11 7 W7011-4.34.17 8.9 W7011-4.34.18 8.9 W7011-4.87.6 8.5 W7011-4.87.8 5.8 W7011-4.87.18 7.2 W7011-4.100.1 14.8 W7011-4.100.14 11.5 W7011-4.100.18 eleven W7011-4.106.3 13.6 W7011-4.106.9 12.7 W7011-4.106.20 16.2 W7011-4.155.8 27.6 W7011-4.155.14 44.9 W7011-4.155.17 30.2 W7011-4.15.10 13.9 W7011-4.15.13 eleven W7011-4.56.1 8.4 W7011-4.56.2 5.2 W7011-4.61.10 23.1 W7011-4.61.12 21.1 W7011-4.61.16 19.4 W7011-4.231.5 8.5 W7011-4.231.6 14.8 W7011-4.231.15 14.7 W7011-4.108.3 20.6 W7011-4.108.6 15.7 W7011-4.108.11 18.7 W7011-4.202.3 28.3 W7011-4.202.8 35.4 W7011-4.202.9 23.6 W7011-4.191.3 18.4 W7011-4.191.6 19.1 W7011-4.191.16 18.2 W7011-4.194.10 11.7 W7011-4.194.11 11.8 W7011-4.194.13 12.8 W7011-4.225.7 15 W7011-4.225.9 15.8

[000357] 3.6 Epitope binning

[000358] CD19-transfected WBP701.CHO-K1.hPro1.B4 cells were seeded in 96-well plates (BD) at a density of 1×10 ⁵ cells/well. Senna dilutions of the antibodies to be tested were prepared and mixed with reference antibodies. The mixtures were added to the plate and incubated for 30 min at 4°C. After washing, PE-conjugated goat anti-mouse anti-Fc IgG (Jackson) was added and incubated at 4°C in the dark for 30 min. Cells were washed twice and resuspended in 100 μl of staining buffer (1X PBS/1% BSA). Fluorescence intensity was measured using flow cytometry (BD Canto II) and analyzed using FlowJo.

[000359] Selected candidate clones were tested for competitive binding to BMK1, BMK2 and BMK3 reference antibodies. Some candidate antibodies may block reference antibodies from binding to CD19. W7011-4.155.8, W7011-4.202.9 and W7011-4.225.7 do not compete with reference antibodies (FIG. 7). Based on the competitive binding results, antibodies are assigned to two epitope bins (Table 9).

Table 9
Epitope bin of candidate antibodies Bin 1 Bin 2 WBP701-BMK1 W7011-4.155.8 WBP701-BMK2 W7011-4.202.9 WBP701-BMK3 W7011-4.225.7 W7011-4.34.11 W7011-4.87.6 W7011-4.100.1 W7011-4.106.3 W7011-4.15.10 W7011-4.56.1 W7011-4.231.5 W7011-4.108.3 W7011-4.191.3 W7011-4.194.10

[000360] After sequencing of the antibody clones, the amino acid sequence of the antibody clones W7011-4.155.8, W7011-4.202.9 and W7011-4.225.7 was found to be identical. Amino acid and nucleic acid sequences for antibody clones are given in the "Detailed Description" section.

Example 4: Antibody Humanization and Affinity Maturation

[000361] 4.1. Hybridoma sequencing

[000362] RNA was isolated from hybridoma cells using the Trizol reagent (Invitrogen-15596018). cDNA was amplified using the 5'-RACE kit (Takara-28001488) followed by PCR amplification using 3' degenerate primers and 3' adapter primers (ExTaq: Takara-RR001B). PCR fragments were inserted into the pMD18-T vector (Takara-D101C) and sent for sequencing (Shanghai Biosune).

[000363] The sequences of antibodies (mouse) from hybridoma are as shown in SEQ ID NO: 94-123.

[000364] 4.2 Humanization

[000365] A "best fit" approach was used to humanize antibody light and heavy chains. For light chains, the amino acid sequences of the respective V genes were degraded against their own database of V genes. human germ line. The sequence of the humanized VL gene was obtained by replacing human CDR sequences in the best variants with mouse CDR sequences using the Kabat CDR determination. For the heavy chain, 4 humanized sequences were generated, for the light chain, 1 humanized sequence was generated according to the method described above, and 3 additional sequences were generated by disrupting the mouse scaffold against the human germline V gene database. Frameworks were defined using the extended CDR definition, where the Kabat CDR1 was extended by 5 amino acids at the N-terminus. The top three variants were used to generate humanized V gene sequences. Humanized genes were reverse translated, codon optimized for expression in mammals, and synthesized using GeneArt Costum Gene Synthesis (Life Technologies). The synthetic genes were re-cloned into an IgG expression vector, expressed and purified.

[000366] 4.3 Affinity maturation

[000367] Each amino acid of the six complementarity determining regions (CDRs) was individually mutated to 20 amino acids using hybridization mutagenesis (Kunkel, 1985). DNA primers containing the NNS codon encoding 20 amino acids were used to introduce a mutation at each target position of the CDR. Individual degenerate primers were used in hybridization mutagenesis reactions. Synthesis products for CDR VH and VL were pooled, respectively. 200 ng of the pooled DNA library was transfected into BL21 to generate scFv fragments.

[000368] The mutants were first screened by capture ELISA using a periplasmic extract of the bacteria. A 96-well Maxisorp Immunoplate (Nunc) was coated with anti-c-myc antibody in coating buffer (200 mM Na ₂ CO ₃ /NaHCO ₃ , pH 9.2) overnight at 4°C. After blocking with casein for 1 h at room temperature, samples of the periplasmic extract were then added to the plate and incubated at room temperature for 1 h. for 1 h. After washing, TMB substrate was added and the reaction was stopped with 2 M HCl. Optical density at 450 nm was determined using a microplate reader (Molecular Device).

[000369] Clones showing an optical density (OD) signal at 450 nm greater than the parent clone were selected for sequencing. Unique clones were verified by FACS at normalized scFv concentration to determine the relative binding affinity of the mutant scFv and the parent antibody.

[000370] Point mutations in VH and VL determined to be beneficial for antigen binding were further pooled to obtain additional binding synergy. Combinatorial mutants were expressed as scFv and screened using capture ELISA. Clones showing an optical density (OD) signal at 450 nm greater than the parent clone were sequenced and further confirmed by FACS binding.

[000371] 4.4 Designed antibody binding affinity

[000372] 4.4.1 WBP7011-4.34.11-z1-m5-IgG1k

[000373] The WBP7011-4.34.11 antibody was humanized and affinity matured. The affinity of the engineered WBP7011-4.34.11-z1-m5 antibody was determined on Ramos cells using FACS (FIG. 8). K _D was calculated using Scatchard analysis. The affinity of WBP7011-4.34.11-z1-m5-IgG1k is 0.23 nM.

[000374] 4.4.2 WBP7011-4.87.6-z1-IgG1k(N-S)

[000375] The WBP7011-4.87.6 antibody was humanized and affinity matured. The affinity of the engineered WBP7011-4.87.6-z1-IgG1k (NS) antibody was determined on Ramos cells by FACS (FIG. 9). K _D was calculated using Scatchard analysis. The affinity of WBP7011-4.87.6-z1-IgG1k(NS) is 0.25 nM.

[000376] 4.4.3 W7011-4.155.8-z1-uIgG1K

[000377] The W7011-4.155.8 antibody was humanized. The affinity of the humanized W7011-4.155.8-z1-uIgG1K antibody was determined on transfected CD19 CHO-K1 cells by FACS (FIG. 10). K _D was calculated using Scatchard analysis. The affinity for W7011-4.155.8-z1-uIgG1K is 0.82 nM.

[000378] 4.5. Engineered antibody sequences

[000379] The sequences of the engineered antibodies are as shown in SEQ ID NOs: 124-135.

Example 5 Preparation of an Antibody-Drug Conjugate (ADC)

[000380] Antibodies were dialyzed against buffer in PBS buffer (pH 7.4) and mixed with DMA (Alfa Aesar). DM1-SMCC (BrightGene) was then added and the mixture was incubated at 22°C with slight rotation for conjugation.

[000381] To remove free drug, the ADC product was dialyzed against buffer in ADC storage buffer using a 30 kDa ultrafilter (Millipore). After 8 fold buffer exchange, the ADC product was filtered through a 0.22 µm membrane filter for final characterization.

[000382] ADC concentration was determined on a UV/Vis spectrophotometer (NanoDrop). The DAR value was determined by UV/Vis spectrophotometry and SEC-HPLC. Aggregation level and purity were determined by SEC-HPLC. The free drug was determined by RP-HPLC. The content of endotoxins was determined by turbidimetric kinetic analysis.

[000383] Basic antibodies were conjugated to DM1. Concentration, purity, DAR, aggregation level and % free drug were evaluated after conjugation (Table 10).

Table 10
Characterization of DM1 Conjugated Antibody Antibody Conc. mg/ml Purity % Free drug % Endotoxins EU/mg UV SEC-DAR Aggr. % DAR W7011-BMK1-DM1 14.2 95.45 0 0.039 3.57 3.57 4.54 W7011-4.87.6-z1-lgG1K(N-S)-DM1 9.71 97.7 1.06 2.36 3.38 2.95 2.31 W7011-4.34.11-z1-m5-lgG1K-DM1 6.45 96.38 0 footprints 3.32 3.38 3.02 IgG1K isotype control-DM1 5.85 98.47 0 0.086 2.86 2.69 1.53

Example 6: ADC Cellular Toxicity Assay

[000384] B-lymphoma cells (5000 per well) were incubated with various concentrations of DM1-conjugated antibodies at 37° C. for 72 hours. Cell cytotoxicity was determined using CellTiter Glo (Promega). Cell viability (%) was calculated as follows: cell viability (%) = sample RLU/control RLU × 100%.

[000385] DM1-conjugated antibodies were tested in a cytotoxicity assay on Daudi, Nalm-6 and WSU-DLCL2 cells (FIGS. 11, 12, 13). EC ₅₀ values are summarized in Tables 11, 12 and 13. WBP7011-4.87.6-z1-IgG1K(NS)-DM1 ADC showed better cytotoxic activity than WBP701-BMK1-DM1 on all tumor cells tested. ADC WBP7011-4.34.11-z1-m5-uIgG1K-DM1 showed comparable cytotoxic activity to WBP701-BMK1-DM1.

Table 11
Cytotoxicity assay on Daudi cells Antibody EC ₅₀ (nM) WBP7011-4.34.11-z1-m5-uIgG1K-DM1 27 WBP7011-4.87.6-z1-IgG1K(N-S)-DM1 1.9 WBP701-BMK1-DM1 22 IgG1k isotype control-DM1 absent

Table 12
Cytotoxicity assay on Nalm-6 cells Antibody EC ₅₀ (nM) WBP7011-4.87.6-z1-IgG1K(N-S)-DM1 0.73 WBP701-BMK1-DM1 absent IgG1k isotype control-DM1 absent

Table 13
Cytotoxicity assay on WSU-DLCL2 cells Antibody EC ₅₀ (nM) WBP7011-4.34.11-z1-m5-uIgG1K-DM1 10.1 WBP7011-4.87.6-z1-IgG1K(N-S)-DM1 1.4 WBP701-BMK1-DM1 9.0

Example 7: ADC antitumor activity assay

[000386] Cell culture

[000387] Nalm-6 tumor cells were maintained in vitro as a suspension culture in RPMI-1640 medium supplemented with 10% fetal calf serum, at 37°C in a humid atmosphere (95% air and 5% CO ₂ ). Tumor cells were usually subcultured twice a week. Cells in the exponential growth phase were harvested and counted for tumor cell inoculation.

[000388] 7.2. Tumor cell inoculation and grouping

Nalm-6 tumor cells (10 million + Matrigel) were subcutaneously implanted on the right flank of each mouse for tumor development. Treatment was started when the average tumor volume reached 113 mm ³ . The administration of the test products and the number of animals in each group is shown in the following table.

Table 14
Administration of test ADCs and number of animals in each group Group Treatment n Dose
(mg/kg) Dose volume
(µl/g) Route of administration Scheme 1 Isotype control-DM1 6 10 10 i/v twice a week×3 weeks 2 W7011-BMK1-DM1 6 1 10 i/v twice a week×3 weeks 3 W7011-BMK1-DM1 6 10 10 i/v twice a week×3 weeks 4 W7011-4.87.6-z1-ulgG1k(N-S)-DM1 6 1 10 i/v twice a week×3 weeks 5 W7011-4.87.6-z1-ulgG1k(N-S)-DM1 6 3 10 i/v twice a week×3 weeks 6 W7011-4.87.6-z1-ulgG1k(N-S)-DM1 6 10 10 i/v twice a week×3 weeks

[000389] 7.3 Observations

[000390] The protocol and any amendment(s) or procedures regarding the housing and use of animals in this study were reviewed and approved by the WuXi AppTec Institutional Animal Care and Use Committee (IACUC) prior to experimentation. During the study, the care and use of animals was carried out in accordance with the provisions of the Association for the Evaluation and Accreditation of Laboratory Animals (AAALAC). After inoculation, the animals were checked daily for signs of disease and mortality. During routine monitoring, animals were checked for any effects of tumor development and treatment on normal behavior such as mobility, food and water intake, weight gain/loss (body weight measured daily), eye/coat changes, and any other abnormal effect. . Animal mortality and observed clinical signs were recorded based on the number of animals in each subgroup.

[000391] 7.4 Tumor measurements and endpoints

[000392] The primary endpoint was tumor growth inhibition or cure in mice. The size of the tumors was measured twice a week in two dimensions using a caliper, and the volume was expressed in mm ³ using the formula: V=0.5 a × b ² where a and b represent the long and short diameters of the tumor, respectively. The size of the tumors was then used to calculate T/C and TGI values. The T/C value (in percent) is an indicator of antitumor efficacy; T and C represent the mean tumor volumes in the treated and control groups on days 21 and 28, respectively. TGI was calculated for each group by the formula: TGI (%) = [1-(T _i -T ₀ )/(V _i - V ₀ ) ] × 100; where Ti represents the mean volume of tumors in the treatment group on days 21 and 28, T ₀ represents the average volume of tumors in the treatment group on the day of initiation of treatment, Vi represents the average volume of tumors in the vehicle control group on days 21 and day 28, and V ₀ represents mean volume of tumors in the vehicle group on the day of treatment initiation.

[000393] The results for all groups were taken on day 28 according to the protocol.

[000394] All animals showed no negative change in body weight during the experiment period.

[000395] 7.5 Efficacy Study in Nalm-6 Lymphoma Tumor Xenograft Model

[000396] In this study, the efficacy of reference antibody drug conjugates W7011-BMK1-DM1 and W7011-4.87.6-z1-uIgG1k(NS)-DM1 was evaluated in Nalm-6 lymphoma tumor xenograft in female CB17-SCID mice. Tumor volumes in all groups at different time points are shown in FIG. 14.

[000397] At PG-D21, the average tumor volume in the isotype control treated group reached 840 mm ³ . W7011-BMK1-DM1 treatment at 1 mg/kg (TV=364 mm ³ , TGI=66%, p<0.01) and 10 mg/kg (TV=327 mm ³ , TGI=71%, p<0.001 ) showed high antitumor activity. ADC W7011-4.87.6-z1-uIgG1k(NS)-DM1 at doses of 1 mg/kg (TV=398 mm ³ , TGI=61%, p<0.01), 3 mg/kg (TV=387 mm ³ , TGI=62%, p<0.01) and 10 mg/kg (TV=332 mm ³ , TGI=70%, p<0.001) showed high antitumor activity in all cases.

[000398] After the introduction of the suspension for 1 week, the average volume of tumors in the group treated with isotype control, reached 1266 mm ³ . W7011-BMK1-DM1 treatment at 1 mg/kg (TV=593 mm ³ , TGI=58%, p<0.01) and 10 mg/kg (TV=499 mm ³ , TGI=67%, p<0.001 ) showed high antitumor activity. ADC W7011-4.87.6-z1-uIgG1k(NS)-DM1 at 1 mg/kg (TV=562 mm ³ , TGI=61%, p<0.01), 3 mg/kg (TV=556 mm ³ , TGI=62%, p<0.01) and 10 mg/kg (TV=502 mm ³ , TGI=66%, p<0.001) showed high antitumor activity in all cases.

[000399] All animals showed no negative change in body weight during the experimental period.

--->

Sequence list

<110> WUXI BIOLOGICS (SHANGHAI) CO. Ltd.

WUXI BIOLOGICS (CAYMAN) INC.

<120> NEW ANTI-CD19 ANTIBODIES

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Asp Arg Val Thr Ile Thr Cys

20

<210> 91

<211> 15

<212> PROTEIN

<213> Homo sapiens

<400> 91

Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

1 5 10 15

<210> 92

<211> 32

<212> PROTEIN

<213> Homo sapiens

<400> 92

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr

1 5 10 15

Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 93

<211> 10

<212> PROTEIN

<213> Homo sapiens

<400> 93

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

1 5 10

<210> 94

<211> 121

<212> PROTEIN

<213> Mus musculus

<400> 94

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Val Ile His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Phe Asn Pro Tyr Asn Asp Gly Thr Glu Tyr Asn Glu Lys Phe

50 55 60

Lys Ala Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Gly Pro Tyr Tyr Tyr Gly Ser Ser Pro Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Leu Thr Val Ser Ser

115 120

<210> 95

<211> 363

<212> DNA

<213> Mus musculus

<400> 95

gaggtccagc tgcagcagtc tggacctgag ctggtaaagc ctggggcttc agtgaagatg 60

tcctgcaagg cttctggata cacattcact aactatgtta ttcactgggt gaagcagaag 120

cctgggcagg gccttgagtg gattggatat tttaatcctt acaatgatgg tactgaatac 180

aatgagaagt tcaaagccaa ggccacactg acttcagaca aatcctccag cacagcctac 240

atggagctca gcagcctgac ctctgaggac tctgcggtct attactgtgc aaaaggtccc 300

tactactacg gtagtagccc ctttgactac tggggccaag gcaccactct cacagtctcc 360

tca 363

<210> 96

<211> 112

<212> PROTEIN

<213> Mus musculus

<400> 96

Asp Ala Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Glu Asn Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Asn Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Leu

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Leu Gln Val

85 90 95

Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 97

<211> 336

<212> DNA

<213> Mus musculus

<400> 97

gatgctgtga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60

atctcttgca ggtctagtca gagccttgaa aacagtaatg gaaacaccta tttgaactgg 120

tacctccaga aaccaggcca gtctccacag ctcctgatct acagggtttc caaccgattt 180

tctggggtcc ttgacaggtt cagtggtagt ggatcaggga cagatttcac actgaaaatc 240

agtagagtgg aggctgagga tttgggagtt tatttctgtc tccaagttac acatgtcccg 300

tacacgttcg gaggggggac caagctggaa ataaaa 336

<210> 98

<211> 121

<212> PROTEIN

<213> Mus musculus

<400> 98

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ser

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Thr Tyr

20 25 30

Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Gln Ile Tyr Pro Gly Asp Asp Asp Thr Lys Tyr Asn Gly Lys Phe

50 55 60

Lys Gly Lys Ala Ser Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ile Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Arg Tyr Phe Arg Tyr Asp Tyr Trp Tyr Ser Asp Val Trp Gly

100 105 110

Ala Gly Thr Thr Val Thr Val Thr Ser

115 120

<210> 99

<211> 363

<212> DNA

<213> Mus musculus

<400> 99

caggttcaac tgcagcagtc tggggctgag ctggtgaggc ctgggtcctc agtgaagatt 60

tcctgcaagg cttctggcta tgcattcagt acctattggga tgaactgggt gaagcagagg 120

cctggacagg gtcttgagtg gattggacag atttatcctg gagatgatga tactaagtac 180

aatggaaagt tcaagggtaa agcctcactg actgcagaca aatcctccag caccgcctac 240

atgcagctca tcagcctaac atctgaggac tctgcggtct atttctgtgc aagaagatac 300

tttaggtacg actactggta ttccgatgtc tggggcgcag ggaccacggt caccgtcacc 360

tca 363

<210> 100

<211> 107

<212> PROTEIN

<213> Mus musculus

<400> 100

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys His Gln Gly Asn Thr Leu Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 101

<211> 321

<212> DNA

<213> Mus musculus

<400> 101

gatatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60

atcagttgca gggcaagtca ggacattagc aattatttaa actggtatca gcagaaaccg 120

gatggaactg ttaaactcct gatctattac acatcaagat tacactcagg agtcccagca 180

agattcagtg gcagtgggtc tggaacagat tactctctca ccattagtaa cctggaacaa 240

gaagatattg ccacttactt ttgccaccag ggtaatacgc ttccgctcac gttcggtgct 300

gggaccaagc tggagctgaa a 321

<210> 102

<211> 116

<212> PROTEIN

<213> Mus musculus

<400> 102

Glu Ile Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Ser Tyr

20 25 30

Asn Met Tyr Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asp Pro Tyr Asn Gly Asp Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met His Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Leu Thr Thr Ala Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val

100 105 110

Thr Val Ser Ser

115

<210> 103

<211> 348

<212> DNA

<213> Mus musculus

<400> 103

gagatccagc tgcagcagtc tggacctgag ctggtgaagc ctggggcttc agtgaaggta 60

tcctgcaagg cttctggtta tgcattcact agctacaaca tgtactggggt gaagcagagc 120

catggaaaga gccttgagtg gattggatat attgatcctt acaatggtga tactacctac 180

aaccagaagt tcaagggcaa ggccacattg actgttgaca agtcctccag cacagcctac 240

atgcatctca acagcctgac atctgaggac tctgcagtct attactgtct cactacggcc 300

tatgctatgg actactgggg tcaaggaacc tcagtcaccg tctcctca 348

<210> 104

<211> 106

<212> PROTEIN

<213> Mus musculus

<400> 104

Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly

1 5 10 15

Glu Glu Ile Thr Leu Thr Cys Ser Ala Ser Ser Thr Val Asn Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Leu Leu Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Phe Tyr Ser Leu Thr Ile Arg Ser Val Glu Ala Glu

65 70 75 80

Asp Ala Ala Asp Tyr Tyr Cys His Gln Trp Ser Ser Tyr Pro Tyr Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 105

<211> 318

<212> DNA

<213> Mus musculus

<400> 105

caaattgttc tcacccagtc tccagcaatc atgtctgcat ctctagggga ggagatcacc 60

ctaacctgca gtgccagctc gactgtaaat tacatgcact ggtaccagca gaagtcaggc 120

acttctccca aactcttgat ttatagcaca tccaacctgg cttctggagt cccttctcgc 180

ttcagtggca gtgggtctgg gaccttttat tctctcacaa tcagaagtgt ggaggctgaa 240

gatgctgccg attattactg ccatcagtgg agtagttatc cgtacacgtt cggagggggg 300

accaagctgg aaataaaa 318

<210> 106

<211> 121

<212> PROTEIN

<213> Mus musculus

<400> 106

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Val Ile His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Glu Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ser Asp Thr Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Ala Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Pro Tyr Tyr Tyr Gly Gly Ser Pro Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Leu Thr Val Ser Ser

115 120

<210> 107

<211> 363

<212> DNA

<213> Mus musculus

<400> 107

gaggtccagc tgcagcagtc tggacctgag ctggtaaagc ctggggcttc agtgaagatg 60

tcctgcaagg cttctggata cacattcact aactatgtta tacactgggt gaagcagaag 120

cctgggcagg gccttgagtg gattggatat attaatcctt acaatgatgg tactgagtac 180

aatgagaagt tcaaaggcaa ggccacactg acttcagaca catcctccag cacagcctac 240

atggcgctca gcagcctgac ctctgaggac tctgcggtct attactgtac aagaggaccc 300

tattactacg gtggtagccc cttcgactac tggggccaag gcaccactct cacagtctcc 360

tca 363

<210> 108

<211> 121

<212> PROTEIN

<213> Mus musculus

<400> 108

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Val Met His Trp Met Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Glu Tyr His Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Phe Tyr Cys

85 90 95

Ala Arg Gly Pro Tyr Tyr Tyr Gly Gly Ser Pro Phe Asp Phe Trp Gly

100 105 110

Gln Gly Thr Thr Leu Thr Val Ser Ser

115 120

<210> 109

<211> 363

<212> DNA

<213> Mus musculus

<400> 109

gaggtccagc tgcagcagtc tgggcctgag ctggtaaagc ctggggcttc agtgaagatg 60

tcctgcaagg cttctggata cacattcact agctatgtta tgcactggat gaaacagaag 120

cctgggcagg gccttgagtg gattggatat attaatcctt acaatgatgg tactgagtac 180

catgagaagt tcaaaggcaa ggccacactg acttcagaca aatcctccag cacagcctac 240

atggagctca gcagcctgac ctctgaggac tctgcggtct tttactgtgc aagaggaccc 300

tattactacg gtggtagccc ctttgacttc tggggccaag gcaccactct cacggtctcc 360

tca 363

<210> 110

<211> 121

<212> PROTEIN

<213> Mus musculus

<400> 110

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Val Ile His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Ala Glu Tyr Thr Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Thr Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Pro Tyr Tyr Tyr Gly Gly Ser Pro Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Leu Thr Val Ser Ser

115 120

<210> 111

<211> 363

<212> DNA

<213> Mus musculus

<400> 111

gaggtccagc tgcagcagtc tggacctgag ctggtaaagc ctggggcttc agtgaagatg 60

tcctgcaagg cttctggata cacattcact agctatgtta tacactgggt gaagcagaag 120

cctgggcagg gccttgagtg gattggatat attaatcctt acaatgatgg tgctgagtac 180

actgagaagt tcaagggcaa ggccacactg acttcagaca aatcctccag tactgcctat 240

atggagctca gcagcctgac ctctgaggac tctacggtct attactgtgc acgaggaccc 300

tattactacg gtggtagccc ctttgactac tggggccaag gcaccactct cacagtctcc 360

tca 363

<210> 112

<211> 121

<212> PROTEIN

<213> Mus musculus

<400> 112

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr

20 25 30

Val Ile His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Ala Glu Tyr Ala Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Ser Ser Ala Tyr

65 70 75 80

Met Glu Leu Gly Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Pro Tyr Tyr Tyr Gly Gly Ser Pro Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Leu Thr Val Ser Ser

115 120

<210> 113

<211> 363

<212> DNA

<213> Mus musculus

<400> 113

gaggtccagc tgcagcagtc tggacctgag ctggtaaagc ctggggcttc agtgaagatg 60

tcctgcaagg cttctggata cacattcagt agttatgtta tacactgggt gaagcagaag 120

cctgggcagg gccttgagtg gattggatat attaatcctt acaatgatgg tgctgagtat 180

gctgagaagt tcaagggcaa ggccacactg acttcagaca aatcctccag ttctgcctat 240

atggagctcg gcagcctgac ctctgaggac tctgcggtct attactgtgc acgaggaccc 300

tattactacg gtggtagtcc ctttgactac tggggccaag gcaccactct cacagtctcc 360

tca 363

<210> 114

<211> 121

<212> PROTEIN

<213> Mus musculus

<400> 114

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Glu Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Val Ile His Trp Leu Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Ala Glu Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Asp Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Pro Tyr Tyr Tyr Gly Ser Ser Pro Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Leu Thr Val Ser Ser

115 120

<210> 115

<211> 363

<212> DNA

<213> Mus musculus

<400> 115

gaggtccagc tgcagcagtc tggacctgag ctggtgaagc ctggggcttc agtggagatg 60

tcctgcaagg cttctggata cacattcact agctatgtta ttcactggtt gaagcagaag 120

cctgggcagg gccttgagtg gattggatat attaatcctt acaatgatgg tgctgagtat 180

aatgagaagt tcaagggcaa ggccacactg acttcagaca aatcctccag tacagcctat 240

atggatctca acagcctgac ctctgaggac tctgcggtct attactgtgc aagaggaccc 300

tattactacg gtagtagccc ctttgactac tggggccaag gcaccactct cacagtctcc 360

tca 363

<210> 116

<211> 121

<212> PROTEIN

<213> Mus musculus

<400> 116

Glu Val Gln Leu Leu Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Val Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Ser Glu Tyr Ser Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Pro Tyr Tyr Tyr Gly Gly Ser Pro Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Leu Thr Val Ser Ser

115 120

<210> 117

<211> 363

<212> DNA

<213> Mus musculus

<400> 117

gaggtccagc tgctgcagtc tggacctgag ctggtaaagc ctggggcttc agtgaagatg 60

tcctgcaagg cttctggata cacattcact gactatgtta tacactgggt gaagcagagg 120

cctgggcagg gccttgagtg gattggatat attaatcctt acaatgatgg ttctgagtac 180

agtgagaagt tcaaaggcaa ggccacactg acttcagaca aatcctccag cacagcctac 240

atggagctca gcagcctgac ctctgaggac tctgcggtct attactgtgc aagaggaccc 300

tattactacg gtggtagtcc ctttgactac tggggccaag gcaccactct cacagtctcc 360

tca 363

<210> 118

<211> 121

<212> PROTEIN

<213> Mus musculus

<400> 118

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Pro Tyr Tyr Tyr Gly Ser Ser Pro Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Leu Thr Val Ser Ser

115 120

<210> 119

<211> 363

<212> DNA

<213> Mus musculus

<400> 119

gaggtccagc tgcagcagtc tggacctgag ctggtaaagc ctggggcttc agtgaagatg 60

tcctgcaagg cttctggata cacattcact agctatgtta tgcactgggt gaagcagaag 120

cctgggcagg gccttgagtg gattggatat attaatcctt acaatgatgg tactaagtac 180

aatgagaagt tcaaaggcaa ggccacactg acttcagaca aatcctccag cacagcctac 240

atggaactca gcagcctgac ctctgaggac tctgcggtct attactgtgc aagaggaccc 300

tattactacg gtagtagccc ctttgactac tggggccaag gcaccactct cacagtctcc 360

tca 363

<210> 120

<211> 112

<212> PROTEIN

<213> Mus musculus

<400> 120

Asp Ala Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Thr Leu Glu Asn Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Asn Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Leu

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Thr Glu Asp Leu Gly Val Tyr Phe Cys Leu Gln Val

85 90 95

Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 121

<211> 336

<212> DNA

<213> Mus musculus

<400> 121

gatgctgtga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60

atctcttgca ggtctagtca gacccttgaa aacagtaatg gaaacaccta tttgaactgg 120

tacctccaga aaccaggcca gtctccacag ctcctgatct acagggtttc caaccgattt 180

tctggggtcc tagacaggtt cagtggtagt ggatcaggga cagatttcac actgaaaatc 240

agcagagtgg agactgagga tttgggagtt tatttctgcc tccaagttac acatgtcccg 300

tacacgttcg gaggggggac caagctggaa ataaaa 336

<210> 122

<211> 121

<212> PROTEIN

<213> Mus musculus

<400> 122

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Gln Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Pro Tyr Tyr Tyr Ser Pro Ser Pro Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Leu Thr Val Ser Ser

115 120

<210> 123

<211> 363

<212> DNA

<213> Mus musculus

<400> 123

gaggtccagc tgcagcagtc tggacctgag ctggtaaagc ctggggcttc agtgaagatg 60

tcctgcaagg cttctggata cacattcact agctatgtca tgcactggggt gaagcagaag 120

cctgggcagg gccttgagtg gattggatat attaatcctt acaatgatgg tactcagtac 180

aatgagaagt ttaaaggcaa ggccacactg acttcagaca aatcctccag cacagcctac 240

atggagctca gcagcctgac ctctgaggac tctgcggtct attactgtgc aagaggaccc 300

tattactaca gtcctagccc ctttgactac tggggccaag gcaccactct cacagtctcc 360

tca 363

<210> 124

<211> 121

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 124

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Val Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Phe Asn Pro Tyr Asn Asp Gly Thr Glu Tyr Asn Glu Lys Phe

50 55 60

Lys Ala Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Pro Tyr Tyr Tyr Gly Ser Ser Pro Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 125

<211> 363

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 125

caggtgcagc ttgtgcagtc tggagctgaa gtgaagaagc caggatcctc cgtgaaggtc 60

tcctgtaagg cttctggcta caccttcacc gattacgtga tccactgggt caggcaggcc 120

cctgggcaag gcttggagtg gatggggtac tttaacccct acaacgatgg gactgagtac 180

aatgagaagt ttaaagcacg ggtgaccatt accgccgaca agagcacaag cacagcctac 240

atggagctgt ccagcctccg cagcgaggat acagccgtct actactgcgc cagaggccct 300

tactactatg ggtccagccc cttcgactat tggggccagg ggactacagt gactgtcagt 360

tca 363

<210> 126

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 126

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Glu Asn Ser

20 25 30

Asn His Asn Thr Tyr Ile Asn Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Arg Val Ser Lys Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys His Gln Val

85 90 95

Thr His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 127

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 127

gatatcgtga tgacccagac tcccctgtcc cttcctgtga ccccaggaga accagcttct 60

atcagctgta ggtcctcaca gagcctggag aactccaacc acaacaccta cataaactgg 120

tacctccaga agcctgggca gtctccccag ttgctgatct acagggtcag caaacgcttc 180

tccggggtgc ccgatcggtt tagtgggagc gggagcggca cagactttac actcaagatt 240

tccagagtgg aggccgagga cgtcggcgtc tattactgcc accaagtgac acacgtgccc 300

tacacattcg gccagggcac taaactggag attaag 336

<210> 128

<211> 121

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 128

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Thr Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gln Ile Tyr Pro Gly Asp Asp Asp Thr Lys Tyr Ser Gly Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Tyr Phe Arg Tyr Asp Tyr Trp Tyr Ser Asp Val Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 129

<211> 363

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 129

caggtccagc ttgtccagtc tggagcagaa gtgaagaagc caggggcttc agtgaaggtg 60

tcttgcaagg cttccggata cgccttctcc acttactgga tgaactggggt gcgccaggcc 120

cctgggcagg gcttggagtg gatgggccag atctatcccg gcgatgacga cacaaaatac 180

agcgggaagt tcaaggggcg ggtgaccatt accgccgata aaagcacctc caccgcctac 240

atggagctca gttccctgag aagcgaggat acagccgtgt actactgtgc caggaggtac 300

tttcggtacg actactggta tagcgacgtc tgggggcaag gcacaactgt cacagtgagc 360

agc 363

<210> 130

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 130

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys His Gln Gly Asn Thr Leu Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 131

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 131

gacatccaaa tgacccagag cccttcctcc ttgtccgcaa gtgtgggaga tagagtgacc 60

atcacctgca gggcttctca ggatatctcc aactacctga actggtatca gcagaagccc 120

ggcaaggtgc caaagctcct tatttactac acctcccggc tgcacagcgg agtcccatct 180

cgcttcagcg ggtcaggcag cggcactgac tttactctga caattagcag cctccagcct 240

gaagacgtcg ccacttacta ctgtcatcag gggaatacac tccccctgac attcgggcag 300

gggacaaaac tggagattaa g 321

<210> 132

<211> 116

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 132

Gln Met Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Thr

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Ser Tyr

20 25 30

Asn Met Tyr Trp Val Arg Gln Ala Arg Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asp Pro Tyr Asn Ala Asp Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Arg Asp Met Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Leu Thr Thr Ala Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 133

<211> 348

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 133

caaatgcagc tcgtccagtc tggacctgaa gtgaagaagc ccgggacatc cgtcaaggtc 60

tcatgtaagg ctagcgggta cgcattcact tcctacaaca tgtactgggt gcgccaggcc 120

agaggacaga ggttggagtg gatcggctac atcgacccat acaacgccga tactacctac 180

aatcagaagt ttaaagggcg ggtgaccatt acccgggata tgtccacctc caccgcctac 240

atggagctga gcagcctgag gagcgaggac acagccgtgt actactgcct gacaacagcc 300

tatgccatgg actattgggg ccagggcaca cttgtgactg tgagcagt 348

<210> 134

<211> 106

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 134

Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Thr Val Asn Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys His Gln Trp Ser Ser Tyr Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 135

<211> 318

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 135

gacatccagc tcacccaatc cccttctttc ctctccgcaa gtgtcggaga tagggtgact 60

atcacctgct cagcttcttc aaccgtgaac tacatgcatt ggtaccagca gaagcccgggg 120

aaagccccaa agctgctgat ctacagcacc tccaatctgg ccagtggagt gccaagccgg 180

tttagcggga gcggctccgg cactgaattc actttgacaa ttagcagcct tcagcctgag 240

gactttgcca catattactg tcaccagtgg tccagctacc cctacacatt cgggcagggc 300

acaaagctgg agattaag 318

<210> 136

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 136

Gly Tyr Thr Phe Thr Asp Tyr Val Ile His

1 5 10

<210> 137

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 137

Arg Ser Ser Gln Ser Leu Glu Asn Ser Asn His Asn Thr Tyr Ile Asn

1 5 10 15

<210> 138

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 138

Arg Val Ser Lys Arg Phe Ser

15

<210> 139

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 139

His Gln Val Thr His Val Pro Tyr Thr

15

<210> 140

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 140

Gln Ile Tyr Pro Gly Asp Asp Asp Thr Lys Tyr Ser Gly Lys Phe Lys

1 5 10 15

gly

<210> 141

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 141

Tyr Ile Asp Pro Tyr Asn Ala Asp Thr Thr Tyr Asn Gln Lys Phe Lys

1 5 10 15

gly

<---

Claims (65)

1. Anti-CD19 antibody or antigen-binding fragment containing: a) heavy chain CDR sequences comprising SEQ ID NO:13, SEQ ID NO:141 and SEQ ID NO:15, and kappa light chain CDR sequences comprising SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18; b) heavy chain CDR sequences comprising SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, and kappa light chain CDR sequences comprising SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6; c) heavy chain CDR sequences comprising SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, and kappa light chain CDR sequences comprising SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12; d) heavy chain CDR sequences comprising SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15, and kappa light chain CDR sequences comprising SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18; e) heavy chain CDR sequences comprising SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21, and kappa light chain CDR sequences comprising SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6; f) heavy chain CDR sequences comprising SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24, and kappa light chain CDR sequences comprising SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6; g) heavy chain CDR sequences comprising SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:27, and kappa light chain CDR sequences comprising SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6; h) heavy chain CDR sequences comprising SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30, and kappa light chain CDR sequences comprising SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6; i) heavy chain CDR sequences comprising SEQ ID NO:31, SEQ ID NO:32 and SEQ ID NO:33, and kappa light chain CDR sequences comprising SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6; j) heavy chain CDR sequences comprising SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36, and kappa light chain CDR sequences comprising SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6; k) heavy chain CDR sequences comprising SEQ ID NO:37, SEQ ID NO:38 and SEQ ID NO:39, and kappa light chain CDR sequences comprising SEQ ID NO:40, SEQ ID NO:41 and SEQ ID NO:42; l) heavy chain CDR sequences comprising SEQ ID NO:43, SEQ ID NO:44 and SEQ ID NO:45, and kappa light chain CDR sequences comprising SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6; m) heavy chain CDR sequences comprising SEQ ID NO:136, SEQ ID NO:2 and SEQ ID NO:3, and kappa light chain CDR sequences comprising SEQ ID NO:137, SEQ ID NO:138 and SEQ ID NO:139; or n) heavy chain CDR sequences comprising SEQ ID NO:7, SEQ ID NO:140 and SEQ ID NO:9, and kappa light chain CDR sequences comprising SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12. 2. The antibody or its antigen-binding fragment according to claim 1, containing: a) a heavy chain variable region comprising SEQ ID NO:132 and a kappa light chain variable region comprising SEQ ID NO:134; b) a heavy chain variable region comprising SEQ ID NO:94 and a kappa light chain variable region comprising SEQ ID NO:96; c) a heavy chain variable region comprising SEQ ID NO:98 and a kappa light chain variable region comprising SEQ ID NO:100; d) a heavy chain variable region comprising SEQ ID NO:102 and a kappa light chain variable region comprising SEQ ID NO:104; e) a heavy chain variable region comprising SEQ ID NO:106 and a kappa light chain variable region comprising SEQ ID NO:96; f) a heavy chain variable region comprising SEQ ID NO:108 and a kappa light chain variable region comprising SEQ ID NO:96; g) a heavy chain variable region comprising SEQ ID NO:110 and a kappa light chain variable region comprising SEQ ID NO:96; h) a heavy chain variable region comprising SEQ ID NO:112 and a kappa light chain variable region comprising SEQ ID NO:96; i) a heavy chain variable region comprising SEQ ID NO:114 and a kappa light chain variable region comprising SEQ ID NO:96; j) a heavy chain variable region comprising SEQ ID NO:116 and a kappa light chain variable region comprising SEQ ID NO:96; k) a heavy chain variable region comprising SEQ ID NO:118 and a kappa light chain variable region comprising SEQ ID NO:120; l) a heavy chain variable region comprising SEQ ID NO:122 and a kappa light chain variable region comprising SEQ ID NO:96; m) a heavy chain variable region comprising SEQ ID NO:124 and a kappa light chain variable region comprising SEQ ID NO:126; or n) a heavy chain variable region comprising SEQ ID NO:128 and a kappa light chain variable region comprising SEQ ID NO:130. 3. An antibody or antigen-binding fragment thereof according to claim 2, additionally containing one or more amino acid residue substitutions, but at the same time retaining a specific binding affinity for CD19, where the substitution takes place in one or more CDR sequences and/or in one or more FR sequences and/or in one or both sequences of the variable region. 4. An antibody or antigen-binding fragment thereof according to claim 1, further comprising an immunoglobulin constant region, optionally an IgG constant region, optionally a human IgG1 constant region. 5. An antibody or antigen-binding fragment thereof according to claim 1, which is a humanized antibody. 6. An antibody or antigen-binding fragment thereof according to claim 1, which is a bispecific antibody, camel single domain antibody, diabody, scFv, scFv dimer, BsFv, dsFv, (dsFv) ₂ , dsFv-dsFv', Fv fragment, Fab, Fab' , F(ab') ₂ , bispecific antibody, diabody, nanobody, domain antibody, or bivalent single domain antibody. 7. An isolated polynucleotide sequence encoding an antibody or antigen-binding fragment thereof according to claim 1. 8. An expression vector containing the isolated polynucleotide according to claim 7. 9. A host cell for producing an antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the cell contains the vector according to claim 8. 10. A method for expressing an antibody or antigen-binding fragment thereof according to claim 1, comprising culturing a host cell containing a vector according to claim 8 containing an isolated polynucleotide sequence encoding an antibody or antigen-binding fragment thereof according to claim 1 under conditions under which the vector is expressed. 11. An antibody-drug conjugate for delivery of cytotoxic agents to tumors, comprising one or more drug molecules covalently linked to the antibody or antigen-binding fragment of claim 1, directly or through a linker, where the drug is a cytotoxin or a radioactive isotope. 12. A pharmaceutical composition for the treatment of a CD19-related disease or condition in an individual, wherein the composition comprises an antibody or antigen-binding fragment thereof according to claim 1 or an antibody-drug conjugate containing one or more drug moieties covalently attached to the antibody or antigen-binding fragment thereof 1, and a pharmaceutically acceptable carrier. 13. A method of treating a CD19-associated disease or condition in an individual, comprising administering to the individual a therapeutically effective amount of the antibody or antigen-binding fragment of claim 1, an antibody-drug conjugate containing one or more drug groups covalently attached to the antibody or its antigen-binding fragment according to claim 1. 14. Chimeric antigen receptor (CAR) for stimulating a T-cell immune response to a CD19-expressing target in a patient, containing an antigen-binding fragment according to claim 1 and a T-cell activation fragment, where the T-cell activation fragment contains a transmembrane domain and an intracellular signaling domain. transduction of the T-cell receptor, where the transmembrane domain contains the transmembrane domain alpha, beta or zeta of the T cell receptor chain, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 or CD154. 15. A nucleic acid sequence molecule encoding a CAR according to claim 14 and containing the polynucleotide sequence of SEQ ID NOs: 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123 , 125, 127, 129, 131, 133 and/or 135, and a polynucleotide sequence encoding a T cell activation fragment, where the T cell activation fragment contains a transmembrane domain and an intracellular signal transduction domain of the T cell receptor, where the transmembrane domain contains the transmembrane domain of the alpha, beta or zeta chain of the T cell receptor, CD28, CD3-epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 or CD154. 16. An expression vector containing the nucleic acid sequence of claim 15. 17. An isolated T cell for stimulating a T cell immune response to a CD19 expressing target in an individual, comprising the nucleic acid sequence of claim 15 or the expression vector of claim 16, wherein the cell expresses the CAR of claim 14. 18. A method of stimulating a T cell immune response to a CD19 expressing target in an individual, wherein the method comprises administering to the individual an effective amount of T cells according to claim 17. 19. A kit for detecting the presence or amount of CD19 in a sample, or for diagnosing a CD19-associated disease or condition in an individual, wherein the kit contains the antibody or antigen-binding fragment of claim 1.

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